U.S. patent application number 13/965090 was filed with the patent office on 2014-05-01 for non-invasive measurement of analytes.
This patent application is currently assigned to Cercacor Laboratories, Inc.. The applicant listed for this patent is Cercacor Laboratories, Inc.. Invention is credited to Robert L. Coleman, Christopher R. Lambert, Jerome J. Workman.
Application Number | 20140120564 13/965090 |
Document ID | / |
Family ID | 34280264 |
Filed Date | 2014-05-01 |
United States Patent
Application |
20140120564 |
Kind Code |
A1 |
Workman; Jerome J. ; et
al. |
May 1, 2014 |
NON-INVASIVE MEASUREMENT OF ANALYTES
Abstract
This invention provides devices, compositions and methods for
determining the concentration of one or more metabolites or
analytes in a biological sample, including cells, tissues, organs,
organisms, and biological fluids. In particular, this invention
provides materials, apparatuses, and methods for several
non-invasive techniques for the determination of in vivo blood
glucose concentration levels based upon the in vivo measurement of
one or more biologically active molecules found in skin.
Inventors: |
Workman; Jerome J.;
(Madison, WI) ; Lambert; Christopher R.; (Hudson,
MA) ; Coleman; Robert L.; (Olive Hill, KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cercacor Laboratories, Inc. |
Irvine |
CA |
US |
|
|
Assignee: |
Cercacor Laboratories, Inc.
Irvine
CA
|
Family ID: |
34280264 |
Appl. No.: |
13/965090 |
Filed: |
August 12, 2013 |
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Application
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11349731 |
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8509867 |
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13965090 |
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10952538 |
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11153263 |
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10712669 |
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10952538 |
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10617915 |
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10712669 |
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10616533 |
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10617915 |
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60516352 |
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Current U.S.
Class: |
435/14 ;
435/288.7; 549/227 |
Current CPC
Class: |
A61B 5/418 20130101;
A61B 5/14532 20130101; A61K 49/0052 20130101; A61B 5/1455 20130101;
A61B 5/415 20130101; A61K 49/0041 20130101; Y10T 436/144444
20150115; A61B 5/14539 20130101; A61B 5/413 20130101; C12Q 1/54
20130101 |
Class at
Publication: |
435/14 ; 549/227;
435/288.7 |
International
Class: |
C12Q 1/54 20060101
C12Q001/54 |
Claims
1. A sensor composition comprising at least one small molecule
metabolic reporter (SMMR), wherein the composition is applied to at
least one surface of living tissue, organs, interstitial fluid, and
whole organisms and transported into the tissue at an effective
concentration, wherein when the at least one small molecule
metabolic reporter is brought in contact with one or more specific
metabolites, a change in fluorescence or absorption of the at least
one small molecule metabolic reporter occurs, thereby allowing
quantification of the change in fluorescence or absorption, thereby
providing detailed in vivo information regarding picomolar through
millimolar levels of cellular metabolites and metabolic precursors
in the living tissue, organs, interstitial fluid, and whole
organisms.
2. The sensor composition of claim 1, wherein the at least one
small molecule metabolic reporter is selected from the group
consisting of a fluorophore, a protein labeled fluorophore, a
protein comprising a photooxidizable cofactor, a protein comprising
another intercalated fluorophore, a mitochondrial vital stain or
dye, and a dye exhibiting at least one of a redox potential, a
membrane localizing dye, a dye with energy transfer properties, a
pH indicating dye, a coumarin dye, a derivative of a coumarin dye,
an anthraquinone dye; a cyanine dye, an azo dye; a xanthene dye; an
arylmethine dye; a pyrene derivative dye and a ruthenium bipyridyl
complex dye.
3. The sensor composition of claim 1, wherein the one or more
specific metabolites are selected from the group consisting of
glucose, lactate, H.sup.+, Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+,
ATP, ADP, P.sub.i, glycogen, pyruvate, NAD(P)+, NAD(P)H, FAD,
FADH.sub.2, and O.sub.2.
4. The sensor composition of claim 1, wherein the information
obtained is selected from the group consisting of assessment of
metabolic function; diagnosis of metabolic disease state;
monitoring and control of disease state; stress status of cells,
tissues and organs; determination of vitality and viability of
cells based on metabolic function; critical care monitoring;
determination of metabolic concentration; cancer diagnosis; cancer
detection; cancer staging; and cancer prognosis.
5. The sensor composition of claim 4, wherein the assessment of
metabolic function provides detailed information about glucose
metabolism, fructose metabolism or galactose metabolism.
6. The sensor composition of claim 4, wherein the diagnosis of
metabolic disease state provides detailed information on
advanced-glycosolated end products.
7. The sensor composition of claim 4, wherein the monitoring and
control of diseases is related to diabetes, cancer, stress or organ
transplantation.
8. The sensor composition of claim 2, wherein the mitochondrial
vital stain or dye is a polycyclic aromatic hydrocarbon dye
selected from the group consisting of: rhodamine 123; di-4-ANEPPS;
di-8-ANEPPS; DiBAC.sub.4(3); RH421; tetramethylrhodamine ethyl
ester, perchlorate; tetramethylrhodamine methyl ester, perchlorate;
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
3,3'-dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide; nonylacridine orange; dihydrorhodamine 123
dihydrorhodamine 123, dihydrochloride salt; xanthene;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
benzenedicarboxylic acid; 2(or
4)[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide.
9. The sensor composition of claim 2, wherein the protein labeled
fluorophore is at least one of Glucose Oxidase-Labeled Fluorophore
(GO-LF) and Glucose Oxidase-Intercalated Fluorophore (GO-IF).
10. The sensor composition of claim 2, wherein the protein
comprising a photooxidizable cofactor is Glucose Oxidase (GOx) with
a flavin adenine dinucleotide (FAD) in the triplet state
(GOx-.sup.3FAD*).
11. The sensor composition of claim 1, wherein the sensor
composition is formulated as a cream, emulsion, ointment, oil,
disposable gel film patch, reservoir device or paint.
12. The sensor composition of claim 1, wherein the sensor
composition is transported within the skin using at least one
technique selected from the group consisting of: electroporation,
solvent transport, tattooing, injecting, and passive transport.
13. The sensor composition of claim 1, wherein the quantification
of the change in fluorescence or absorption is monitored using
fluorescence or absorption spectroscopy.
14. The sensor composition of claim 1, wherein the effective
concentration is selected from the group consisting of at least
between 0.01 to 500 .mu.g/ml, between 0.1 to 500 .mu.g/ml, between
1.0 to 150 .mu.g/ml, between 1 to 100 .mu.g/ml, and between 10 to
100 .mu.g/ml.
15. The sensor composition of claim 1, wherein a specific
application comprises a 5 .mu.L volume of a 400 .mu.M SMMR
solution, or a 10 .mu.L volume at 200 .mu.M concentration.
16. The sensor composition of claim 1, wherein the one or more
metabolites directly report on and relate to in vivo blood glucose
levels.
17. The sensor composition of claim 1, wherein the at least one
small molecule metabolic reporter is chosen based on one or more
properties selected from the group consisting of molecular size,
charge, structure, pKa, solubility, polarity, and solvent system
used to transport the one or more small molecule metabolic
reporters to living tissue.
18. A method for identifying a small molecule metabolic reporter
(SMMR) suitable for use in a sensor composition, the method
comprising: delineating the one or more metabolites required to
characterize a selected metabolic pathway in a living system;
determining a basic mechanism of action for the SMMR; selecting one
or more wavelength choices for excitation and emission of the SMMR
for analysis of absorption and fluorescence measurements; selecting
a molecular structure to meet quantum efficiency and yield
requirements; selecting location, diffusion rate, and duration or
lifetime of the SMMR within a tissue or organ layers; selecting
toxicity requirements and limitations; and optionally relating
measured real-time metabolic conditions to disease state for
diagnostics or patient care, thereby identifying a small molecule
metabolic reporter for use in a sensor composition.
19. An in vivo method for determining the metabolic health and
well-being in living organisms, the method comprising: applying at
least one small molecule metabolic reporter (SMMR) to a surface of
an organ for a predetermined period of time; causing penetration of
the SMMR to a depth of about 10 .mu.m to about 300 .mu.m;
monitoring a change in the fluorescence or absorption based upon
peripheral or epithelial tissue metabolite levels; and correlating
the metabolite levels within peripheral or epithelial tissue with
cellular metabolite levels, thereby determining the metabolic
health and well-being in living organisms.
20. An in vivo method for monitoring and controlling disease states
that affect metabolic processes in living organisms, the method
comprising: applying at least one small molecule metabolic reporter
(SMMR) to at least one surface of a living tissue, organs, and/or
whole organisms for a predetermined period of time; causing
penetration of the SMMR to a depth of about 10 .mu.m to about 300
.mu.m; monitoring a change in the fluorescence or absorption based
upon peripheral or epithelial tissue metabolite levels; and
correlating the metabolite levels within peripheral or epithelial
tissue with cellular metabolite levels, thereby monitoring and
controlling disease states that affect metabolic processes in
living organisms.
21. The method of claim 20, wherein the disease states are selected
from the group consisting of diabetes, diabetes progression, aging,
critical care states, organ transplantation, tissue and cell
viability and vitality, cancer diagnosis, cancer detection, cancer
staging and cancer prognosis.
22. An in vivo method for monitoring the concentration of one or
more metabolites or analytes, the method comprising: applying at
least one small molecule metabolic reporter (SMMR) to at least one
surface of a living tissue, organs, and/or whole organisms for a
predetermined period of time; causing penetration of the SMMR to a
depth of about 10 .mu.m, wherein said depth corresponds with the
bottom of the dead stratum corneum layer, to about 175 .mu.m,
wherein said depth corresponds with the top of the dermal layer,
into the epidermis; and monitoring a change in the concentration of
the one or more metabolites or analytes in a metabolic pathway by
detecting changes in the at least one SMMR at one or more time
points using an optical reader.
23. The method of claim 22, wherein the SMMR comprises a
mitochondrial stain sensitive to membrane potential or chemical
gradient.
24. The method of claim 23, wherein the mitochondrial stain is a
polycyclic aromatic hydrocarbon dye selected from the group
consisting of: rhodamine 123; di-4-ANEPPS; di-8-ANEPPS;
DiBAC.sub.4(3); RH421; tetramethylrhodamine ethyl ester,
perchlorate; tetramethylrhodamine methyl ester, perchlorate;
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
3,3'-dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide; nonylacridine orange; dihydrorhodamine 123
dihydrorhodamine 123, dihydrochloride salt; xanthene;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
benzenedicarboxylic acid; 2(or
4)-[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide.
25. The method of claim 22, wherein the at least one SMMR comprises
a dye or stain that transfers energy from a molecule generated as a
result of the oxidative metabolic pathway and that has a
stoichiometric or highly correlated relationship with glucose
concentration.
26. The method of claim 22, wherein the at least one SMMR comprises
a dye selected from the group consisting of: coumarin; derivatives
of coumarin; anthraquinones; cyanine dyes; azo dyes; xanthene dyes;
arylmethine dyes; pyrene derivatives; and ruthenium bipyridyl
complexes.
27. The method of claim 22, wherein the at least one SMMR comprises
a protein labeled fluorophore.
28. The method of claim 27, wherein the protein labeled fluorophore
is at least one of Glucose Oxidase-Labeled Fluorophore (GO-LF) and
Glucose Oxidase-Intercalated Fluorophore (GO-IF).
29. The method of claim 22, wherein the at least one SMMR comprises
a protein comprising a photooxidizable cofactor.
30. The method of claim 29, wherein the protein comprising a
photooxidizable cofactor is Glucose Oxidase (GOx) with a flavin
adenine dinucleotide (FAD) in the triplet state
(GOx-.sup.3FAD*).
31. The method of claim 22, wherein the one or more metabolites or
analytes is selected from the group consisting of: glucose;
lactate; hydrogen ion (H.sup.+); calcium ion (Ca.sup.2+) pumping
rate; magnesium ion (Mg.sup.2+) pumping rate; sodium ion (Na.sup.+)
pumping rate; potassium ion (K.sup.+) pumping rate; adenosine
triphosphate (ATP); adenosine diphosphate (ADP); the ratio of ATP
to ADP; inorganic phosphate (P.sub.i); glycogen; pyruvate;
nicotinamide adenine dinucleotide phosphate, oxidized form
(NAD(P)+); nicotinamide adenine dinucleotide phosphate, reduced
form (NAD(P)H); flavin adenine dinucleotide, oxidized form (FAD);
and flavin adenine dinucleotide, reduced form (FADH.sub.2); and
oxygen (O.sub.2) utilization.
32. The method of claim 22, where monitoring the change in
metabolite or analyte concentration comprises detecting at least
one wavelength above 350 nm.
33. The method of claim 22, wherein the SMMR is formulated as a
cream, emulsion, ointment, oil, disposable gel film patch,
reservoir device or paint.
34. The method of claim 22, wherein the SMMR penetrates within the
skin using at least one technique selected from the group
consisting of: electroporation, solvent transport, tattooing,
injecting, and passive transport.
35. The method of claim 22, wherein the quantification of the
change in fluorescence or absorption is monitored using
fluorescence or absorption spectroscopy.
36. An in vivo method for measuring metabolite levels, said method
comprising monitoring in a population of cells one or more relevant
metabolites, parameters or analytes in at least one metabolic
pathway, wherein the monitoring comprises measuring the
fluorescence spectrum emitted by at least one small molecule
metabolic reporter (SMMR), wherein at least one fluorescence
spectrum emitted by the SMMR is stoichiometrically related to the
metabolite, parameter or analyte concentration in the population of
cells, whereby analyzing the relatedness provides the in vivo
metabolite level.
37. The method of claim 36, wherein the population of cells has a
predominantly glycolytic metabolism or can be induced to have a
glycolytic metabolism.
38. The method of claim 37, wherein the population of cells is
located in the epidermis, wherein the epidermis comprises a
dynamic, metabolically homogeneous, and homeostatic population of
cells.
39. The method of claim 37, wherein the population of cells having
a glycolytic metabolism comprise live keratinocytes.
40. The method of claim 39, wherein the live keratinocytes are
present in the epidermal layer of skin.
41. The method of claim 40, wherein the live keratinocytes are
present at a depth from the surface of the skin from about 10
.mu.m, wherein said depth corresponds with the bottom of the dead
stratum corneum layer, to about 175 .mu.m, wherein said depth
corresponds with the top of the dermal layer.
42. The method of claim 36, wherein the metabolic pathway is
monitored within the population of cells via measurement of one or
more specific metabolite or analyte of the glycolytic pathway that
has a stoichiometric or highly correlated relationship with glucose
concentration.
43. The method of claim 42, wherein the one or more relevant
metabolites or analytes are selected from the group consisting of:
glucose; lactate; hydrogen ion (H.sup.+); calcium ion (Ca.sup.2+)
pumping rate; magnesium ion (Mg.sup.2+) pumping rate; sodium ion
(Na.sup.+) pumping rate; potassium ion (K.sup.+) pumping rate;
adenosine triphosphate (ATP); adenosine diphosphate (ADP); the
ratio of ATP to ADP; inorganic phosphate (P.sub.i); glycogen;
pyruvate; nicotinamide adenine dinucleotide phosphate, oxidized
form (NAD(P)+); nicotinamide adenine dinucleotide (phosphate),
reduced form (NAD(P)H); flavin adenine dinucleotide, oxidized form
(FAD); flavin adenine dinucleotide, reduced form (FADH.sub.2); and
oxygen (O.sub.2) utilization.
44. The method of claim 36, wherein the metabolic pathway is
monitored within the population of cells, said monitoring
comprising measuring a physico-chemical parameter that is related
to the glycolytic pathway, wherein said parameter comprises a
stoichiometric or highly correlated relationship with glucose
concentration.
45. The method of claim 36, wherein the population of cells
comprises a predominantly oxidative metabolism or can be induced to
comprise a metabolism predominantly based on oxidative
phosphorylation.
46. The method of claim 45, wherein the metabolic pathway is
monitored within the population of cells, said monitoring
comprising measuring a metabolite or analyte that is generated as a
result of the oxidative metabolic pathway, wherein said metabolite
or analyte comprises a stoichiometric or highly correlated
relationship with glucose concentration.
47. The method of claim 45, wherein the metabolic pathway is
monitored within the population of cells, said monitoring
comprising measuring a physico-chemical parameter that is generated
as a result of the oxidative metabolic pathway and that comprises a
stoichiometric or highly correlated relationship with glucose
concentration.
48. A noninvasive method for monitoring in vivo blood glucose
levels, the method comprising: applying at least one small molecule
metabolic reporter (SMMR) to at least one surface of skin for a
predetermined period of time; causing penetration of the one or
more SMMR to a depth of about 10 .mu.m, wherein said depth
corresponds with the bottom of the dead stratum corneum layer, to
about 175 .mu.m, wherein said depth corresponds with the top of the
dermal layer, into the epidermis; contacting the one or more SMMR
with one or more metabolites or analytes; monitoring a change in
the concentration of the one or more metabolites or analytes by
detecting changes in the SMMR using an optical reader, and
correlating the change in the concentration of the one or more
metabolites or analytes with in vivo blood glucose levels.
49. The method of claim 48, wherein the at least one small molecule
metabolic reporter is selected from the group consisting of a
fluorophore, a protein labeled fluorophore, a protein comprising a
photooxidizable cofactor, a protein comprising another intercalated
fluorophore, a mitochondrial vital stain or dye, and a dye
exhibiting one or more of a redox potential, a membrane localizing
dye, a dye comprising energy transfer properties, a pH indicating
dye, a coumarin dye, a derivative of a coumarin dye, an
anthraquinone dye; a cyanine dye, an azo dye; a xanthene dye; an
arylmethine dye; a pyrene derivative dye and a ruthenium bipyridyl
complex dye.
50. The method of claim 48, wherein the one or more specific
metabolites are selected from the group consisting of glucose,
lactate, H.sup.+, Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, ATP,
ADP, P.sub.i, glycogen, pyruvate, NAD(P)+, NAD(P)H, FAD,
FADH.sub.2, and O.sub.2.
51. The method of claim 49, wherein the protein labeled fluorophore
is Glucose Oxidase-Labeled Fluorophore (GO-LF) and the metabolite
is glucose.
52. The method of claim 49, wherein the protein comprising a
photooxidizable cofactor is Glucose Oxidase (GOx) with a flavin
adenine dinucleotide (FAD) in the triplet state
(GOx-.sup.3FAD*).
53. The method of claim 49, wherein the mitochondrial vital stain
or dye is at least one polycyclic aromatic hydrocarbon dye selected
from the group consisting of: Rhodamine 123, Di-4-ANEPPS;
Di-8-ANEPPS, DiBAC.sub.4(3), RH421, Tetramethylrhodamine ethyl
ester, perchlorate, Tetramethylrhodamine methyl ester, perchlorate,
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide,
3,3'-Dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide, Nonylacridine Orange, Dihydrorhodamine 123 and
Dihydrorhodamine 123, dihydrochloride salt; xanthene;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
benzenedicarboxylic acid; 2(or
4)-[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide.
54. The method of claim 49, where monitoring the change in the one
or more metabolite or analyte concentrations comprises measuring at
least one spectral emission at a wavelength above 450 nm.
55. A reagent strip for use in a glucose measuring instrument
comprising: a polymer strip; and a known concentration of at least
one small molecule metabolic reporter (SMMR), wherein when a sample
of a biological fluid containing an amount of glucose is interacted
with the reagent strip, a change in fluorescence or absorption of
the at least one SMMR occurs, wherein said change is measured by
the glucose measuring instrument, thereby detecting the glucose
concentration of the biological fluid.
56. The reagent strip of claim 55, wherein the at least one SMMR is
selected from the group consisting of Glucose Oxidase-Labeled
Fluorophore (GO-LF) and Glucose Oxidase (GOx) with a flavin adenine
dinucleotide (FAD) in the triplet state (GOx-.sup.3FAD*).
57. The reagent strip of claim 55, wherein the change in
fluorescence or absorption is monitored using fluorescence or
absorption spectroscopy.
58. A reagent strip for use in calibrating a glucose measuring
instrument comprising: a polymer strip; a known concentration of at
least one small molecule metabolic reporter (SMMR); and at least
one sample containing a known concentration of glucose, wherein
when the at least one sample is interacted with the reagent strip,
a change in fluorescence or absorption of the at least one SMMR
occurs, wherein said change is measured by the glucose measuring
instrument and wherein the calculated glucose is compared to the
known concentration, thereby calibrating the instrument.
59. The reagent strip of claim 58, wherein the at least one SMMR is
selected from the group consisting of Glucose Oxidase-Labeled
Fluorophore (GO-LF) and Glucose Oxidase (GOx) with a flavin adenine
dinucleotide (FAD) in the triplet state (GOx-.sup.3FAD*).
60. The reagent strip of claim 59, wherein the change in
fluorescence or absorption is monitored using fluorescence or
absorption spectroscopy.
61. A sensor system, the system comprising: a device comprising a
component that transmits radiation to a material or tissue, a
component that detects radiation emitted from a material or tissue,
and a component to display the detection results, wherein each
component is operably linked; an applicator that delivers the
sensor composition of claim 1 to the material or tissue; and an
interface between the device and the material or tissue, wherein
the interface measures a resulting excitation radiation emitted
from the irradiated sensor composition.
62. The sensor system of claim 61, wherein said system comprises a
device that emits radiation at one or more wavelengths chosen to
specifically excite the sensor composition that is applied to the
material or tissue, wherein the sensor composition comprises at
least one small molecule metabolic reporter (SMMR), wherein the
sensor composition is present at a depth from the surface of the
skin of about 10 .mu.m, wherein said depth corresponds with the
bottom of the dead stratum corneum layer, to about 175 .mu.m,
wherein said depth corresponds with the top of the dermal layer, in
the epidermis at an effective concentration for detection of one or
more metabolites or analytes in a biological sample.
63. The sensor system of claim 62, wherein said system detects
radiation at one or more wavelengths chosen to specifically
identify remitted energy fluorescence scattered to the system from
the sensor composition.
64. A method for determining in vivo blood glucose concentration,
comprising the steps of: performing an instrument response
measurement on a calibration target and recording the response
data; applying at least one SMMR mixture to the skin in a first
controlled area such that the SMMR resides in the epidermal layer
of the skin; applying a second SMMR mixture to the skin in a second
controlled area; perturbing the second area such that one or more
extreme changes that the mixture may undergo are achieved;
performing a calibration measurement on the perturbed area and
recording the calibration data; performing a background measurement
on an area of skin that has no SMMR and recording this background
data; illuminating the first area with light and performing a first
measurement on the first area; detecting at least one wavelength
spectrum of light reflected back from the first area; performing at
least a second measurement on the first area at wavelengths
suitable for each SMMR present; calculating at least one parameter
from the response data to normalize the background data,
calibration data and measurement data for the response using a
spectrometer; calculating at least one parameter from the
background data to correct the calibration data and measurement
data for emission, absorption and scattering properties of the
tissue; and calculating at least one metabolite parameter from the
calibration data to relate the measurement data to the blood
glucose concentration; thereby determining in vivo blood glucose
concentration.
65. The method of claim 64, wherein the one or more extreme changes
is a change in concentration of the metabolite or analyte between a
zero or low measurable concentration and a saturation level or high
measurable concentration.
66. A method of calculating a blood glucose concentration, said
method comprising: measuring at least one background response and
at least one autofluorescence tissue response from a calibration
target comprising an epidermal layer of skin; providing a first
SMMR mixture to a first skin location and causing portions of the
first SMMR mixture to transfer into the epidermal layer of the
skin; providing a second SMMR mixture to a second skin location and
causing and recording at least one extreme change in the mixture;
illuminating the first skin location with a radiative emission;
detecting at least one resulting wavelength spectrum reflected from
the first skin location; optionally repeating the illuminating and
detecting steps using at least one irradiation and wavelength
spectrum associated with each SMMR provided; and detecting at least
one physico-chemical parameter that is related to the glycolytic
pathway, wherein said parameter comprises a stoichiometric or
highly correlated relationship with glucose concentration; thereby
determining the blood glucose concentration.
67. The method of claim 66, wherein the SMMR mixture comprises a
bloodless calibration procedure as outlined in one or more of
equations 13, 16, 17, 18, 19, 20 or 21.
68. The method of claim 66, wherein the at least one extreme change
is a change in the blood glucose concentration between a zero or
low measurable concentration and a saturation level or high
measurable concentration.
69. A method for determining the concentration of at least one
metabolite or analyte in skin tissue, the method comprising:
administering to the skin tissue at least one small molecule
metabolite reporter (SMMR) agent; causing penetration of the at
least one SMMR agent to a region of the skin at a depth between the
dermis and the epidermis, wherein the depth from the surface of the
skin is from about 10 .mu.m, wherein said depth corresponds with
the bottom of the dead stratum corneum layer, to about 175 .mu.m,
wherein said depth corresponds with the top of the dermal layer;
irradiating the at least one SMMR agent in the skin tissue with a
source of electromagnetic radiation; measuring at least one
fluorescence spectrum emitted from the at least one SMMR agent; and
analyzing the emitted fluorescence spectra; wherein the analysis
results in a determination of the concentration of the metabolite
or analyte.
70. The method of claim 63, wherein the measuring of at least one
fluorescence spectrum comprises a bloodless calibration procedure
as outlined in one or more of equations 13, 16, 17, 18, 19, 20 and
21.
71. The method of claim 32, where monitoring the change in
metabolite or analyte concentration comprises detecting at least
one wavelength above 450 nm.
Description
RELATED APPLICATIONS
[0001] This invention is a continuation in part of U.S. Ser. No.
10/617,915, filed on Jul. 10, 2003, which is a continuation in part
of U.S. Ser. No. 10/616,533, filed on Jul. 9, 2003, which claims
priority to U.S. provisional patent application Ser. No.
60/425,488, filed Nov. 12, 2002, and this application also claims
priority to Ser. No. 60/438,837, filed Jan. 9, 2003, Ser. No.
60/439,395, filed Jan. 10, 2003, Ser. No. 60/447,603, filed Feb.
13, 2003, and Ser. No. ______, filed on Oct. 31, 2003, each of
which is incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention provides devices, compositions and methods
for determining the concentration of one or more metabolites or
analytes in a biological sample, including cells, tissues, organs,
organisms, and biological fluids. In particular, this invention
provides materials, apparatuses, and methods for several
non-invasive techniques for the determination of in vivo blood
glucose concentration levels based upon the in vivo measurement of
one or more biologically active molecules found in skin.
BACKGROUND OF THE INVENTION
[0003] Identifying and understanding the risk factors associated
with diabetes is invaluable for the development and evaluation of
effective intervention strategies.
[0004] Lacking normal regulatory mechanisms, diabetics are
encouraged to strive for optimal control through a modulated life
style approach that focuses on dietary control, exercise, and
glucose self-testing with the timely administration of insulin or
oral hypoglycemic medications. Invasive forms of self-testing are
painful and fraught with a multitude of psychosocial hurdles, and
are resisted by most diabetics. Alternatives to the currently
available invasive blood glucose testing are highly desirable.
[0005] Conventional approaches to non-invasive alternatives seek to
reduce or eliminate the skin trauma, pain, and blood waste
associated with traditional invasive glucose monitoring
technologies. In general, though never effectively demonstrated
prior to this invention, noninvasive optical blood glucose
monitoring requires no bodily fluid samples be withdrawn from
tissue and involves external irradiation with electromagnetic
radiation and measurement of the resulting optical flux (e.g.,
fluorescence or diffuse reflectance). In theory, but not in
practice, glucose levels would be derived from the spectral
information following comparison to reference spectra for glucose
and background interferants, reference calibrants, and/or
application of advanced signal processing mathematical
algorithms.
[0006] Radiation-based technologies, which are often referred to as
potential candidates for solving the non-invasive glucose problem,
have included variations of sampling and data processing methods
including: 1) mid-infrared (MIR) spectroscopy, 2) near-infrared
radiation (NIR) spectroscopy, 3) radio wave impedance, 4)
autofluorescence and white light scattering, and 5) Raman
spectroscopy. Each of these methods uses optical sensors and relies
on the premise that the absorption or fluorescence pattern of
electromagnetic radiation can be quantitatively related to a change
in blood glucose concentration. However, other endogenous
substances including, but not limited to, water, lipids, proteins,
and hemoglobin are known to absorb energy, particularly infrared
light and can easily obscure the relatively weak glucose
signal.
[0007] Other approaches to non-invasive glucose measurements are
based on microvascular changes in the retina, acoustical impedance,
nuclear magnetic resonance (NMR) spectroscopy and optical hydrogels
that quantify glucose levels in tear fluid. While putatively
non-invasive, these technologies have yet to be demonstrated as
effective in clinical testing.
[0008] Nearly noninvasive techniques tend to rely on interstitial
fluid extraction from skin. This can be accomplished using
permeability enhancers, sweat inducers, and/or suction devices with
or without the application of electrical current. One device
recently approved by the FDA relies on reverse iontophoresis,
utilizing an electrical current applied to the skin. The current
pulls out salt, which carries water, which, in turn, carries
glucose. The glucose concentration of this recovered fluid is
measured and is proportional to that of blood. In keeping with its
nearly noninvasive description, this technology is commonly
associated with some discomfort and requires at least twice daily
calibrations against conventional blood glucose measurements (e.g.
invasive lancing).
[0009] Other nearly noninvasive blood glucose monitoring techniques
similarly involve transcutaneous harvesting for interstitial fluid
measurement. Other technologies for disrupting the skin barrier to
obtain interstitial fluid include: 1) dissolution with chemicals;
2) microporation with a laser; 3) penetration with a thin needle;
and/or 4) suction with a pump. Minimally invasive blood glucose
monitoring can also involve the insertion of an indwelling glucose
monitor under the skin to measure the interstitial fluid glucose
concentration. These monitors typically rely on optical or
enzymatic sensors. Although technologically innovative, these in
situ sensors have had limited success. Implantable glucose oxidase
("GO") sensors have been limited by local factors causing unstable
signal output, whereas optical sensors must overcome signal
obfuscation by blood constituents as well as interference by
substances with absorption spectra similar to glucose. Moreover,
inflammation associated with subcutaneous monitoring may contribute
to systematic errors requiring repositioning, recalibration or
replacement, and more research is needed to evaluate the effects of
variable local inflammation at the sensor implantation site on
glucose concentration and transit time.
[0010] Interstitial fluid glucose concentrations have previously
been shown to be similar to simultaneously measured fixed or
fluctuating blood glucose concentrations. See, e.g., Bantle et al.,
Journal of Laboratory and Clinical Medicine 130:436-441, 1997;
Sternberg et al., Diabetes Care 18:1266-1269, 1995. Such studies
helped validate noninvasive/minimally invasive technologies for
blood glucose monitoring, insofar as many of these technologies
measure glucose in blood as well as interstitial fluid.
[0011] A noninvasive glucose monitor that is portable, simple and
rapid to use, which provides accurate clinical information is
desirable. In particular, the ability to derive first and second
order information in real-time for dynamic glucose metabolism, such
as the direction and rate of change of bioavailable glucose
distributed within the blood and interstitial fluid space, would be
extremely important for continuous and discrete glucose
monitoring.
SUMMARY OF THE INVENTION
[0012] The methods and compositions of the present invention
effectively determine the glucose concentration in blood for a
living organism by non-invasive, in vivo measurement of the glucose
level in skin by means of fluorescence measurements of metabolic
indicators/reporters of glucose metabolism. Disclosed are dyes used
as metabolic indicators that allow for specific in vivo monitoring
of metabolites, which are used as indicators of metabolic activity.
Dyes characterized by this invention are referred to herein as a
small molecule metabolite reporters ("SMMRs").
[0013] This invention also provides for fluorescence measurements
of extracellular and intracellular reporter molecules placed into
the cytosol, nucleus, or organelles of cells within intact, living,
tissue that track the concentration of blood glucose in an
organism. When any one of a series of metabolites is measured using
this technique, the molar concentration of blood glucose can be
calculated. Direct or indirect fluorescence measurements of glucose
using one or more of the following measurements is described: pH
(as lactate/H), membrane reduction-oxidation electric potential,
NAD(P)H (nicotinamide adenine dinucleotide (phosphate), reduced
form) for energy transfer, FAD.sup.+ (flavin adenine dinucleotide,
oxidized form) for energy transfer, ATP/ADP ratio,
Ca.sup.2+-pumping rate, Mg.sup.2+-pumping rate, Na.sup.+-pumping
rate, K.sup.+-pumping rate, and vital mitochondrial membrane
stains/dyes/molecules fluorescence response. These analytes,
measured in skin using the techniques taught herein, are used to
provide a complete picture of epidermal skin glycolytic metabolism
where local epidermal analyte (glucose) quantities are proportional
to the concentration of glucose in systemic blood, specifically the
capillary fields within the papillary layer of the dermis (corium).
Temperature and/or nitric oxide measurement may also be combined
with the above measurements for better calibration and
determination of glucose concentrations.
[0014] The invention further provides sensor compositions that are
applied to at least one surface of living tissue, organs,
interstitial fluid, and whole organisms and transported into the
tissue at an effective concentration. The sensor composition can
include at least one small molecule metabolic reporter (SMMR) at an
effective concentration such that when the at least one SMMR is
brought in contact with one or more specific metabolites or
analytes, a change in fluorescence or absorption occurs, thereby
allowing quantification of the change in fluorescence or
absorption.
[0015] For example, the at least one small molecule metabolic
reporter used in the sensor composition can be a fluorophore, a
protein labeled fluorophore, a protein comprising a photooxidizable
cofactor, a protein comprising another intercalated fluorophore; a
mitochondrial vital stain or dye, a dye exhibiting at least one of
a redox potential, a membrane localizing dye, a dye with energy
transfer properties, a pH indicating dye; a coumarin dye, a
derivative of a coumarin dye, an anthraquinone dye, a cyanine dye,
an azo dye, a xanthene dye, an arylmethine dye, a pyrene derivative
dye, or a ruthenium bipyridyl complex dye.
[0016] Examples of suitable mitochondrial vital stains or dyes
include, but are not limited to, a polycyclic aromatic hydrocarbon
dye, such as, for example, rhodamine 123; di-4-ANEPPS; di-8-ANEPPS;
DiBAC.sub.4(3); RH421; tetramethylrhodamine ethyl ester,
perchlorate; tetramethylrhodamine methyl ester, perchlorate;
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
3,3'-dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide; nonylacridine orange; dihydrorhodamine 123
dihydrorhodamine 123, dihydrochloride salt; xanthene;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
benzenedicarboxylic acid; 2(or
4)[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide.
[0017] Examples of suitable protein labeled fluorophores include,
but are not limited to, Glucose Oxidase-Labeled Fluorophore (GO-LF)
and Glucose Oxidase-Intercalated Fluorophore (GO-IF). Examples of a
suitable protein include a photooxidizable cofactor includes
Glucose Oxidase (GOx) with a flavin adenine dinucleotide (FAD) in
the triplet state (GOx)-.sup.3FAD*).
[0018] The one or more specific metabolites or analytes to be
detected in a surface of living tissue, organs, interstitial fluid,
and whole organisms include, for example, glucose, lactate,
H.sup.+, Ca.sup.2+, Mg.sup.2+, Na.sup.+, K.sup.+, ATP, ADP,
P.sub.i, glycogen, pyruvate, NAD(P).sup.+, NAD(P)H, FAD,
FADH.sub.2, and O.sub.2.
[0019] The in vivo information obtained when the SMMR is brought in
contact with the one or more metabolites or analytes can include,
but is not limited to, assessment of metabolic function; diagnosis
of metabolic disease state; monitoring and control of disease
state; stress status of cells, tissues and organs; determination of
vitality and viability of cells based on metabolic function;
critical care monitoring; diagnosis and monitoring of
cardiovascular diseases, autoimmune disorders, neurological
disorders, degenerative diseases; determination of metabolic
concentration; and cancer diagnosis, detection, staging and
prognosis.
[0020] For example, the in vivo information obtained may provide
detailed information on glucose metabolism, fructose metabolism and
galactose metabolism; advanced-glycosolated end products;
monitoring and control of diseases such as diabetes, cancer, stress
and organ transplantation.
[0021] The sensor compositions used in these methods for monitoring
the concentration of one or more metabolite(s) or analyte(s) can be
formulated as, but are not limited to, emulsions, ointments,
disposable gel film patches, reservoir devices, creams, paints,
polar solvents, non-polar solvents, or any combination thereof.
[0022] Penetration of the sensor composition can be accomplished
using an active transport technique or a passive transport
technique, such as, for example, electroporation, laser poration,
sonic poration, ultrasonic poration, iontophoresis,
mechanical-poration, solvent transport, tattooing, wicking,
microneedle or pressurized delivery. In addition, penetration of
the sensor composition to the desired depth can be accomplished by
combining the composition with various molecular size
attachments.
[0023] Typically, the quantification of the change in fluorescence
or absorption is monitored using fluorescence or absorption
spectroscopy.
[0024] An effective concentration of the sensor composition is, for
example, at least between 0.01 to 500 .mu.g/ml, between 0.1 to 500
.mu.g/ml, between 1.0 to 150 .mu.g/ml, between 1 to 100 .mu.g/ml,
and between 10 .mu.M to 100 .mu.g/ml. The SMMR can be introduced in
a low concentration in a range from 10 .mu.M to 1000 .mu.M and in a
volume from 200 .mu.L to 0.1 .mu.L, respectively (e.g., introducing
the SMMR at a concentration in the range of 200 .mu.L of a 10 .mu.M
SMMR solution to 0.1 .mu.l of a 1000 .mu.M SMMR solution). One
specific application of the sensor composition is, for example, a 5
.mu.L volume of a 400 .mu.M SMMR solution, or a 10 .mu.L volume at
200 .mu.M concentration.
[0025] The one or more metabolite(s) or analyte(s) can directly
report on, and/or relate to, in vivo blood glucose levels. Suitable
metabolites or analytes include any of the metabolites or analytes
listed herein.
[0026] The SMMR may be chosen based on one or more properties
selected from the group consisting of molecular size, charge,
structure, pKa, solubility, polarity, and solvent system used to
transport the one or more small molecule metabolic reporters to
living tissue.
[0027] The invention also provides methods for identifying a small
molecule metabolic reporter (SMMR). According to these methods, one
or more metabolites required to characterize a selected metabolic
pathway in a living system are delineated. A basic mechanism of
action for the SMMR is determined. One or more wavelength choices
for excitation and emission of the SMMR are selected by analysis of
absorption and fluorescence measurements. A molecular structure to
meet quantum efficiency and yield requirements is selected, as well
as location, diffusion rate, and duration or lifetime of the SMMR
within a tissue or organ layers, as well as toxicity requirements
and limitations. Optionally, measured real-time metabolic
conditions are related to disease state for diagnosis or patient
care.
[0028] The invention also provides in vivo methods for determining
the metabolic health and well-being in living organisms by applying
at least one small molecule metabolic reporter (SMMR) to a surface
of an organ for a predetermined period of time. The SMMR penetrates
to a depth of about 10 .mu.m to about 300 .mu.m. A change in the
fluorescence or absorption is measured based upon peripheral or
epithelial tissue metabolite levels. The metabolite levels within
peripheral or epithelial tissue are then correlated with cellular
metabolite levels.
[0029] Also provided by this invention are in vivo methods for
monitoring and controlling disease states that affect metabolic
processes in living organisms. According to these methods, at least
one small molecule metabolic reporter (SMMR) is applied to at least
one surface of a living tissue, organs, and/or whole organisms for
a predetermined period of time. The SMMR penetrates to a depth of
about 10 .mu.m to about 300 .mu.m. A change in the fluorescence or
absorption is monitored based upon peripheral or epithelial tissue
metabolite levels. The metabolite levels within peripheral or
epithelial tissue is then correlated with cellular metabolite
levels.
[0030] For example, disease states may include diabetes, diabetes
progression, aging, critical care states, organ transplantation,
tissue and cell viability and vitality, cardiovascular disease,
autoimmune disorders, neurological disorders, degenerative disease;
and cancer diagnosis, detection, staging and prognosis.
[0031] Also provided are in vivo methods for monitoring the
concentration of one or more metabolites or analytes. According to
these methods, at least one small molecule metabolic reporter
(SMMR) is applied to at least one surface of a living tissue,
organs, and/or whole organisms for a predetermined period of time.
The SMMRs then penetrate to a depth of about 10 .mu.m, wherein the
depth corresponds with the bottom of the dead stratum corneum
layer, to about 175 .mu.m, wherein the depth corresponds with the
top of the dermal layer, into the epidermis. A change in the
concentration of the one or more metabolites or analytes in a
metabolic pathway is monitored by detecting changes in the at least
one SMMR at one or more time points using an optical reader by
detecting at least one wavelength above 350 nm.
[0032] The SMMR can include a mitochondrial vital stain or dye
sensitive to membrane potential or chemical gradient. For example,
the mitochondrial stain can be a polycyclic aromatic hydrocarbon
dye, such as, for example, rhodamine 123; di-4-ANEPPS; di-8-ANEPPS;
DiBAC.sub.4(3); RH421; tetramethylrhodamine ethyl ester,
perchlorate; tetramethylrhodamine methyl ester, perchlorate;
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
3,3'-dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide; nonylacridine orange; dihydrorhodamine 123
dihydrorhodamine 123, dihydrochloride salt; xanthene;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
benzenedicarboxylic acid; 2(or
4)-[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide.
[0033] The SMMR can include a dye or stain that transfers energy
from a molecule generated as a result of the oxidative metabolic
pathway and that has a stoichiometric or highly correlated
relationship with glucose concentration.
[0034] Alternatively, the SMMR includes a dye selected from the
group consisting of: coumarin; derivatives of coumarin;
anthraquinones; cyanine dyes; azo dyes; xanthene dyes; arylmethine
dyes; pyrene derivatives; and ruthenium bipyridyl complexes.
[0035] The SMMR may be a protein labeled fluorophore. For example,
Glucose Oxidase-Labeled Fluorophore (GO-LF) and Glucose
Oxidase-Intercalated Fluorophore (GO-IF). The SMMR may also be a
protein comprising a photooxidizable cofactor, such as, for example
Glucose Oxidase (GOx) with a flavin adenine dinucleotide (FAD) in
the triplet state (GOx-.sup.3FAD*).
[0036] The one or more metabolites or analytes that can be
monitored can be one of glucose; lactate; hydrogen ion (H.sup.+);
calcium ion (Ca.sup.2+) pumping rate; magnesium ion (Mg.sup.2+)
pumping rate; sodium ion (Na.sup.+) pumping rate; potassium ion
(K.sup.+) pumping rate; adenosine triphosphate (ATP); adenosine
diphosphate (ADP); the ratio of ATP to ADP; inorganic phosphate
(P.sub.i); glycogen; pyruvate; nicotinamide adenine dinucleotide
phosphate, oxidized form (NAD(P)+); nicotinamide adenine
dinucleotide phosphate, reduced form (NAD(P)H); flavin adenine
dinucleotide, oxidized form (FAD); and flavin adenine dinucleotide,
reduced form (FADH.sub.2); and oxygen (O.sub.2) utilization.
[0037] The SMMRs of the invention can be formulated as emulsions,
ointments, disposable gel film patches, reservoir devices, creams,
paints, polar solvents, non-polar solvents, or any combination
thereof.
[0038] Penetration of the SMMR can be accomplished using an active
transport technique or a passive transport technique, such as, for
example, electroporation, laser poration, sonic poration,
ultrasonic poration, iontophoresis, mechanical-poration, solvent
transport, tattooing, wicking, microneedle or pressurized delivery.
In addition, penetration of the sensor composition to the desired
depth can be accomplished by combining the composition with various
molecular size attachments.
[0039] The invention also provides in vivo methods for measuring
metabolite levels by monitoring in a population of cells one or
more relevant metabolites, parameters or analytes in at least one
metabolic pathway, wherein the monitoring involves measuring the
fluorescence spectrum emitted by at least one small molecule
metabolic reporter (SMMR), wherein at least one fluorescence
spectrum emitted by the SMMR is stoichiometrically related to the
metabolite, parameter or analyte concentration in the population of
cells, whereby analyzing the relatedness provides the in vivo
metabolite level.
[0040] The population of cells can have a predominantly glycolytic
metabolism, or alternatively, the population of cells can be
induced to have a glycolytic metabolism. The population of cells in
the skin can be located in the epidermis, which contains a dynamic,
metabolically homogeneous, and homeostatic population of cells. For
example, the population of cells having a glycolytic metabolism can
include live keratinocytes. These live keratinocytes can be present
in the epidermal layer of skin. In some cases, the live
keratinocytes can be present in the skin at a depth, from the
surface of the skin, of about 10 .mu.m, which corresponds to the
bottom of the dead stratum corneum layer, to about 175 .mu.m, which
corresponds to the top of the dermal layer.
[0041] The metabolic pathways monitored within the population of
cells, according to these methods for measuring in vivo blood
glucose levels through the skin, can be monitored by measuring a
specific metabolite or analyte of the glycolytic pathway, wherein
the specific metabolite or analyte has a known stoichiometric or
highly correlated relationship with glucose concentration. The
metabolic pathways can also be monitored within the population of
cells by observing a physico-chemical parameter that is related to
the glycolytic pathway, wherein the selected physico-chemical
parameter has a stoichiometric or highly correlated relationship
with glucose concentration.
[0042] For example, the relevant metabolites or analytes that are
monitored in these methods for measuring in vivo blood glucose
levels through the skin can be lactate; hydrogen ion (H.sup.+);
calcium ion (Ca.sup.2+) pumping rate; magnesium ion (Mg.sup.2+)
pumping rate; sodium ion (Na.sup.+) pumping rate; potassium ion
(K.sup.+) pumping rate; adenosine triphosphate (ATP); adenosine
diphosphate (ADP); the ratio of ATP to ADP; inorganic phosphate
(P.sub.i); glycogen; pyruvate; nicotinamide adenine dinucleotide
phosphate, oxidized form (NAD(P)+); nicotinamide adenine
dinucleotide (phosphate), reduced form (NAD(P)H); flavin adenine
dinucleotide, oxidized form (FAD); flavin adenine dinucleotide,
reduced form (FADH.sub.2); or oxygen (O.sub.2) utilization.
[0043] The population of cells to be monitored in these methods for
measuring in vivo blood glucose levels through the skin can have a
predominantly oxidative metabolism, or alternatively, the
population of cells can be induced to have a metabolism
predominantly based on oxidative phosphorylation. The metabolic
pathways monitored within the population of cells can be monitored
by measuring a metabolite or analyte that is generated as a result
of the oxidative metabolic pathway, wherein the specific metabolite
or analyte has a stoichiometric or highly correlated relationship
with glucose concentration. Alternatively, the metabolic pathways
can be monitored within the population of cells by observing a
physico-chemical parameter that is generated as a result of the
oxidative metabolic pathway, wherein the physico-chemical parameter
has a stoichiometric or highly correlated relationship with glucose
concentration.
[0044] Also provided are noninvasive methods for monitoring in vivo
blood glucose levels. According to these methods at least one small
molecule metabolic reporter (SMMR) is applied to at least one
surface of skin for a predetermined period of time causing
penetration of the one or more SMMRs to a depth of about 10 .mu.m,
wherein the depth corresponds with the bottom of the dead stratum
corneum layer, to about 175 .mu.m, wherein the depth corresponds
with the top of the dermal layer, into the epidermis. The one or
more SMMRs come in contact with one or more metabolites or analytes
and a change in the concentration of the one or more metabolites or
analytes is monitored by detecting changes in the SMMRs using an
optical reader. The change in the concentration of the one or more
metabolites or analytes is then correlated with in vivo blood
glucose levels.
[0045] The at least one small molecule metabolic reporter can be
selected from the group consisting of a fluorophore, a protein
labeled fluorophore, a protein comprising a photooxidizable
cofactor, a protein comprising another intercalated fluorophore, a
mitochondrial vital stain or dye, and a dye exhibiting one or more
of a redox potential, a membrane localizing dye, a dye comprising
energy transfer properties, a pH indicating dye, a coumarin dye, a
derivative of a coumarin dye, an anthraquinone dye; a cyanine dye,
an azo dye; a xanthene dye; an arylmethine dye; a pyrene derivative
dye and a ruthenium bipyridyl complex dye.
[0046] The one or more specific metabolites can be selected from
the group consisting of glucose, lactate, H.sup.+, Ca.sup.2+,
Mg.sup.2+, Na.sup.+, K.sup.+, ATP, ADP, P.sub.i, glycogen,
pyruvate, NAD(P)+, NAD(P)H, FAD, FADH.sub.2, and O.sub.2.
[0047] For example, when the SMMR is a protein labeled fluorophore,
Glucose Oxidase-Labeled Fluorophore (GO-LF) is used and the
metabolite monitored is glucose.
[0048] Alternatively, the SMMR can be a protein comprising a
photooxidizable cofactor such as Glucose Oxidase (GOx) with a
flavin adenine dinucleotide (FAD) in the triplet state
(GOx-.sup.3FAD*).
[0049] The SMMRs used in these methods for monitoring in vivo blood
glucose levels can include, for example, a mitochondrial stain
sensitive to membrane potential or chemical gradient. Examples of
suitable mitochondrial stains include a polycyclic aromatic
hydrocarbon dye, such as, for example, rhodamine 123; di-4-ANEPPS;
di-8-ANEPPS; DiBAC.sub.4(3); RH421; tetramethylrhodamine ethyl
ester, perchlorate; tetramethylrhodamine methyl ester, perchlorate;
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
3,3'-dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide; nonylacridine orange; dihydrorhodamine 123
dihydrorhodamine 123, dihydrochloride salt; xanthene;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
benzenedicarboxylic acid; 2(or
4)-[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide. Monitoring the change in the one or
more metabolite or analyte concentrations can be accomplished by
measuring at least one spectral emission at a wavelength above 350
nm.
[0050] Also included in the invention is a reagent strip for use in
a glucose measuring instrument comprising a polymer strip and a
known concentration of at least one small molecule metabolic
reporter (SMMR), wherein when a sample of a biological fluid
containing an amount of glucose is interacted with the reagent
strip, a change in fluorescence or absorption of the one or more
molecular sensor proteins occurs, and the change is measured by the
glucose measuring instrument, thereby detecting the glucose
concentration of the biological fluid.
[0051] The at least one SMMR can be selected from Glucose
Oxidase-Labeled Fluorophore (GO-LF), Glucose Oxidase-Intercalated
Fluorophore (GO-IF) and Glucose Oxidase (GOx) with a flavin adenine
dinucleotide (FAD) in the triplet state (GOx-.sup.3FAD*).
[0052] The change in fluorescence or absorption can be monitored
using fluorescence or absorption spectroscopy. Those of ordinary
skill in the art will recognize that any fluorescence or absorption
spectroscopic techniques can be used in accordance with the
invention.
[0053] Also provided is a reagent strip for use in calibrating a
glucose measuring instrument comprising a polymer strip, a known
concentration of at least one small molecule metabolic reporter
(SMMR), and at least one sample containing a known concentration of
glucose, wherein when the at least one sample is interacted with
the reagent strip, a change in fluorescence or absorption of the
one or more molecular sensor proteins occurs, wherein the change is
measured by the glucose measuring instrument, thereby calibrating
the instrument.
[0054] The at least one SMMR may be selected from the group
consisting of Glucose Oxidase-Labeled Fluorophore (GO-LF) and
Glucose Oxidase (GOx) with a flavin adenine dinucleotide (FAD) in
the triplet state (GOx-.sup.3FAD*).
[0055] The change in fluorescence or absorption can be monitored
using fluorescence or absorption spectroscopy.
[0056] The invention also provides sensor systems that include a
device having a component that transmits radiation to a material or
tissue, a component that detects radiation emitted from a material
or tissue, and a component to display the detection results, each
component is operably linked. The sensor systems further include an
applicator that delivers the sensor composition of the invention to
the material or tissue. Typically, there is an air interface
between the device and the material or tissue, wherein the air
interface measures a resulting excitation radiation emitted from
the irradiated sensor composition.
[0057] The device included in the sensor system can emit radiation
at one or more wavelengths that have been chosen to specifically
excite the SMMR mixture that is applied to the material or tissue.
The sensor composition can include a reporter dye and a marker dye,
or alternatively, a dye exhibiting a wavelength shift in absorption
or fluorescence emission in the presence of a metabolite. The
sensor composition can be present at a depth from the surface of
the skin of about 10 .mu.m to about 175 .mu.m in the epidermis in a
concentration that is effective for detection of one or more
metabolites or analytes in a biological sample.
[0058] The sensor system can detect radiation at one or more
wavelengths that have been chosen to specifically identify
fluorescence emission that has been scattered back to the system
from the sensor composition.
[0059] The invention also provides additional methods for
determining in vivo blood glucose concentration. According to these
methods, an instrument response measurement is performed on a
calibration target, and the response data is recorded. At least one
SMMR mixture is applied to the skin in a first, controlled area,
such that the SMMR resides in the epidermal layer of the skin, and
a second SMMR mixture is applied to the skin in a second controlled
area. The second area is perturbed, such that one or more extreme
changes that the mixture may undergo is achieved. A calibration
measurement is performed on the perturbed area, and the calibration
data is recorded. A background measurement is made on an area of
skin that has no SMMR, and this background data is recorded. A
measurement on the first area is performed by illuminating the
first area with light, and at least one wavelength spectrum of
light reflected back from the first area is detected. Further
measurements on the first area are performed at wavelengths
suitable for each SMMR present. At least one parameter from the
response data is calculated to normalize the background data,
calibration data and measurement data for the response of the
spectrometer. At least one parameter from the background data is
calculated to correct the calibration data and measurement data for
emission, absorption and scattering properties of the tissue. At
least one metabolite parameter from the calibration data is
calculated to relate the measurement data to the blood glucose
Concentration, thereby determining in vivo blood glucose
concentration. The one or more extreme changes can be, for example,
a change in concentration of the metabolite or anal yte between a
zero or low measurable concentration and a saturation level or high
measurable concentration.
[0060] The invention also provides methods of calculating blood
glucose concentration. According to these methods, at least one
background response and at least one autofluorescence tissue
response and measured from a calibration target comprising an
epidermal layer of skin. A first SMMR mixture is provided to a
first skin location, and portions of the first SMMR mixture are
transferred into the epidermal layer of the skin. A second SMMR
mixture is provided to a second skin location, and at least one
extreme change in the mixture is triggered and recorded. The
extreme change can be, for example, a change in concentration of
the analyte comprising a zero or low measurable concentration and a
saturation level or high measurable concentration. These extremes
are used to calibrate the sensor enabling it to measure a test
sample accurately with a concentration between the extremes. See
e.g., equations (13) through (21), as described herein. The first
skin location is illuminated with a radiative emission, and a
resulting wavelength spectrum reflected from the first skin
location is detected. The illuminating and detecting can be
repeated using at least one irradiation and wavelength spectrum
associated with each SMMR provided. At least one physico-chemical
parameter that is related to the glycolytic pathway is detected.
Preferably, the physico-chemical parameter has a stoichiometric or
highly correlated relationship with glucose concentration, which is
used in determining the blood glucose concentration. The sensor
system can include a bloodless calibration procedure such as, for
example, the procedure(s) outlined in equations 13, 16, 17, 18, 19,
20 or 21 set forth herein.
[0061] The invention also provides methods for determining the
concentration of at least one metabolite or analyte in skin tissue.
According to these methods, a small molecule metabolite reporter
(SMMR) agent is administered to the skin tissue. The SMMR agent
penetrates to a region of the skin at a depth between the dermis
and the epidermis, wherein the depth from the surface of the skin
is from about 10 .mu.m to about 175 .mu.m. The SMMR agent is
irradiated with a source of electromagnetic radiation, and the
fluorescence spectra emitted from the SMMR agent are detected. The
emitted fluorescence spectra are then analyzed, which results in a
determination of the concentration of the metabolite or analyte.
Measuring the fluorescence spectra according to these methods can
include a bloodless calibration procedure, such as, the
procedure(s) outlined in equations 13, 16, 17, 18, 19, 20 and 21
set forth herein.
[0062] Unless otherwise defined, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which the invention belongs. Specific
technical and scientific terms used herein have the following
meanings:
[0063] As used herein and in the claims, the singular forms "a",
"and" and "the" include plural referents unless the context clearly
dictates otherwise. For example, the term small molecule metabolic
reporter "SMMR" includes one or more small molecule metabolic
reporters "SMMRs". Those skilled in the art will recognize that the
terms "SMMR" and "SMMRs" are used interchangeably herein.
[0064] As used herein, the term "biologically active molecule"
includes, but is not limited to, enzymes, coenzymes, metabolites,
analytes, reactive species, polypeptides, proteins, cofactors,
small molecules and other macromolecules of physiological
significance including mixtures or active fragments or subunits
thereof. A "small molecule" is defined as a molecule from 100 Da to
250 kDa. Molecules of this molecular weight range have a
demonstrated ability for use as quantitative reporters of glucose
activity.
[0065] The terms "small molecule metabolic reporter(s)", "SMMR
(s)", "analyte enhancing molecules", "reporter" and "reporters"
include, but are not limited to, fluorophores, protein-labeled
fluorophores, proteins with a photooxidizable cofactor (such as
FADH contained in a glucose oxidase), and proteins with another
intercalated fluorophore.
[0066] As used herein, a "chromophore" is defined as a molecule
exhibiting specific absorption or fluorescence emission when
excited by energy from an external source. This is a more generic
term than fluorophore.
[0067] As used herein, a "fluorophore" is defined as a molecule
exhibiting specific fluorescence emission when excited by energy
from an external source.
[0068] As used herein, an "intercalated fluorophore" is defined as
a fluorophore that will fluoresce when intercalated with a
molecule. For example, Glucose Oxidase-Intercalated Fluorophore
(GO-IF) is a molecule with specific glucose binding sites. The
fluorescent properties will change when glucose binds to the
molecule, causing a measurable change.
[0069] As used herein, a "dye" is defined as a molecule having
large absorptivity or high fluorescence quantum yield and which
demonstrates affinity for certain materials or organic (cellular)
structures.
[0070] As used herein, a "xanthene dye" is defined as a molecule
having a xanthene-like skeletal structure, which exhibits large
absorptivity and high fluorescence quantum yield and which
demonstrates affinity for certain materials or organic (cellular)
structures.
[0071] The phrase "energy transfer from reducing equivalents (e.g.,
NAD/NADH, NAD(P)/NAD(P)H, FAD/FADH.sub.2) indicating SMMRs" refers
to a use of SMMRs whereby the presence of these reducing
equivalents molecules, is detected by excitation of the reducing
equivalents molecules from an external source, energy transfer from
the reducing equivalents molecule(s) to an SMMR, and detection of
the fluorescence emission at the SMMR emission wavelength.
[0072] The phrase "transmembrane redox potential indicating SMMRs"
refers to the use of SMMRs to indicate the degree of
reduction-oxidation electric potential occurring within the cell,
including such organelle structures as the inner mitochondrial
membrane. In one such case, the degree of reduction-oxidation
electric potential is indicated by the number of SMMR molecules
bound to the inner mitochondrial membrane. In this case, SMMR
binding is proportional to the membrane potential as indicated by
quantitative fluorescence quenching. Thus, an increase in glucose
brings about an increase in glycolysis and membrane potential,
thereby reducing the fluorescence signal. This phrase refers to the
generic use of SMMRs as a means for detecting intracellular
reduction-oxidation electric potential.
[0073] The phrase "mitochondrion-selective vital SMMRs" refers to
SMMRs that bind selectively to the inner mitochondrial membrane of
living cells.
[0074] The phrase "pH:lactate/H.sup.+ indicating SMMRs" refers to
SMMRs that report on the local intra- or extracellular environment
with respect to hydrogen ion concentration, pH, or lactate
concentration.
[0075] The phrase "enzyme-based SMMR, including a fluorescent
protein SMMR" refers to a protein-based SMMR that is capable of
reacting directly with glucose to form a fluorescence response,
whether measured directly as fluorescence emission intensity or
fluorescence lifetime.
[0076] The phrase "intracellular pH sensitive SMMRs" refers to
SMMRs that enter the cell membrane and report on intracellular pH
within the cytosol. Other pH SMMRs are distinguished, as they
report on organelle pH or extracellular pH, independent of
cytosolic pH.
[0077] The phrase "extracellular pH sensitive SMMRs" refers to
SMMRs that remain on the outside of the cell and report on
extracellular pH within the interstitial fluid or extracellular
environment. Other pH SMMRs are distinguished, as they report on
intracellular pH, independent of extracellular pH.
[0078] The phrase "absorption/diffuse reflection or fluorescence
spectrum" refers to two types of spectra measured independently.
The absorption/diffuse reflection spectrum refers to the energy
reflection spectrum from a material reported in either the
dimensions of reflectance or absorbance versus wavelength. The
fluorescence spectrum is measured independently as the fluorescence
emission intensity or the fluorescence lifetime of a fluorophore
following excitation from an external source.
[0079] The phrase "molecular size attachment" refers to the
molecular size in Angstroms (.ANG.), which is related to molecular
weight in Daltons (Da), of an attachment added as an adjunct to an
SMMR. As used herein, "molecular size attachments" is defined as
adducts to the fluorescent moieties of SMMRs that include, but are
not limited to, structural modifications of fluorescence SMMRs as
the additions to the fluorescence structure of: acetoxy methyl
esters, chloro-methyl derivatives, alkyl chain adducts, highly
charged moieties, enzyme substrate mimics, enzyme cofactor tethers,
and membrane binding tethers.
[0080] As used herein, a "reporter" is defined as an SMMR having
the property of optical or fluorescence signal related to the
quantity of analyte in the immediate vicinity of the SMMR. Thus, as
the analyte quantity increases, the fluorescence signal changes (up
or down) in proportion.
[0081] As used herein, a "marker" is defined as a molecule having
the property of yielding a fluorescence signal that is constant
when applied to target cells or tissues. Its main purpose is for
use as a reference signal channel. As such, it is applied in a
ratiometric measurement for correction of a reporter signal. The
variation in physiological and optical characteristics of
individual subjects requires a reference channel signal to correct
or normalize a reporter channel signal when the ratio of reporter
to marker is used for quantitative applications.
[0082] As used herein, a "sensor" is defined as a handheld device
capable of making absorption or fluorescence measurements at one or
more wavelengths, and converting the ratios and sums of these
measurements into analyte concentrations. These analyte
concentrations are used to infer the rate or quantity of a specific
metabolic process.
[0083] As used herein, a "metabolite" is defined as a substance
produced by a metabolic process, such as glycolysis, which can be
quantitatively measured as an indication of the rate or quantity of
a specific metabolic process.
[0084] As used herein, an "analyte" is defined as a measurable
parameter, using analytical chemistry, which can be quantitatively
measured as an indication of the rate and quantity of a specific
metabolic process. The term analyte is a generic term describing
such concepts as metabolites, ions, processes, conditions,
physico-chemical parameters, or metabolic results that can be used
to infer the rate or quantity of specific metabolic processes.
[0085] As used herein, a "response range" is defined as an analyte
range (lower and upper limits) over which a metabolic process, and
its measured absorption or fluorescence signal, follow a linear or
defined mathematical function.
[0086] The phrase "physico-chemical parameter" refers to a subset
of broadly defined analyte parameters specifically related to the
physical chemistry constants of materials. These constants can be
used in combination with the measurement of other analytes to infer
the rate or quantity of specific metabolic processes. Such
constants refer specifically to, e.g., atomic mass, Faraday
constant, Boltzmann constant, molar volume, dielectric properties,
and the like.
[0087] As used herein, "wicking" is defined as the flow of a liquid
into a solid material via the pull of gravity, Brownian motion,
adhesion, mass transport, or capillary action such that a natural
movement of a liquid occurs into a solid material.
[0088] The phrases "direct metabolic reporters," and "indirect
metabolic reporters" refer to the mechanism of action of SMMR for
reporting glucose concentration. Direct metabolic reporters report
the concentration of glucose directly, whereas indirect metabolic
reporters report the concentration of analytes used to infer the
concentration of glucose.
[0089] As used herein, an "octanol-water coefficient (K.sub.ow)" is
defined as a measure of the extent to which a solute molecule is
distributed between water and octanol in a mixture. The
octanol-water partition coefficient is the ratio of a chemical's
solubility (concentration) in octanol to that in water using a
two-phase mixture at equilibrium.
[0090] As used herein, "toxicity" is defined as the degree or
quality of being toxic or hazardous to the health and well being of
human and other mammalian organisms, organs, tissues, and
cells.
[0091] The phrase "specialized tattoo" or more precisely the
"active viewing window" refers to an area of tissue treated with an
SMMR. That area is used for viewing the fluorescence ratio
measurements of the SMMR interaction with tissue, in order to
directly measure, calculate, or otherwise infer the concentration
of skin and blood glucose or other metabolites of interest.
[0092] As used herein, "organ" is defined as a structure that
contains at least two different types of tissue functioning
together for a common purpose. Examples of organs in the body
include, but are not limited to, the brain, heart, liver, kidneys,
pancreas, stomach, intestines, lungs, skin.
[0093] As used herein, a "keratinocyte" is defined as a living cell
comprising the majority of the epidermis of mammalian skin. The
keratinocyte is unique in both its proximity to the surface of an
organism as well as in its glycolytic behavior. The keratinocyte
metabolizes glucose in such a way as to produce a number of
analytes whereby the glucose concentration within the cell can be
inferred.
[0094] As used herein, "Rt (in ohms)" is defined as the sum of a
5-ohm series resistor and the resistance (impedance) of the skin in
parallel with a 50-ohm resistor. As used herein, "Rskin" is defined
as impedance representing a function of the electrode contact
resistance, the distance between electrodes, and the applied pulse.
Rskin is typically in the range of 30 to 100 kohm/cm.sup.2.
[0095] As used herein, a "mammal" includes both a human and a
non-human mammal (e.g., rabbit, mouse, rat, gerbil, cow, horse,
sheep, etc.). Transgenic animals are also encompassed within the
scope of the term.
[0096] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and not intended to be
limiting. Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0097] FIG. 1 is a schematic showing the preferred location for
small molecule metabolic reporters (SMMRs) as they are introduced
into the stratum germinativum or dermis near the surface of
peripheral tissue or skin using one of many possible techniques
disclosed for monitoring of blood borne metabolites which move to
peripheral cells and tissues;
[0098] FIG. 2A and FIG. 2B are schematics showing the method for
coloring epidermal skin cells (i.e., keratinocytes) of the
fingertip (FIG. 2A) using a sensor composition of the invention,
wherein one or more SMMRs are applied to the skin surface and
transported up to 1500 microns (.mu.m) through the top of the skin
using passive or active transport (FIG. 2B);
[0099] FIG. 3A and FIG. 3B are schematics showing the fluorescence
response to D-glucose using a Lactate/H.sup.+ small molecule
metabolite reporter (FIG. 3A), and the corresponding epidermal
location of the SMMR in the stratum germinativum near the surface
of the skin (FIG. 3B) and demonstrates a spectral response to
changes in D-glucose as measured by lactate/H.sup.+ reporting shown
in FIG. 3A;
[0100] FIG. 4A and FIG. 4B are schematics showing a measurement
technique for determining D-glucose concentrations utilizing one or
more wavelengths. FIG. 4A depicts Reporter and Marker channel
detection using a dual wavelength measurement technique. FIG. 4B
depicts measurement of the Total Integrated Fluorescence Signal
(gray region). The initial signal measured to determine glucose
concentration [Glucose.sub.I] is derived as a function of the ratio
of the fluorescence signal from the reporter to marker such that
[Glucose.sub.I]=f(Reporter/Marker). A multichannel wavelength
correction is applied later. As designated in the FIG. 4A,
FL*=Fluorescence detection;
[0101] FIG. 5A and FIG. 5B are schematics showing a broad
wavelength correction technique for correcting the fluorescence
ratio. Corrected signal for Glucose concentration [Glucose.sub.C]
is a function of the ratio of reporter to marker signal corrected
for variation in reflection (i.e., broad wavelength reflection)
unique for each individual, such that
[Glucose.sub.C]=f(Reporter/Marker).times.(Broad Wavelength
Reflection Correction). FIG. 5A depicts light correction profile
detection. FIG. 5B depicts broad wavelength reflection signal
measurement (gray area). As designated in the FIG. 5A, DR*=Diffuse
Reflection;
[0102] FIG. 6 is a flow chart showing signal processing logic for
determining metabolite levels. The Detector signal (as fluorescence
or diffuse reflectance) is pre-amplified and the initial
calculation is made. One or more of a series of demographic
functions (e.g., empirical modeling of different demographic
clusters of the population, as shown in the figure) are applied to
the initial calculation. A physiological correction is then further
applied, as well as a metabolite model to derive the corrected
metabolite computation (i.e., in a preferred embodiment, glucose
concentration is determined);
[0103] FIG. 7 is a flow chart showing determination of metabolite
concentration. The Detector signal (as fluorescence or diffuse
reflectance) is pre-amplified and the total fluorescence counts are
determined. The initial calculation is made and is corrected using
the diffuse reflection information as per FIGS. 5A and 5B.
Demographic and physiology functions (e.g., empirical modeling of
different Physiological clusters of the population, as shown in the
figure) are then applied to correct for individual skin optical
properties and unique physiology. The corrected metabolite levels
are then subjected to a final correction model relating measured
skin metabolite levels to blood metabolite levels (lag correction).
The result is a blood metabolite computation derived from a
measurement of skin fluorescence (i.e., in a preferred embodiment,
glucose concentration is determined);
[0104] FIG. 8 is a schematic showing blood glucose concentration
determination using measured fluorescence ratio versus D-glucose.
The measured ratio response versus D-glucose changes as a function
of changing blood glucose concentration. Also shown is the
corresponding relative lactate/H.sup.+ concentration;
[0105] FIG. 9A and FIG. 9B are schematics showing blood glucose
concentration results determined for actual versus measured SIAM
ratios for a timed rat-clamp study. Blood glucose concentration
determination using measured fluorescence ratio versus blood
D-glucose ranges from 118 to 249 mg/dL blood D-glucose
concentration obtained using the YSI method (YSI Incorporated, PO
Box 279, Yellow Springs, Ohio 45387 USA) (FIG. 9A). Glucose is
infused at 2:28, 25 g/dL at 7.5 ml/hr. The results of this study
are plotted in FIG. 9B on a standard Clarke Error grid showing all
data points from the experiment in Region A (center diagonal
labeled "A") having 6.76% total error, 1 sigma. As shown, the
Clarke Error grid analysis divides the correlation plot into five
regions. Region A represents glucose values that deviate from the
comparative value by <20%, or are <70 mg/dL when the
comparative value is <70 mg/dL. The B regions (broader center
diagonal labeled "B") represent values that deviate by greater than
20%, and if heeded would lead to benign treatment. Deviations
within Regions A and B are considered clinically acceptable. Region
C (mid-axis near top and bottom labeled "C") values are described
as those deviations that would overcorrect an acceptable glucose.
Region D (mid-axis left and right labeled "D") consists of those
deviation values that would result in a dangerous failure to detect
and treat a blood sugar condition. Region D values below 70 mg/dL
are particularly common among the majority of consumer use glucose
measurement devices. Many home blood glucose meters have up to 20%
of data points in the D region. Region E deviations (left vertical
and bottom labeled "E") are described as those points that if
heeded would result in a potentially erroneous and dangerous
treatment;
[0106] FIG. 10A and FIG. 10B are a schematic and a magnified
insert, respectively, showing how small metabolites present in the
blood are transported from the blood vessels of the dermis into the
interstitial fluid of the epidermis. This occurs as the metabolites
move from small blood vessels in the subcutaneous layers of the
integument into the capillary fields of the dermis. Metabolite
molecules useful for tracking glucose include D-glucose; lactate;
H.sup.+; NAD(P)H; Ca.sup.2+; FAD.sup.+; redox potential
(mitochondrial membrane); ATP/ADP; and O.sub.2 (aerobic). Such
small metabolite molecules move from the capillaries to the
interstitial fluid surrounding the epidermal keratinocytes via mass
transport. Thus the metabolite concentration for interstitial fluid
outside the keratinocytes is proportional to the concentration of
metabolites in peripheral dermal blood vessels. The only exception
to this is oxygen, which decreases with distance from the
subcutaneous blood vessels. At approximately 50 to 100 .mu.m down
from the surface of the skin, there is very little oxygen, which is
why the keratinocytes function using anaerobic glycolysis.
Applications of SMMRs as reporters for blood metabolite and
precursor levels can be inferred from peripheral tissue metabolite
levels (i.e., why measurements of skin are useful for measurement
of some blood metabolites). Small metabolite molecules move from
the capillaries to the interstitial fluid via non-insulin
regulated, concentration dependent, mass transport (i.e., a
diffusion rate of .about.4 to 10% per minute of the difference in
concentration between capillary and skin metabolite levels). The
skin cells transport via GluT1 (GenBank Accession Number: K03195),
not GluT4 (GenBank Accession Number: M91463);
[0107] FIG. 11 is a schematic showing the placement of at least one
SMMR into a keratinocyte. SMMRs are added to the skin surface with,
e.g., a disposable patch, and are passively or actively transported
to a keratinocyte. Indirect Mechanisms 1-3 and Direct Mechanisms
4-5 for fluorescence measurement are further detailed in FIGS.
12-16;
[0108] FIG. 12 is a schematic of SMMR Mechanism 1 for an indirect
energy transfer reporter. SMMR energy transfer reporter mechanism
for fluorescence signal detection is based upon energy transfer
from a metabolite molecule to the SMMR. The metabolite molecule is
excited and transfers energy to the SMMR. Then, the emission from
the SMMR is detected with a sensor. Under conditions where energy
transfer is a significant route for the decay of the excited
metabolite, and where there is present a non-rate-limiting excess
of SMMR, the emission intensity is then proportional to the
concentration of metabolite present;
[0109] FIG. 13 is a schematic of SMMR Mechanism 2 for an indirect
metabolite reporter. SMMR metabolite reporter mechanism for a
fluorescence signal is based upon the influence of a metabolite
molecule on the SMMR. The fluorescent SMMR is excited wherever the
influence of the metabolite alters the fluorescence properties of
the SMMR. This altered fluorescence emission from the SMMR is
detected with a sensor. Where there is a non-rate-limiting excess
of SMMR, the emission intensity is proportional to the
concentration of metabolite present;
[0110] FIG. 14 is a schematic of SMMR Mechanism 3 for an indirect
membrane potential reporter. SMMR membrane potential reporter
mechanism for a fluorescence signal is based upon the fluorescence
properties of an SMMR when bound to a cellular membrane, such as
the inner membrane of mitochondria. A fluorescent SMMR is excited
wherever the influence of the membrane potential at the
membrane-binding site alters the fluorescence properties of the
SMMR. This altered fluorescence emission from the SMMR is detected
with a sensor. Where there is a non-rate-limiting excess of SMMR,
the emission intensity is proportional to the concentration of
metabolite present;
[0111] FIG. 15 is a schematic of SMMR Mechanism 4 for a direct
complex intensity reporter. SMMR direct complex intensity reporter
mechanism for a fluorescence signal is based upon the specific
binding of a metabolite molecule (e.g., D-glucose) into a larger
protein (e.g., enzyme-based) SMMR. The fluorescent protein SMMR is
excited wherever the influence of the specifically bound metabolite
alters the fluorescence properties of the SMMR. This altered
fluorescence emission from the SMMR is detected with a sensor.
Where there is a non-rate-limiting excess of SMMR, the emission
intensity is proportional to the concentration of metabolite
present. This mechanism is effective for intracellular,
extracellular, and in vitro glucose quantitative measurements. This
mechanism could also be useful for various in vitro diagnostic
uses;
[0112] FIG. 16 is a schematic of SMMR Mechanism 5 for a direct
complex lifetime reporter. SMMR direct complex lifetime reporter
mechanism for an absorption signal is based upon the specific
binding of a metabolite molecule (e.g., D-glucose) into a larger
protein (e.g., enzyme-based) SMMR. The protein-based SMMR is
excited by irradiation using modulated light, whereas irradiation
with a second wavelength of light is used to monitor the transient
absorption lifetime using a detection system that can include a
lock-in amplifier. The influence of the specifically bound
metabolite such as glucose alters the excited state lifetime
properties of the SMMR. This altered lifetime from the SMMR is
detected with a sensor. Under conditions where the influence of the
metabolite is significant, and where there is a non-rate-limiting
excess of SMMR, the fluorescence lifetime signal is proportional to
the concentration of metabolite present. As in the case of
Mechanism 4, this mechanism is effective for intracellular,
extracellular, and in vitro glucose quantitative measurements. It
would be clear to one skilled in the art that this mechanism could
also be useful for in vitro diagnostic uses;
[0113] FIGS. 17A, 17B, 17C and 17D are schematics depicting
mechanisms operating in skin metabolism, which are referred to
herein as Scheme 1, Scheme 2, Scheme 3 and Scheme 4, respectively.
FIG. 17A depicts mechanisms operating in skin metabolism and points
of measurement using SMMRs (Scheme 1). FIG. 17B depicts an overview
of the metabolic pathways for glucose in epidermis (Scheme 2). FIG.
17C depicts the structure of a generic chemical backbone for
designing a pH sensitive dye for specific action as a
lactate/H.sup.+ SMMR (Scheme 3). FIG. 17D illustrates fluid issues
related to in vivo skin calibration (Scheme 4);
[0114] FIG. 18 illustrates the X-Ray Crystal structure of glucose
oxidase from Aspergillus niger refined at 2.3 Angstrom
resolution;
[0115] FIG. 19 illustrates the molecular structure of glucose
oxidase and depicts glucose insertion;
[0116] FIG. 20 illustrates the molecular structure of glucose
oxidase and depicts replacement of FAD with reagent FL SubMol
(fluorophore labeled substituent molecule) inclusion;
[0117] FIG. 21 illustrates a generic protein (e.g., an enzyme) with
analyte molecule and SubMol intercalated into the FAD position
(specific case). Note that the SubMol could also be attached to the
periphery of the protein molecule to produce an optical response in
the presence of an analyte molecule (e.g., glucose);
[0118] FIG. 22 is a schematic showing the steps of glucose
metabolism;
[0119] FIG. 23 is a schematic of glucose metabolism showing the
specific analytes where glucose measurements are made for the
invention, shown as bold, underlined and italicized*. SMMR are used
by measuring glucose directly, or by measuring metabolites as
indirect indicators of the quantity of glucose entering the
cellular glycolytic pathway. Such metabolites are described in
detail for the invention and examples are given here as: reducing
equivalents molecules (e.g., NAD(P)H, NADH, FAD, FADH.sub.2);
changes in ATP-driven processes (e.g., cation pumping, transport at
membranes, membrane reduction-oxidation electric potential, and pH
gradient); and stoichiometric products of glucose utilization in
glycolysis (e.g., lactate, hydrogen ion, pH, and pyruvate);
[0120] FIG. 24 is a schematic showing fructose metabolism;
[0121] FIG. 25 is a schematic of fructose metabolism showing the
specific analytes where glucose measurements are made for the
invention, shown as bold, underlined and italicized*. SMMR are used
by measuring glucose directly, or by measuring metabolites as
indirect indicators of the quantity of glucose entering the
cellular glycolytic pathway. Such metabolites are described in
detail for the invention and examples are given here as: reducing
equivalents molecules (e.g., NAD(P)H, NADH, FAD, FADH.sub.2);
changes in ATP-driven processes (e.g., cation pumping, transport at
membranes, membrane reduction-oxidation electric potential, and pH
gradient); and stoichiometric products of glucose utilization in
glycolysis (e.g., lactate, hydrogen ion, pH, and pyruvate);
[0122] FIG. 26 is a schematic showing galactose metabolism;
[0123] FIG. 27 is a schematic of galactose metabolism showing the
specific analytes where glucose measurements are made for the
invention, shown as bold, underlined and italicized*. SMMR are used
by measuring glucose directly, or by measuring metabolites as
indirect indicators of the quantity of glucose entering the
cellular glycolytic pathway. Such metabolites are described in
detail for the invention and examples are given here as: reducing
equivalents molecules (e.g., NAD(P)H, NADH, FAD, FADH.sub.2);
changes in ATP-driven processes (e.g., cation pumping, transport at
membranes, membrane reduction-oxidation electric potential, and pH
gradient); and stoichiometric products of glucose utilization in
glycolysis (e.g., lactate, hydrogen ion, pH, and pyruvate);
[0124] FIG. 28 lists mechanisms of action of the reporters of the
invention. The mechanisms of action are threefold: (1) as a
technology to increase the signal-to-noise of native
autofluorescence signals indicative of human glucose metabolism
[for FAD, NADH, and NAD(P)H], (2) for the enhancement of specific
metabolite and precursor signals in tissue that are indicative of
glucose metabolism and allow determination of changes in blood
glucose [Ca.sup.2+, lactate, oxygen], and (3) as a technology to
directly measure the presence of intracellular or extracellular
molecular glucose [GOx-LF, and GOx-.sup.3FAD*];
[0125] FIG. 29 is a diagram demonstrating the mechanism of action
for energy transfer reporters;
[0126] FIG. 30 is a diagram demonstrating the mechanism of action
for redox potential reporters;
[0127] FIG. 31 is a diagram demonstrating the mechanism of action
for lactate reporters;
[0128] FIG. 32 is a diagram demonstrating the mechanism of action
for ion pump reporters (via calcium ion tracking);
[0129] FIG. 33 is a diagram demonstrating the mechanism of action
for oxygen utilization reporters;
[0130] FIG. 34 is a schematic showing the use of SMMRs to establish
analytical methods for measurement of each glucose pathway for a
variety of cell types;
[0131] FIG. 35 is a diagram summarizing various applications using
the SMMRs of the invention;
[0132] FIG. 36 is a schematic showing the method for adding SMMRs
to peripheral epithelial cells in tissues and organs;
[0133] FIG. 37 is a schematic showing how to use SMMRs for
metabolite or precursor discrimination or imaging (i.e.,
qualitative measurement);
[0134] FIG. 38 is a graph showing how the intensity of fluorescence
is indicative of the glucose concentration and this measurement may
be combined with the dynamic measurements to determine glucose
concentration.
[0135] FIG. 39 is a graph showing phase shift as a function of
transient lifetime at a modulation frequency of 20 kHz using an
instrument that operates in the time domain for quantifying the
triplet state.
DETAILED DESCRIPTION
[0136] The non-invasive devices, compositions, and methods of the
present invention directly yield in vivo information for the
assessment of intracellular and extracellular metabolic state, as
well as the stress status of cells, tissues, and organisms. In a
preferred embodiment, the devices, compositions and methods of the
invention can be used to monitor and determine metabolite
concentration levels, and more specifically, determine blood
glucose concentration levels.
[0137] Truly non-invasive methods require that no device is placed
into or under the tissue; that no probe is used to remove fluid or
to inject materials into the tissue; and that the protective layers
of tissue, such as the stratum corneum of skin, or outer membrane
layers of organs, are not mechanically penetrated or otherwise
physically compromised.
[0138] Procedures that create pores or holes in the tissue for
introducing molecules or extracting fluid are considered somewhat
invasive. Ideally, a non-invasive monitoring device would supply
continuous, accurate monitoring of intracellular activity,
extracellular state, and whole organism or tissue metabolic status.
In this way direct, real-time information regarding tissue, organ,
and organism metabolic status is produced. In contrast, chemical
sensors making measurements of highly buffered and highly regulated
body fluids such as interstitial fluids and blood provide less
responsive, more indirect data regarding tissue and overall subject
status.
[0139] The invention provides non-invasive sensor compositions that
comprise one or more small molecule metabolic reporters ("SMMRs" or
"reporters"). When applied topically to skin, peripheral tissues,
or organs, these reporters are able to penetrate the upper tissue
layers and interact with a specific biologically active molecule in
such a way as to report metabolic or health status, while not
interfering with metabolic function. The reporters provide a
metabolic signal that can be used for multiple purposes including,
but not limited to, assessment of metabolic function (e.g.,
particularly as related to glucose metabolism); diagnosis of
metabolic disease states (e.g., as related to advanced glycosylated
end-products); monitoring and control of disease state; stress
status of cells, tissues and organs; determination of vitality and
viability of cells based on metabolic function; critical care
monitoring; diagnosis and monitoring of cardiovascular diseases,
autoimmune disorders, neurological disorders, degenerative
diseases; determination of metabolic concentration; and cancer
diagnosis, detection, staging and prognosis. Specifically, applying
the reporters of the invention to living peripheral or epithelial
tissue provides detailed information on the state of multiple
metabolic pathways in living organisms that can be analyzed using
low-cost, hand held instrumentation.
[0140] The advantages of the mechanisms of action of the reporters
of the invention are threefold: (1) as a technology to increase the
signal-to-noise of native autofluorescence signals indicative of
human glucose metabolism [for FAD, NADH, and NAD(P)H], (2) for the
enhancement of specific metabolite and precursor signals in tissue
that are indicative of glucose metabolism and allow determination
of changes in blood glucose [Ca.sup.2+, lactate, oxygen], and (3)
as a technology to directly measure the presence of intracellular
or extracellular molecular glucose [GOx-LF, and GOx-.sup.3FAD*].
The mechanisms of action for these small molecule metabolic
reporters are described in FIGS. 28-35.
[0141] The invention provides techniques whereby one or more
reporters are applied to solid tissue (i.e., are introduced to the
upper cell layers of tissues and organisms following local and/or
topical administration). The reporters are added in trace
quantities (from about 10 to about 1000 .mu.L of 0.1 to 200 .mu.M,
preferably from about 5 to about 100 .mu.L), using a substance that
is transparent to visible light and that has a pre-specified
temporary residence at the application site (e.g., 2 days-up to 30
days, 24-48 hours, preferably 2-6 hours, more preferably 30 seconds
to 5 minutes, and most preferably 5 seconds to 5 minutes).
Contemplated diffusion times include periods less than 48 hrs, 24
hrs, 10 hrs, 6 hrs, 2 hrs, 1 hr, 30 min, 15 min, 10 min, 5 min, 1
min, 30 sec, 10 sec, or 1 sec. Reporters that are placed on skin
are able to penetrate the skin and be transported to a depth from
the surface of from about 10 .mu.m to about 300 .mu.m into the
tissue and are brought in contact with a specific metabolite,
wherein a change in fluorescence or absorption (e.g., measured
using fluorescence or absorption spectroscopy) of the one or more
reporters occurs, thereby allowing quantification of the change in
fluorescence or absorption that provides detailed in vivo
information regarding picomolar through millimolar cellular
metabolite and precursor levels for living tissue, organs,
interstitial fluid, and whole organisms.
[0142] The reporters can be monitored non-invasively using any
low-cost instrumentation capable of directly analyzing the
metabolic state in tissue (e.g., using optical instrumentation).
The reporters are chosen to specifically enhance the signal of
pre-specified analytes in order to assess metabolic state of a
tissue or organism and to yield detailed, real-time information
regarding the state of intracellular and extracellular
metabolism.
[0143] Methods are provided for the direct measurement of
intracellular and extracellular metabolism in epidermal or
epithelial cells using these reporters in combination with
fluorescence or absorption detection. The specific optical signal
used to measure metabolite or precursor levels is derived from
emission or reflection using fluorophores or analyte-binding
proteins with fluorescence labels. These analyte-enhancing
molecules, e.g., SMMRs, have specific properties as described
herein.
[0144] In one embodiment, the invention provides methods for
deriving SMMRs as follows: (1) delineating the metabolites or
precursors (analytes) required to characterize a metabolic pathway
in a living system (e.g., see FIG. 34 for various/optional
alternative glucose metabolism pathways); (2) selecting a basic
mechanism of action for the SMMR (see FIGS. 22-27 for examples of
glycolytic activities); (3) selecting the wavelength options for
excitation and emission of the SMMR by absorption and fluorescence
measurements; (4) selecting molecular structure to meet quantum
efficiency and yield requirements; (5) selecting location,
diffusion rate, and duration or lifetime of the SMMR within the
tissue or organ layers; (6) selecting toxicity requirements and
limitations; and (7) relating measured real-time metabolic
conditions to normal versus disease state for diagnostics or
patient care.
[0145] In order to accomplish this, the reporter is derived using a
combination of molecular properties including, but not limited to,
specific molecular size, polarity, charge, structure, pKa,
solubility, and the size and type of molecular attachments or
anchors. Each of the steps are provided to optimize the real-time
monitoring of metabolic conditions in living cells using
non-invasive, in vivo, and low-cost instrumentation, as described
herein.
[0146] Metabolic pathways for glucose, fructose, and galactose have
been described in detail in numerous references delineating
biochemical pathways (See, Metzler, D. E., 1977, Biochemistry: The
Chemical Reactions of Living Cells, Academic Press, New York, pp.
539-543, 673; Stryer, L., 1988, Biochemistry, 3.sup.rd Ed., W.H.
Freeman and Company, New York, pp. 349-370; Champe, P. C., Harvey,
R. A., 1994, Biochemistry, 2.sup.nd Ed., Lippincott Williams &
Wilkins, Philadelphia, pp. 61-157).
[0147] Metabolic monitoring, as provided using the reporters of the
invention, requires a detailed understanding of the metabolic
pathways and analytes required to understand the relationship
between a measured analyte and the metabolic or disease state.
Simply measuring a specific analyte does not necessarily give
detailed information on disease or metabolic state of cells,
tissues, or organisms. FIGS. 22-27 show various metabolic pathways
of interest, and depict how the reporters of the invention can be
used to analyze specific analytes for the assessment of metabolic
function, providing detailed information on glucose metabolism,
fructose metabolism and/or galactose metabolism. Examples of
specific analytes include, but are not limited to, glucose,
NAD(P)H, ATP, NADH, FAD, lactate, Ca.sup.2+, and O.sub.2.
[0148] The invention provides sensor compositions that can be
present in the epidermis at a depth from the surface of the skin of
about 10 .mu.m, which corresponds to the bottom of the dead stratum
corneum layer, to about 175 .mu.m, which typically corresponds to
the top of the dermal layer. However, those skilled in the art will
recognize that depths up to about 1500 .mu.m are also contemplated
as part of the invention. In a preferred embodiment, the sensor
compositions of the invention can be present in the epidermis at a
depth from the surface of the skin to about 175 .mu.m. However, in
certain embodiments, the sensor composition can be present in the
epidermis at a depth from the surface of the skin to about 300
.mu.m, about 500 .mu.m, about 1000 .mu.m and about 1500 .mu.m. For
example, when the sensor compositions are present on the eyelids,
the sensor composition may be present in the epidermis at a depth
from the surface of the skin of about 50 .mu.m. When the sensor
compositions of the invention are present on the soles of the feet,
it may be desirable for the compositions to be present in the
epidermis at a depth from the surface of the skin of up to about
1500 .mu.m. Thus, those skilled in the art will recognize that the
sensor composition may be present in the epidermis at varying
depths from the surface of the skin depending on site of
measurement and variation among individuals.
[0149] The skin SMMR compositions are present in the epidermis at
an effective concentration that allows one or more metabolites or
analytes in a metabolic pathway to be detected in a subject or
biological sample.
[0150] The invention is designed to target analytes capable of
providing detailed information for peripheral tissue metabolic
pathways that are driven specifically by the measured analyte or
analytes. Where these biosynthetic processes require multiple
analytes, or are for metabolic systems that are distinctly
non-linear, analytes representing more than one pathway may be
combined to model such systems. A final measurement system for
multiple analytes provides a wide dynamic range and is less prone
to interference. For human subjects, first principle mathematical
models can be developed, preferably for individual subjects, more
preferably for small local populations, and most preferably for the
universal case.
[0151] The mechanism of action of any specific reporter of the
invention is related to its unique properties in interacting in
real-time with a known metabolic biochemical reaction for the
explicit purpose of instantaneously defining metabolic function in
living tissue. It is noted that one skilled in the art could easily
adapt this invention for either additional in vivo or in vitro
applications on other tissue, if desired, by using the same
principles taught herein.
[0152] In one embodiment, the invention provides in vivo methods
for monitoring and controlling disease states that affect metabolic
processes in living organisms by applying one or more reporters to
a surface of the skin for a predetermined period of time; causing
penetration of the reporter to a depth of about 10 .mu.m to about
175 .mu.m; monitoring a change in the fluorescence or absorption
based upon peripheral or epithelial tissue metabolite levels; and
correlating the metabolite levels within peripheral or epithelial
tissue with cellular metabolite levels, thereby monitoring and
controlling disease states that affect metabolic processes in
living organisms.
[0153] In another embodiment, the invention provides in vivo
methods for determining the metabolic health and well-being in
living organisms by applying one or more reporters to a surface of
the skin for a predetermined period of time; causing penetration of
the reporter to a depth of about 10 .mu.m to about 175 .mu.m;
monitoring a change in the fluorescence or absorption based upon
peripheral or epithelial tissue metabolite levels; and correlating
the metabolite levels within peripheral or epithelial tissue with
cellular metabolite levels, thereby determining the metabolic
health and well-being in living organisms.
[0154] The invention also provides methods for monitoring the
concentration of one or more metabolite(s) or analyte(s) in a
metabolic pathway using the sensor compositions of the invention.
According to these methods, the sensor composition is applied to
the surface of the skin for a predetermined period of time. The
sensor composition penetrates the epidermis to a depth of about 10
.mu.m, which corresponds to the bottom of the dead stratum corneum
layer, to about 175 .mu.m, which corresponds to the top of the
dermal layer. An optical reader is used to monitor changes in the
concentration of the one or more metabolite(s) or analyte(s) in a
metabolic pathway. These changes in concentration are monitored by
detecting changes in one or more reporter dyes, at one or more
points in time. Monitoring the change in metabolite or analyte
concentration can be accomplished by detecting at least one
wavelength above 350 nm, including wavelengths above 400 nm, 450
nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm,
900 nm and above. In a preferred embodiment, the change in
metabolite or analyte concentration can be accomplished by
detecting at least one wavelength above 450 nm.
[0155] The invention provides methods for determining the
concentration of at least one metabolite or analyte in skin tissue.
According to these methods, a small molecule metabolite reporter
SMMR is administered to the skin tissue. The SMMR penetrates to a
region of the skin at a depth between the dermis and the epidermis,
wherein the depth from the surface of the skin is from about 10
.mu.m to about 175 .mu.m. The SMMR is irradiated with a source of
electromagnetic radiation, and the fluorescence spectra emitted
from the SMMR is detected. The emitted fluorescence spectra are
then analyzed, which results in a determination of the
concentration of the metabolite or analyte. Measuring the
fluorescence spectra according to these methods can include a
bloodless calibration procedure, such as, for example, the
procedure(s) outlined in equations 13, 16, 17, 18, 19, 20 and 21
set forth herein (See Examples 5-8).
[0156] The ability to derive primary and secondary order
information regarding real time, dynamic glucose metabolism (such
as the direction and rate of change of bioavailable glucose
distributed within the blood and interstitial fluid space) is
desirable. In vivo fluorescence (autofluorescence) has been used
for a number of years to determine the metabolic state and to
monitor pharmaceutical effects in cells and tissues. See, e.g.,
Dellinger et al., Biotechnol Appl Biochem, 28 (Pt. 1): 25-32,
(1998). Consideration of the photophysics involved in
autofluorescence rapidly leads one to the conclusion that the use
of autofluorescence alone, as the analytic probe or mechanism,
imposes some severe limitations on any measurement technique.
Specifically, signal-to-noise is not sufficient to meet the
requirements for an accurate, low-cost, quantitative
measurement.
[0157] Recently, the state-of-the-art in making time resolved
fluorescence measurements have advanced to a degree whereby robust
and low-cost instrumentation can be readily assembled. However,
effective measurements have only been made in vitro for specific
analytes, and real-time in vivo analysis has yet to be reported.
Researchers have used phase-modulation fluorometry in vitro to
demonstrate first generation sensing devices for a number of
analytes (pO.sub.2, pH, pCO.sub.2, NH.sub.3, etc.). See e.g.,
Dalbey, R. E., et al., J. Biochem. Biophys. Meth., 9: 251-266,
(1984). The use of long lifetime red-sensitive probes has resulted
in transdermal sensing becoming a reality since human skin is
translucent at wavelengths above 630 nm.
[0158] Fluorescent lifetime-based sensing offers novel applications
in the bioprocessing and biomedical arenas. For instance, in
measurement of Green Fluorescent Protein (GFP) as a marker for
expression of heterologous proteins does not require any additional
co-factors for its visualization. GFP-fusion proteins have been
expressed in a variety of cell lines and in situ measurements in
bioreactors have been made. Fluorescence polarization measurements
for the quantitation of large antigens, such as antibodies labeled
with long-lived fluorescent labels, can, in principle, directly
measure antigens of several million Daltons (Da).
[0159] Fluorescence techniques are capable of detecting molecular
species at picomole (pm) levels or less. This sensitivity arises
because of the simplicity of detecting single photons against a
dark background. This advantage disappears if there are other
fluorescent species in the detection volume that is obtained from
the sample material being measured. Furthermore, fluorescence
intensity is not an absolute technique and measurements must be
referenced to an internal standard using a ratiometric or
comparative method.
[0160] Autofluorescence arises from the innate fluorescence of
compounds that are not particularly efficient fluorophores and that
are not photostable. Because of these properties, detectors for
autofluorescence need to have an excellent signal-to-noise ratio,
with sufficient dynamic range, and require that the excitation
source be of low enough power so as not to cause
photosensitization. In addition, there are a number of fluorescing
species present in the skin that constitute a significant
background signal. The situation is further complicated in that it
is quite difficult to identify or introduce a standard optical
reference material or apparatus into the skin.
[0161] Fluorophores, or colored dyes utilizing absorption
spectroscopy, can be used to measure glucose in solution or serum
by using a series of separate generic reagents. These generic
reagents include, but are not limited to, glucose oxidase (which
oxidizes glucose forming hydrogen peroxide) or peroxidase
(generally horseradish peroxidase ("HRP") used to create an
oxidizing reaction in the presence of hydrogen peroxide with the
dye or fluorophore), and a dye reagent or fluorophore, which
changes its color or fluorescence spectrum when brought in contact
with hydrogen peroxide and peroxidase. The resultant colored or
fluorescent species is measured with a colorimeter or fluorometer,
and the amount of glucose in solution is calculated. In addition,
other standard analytical techniques have been shown to be
commercially useful for measuring hydrogen peroxide generated from
the reaction of glucose oxidase and glucose.
[0162] Tissues derive free energy from the oxidation of fuel
molecules, including glucose and fatty acids. In energy releasing
metabolic processes, fuel molecules transfer electrons to carrier
molecules for transport and conservation. These basic carrier
molecules are either pyridine nucleotides or flavins. The carrier
molecules, in their reduced form, transfer high-energy electrons to
molecular oxygen by means of an electron transport chain located in
the inner membrane of mitochondria. Upon electron transport,
adenosine diphosphate (ADP) and orthophosphate (P.sub.i) yield
adenosine triphosphate (ATP) useful as an energy source in many
metabolic processes.
[0163] Aerobic glycolysis results in the biosynthesis of pyruvate
which serves as a substrate for the mitochondria. In turn, this
substrate feeds oxidative phosphorylation resulting in ATP
production. Mitochondrial inner membrane redox potential can be
measured using the reporters of the invention as an indication of
healthy or perturbed aerobic cell function as exemplified by
oxidative phosphorylation. The mitochondrial membrane potential
indicates status of the biosynthetic process of ATP production for
powering cellular metabolism. This ATP synthesis is directly
coupled to the flow of electrons from the reduced forms of the
coenzymes nicotinamide adenine dinucleotide (NADH), nicotinamide
adenine dinucleotide phosphate (NAD(P)H), and flavin adenine
dinucleotide (FADH.sub.2) to molecular oxygen (O.sub.2) by a proton
gradient across the inner mitochondrial membrane.
[0164] Nicotinamide adenine dinucleotide (NAD.sup.+) is a major
electron acceptor in the oxidation of fuel molecule (e.g., glucose,
fructose, galactose) oxidation. The nicotinamide ring of NAD.sup.+
(oxidized form) accepts a hydrogen ion plus two electrons becoming
NADH (reduced form). Another major electron acceptor is flavin
adenine dinucleotide (FAD) due to its isoalloxazine ring. The
oxidized form of FAD is denoted as FAD, whereas the reduced form is
FADH.sub.2. The major electron donor in most reductive biosyntheses
is NAD(P)H (reduced form). The oxidized form of this electron donor
is NAD(P).sup.+. NADH is used for ATP production, whereas NAD(P)H
is used for reductive biosynthesis. SMMRs are useful for energy
transfer enhancement for direct detection of NADH and NAD(P)H as
well as FADH.sub.2 thereby indicating biosynthetic activity levels.
An increase in the formation of these electron transfer species can
be measured and is indicative of substrate concentration (e.g.
glucose) and overall metabolic health and activity.
[0165] In one preferred embodiment of the invention, the devices,
compositions, and methods effectively determine and monitor the
glucose concentration in blood for a living organism by
non-invasive, in vivo measurement of the glucose level in skin by
means of fluorescence measurements of metabolic
indicators/reporters of glucose metabolism, or by means of direct
measurement of glucose levels in the skin, as described below.
Indirect Measurements using the Sensor Compositions of the
Invention
[0166] This invention provides for fluorescence measurements of
extracellular and intracellular reporter molecules placed into the
cytosol, nucleus, or organelles of cells within intact, living,
tissue that track the concentration of blood glucose in an
organism. When any one of a series of metabolites or analytes is
measured using this technique, the molar concentration of blood
glucose can be calculated. The one or more metabolite(s) or
analyte(s) can directly report on, and relate to, in vivo blood
glucose levels. Suitable metabolites or analytes include for
example, lactate; hydrogen ion (H.sup.+); pH (as lactate/H.sup.+);
calcium ion (Ca.sup.2+) pumping rate; magnesium ion (Mg.sup.2+)
pumping rate; sodium ion (Na.sup.+) pumping rate; potassium
(K.sup.+) pumping rate; adenosine triphosphate (ATP); adenosine
diphosphate (ADP); the ratio of ATP to ADP; glycogen; pyruvate;
nicotinamide adenine dinucleotide phosphate, oxidized form
(NAD(P)+); nicotinamide adenine dinucleotide phosphate, reduced
form (NAD(P)H); flavin adenine dinucleotide, oxidized form
(FAD.sup.+); flavin adenine dinucleotide, reduced form
(FADH.sub.2); or oxygen (O.sub.2) utilization. These analytes,
measured in skin using the techniques taught herein, provide a
complete picture of epidermal skin metabolism where local epidermal
analyte (glucose) quantities are proportional to the concentration
of glucose in systemic blood, specifically the capillary fields
within the papillary layer of the dermis (corium). Temperature
and/or nitric oxide measurement may also be combined with the above
measurements for better calibration and determination of glucose
concentrations.
[0167] The invention provides methods for monitoring in vivo blood
glucose levels by applying the sensor composition of the invention
to a surface of the skin for a predetermined period of time. The
sensor composition penetrate the epidermis to a depth of about 10
.mu.m, which corresponds to the bottom of the dead stratum corneum
layer, to about 175 .mu.m, which corresponds to the top of the
dermal layer. However, depths up to about 300 .mu.m are also
contemplated as part of the invention. An optical reader is used to
detect changes in the reporter dye by monitoring changes in the
concentration of the one or more metabolites or analytes. The
change in the concentration of the one or more metabolites or
analytes is then correlated with in vivo blood glucose levels.
Monitoring the change in metabolite or analyte concentration can be
accomplished by detecting at least one wavelength above 350 nm.
[0168] The invention also provides methods for measuring in vivo
blood glucose levels through the skin by monitoring, in a
population of cells, one or more relevant metabolites or analytes
in at least one metabolic pathway. The one or more metabolite(s),
parameter(s) or analyte(s) is monitored by measuring the
fluorescence spectrum emitted by a reporter composition located in
the skin. The fluorescence spectrum emitted by the reporter is
stoichiometrically related to the metabolite, parameter or analyte
concentration in the population of cells. The in vivo blood glucose
level is determined by analyzing the fluorescence spectrum, using
the known stoichiometric relationship between the fluorescence
spectrum of the reporter and the metabolite, parameter or analyte
concentration.
[0169] The population of cells can have a predominantly glucose
metabolism, or alternatively, the population of cells can be
induced to have a glucose metabolism. The population of cells in
the skin can be located in the epidermis, which contains a dynamic,
metabolically homogeneous, and homeostatic population of cells. For
example, the population of cells having a glucose metabolism can
include live keratinocytes. These live keratinocytes can be present
in the epidermal layer of skin. In some cases, the live
keratinocytes can be present in the skin at a depth, from the
surface of the skin, of about 10 .mu.m, which corresponds to the
bottom of the dead stratum corneum layer, to about 175 .mu.m, which
corresponds to the top of the dermal layer.
[0170] The metabolic pathways monitored within the population of
cells, according to these methods for measuring in vivo blood
glucose levels through the skin, can be monitored by measuring a
specific metabolite or analyte of the glycolytic pathway, wherein
the specific metabolite or analyte has a known stoichiometric or
highly correlated relationship with glucose concentration. The
metabolic pathways can also be monitored within the population of
cells by observing a physico-chemical parameter that is related to
the glycolytic pathway, wherein the selected physico-chemical
parameter has a stoichiometric or highly correlated relationship
with glucose concentration.
[0171] Metabolites produced as the result of glycolysis that are
present in the cell can also be measured in vivo, using the
reporters of the invention. These metabolites include lactate;
hydrogen ion (H.sup.+); calcium ion (Ca.sup.2+) pumping rate;
magnesium ion (Mg.sup.2+) pumping rate; sodium ion (Na.sup.+)
pumping rate; potassium ion (K.sup.+) pumping rate; adenosine
triphosphate (ATP); adenosine diphosphate (ADP); the ratio of ATP
to ADP; inorganic phosphate (P.sub.i); glycogen; pyruvate;
nicotinamide adenine dinucleotide phosphate, oxidized form
(NAD(P)+); nicotinamide adenine dinucleotide (phosphate), reduced
form (NAD(P)H); flavin adenine dinucleotide, oxidized form (FAD);
flavin adenine dinucleotide, reduced form (FADH.sub.2); or oxygen
(O.sub.2) utilization. Individually or in combination, these
metabolites measured in skin using the techniques taught herein
give a complete picture of epidermal skin glucose metabolism, and
an indirect measure of the quantity of glucose molecules entering
the cells.
[0172] The population of cells to be monitored in these methods for
measuring in vivo blood glucose levels through the skin can have a
predominantly oxidative metabolism, or alternatively, the
population of cells can be induced to have a metabolism
predominantly based on oxidative phosphorylation. The metabolic
pathways monitored within the population of cells can be monitored
by measuring a metabolite or analyte that is generated as a result
of the oxidative metabolic pathway, wherein the specific metabolite
or analyte has a stoichiometric or highly correlated relationship
with glucose concentration. Alternatively, the metabolic pathways
can be monitored within the population of cells by observing a
physico-chemical parameter that is generated as a result of the
oxidative metabolic pathway, wherein the physico-chemical parameter
has a stoichiometric or highly correlated relationship with glucose
concentration.
[0173] The invention also provides methods for determining blood
glucose concentration. According to these methods, a first
instrument response measurement is performed using a calibration
target, and the response data is recorded. A first SMMR mixture is
applied to the skin in a first, controlled area, such that the SMMR
resides in the epidermal layer of the skin, and a second SMMR
mixture is applied to the skin in a second, controlled area. The
second area is perturbed, such that extreme changes that the
mixture may undergo are achieved. The extreme change can be, for
example, a change in concentration of the analyte comprising a zero
or low concentration and a saturation level or high concentration;
or the extreme change can be, for example, a change in temperature,
as described herein. A second calibration measurement is then
performed on the perturbed area, and the calibration data is
recorded. A third background measurement is made on an area of skin
that has no SMMR, whereby this background data is recorded. A
measurement on the first area is performed by illuminating the
first area with light, and the wavelength spectrum of light
reflected back from the first area is detected. Further
measurements on the first area are performed at wavelengths
suitable for each SMMR present. A parameter from the response data
is calculated in order to normalize the background data,
calibration data and measurement data for the response of the
spectrometer. A parameter from the background data is calculated in
order to correct the calibration data and measurement data for
emission, absorption and scattering properties of the tissue. A
metabolite parameter from the calibration data is calculated in
order to relate the measurement data to the blood glucose
concentration.
[0174] The invention also provides methods of calculating a blood
glucose concentration. Accurate direct or indirect in vivo
measurement of glucose concentration in immortal cell lines, human
keratinocyte cell cultures, and mammalian (including human) skin
are achieved by using this application of in vivo fluorescence
labeling and detection of SMMRs in skin. According to these
methods, a background response and an autofluorescence tissue
response is measured from a calibration target that includes an
epidermal layer of skin. A first dye is provided to a first skin
location, and residues of the first dye mixture are transferred
into the epidermal layer of the skin. A second dye is provided to a
second skin location, and at least one extreme change in the
mixture is triggered and recorded. The extreme change can be, for
example, a change in concentration of the analyte comprising a zero
or low concentration and a saturation level or high concentration;
or the extreme change can be, for example, a change in temperature,
as described herein. These extremes are used to calibrate the
sensor enabling it to measure a test sample accurately with a
concentration between the extremes. See e.g., equations (13)
through (21), as described herein (Examples 5-8). The first skin
location is illuminated with a radiative emission, and a resulting
wavelength spectrum reflected from the first skin location is
detected. The illuminating and detecting can be repeated using
irradiation and wavelength spectra associated with each dye
provided. At least one physico-chemical parameter that is related
to the glycolytic pathway is then detected. Preferably, the
physico-chemical parameter has a stoichiometric or highly
correlated relationship with glucose concentration, which is used
in determining the blood glucose concentration. The sensor system
can include a bloodless calibration procedure such as, the
procedure(s) outlined in equations 13, 16, 17, 18, 19, 20 or 21 set
forth herein (Examples 5-8).
[0175] The methods and compositions of the present invention use
reporters such that two basic techniques are available for
obtaining ratiometric measurements of glucose concentration or
exemplary utilization versus fluorescence response. Mechanism 1
utilizes a combination of a reporter dye having a specific and
fluorescence response proportional to a change in metabolite
concentration, where that metabolite has a direct stoichiometric
relationship to a change in glucose concentration. Mechanism 1 also
utilizes a marker dye, that is stable but unresponsive to changes
in glucose and is used explicitly to produce a reference signal. In
this case, the marker dye is used as a reference wavelength for the
reporter dye, which changes emission at only one wavelength in
response to glucose. An example of a suitable marker dyes includes
the class of coumarins, which fluoresce in the blue region of the
spectrum and localize in the cytosol of the cell, but do not
respond to a change in glucose or metabolite concentration. In
certain embodiments, the reporter dye can be located in the cytosol
of the cell, and the marker dye can be in a different cellular
compartment. One skilled in the art of photochemistry (including
synthetic organic chemistry) can readily synthesize derivatives of
these dyes that have these altered properties. For example, alkyl
coumarins maintain the fluorescent properties of the coumarin
parent but localize in the membranes of cell.
[0176] Alternatively, the sensor composition can include a dye that
exhibits a wavelength shift in absorption or fluorescence emission
in the presence of a metabolite, such as, for example, glucose. In
this second case, Mechanism 2, only one dye is used that has two
wavelengths where fluorescence signal varies with the introduction
of D-glucose concentration to living cells (i.e., a first emission
wavelength of the fluorescence spectrum increases with glucose,
while a second emission wavelength decreases). The ratio of the
first and second emission wavelengths can be determined, thereby
allowing the selected dye to act as a self-referencing reporter.
This phenomenon is illustrated in FIGS. 3-5, and 8 with analytical
results demonstrated in FIG. 9. The mechanisms by which SMMRs
report on the rate and quantity of metabolic activity, particularly
glycolytic processes for the invention are described herein.
Prediction of Blood Glucose from Skin Glucose
[0177] The fluorescence measurement of extracellular and
intracellular reporter molecules placed into the cytosol, nucleus,
or organelles of cells within intact living tissue will track the
concentration of blood glucose in an organism. When any one of a
series of analytes or metabolites is measured using this technique,
the molar concentration of blood glucose can be calculated.
Fluorescence measurements of metabolite reporters described for
this invention in a metabolic pathway of interest can be taken from
one or more of the following parameters: pH (e.g., as lactate/H+);
redox potential; NAD(P)H (nicotinamide adenine dinucleotide
phosphate, for the reduced form using energy transfer); FAD.sup.+
(flavin adenine dinucleotide, for the oxidized form using energy
transfer); ATP/ADP ratio; Ca.sup.2+-pumping rate; Mg.sup.2+-pumping
rate; Na.sup.+-pumping rate; K.sup.+-pumping rate; and redox
potential of mitochondrial and other cellular membranes.
[0178] Previous studies have demonstrated that the lag time between
blood glucose levels and glucose levels in non-perturbed epidermis
is 2.9 to 4 percent per minute for the differential concentrations.
Thus, the time required for the epidermis to reach an equilibrium
with blood glucose at steady-state is 25 to 35 minutes as described
by K. Jungheim and T. Koschinsky Diabetes Care 25(6), 956, 2002;
and J. Ellison et al. Diabetes Care 25(6), 961, 2002. When blood
glucose is rapidly increasing (hyperglycemia) or decreasing
(hypoglycemia), this lag time becomes a critical issue for
determining the response of any blood glucose monitor.
[0179] Thus, a rapid response is required for identifying important
health related changes in glucose level and to avoid critical blood
glucose scenarios. In one embodiment, issues of rapid response are
addressed by using elevated temperatures at the measurement site to
increase blood flow to these regions. The sensors are calibrated by
comparing actual blood glucose to the sensor output. The zero and
slope of the sensor calibration are determined by measuring an
initial glucose level and a later glucose level to determine the
change in glucose. The sensor calibration is then measured as
[G]=K.sub.1(sensor response)+K.sub.0. The K.sub.1 and K.sub.0
values are entered into the sensor and calibration is checked
against a reference standard material. The reference standard
material is comprised of a matrix, which responds to glucose
concentration in such a way as to provide primary standard
concentration and response data.
[0180] In addition, the measurement of temperature may be combined
with direct or indirect fluorescence measurements of glucose using
one or more of the following parameter measurements: pH (as lactate
and/or H.sup.+); redox potential; inorganic phosphate (P.sub.i);
glycogen; pyruvate; nicotinamide adenine dinucleotide phosphate,
oxidized form (NAD(P).sup.+); nicotinamide adenine dinucleotide
(phosphate), reduced form (NAD(P)H); flavin adenine dinucleotide,
oxidized form (FAD.sup.+) for energy transfer; flavin adenine
dinucleotide, reduced form (FADH.sub.2) for energy transfer;
adenosine triphosphate (ATP); adenosine diphosphate (ADP); the
ATP/ADP ratio; Ca.sup.2+-pumping rate; Mg.sup.2+-pumping rate;
Na.sup.+-pumping rate; K.sup.+-pumping rate; oxygen (O.sub.2)
utilization and vital mitochondrial membrane stains/dyes/molecules
fluorescence response. These analytes measured in skin using the
techniques taught herein give a complete picture of epidermal skin
glycolytic metabolism where local epidermal analyte (glucose)
quantities are proportional to the concentration of glucose in
systemic blood, specifically the capillary fields within the
papillary layer of the dermis (corium). The control of temperature
at the measurement site, or the additional measurement of
temperature, can be useful to correct measured fluorescence for
optical pathlength, vasodilation, perfusion, and local
physiology.
[0181] The fluorescence response of the reporter protein is then
related to blood glucose level by the relationships shown in
equations M1 and M2. The action of a reporter meeting the
requirements of this invention include those molecules that are
reactive with glucose following the mechanisms described herein.
The reporter (used singly or in combination) has an affinity for
and a response to the presence of glucose in a quantity that is
directly proportional to the concentration of glucose within the
individual cells or interstitial fluids, including blood. All such
reporters useful for this invention are preferably nontoxic,
non-carcinogenic, non-teratogenic, and do not deleteriously affect
the skin when exposed to ultraviolet light or natural sunlight. The
reporters included in the present invention are highly fluorescent
or absorptive, evenly dispersible in the cell and interstitial cell
fluid, do not aggregate or agglomerate, and do not exhibit
binding-dependent fluorescent efficiency and quantum yields.
Preferably, the reporters do not inhibit or restrict normal cell
metabolism nor adversely affect cell viability or health in the
concentrations and manner used.
[0182] Indirect measurement of blood glucose concentration is made
as follows. A first molecule that exhibits no fluorescence or
absorptive change with a change in glucose or other specific
metabolites (i.e., the marker molecule) and a second molecule that
exhibits direct changes in fluorescence intensity with a change in
glucose (i.e., the reporter molecule) are measured individually.
The molecules are safe, relatively permanent, and non-absorbing
into the dermal tissue. Individual molecule fluorescence intensity
measurements are ideally made using an ultraviolet or visible light
emitting diode (LED) or laser diode for an excitation source or an
equivalent known to those skilled in the art. The emission detector
collects the light from the emission of the molecule signal within
the skin and calculates the ratio of reporter dye fluorescence or
absorption (following a predetermined lag time as lagt) to the
marker dye fluorescence or absorption (following the same lag
period lagt). A linear univariate computational formula for
calibrating such an analyzer for blood glucose is given in equation
M1 as:
[ Glucose Blood ] = k 4 .times. Reporter signal lagt Marker signal
lagt + k o ( M1 ) ##EQU00001##
where k.sub.1 is the regression coefficient (slope) for the line
describing a change in fluorescence or absorption signal for the
Reporter to Marker ratio versus glucose concentration in the blood,
and k.sub.0 is the calibration line intercept. Additionally, a
change in glucose concentration over a time interval from T.sub.1
to T.sub.2 involves the following relationship shown in equation
M2:
.DELTA. [ Glucose Blood ] = k 4 .times. Reporter signal lagt ( T 2
- T 1 ) Marker signal lagt ( T 2 - T 1 ) + k o ( M 2 )
##EQU00002##
where .DELTA.[Glucose.sub.Blood] represents the change in blood
glucose concentration and the terms (T.sub.2-T.sub.1) represent the
change in reporter or marker dye fluorescence or absorption over
the time interval measured.
[0183] The dyes described within this invention may also exhibit an
exponential relationship between fluorescence or absorption
intensity and glucose concentration such that the computational
formula for calibrating such an analyzer for blood glucose is given
as equation M3:
[Glucose.sub.blood]=k.sub.0e.sup.k.sub.1.sup.R (M3)
where R is the ratio of Reporter signal.sub.lagt to Marker
signal.sub.lagt.
[0184] Once activated, the response of the tissue cells to
metabolite content or metabolic state is monitored directly using
an optical reader. The optical reader calculates the tissue
response to metabolite levels, applies first principles
mathematical models to the response, and provides a determination
of the organ, system, or organism metabolite levels.
[0185] A quality value is simultaneously calculated, which tells
the user the quality of the measurement taken and of the resultant
metabolite value reported. Based on this quality value, the user
may be instructed to make one or more additional measurements until
the quality value is indicative of an accurately reported
metabolite value result.
[0186] It should be noted that an extension of this embodiment is
the addition of other reporters, which are allowed to penetrate
more deeply into the tissue; in some cases penetrating as far as
300 .mu.m of the tissue. In some applications, reporters may be
applied into the deeper layers of tissues and organs. In other
embodiments of this invention, injection or ingestion of reporters
into the bloodstream, or into specific organs or tissues may be
utilized and the resultant fluorescence or absorption response
measured at the site of application using an optical reader having
remote optics.
[0187] Those skilled in the art will recognize that FAD and
FADH.sub.2 are formed in the citric acid cycle during aerobic
(oxidative) biosynthesis and are used for electron transport in
this pathway. FIG. 24 shows anaerobic glycolysis, where NAD(P)H is
the major electron donor for reductive biosynthesis.
[0188] The invention also provides SMMRs that are combined with a
small reagent strip in order to calibrate a sensor used for direct
in vivo, non-invasive glucose measurement. This calibration strip
is used for a single reaction to adjust the glucose sensor response
and is then depleted. Each calibration ideally requires a new
calibration strip for adjusting the sensor response. Those skilled
in the art will appreciate that such a reagent strip can be used to
detect glucose in fluids withdrawn from the body.
Cellular Respiration
[0189] The implications and status of cellular respiration can be
determined by molecular oxygen consumption. The classic method for
determining oxygen consumption in living organisms is to cut small
pieces of living tissue and study respiration rate using a
Fenn-Winterstein type respirometer (See Wennesland, R., Science
114: 100-103, 1951). This method has obvious drawbacks for
day-to-day monitoring of human subjects. Thus, SMMRs that detect
oxygen levels are also provided.
[0190] Some tissues and organisms vary widely in respiration rate.
The respiration rate is also known to vary with cell size in terms
of surface area-to-volume ratio. Oxygen consumption is proportional
to cell surface area. When overall cell size increases, respiration
rate decreases. The respiration rate of a tissue or whole organisms
is the arithmetic sum of the rates of its component cells.
Metabolic rate changes for some cell types may vary by a factor of
100 as a function of cell activity levels. Temperature affects
respiratory rate such that a 10.degree. C. rise in temperature
increases rate by 2 to 4 times. Oxygen partial pressure and water
concentration also affect respiration rate, but only when levels
are abnormally high or low.
Redox Potential and Ion Pumping
[0191] Reduction potential in cells, tissues, and organisms is
indicative of glycolytic activity and respiration health of cells,
tissues, and whole organisms. Direct measurement of intracellular
redox potential in cells indicates mitochondrial health and levels
of aerobic (i.e., increased mitochondrial activity due to oxidative
phosphorylation, reduction or cessation of lactate production,
increased oxygen consumption) versus anaerobic respiration (i.e.,
cessation or decrease in mitochondrial activity due to inhibited
oxidative phosphorylation, as well as increased lactate production,
and decreased oxygen consumption). These indicators yield direct
ability to assess the health state of cells in real-time.
[0192] Biochemical reactions for respiration, glycolysis, and other
basic metabolic processes require the transfer of electrons from
one molecule (or atom) to another. These are termed
oxidation-reduction (redox) reactions. Oxidation is a term used to
denote loss of electrons from a molecule, whereas reduction is the
term used to denote a gain of electrons in a molecule. Electrons
are neither created or destroyed in redox reactions and, thus, when
one molecule is oxidized, another is reduced. The transfer of a
single hydrogen atom is equivalent to a transfer of one proton and
one electron.
[0193] Many important redox reactions that occur in living systems
involve the transfer of hydrogen rather than the transfer of
isolated electrons. The affinity of a molecule to accept electrons
is termed its reduction potential, and when measured under standard
conditions it is denoted by the symbol (E.sub.0'). Reduction
potential is measured in volts (V) on a scale relative to a value
of 0.0 V for the half-reaction of hydrogen at standard conditions
(i.e., 1 atmosphere pressure, 1 molar concentration of reactants,
and 25.degree. C.). Values for the redox potential in living cells
may vary because the reactants are not normally at 1 M
concentration. A positive redox potential indicates that a molecule
has more affinity for electrons than the hydrogen ion (H.sup.+).
Furthermore, in redox reactions electrons move toward the molecule
with a positive reduction potential.
[0194] In redox reactions, the total electric potential or voltage
change (.DELTA.E) is equal to the arithmetic sum of the individual
oxidation or reduction steps. The voltage change can also be
denoted as equivalent to a change in the chemical free energy
(.DELTA.G). This chemical free energy is calculated using a
constant specifying the charge in 1 mole of electrons as 96,500
joules per volt, referred to as the Faraday constant (I). It should
be noted that the oxidation potential is simply the negative value
of the reduction potential. A positive .DELTA.G indicates a
reaction will not occur spontaneously, however in biochemical
reactions, a positive .DELTA.G reaction is often coupled with a
negative .DELTA.G reaction of greater magnitude, thus the reaction
proceeds.
[0195] Biological reduction-oxidation (redox) potential (E.sub.0)
is affected by the presence of molecular oxygen (O.sub.2) and by
hydrogen ion concentration, which is measured as pH. Many cellular
redox reactions, such as those in glycolysis, involve electron
transfer and hydrogen transfer. In these reactions, E.sub.0 (the
reduction potential, in volts) changes with pH. An increase in pH
creates a decrease in E.sub.0' (the standard reduction potential)
whenever the concentration of the oxidant equals the concentration
of the reductant. See, Hewitt, L. F., Oxidation-Reduction
Potentials in Bacteriology and Biochemistry, 2.sup.1 Ed. Williams
& Wilkins, Baltimore, Md., USA, 1950.
[0196] Dyes have been proposed as an in vitro means for measuring
redox potential in living cells. However, such dyes have not been
used or specified for use to indicate in vivo metabolic pathway
delineation in living organisms for the expressed purpose of
assessing health and well-being of tissues or organs. Furthermore,
in vitro dyes using absorption spectroscopy are typically less
sensitive by two orders of magnitude to metabolic changes compared
to fluorescent. Current commercial absorption and fluorescent dyes
have not been optimized for molecular characteristics. Thus, they
are not optimal for use in non-invasive, in vivo monitoring of
metabolic conditions derived from in situ measurements made on
living subjects.
[0197] Previous work with in vitro dyes and fluorophores used in
cell or tissue cultures has specified that, in order to use these
dyes for in vitro membrane potential measurement of cells, one must
determine: (1) the reduction potential (E.sub.o), (2) the standard
reduction potential (E.sub.0'), and (3) the titration curves for
each oxidizable dye used (See, Giese, A. C., 1973, Cell Physiology,
4.sup.th Ed. W.B. Saunders Company, Philadelphia, pp. 420-429). In
this invention, a specific SMMR is designed such that when the SMMR
comes in contact (in vivo and in situ) with the analyte or
metabolite of interest, the appropriate optical response occurs.
Preferably, this optical response is fluorescence, but alternative
absorption mechanisms are not excluded where signal is sufficient
for measurement using low-cost instrumentation.
[0198] SMMR have the above basic properties as well as the ability
to be applied locally and topically, in trace quantities (from
about 10 to about 400 .mu.L of a 1 to 50 .mu.M mixture), using a
small molecule in solvent solutions that are transparent to visible
light, and that have a pre-specified temporary residence at the
application site (from about 5 seconds to about 30 days).
[0199] As noted, a negative reduction potential indicates a
substance has lower affinity for electrons than hydrogen (H.sub.2),
whereas a positive reduction potential indicates a substance has
higher affinity for electrons than H.sub.2. Thus, the coenzymes
NADH, NAD(P)H, and FADH.sub.2 are strong reducing agents and have
negative reduction potentials. Molecular oxygen (O.sub.2) is a
strong oxidizing agent having a positive reduction potential.
NAD(P)H, NADH, and FADH.sub.2 are coenzymes acting as reducing
agents and have electron transfer potential useful for providing
electrons for biological metabolic pathways. The electron transfer
potential is Converted to phosphate transfer potential
(.DELTA.G.sup.0) in the form of ATP. In redox reactions, an
oxidized form of a substance (X.sup.+) and a reduced form (X.sup.-)
make up a redox couple. Therefore, NAD(P)H, NADH, and FADH.sub.2
can be detected using reporters of the invention for sensing either
energy transfer or redox potential. The energy transfer for these
coenzymes is demonstrated in FIG. 29 and the redox potential
measurement is made as illustrated in FIG. 30. The redox potential
is measured at the inner mitochondrial membrane.
[0200] In energy transfer measurements using SMMR methodology,
external energy from a handheld sensor is applied to target cells
containing naturally occurring fluorophores such as the coenzymes
NADH, NAD(P)H, or FADH.sub.2. A small molecule metabolic reporter
is added to the target tissue to provide an energy transfer vehicle
for enhancement of fluorescent yield and efficiency. The excitation
energy is absorbed by the natural fluorophore and emitted at the
absorption frequency of the reporter. The reporter, in turn, emits
enhanced signal at a pre-specified frequency. This emission
frequency is preselected in order to be compatible and
non-interfering with respect to other measurements made
sequentially at the same target tissue site. Thus, the
amplification factor and emission wavelength of the reporter can be
optimized for the measurement regime selected.
[0201] The implications of measuring intracellular redox potential
(whether or not combined with other metabolites or ions) in living
cells in vivo using SMMRs include, but are not limited to, the
following:
[0202] A) Research in mammals has shown that orthotopic liver
transplantation is associated with significant variations over time
in the redox potential of the cytosol. Postoperative mortality is
related to redox state of the liver cell mitochondria. Research has
suggested that abnormal tissue oxygenation can occur during liver
transplantation (A. de Jaeger et al., Intensive Care Medicine,
24(3): 268-275, 1998). Thus, the physician would choose to monitor
both intracellular oxygenation and mitochondrial redox state both
during and after transplant surgery. SMMRs can be used to monitor
both intracellular oxygenation and mitochondrial redox state using
both an intracellular tissue oxygen reporter and a mitochondrial
redox reporter.
[0203] B) According to the literature pertaining to emergency
medicine, a variety of pathogenic mechanisms of cellular injury
occur during shock in humans. See, e.g., Jeffrey A. Kline M D,
Pathogenic mechanisms of cellular injury during shock, May 8, 2001,
Society for Academic Emergency Medicine Annual Meeting, Atlanta,
Ga., oral abstract presentation found at
http://www.saem.org/download/Olkline.pdf. Such cellular metabolic
changes include, e.g., (1) a transformation of cells from fatty
acid to carbohydrate utilization, (2) an increase in lactate
production in cells due to metabolic conversion from aerobic to
anaerobic glycolysis, and (3) an overall decrease in ATP production
with a resultant decrease in calcium ion pumping and associated ATP
driven processes. An advanced stage of injury moving to shock in
humans involves a hypoxic condition occurring in mitochondria of
affected cells, which creates (a) a cessation of pyruvate
oxidation, (b) a cessation of calcium ion pumping and ATP
production, and (c) a leaking of electrons across organelle and
outer cellular membranes. Further metabolic stress leads to lactic
acidosis, a drastic change in intracellular redox potential, and
increased leakage of calcium ion into the cytosol. SMMRs enable
direct measurement of real-time changes in the concentrations of
intracellular lactate, calcium ion, and redox potentials in
affected cells and tissues. This information allows real-time
detection of changes at the cellular level and would provide rapid
information relative to organ or whole organism health status for
critical care monitoring.
[0204] C) In the case of cardiac muscle under stress, the muscle
cells exhibit rapid changes in glycolysis, oxygen consumption,
lactic acidosis, drastic changes in intracellular redox potentials,
and changes in intracellular versus extracellular calcium ion
concentrations within cardiac muscle cells. Again, SMMRs enable
direct measurement of real-time changes in the concentrations of
intracellular lactate, calcium ion, and the redox potentials in
affected muscle cells and tissues. This information can provide
immediate detection of changes at the cellular level for cardiac
subject monitoring and would provide rapid information relative to
health status for critical care monitoring.
[0205] D) Intracellular calcium ion (Ca.sup.2+) performs critical
functions in muscle contraction, nerve impulse transmission, ion
transport, and transmission of signals across membranes. For normal
cells, the concentration of extracellular and intracellular calcium
is closely regulated. A perturbation in normal calcium ion balance
is indicative of metabolic stress, pre-shock, cell viability
concerns, and cell mortality. SMMRs provide direct concentration
information regarding intracellular and extracellular calcium ion
levels.
[0206] In addition, the measurement of electric oxidation-reduction
potential across cell membranes in vivo is an accurate indirect
indicator of glucose quantities entering the cell to fuel
glycolytic processes. SMMRs reporting on changes in membrane
potential are attached to cell membranes including the inner and
outer cell membranes, the nuclear membranes, as well as those of
organelles, such as mitochondrial membranes. Membrane potential
measured in skin cells using the techniques taught herein give a
complete picture of epidermal skin glycolysis.
[0207] Specific SMMRs, e.g., those acting as vital mitochondrial
membrane stains, require that a fluorescence response occurs upon a
change in membrane potential. Several fluorophores are know to
comply with this requirement. These fluorophores behave in such a
way as to change fluorescent intensity and emission spectral line
shape in response to changes in mitochondrial membrane potential.
In the present invention, an increase in intracellular glucose
concentration increases the mitochondrial membrane potential and
causes additional SMMR units to attach to the inner mitochondrial
membrane. This increased SMMR binding to the inner membrane causes
fluorescence quenching of the SMMR proportional to changes in
glucose concentration. This response is based upon the interaction
of the redox coupling of NAD.sup.+ and NADH, NAD(P).sup.+ and
NAD(P)H, FAD and FADH.sub.2, and ion transport. Thus, optical flux
changes detected by a hand-held sensor provide detailed information
regarding intracellular redox potential at the mitochondrial level
suitable for an assessment of cell health and well-being.
Reversible interaction between the SMMR and the mitochondrial
membrane allow real-time monitoring of these processes.
[0208] Traditional redox potential sensors involve electrodes and
invasive procedures. Moreover, these instruments measure analytes
present in solution and are not able to detect intracellular
activity in a non-invasive manner. In contrast, SMMRs yield a
direct, real-time measurement of intracellular activity relative to
cellular metabolism, as well as a measurement of the direct state
of health of tissues as compared to buffered solutions surrounding
tissues. Immediate and real-time information of the intracellular
metabolic state gives a more rapid and accurate indication of
organism health for diagnostic-based, corrective treatment.
Diagnosis of Disease State
[0209] Many disease states in cells and organisms affect a
host's/subject's metabolic condition and efficiency. Thus a
non-invasive, in vivo method for directly measuring intercellular
and intracellular metabolic changes in tissues and organisms is
valuable in assessing health versus disease or stress state
conditions of cells, tissues, and whole organisms.
[0210] Metabolic disease states may be monitored using the
reporters of the invention by: (1) measuring NADH, NADPH, and FAD
using energy transfer fluorescence measurements (to validate the
presence of coenzyme activity as an indication of glucose
metabolism) and (2) measuring cellular reduction-oxidation
potentials (indicating cellular activity); lactate formation
(indicating anaerobic glycolysis in the stress state); calcium ion
pumping (as an indication of ATP availability); and oxygen
consumption (indicating healthy cellular respiration and aerobic
glycolysis). Metabolic diseases affecting cellular respiration, ion
pumping, and energy production can be monitored non-invasively for
cells, tissues, organs, and systems using SMMR technology, as
described herein.
[0211] Cationic transport diseases include, but are not limited to,
potassium-channel disease affecting heartbeat, epileptic
tendencies, and deafness. Sodium-channel disease can result in,
e.g. muscle spasms, or osmotic imbalance leading to
hypertension.
[0212] The onset of disease states affecting metabolism of glucose,
accumulation of lactate, deficiencies in ion pumping and ATP
formation, and changes in oxygen consumption can be detected in
real-time using SMMRs. The SMMRs are synthesized or constructed
with unique and specific molecular properties, such that a known
optical signal is produced when the SMMR is reacted with precisely
identified metabolites or precursors. The resultant optical flux is
an indication of the in vivo health, stress, disease state, or
necrotic conditions of tissues and organ systems. Specifically,
abnormalities in glucose utilization from anaerobic or aerobic
glycolysis can be identified using SMMRs, as illustrated in FIGS.
22-27 and 34, according to the mechanisms described in FIGS. 29-33,
and 10.
[0213] For example, the details for glycolytic metabolism can be
identified using SMMR technology. Examples include the cellular
utilization of glucose, fructose, and galactose. Metabolic disease
conditions related to glycolysis include diabetes mellitus (a
disease condition related to insulin regulated glucose transport or
utilization/response deficiency); essential fructosuria (a
deficiency in fructokinase); hereditary fructose intolerance (a
deficiency in aldolase B); and hereditary fructose-1,6-biphosphate
deficiency results in hypoglycemia, apnea, hyperventilation,
ketosis and lactic acidosis due to impaired hepatic
gluconeogenesis. These symptoms can take on a lethal course in
neonates. For galactose metabolism, a deficiency in
Galactose-1-phosphate uridyl transferase, galactokinase, or
UDP-galactose-4-epimerase results in galactosemia.
[0214] Deficiencies of normal metabolic activity related to
glucose, fructose, or galactose metabolism can be detected in vivo
by applying SMMRs to the target tissue and adding the appropriate
sugar substrate molecules into the immediate target area where the
SMMR has entered the cells. Tracking the metabolic rates using the
SMMR in this manner allows the detection of a normal versus
abnormal metabolic state. Moreover, this test is rapid and can be
accomplished using low cost hand held sensors specific for the type
of SMMR used.
Determination of Vitality and Viability of Cells Based on Metabolic
Function
[0215] The health of cells can be determined based upon their
normal utilization of glucose as well as by calcium ion transport
(an ATP-driven cellular ion pump), ATP formation, lactate
formation, redox state, electro-motive potential, NADH.sup.+ or
NAD(P)H.sup.+ or FADH.sub.2 utilization, and oxygen consumption.
For example, necrotic tissue relative to surgical procedures such
as bowel resection; acute appendicitis; frost bite; septicemia;
leprosy; restricted circulation; burns from heat, chemical, or
radiation exposure; trauma damage to tissue; or any other condition
where viability and vitality are essential considerations, may need
to be assessed. There are a number of reporter molecules that when
placed into tissue, provide precise information on cellular
respiration, metabolic rate, relative health (vitality), and
viability.
[0216] Healthy tissue performs a number of specialized and general
functions that may form the basis of targeted SMMR technology.
Specialized functionality includes, but is not limited to, the
synthesis and utilization of biochemicals unique to that tissue.
General functionality includes, but is not limited to, maintaining
the integrity of the cell membrane, the utilization of glucose and
other metabolic substrates, the synthesis of lactate in anaerobic
tissue and the consumption of oxygen in aerobic tissue.
[0217] SMMRs may be diffused directly into cells and tissues to
detect viability based upon active metabolism indicated by the
presence of glycolysis, ion pumping, redox potential, lactate
formation and accumulation, and oxygen consumption rates. Any of
these metabolic indicators can all be measured using SMMRs as
described in detail above. In addition, direct SMMRs are available
for viability monitoring based on other cellular mechanisms.
Critical Care Monitoring
[0218] The viability and metabolic health of cells can be
determined by oxygen consumption; by lactate formation; by calcium
ion transport; by glucose metabolism; by ATP production and
utilization; by NADPH, NADH or FADH.sub.2 utilization; by
measurements of electron transfer potential; and/or by measuring
changes in both intracellular and extracellular resting potential.
SMMRs allow detection and real-time tracking for each metabolite
(analyte) and allows intracellular tracking of metabolic
conditions.
[0219] Thus, SMMRs, combined with low-cost spectroscopic
techniques, can provide the next generation of critical care
monitoring. Advantages to the subject and physician include: (1)
obtaining information directly from within the cells instead of
looking at footprints, reflections or shadows of processes that
affect or predict morbidity/viability, (2) exploiting the
combination of new direct information with dramatic improvements in
the time constants for either degradation or improvement of subject
status, (3) exploiting the ability to differentially monitor
central and peripheral tissues to better characterize subject
status, and (4) monitoring the real-time effect of anesthesia
and/or therapeutics at the intracellular level.
[0220] Skilled deployment of the SMMR/instrument platforms of the
invention, which exploit these advantages, will improve subject
outcomes at lower cost to the healthcare system while providing
physicians with real-time cellular and organism status.
[0221] The current state-of-the art in critical care monitoring
involves assessment of the status of selected parameters in blood,
e.g., glucose and oxygen supply, and parameters such as pH and
lactate for evidence of dysfunction at the cellular, tissue, and
organ level. Because blood is highly buffered and in large volume,
it is a poor source of early warning information and does not
provide the opportunity to assess the metabolic state of cells,
organs, or systems in real-time. Providing real-time intracellular
status using appropriate SMMR/instrument technology (including the
methods and compositions of the invention) can provide life-saving
information to the critical care medical staff and can give
appropriate and timely diagnostic warning for life saving
actions.
[0222] When injury or stress occurs to a cell the electric
potential changes. The magnitude of the change on electric
potential is calculated using the well-known Nernst equation and
simplified derivations thereof as follows in equation M4:
E = 59.5 log 10 ( [ e h ] [ e 1 ] ) ( M4 ) ##EQU00003##
where E is the electric potential in millivolts, and e.sub.h and
e.sub.l represent the higher concentration and lower concentration
(molar) of the electrolyte, respectively. From this relationship,
it is possible to calculate the ionic diffusion coefficients from
measurements of electric potential from which molar concentration
of transported ions can be determined. SMMRs are useful for
measuring electric potential as well as direct ion
concentrations.
[0223] There are multiple methods available to evaluate the type of
stress occurring to a cell using SMMR technology of the invention.
For example, lack of molecular oxygen (O.sub.2) reduces the resting
potential and changes the intracellular versus extracellular ion
concentration. A decrease in heat liberation also occurs when the
cell is under metabolic stress. Inhibition of any glycolytic
function also reduces the resting potential. In fact, the potential
may fall toward zero as poisoning or death become imminent. Both
the potential and the ion transport effects can be calculated. The
energy required to move 1M of cation from inside the cell to the
outside of the cell for a single and two compartment cell is also
calculated using modified forms of the Nernst equation shown in
equation M5 and M6. See Giese, A. C., Cell Physiology, 4.sup.th Ed.
W.B. Saunders Company, Philadelphia, pp. 571-582, 1973). The
modified Nernst equation is as follows: For the single cell
(compartment):
W = RT ( ln [ A out + ] [ A in + ] ] ( M5 ) ##EQU00004##
For the two cell (compartment)
W = RT ( ln [ A out + ] [ A in + ] + ln [ B in + ] [ B out + ] ) (
M6 ) ##EQU00005##
where W is the energy required, A.sup.+ and B.sup.+ are cations, R
is the gas constant (i.e., 8.312 joules per degree per mole), T is
the absolute temperature in degrees Kelvin, and I is the Faraday
constant (i.e., 96,500 coulombs per gram equivalent), and ln is the
natural logarithm (2.3.times.log.sub.10).
Cancer Diagnosis, Detection, and Prognosis
[0224] Tumor cells engage in anaerobic glycolysis, as do epidermal
keratinocytes, and thus metabolic activity differences between
metastatic cells and normal cells are quite pronounced and obvious,
because tumor cells are known to 1) have higher metabolic rates
than normal cells, 2) accumulate dye molecules at higher levels
than nominal tissue, 3) have lower pH then normal tissue, and 4)
frequently undergo glycolysis at much higher rates.
[0225] Current and commercial spectroscopic characterization of
cancer cells is limited to discriminant analysis of raw
spectroscopic data. These data yield limited signal-to-noise
differences between metastatic and normal cells when applying
measurements using molecular spectroscopy, or native
autofluorescence and white light reflection. These techniques
provide only weak differentiating power to distinguish cancerous
tissue from normal surrounding tissue due to the low
signal-to-noise molecular absorption, or autofluorescence signals
within the metastatic versus normal tissues.
[0226] A large number of in vitro molecular probe/limited
wavelength fluorescent microscope techniques for characterizing
cancer cells are available. Simultaneously, dramatic improvements
in the ability to separate, capture and present cancer cells for
characterization are occurring. Thus, a technique that enables the
selection and measurement of specific intracellular metabolic
pathway signals would be valuable for distinguishing normalcy,
malignancy, or pre-malignancy as the result of non-invasive, in
vivo measurements. The necessary SMMR materials could simply be
"painted" or sprayed onto the targeted area to discriminate
malignant cells (i.e., hyper-metabolic), or pre-malignant cells
(i.e., semi-hyper-metabolic), from normal cells.
[0227] Cancer screening is often an invasive process. A number of
techniques are currently utilized, including physical examination,
biopsy, and some fluorescence imaging. Additionally, the drugs used
for photodynamic therapy have been used to delineate cancerous
tissue with some success. Photodynamic therapy has been used since
the late nineteen fifties as an anti-cancer treatment. Briefly, a
drug that selectively binds to tumor cells is applied either
topically or intravenously. A red light is then shone on the
tissue, and the drug generates active oxygen species that destroy
the cells. Red light is most often used for the therapy, since it
has an improved penetration into tissue. The drugs most commonly
used in these therapies are porphyrins and common derivatives
include hematoporphyrin, benzoporphyrin and commercial preparations
such as photofrin that consist of mixtures of porphyrins and
oligomeric porphyrins.
[0228] Porphyrins typically generate long lived excited states with
a quantum yield of about 0.6. They fluoresce in the red region of
the spectrum above 620 nm with quantum yields of about 0.1. In the
blue region of the spectrum, they have large molar absorption
coefficients of about 10.sup.5 M.sup.-1 cm.sup.-1 and some
derivatives including benzoporphyrin and chlorophylls have
similarly high molar absorption coefficients in the red. The
molecules are extremely sensitive to their microenvironment and
lose many of their photophysical properties when aggregated.
[0229] The photophysics of these molecules have assured their use
as molecules to detect oxygen concentrations, to delineate cancer
cells in vivo as well as their use in photodynamic therapy (PDT).
As molecules to detect cancerous tissue, advantage is taken of
their selective uptake by tumors. Typical uptake ratios for the dye
in cancer versus normal tissue are about four to one. Provided that
the molecule is monomerized once inside the cell, it can be
detected by its fluorescence. However, there are a number of
disadvantages associated with using porphyrins for this technology.
The molecules are photosensitive and can be destroyed by the
reactive oxygen species they generate. If the molecules aggregate
inside the cell, they will not fluoresce. This aggregation is
dependent both on the microenvironment of the molecule and on its
effective localized concentration. Hematoporphyrin, for example,
starts to aggregate in water at concentrations as little as 1 .mu.M
and is predominantly aggregated at concentrations above 10 .mu.M.
There is also a disadvantage in using the same drugs for therapy as
for cancer detection in that the quantum yield of fluorescence of
these compounds is not particularly high, as only about 10 percent
of absorbed photons are returned as fluorescence.
[0230] The wavelengths used to excite the dye depend on the purpose
of the treatment. For photodynamic therapy, red light is used since
this gives the best penetration of scattering tissue. For detecting
cancer cells, green or blue light is often used since the drug has
a higher molar absorption coefficient at these wavelengths and
light penetration is not an issue. See, Photochem. Photobiol. 73:
278-282, 2001.
[0231] The rational behind these techniques is that the cancer cell
preferentially takes up the dye over normal tissue. Strictly
speaking, it is not accurate to say that the dye selectively binds
to the cancer cell. Dye uptake is more closely related to the fact
that the cells have a higher metabolic rate. Truly selective uptake
or binding would involve exploitation of some chemical difference
in the metabolic pathways or genetic expression that a cancer cell
demonstrates. For this reason, an unfortunate side effect of
porphyrin type dyes is that normal tissue that has a high metabolic
rate may preferentially accumulate the dye.
[0232] Malignant changes result in modified rates of metabolic
activity and in cellular proliferation. These changes result in
biochemical changes, which may be monitored using changes in
autofluorescence. See Cancer Res. 62: 682-687, 2002; Photochem.
Photobiol. 68: 603-632, 1998; Neoplasia 2: 89-117, 2000. However,
the underlying problem behind all these techniques is that the
autofluorescence is extremely weak and subject to interferences
from photooxidation and variability in the spectral shape of the
autofluorescence.
[0233] Cancer cells exhibit hyperglycolytic activity as compared to
normal cells. In addition cells moving into the cancerous state
covert glycolytic activity from aerobic to anaerobic glycolysis.
Hyperglycolytic cancer cells exhibit increased glucose uptake and
transport; changes in ion pumping; decreased ATP production;
decreased oxygen utilization; and increased lactate production due
to the conversion of pyruvate to lactate in anaerobic glycolysis.
Thus, a measurement technique capable of monitoring and comparing
glucose utilization and transport, ion pumping rate changes and
concentration gradients, oxygen utilization, and lactate
production, either as individual data points or combined, can be
used to detect hyperactive pre- and post-cancerous activity. The
metabolic state, kinetics, and aggressiveness of these cells can be
characterized and classified. Furthermore, the velocity of
glycolysis, the maximum velocity, and the Michaelis-Menten constant
can be calculated and compared with normal cell data.
[0234] In one embodiment of the invention, SMMR technology
described herein provides an opportunity for detailed exploration
and spectroscopic monitoring of cellular metabolic pathways with
novel low-cost instrumentation, which may lead to substantial
improvements in the identification and the characterization of
cancer cells. The ability to improve diagnosis, staging,
therapeutic selection/effectiveness assessment and monitoring for
metastatic cell recurrence would represent a significant
advancement in cancer diagnosis, and potentially for improved
differentiation and classification of solid tumors for selection
and optimization of treatment regimes.
[0235] Tumor Markers
[0236] Potentially the most powerful screening technique for cancer
would involve a class of compounds that have been designated as
tumor markers. The presence, or less optimally the absence of these
species in blood or other readily accessible tissue would indicate
a high probability of a cancer in a subject. The marker would be
detectable preferably by the use of spectroscopy of the skin or
peripheral tissues, and the monitoring of such a compound would
correlate with the development of the cancer and also indicate the
type of disease. Cancer staging, assessing the extent of local and
distant disease, can also be accomplished using the SMMR of the
invention. To date, no one marker has been identified that can
definitively signal the presence of a tumor. There are however a
number of biochemicals and genetic markers that together can
improve the diagnosis of a cancerous condition.
[0237] The markers may be generated either by the cancer cells
themselves or by the body in response to the tumor. The marker may
be a normal biochemical or may be a material that is only generated
when a tumor is present. A number of possible markers have been
identified including, but not limited to, antigens, some
antibodies, hormones and enzymes.
[0238] Antigens that indicate carcinogenesis include oncofetal
antigens, which are antigens that are normally only present in an
embryo or fetus. However, the presence or increased concentrations
in the adult are a good indication of tumor formation. Hormone
production at poorly controlled concentrations may arise from a
tumor of a particular endocrine gland. Some pancreatic tumors for
example cause the synthesis of high concentrations of insulin.
Hormones may also be produced by the tumor cell expressing a
synthetic pathway that the normal cell type would not produce.
Examples of enzymes that may serve as markers for cancer growth
include the over production of acid phosphatase associated with the
development of prostrate cancer, as well as increased levels of
galactosyl transferase II associated with colon cancer.
[0239] The in vivo, non-invasive techniques described herein,
enable the selection and measurement of specific intracellular
metabolic pathway signals for cells, tissues, organs, and organ
systems. This technique would be valuable for distinguishing
normalcy, malignancy, or pre-malignancy from non-invasive, in vivo
measurements. For example, the SMMR materials delineated in this
invention can simply be "painted" or sprayed onto the targeted area
to discriminate malignant cells (i.e., hyper-metabolic), or
pre-malignant cells (i.e., semi-hyper-metabolic), from normal
cells. This discriminative measurement can be accomplished using a
low cost fluorescence detection system or devices, as described
herein.
[0240] The changes that occur in cells with the onset of
carcinogenesis in terms of the active biochemical pathways, the
rate of metabolism and the synthesis of marker compounds all
provide target mechanisms that may be exploited by the use of
fluorescent Monitoring compounds such as SMMR. The use of these
compounds, some of which have been suggested to be used for the
monitoring of blood glucose levels in diabetics, provides a level
of sensitivity and selectivity that has not been possible using
current technology, i.e. porphyrin dyes and UV or green light.
[0241] There are two exemplary methods by which the present
technology may be used, including carrying out metabolic monitoring
of the whole body by the use of fluorophores applied to the skin,
and targeting these changes using fluorescent dyes that respond in
a well-characterized mechanism to the altered metabolism. The
fluorophores that will be used to monitor these processes include,
but are not limited to, compounds described for each targeted
pathway.
Metabolic Rate
[0242] Current technologies exploit the enhanced metabolic rate of
tumor tissue to delineate it from normal tissue. SMMR technology
presents a more selective means to monitor tissue having increased
metabolic rate. By using dyes that report the activity of a
specific pathway, SMMR technology registers an increased level of
fluorescence for the increased uptake of the dye and the
enhancement due to the activity of the metabolic pathway targeted.
For example, pH sensing dyes such as 3-oxo-3H benzoxanthene
derivatives undergo a wavelength shift in their emission as a
function of pH. Such dyes, when used to delineate tumor tissue,
show an increased level of fluorescence and a change in the ratio
of their emission bands, which indicate an increased metabolic rate
and a lower pH on the tumor tissue.
Glycolysis
[0243] Metabolic markers that may be targeted to monitor glycolysis
include lactate and oxygen consumption. In tissue that undergoes
primarily anaerobic metabolism, the products of this reaction
pathway are lactate and adenosine triphosphate (ATP). ATP is
synthesized from ADP, the diphosphate analog and phosphate. Lactate
is generated as a waste product of the pathway. The ability to
determine the relative importance of glycolysis is achieved by
monitoring lactate, pH, or NAD(P)H production, as a function of
oxygen concentration. It is possible to perturb the oxygen
concentration by clamping or cooling the tissue. The relative
change in lactate to NAD(P)H ratio then indicates the fraction of
metabolism that is carried out via mitochondrial activity. The use
of NMR techniques using phosphorous and proton probes would allow
the measurement of phosphate, pH and lactate simultaneously.
[0244] Lactate Production
[0245] Lower pH values of tumors are associated with the synthesis
and export of lactic acid by the cell and a higher rate of
glycolysis. The generation of a large amount of lactate occurs,
even under aerobic conditions. Such behavior is unusual, since in a
typical cell the fate of pyruvate, the product of glycolysis, in
the presence of oxygen is to be oxidized within the mitochondria.
The reasons why the pyruvate generated in a tumor that is not
anoxic is not further metabolized by the mitochondria is not
clearly understood. It is known that the hexokinase responsible for
the initial steps in glycolysis is found bound to the surface of
the mitochondrial membrane.
Oxygen Consumption
[0246] The use of probes such as Ruthenium tris bipyridyl and
related derivates provides a well-proven technique for monitoring
oxygen concentration. Other dyes that have also been used to
monitor oxygen concentration include porphyrin and phthalocyanine
derivatives. The emission of these molecules is sensitive to oxygen
concentration. It is possible to monitor the intensity or the
lifetime of the emission of these dyes to determine the oxygen
concentration. Technically, the simplest apparatus that may be used
to monitor the emission lifetime of these dyes contains a modulated
light source and a phase-sensitive detector. The phase angle shift
of the dye and the degree to which the dye emission is modulated
allow the lifetime of the dye to be determined. The parameters
measured by the device are related to the lifetime by the
expressions shown in equation M7 and M8
Tan .phi. = .omega. .tau. and m = 1 ( 1 + .omega. 2 .tau. 2 ) ( M7
; M8 ) ##EQU00006##
where .phi. is the phase shift, m is the degree of modulation,
.omega. is the circular modulation frequency and .tau. is the
lifetime.
[0247] However, there are metabolic differences in the cancer cell
that can be targeted using metabolic monitoring technology. The
methods and compositions of the invention can be used to monitor
those parts of metabolism that are altered when a cell becomes
cancerous. For example, such a system can be used to monitor
high-risk individuals or populations for particular cancers, and to
determine the progress of disease in a subject undergoing
treatment.
Antigens and Hormones
[0248] Antigens may be present in circulating blood at extremely
low concentrations. To detect them and to monitor their
concentrations requires a high degree of selectivity and
sensitivity. These molecules can be targeted by the use of antibody
bound SMMR fluorophores. Antibodies are large protein molecules
that have a high degree of specificity for a particular antigen.
They are synthesized by the immune system specifically to target an
antigen of interest. The antibody provides the selectivity required
to eliminate false positive detection. The binding of the antigen
causes a conformational change in the antibody that results in a
large fluorescent change in the dye molecule. Those skilled in the
art will recognize that hormones may be monitored in a similar
fashion using fluorescent modified hormone receptor molecules in
the same way as one would use the antibody.
Enzymes
[0249] Common techniques for the monitoring of enzyme activity
include monitoring the substrate, the cofactor or a product of the
enzyme reaction. To monitor enzyme activity through the skin, SMMR
technology uses fluorescent substrate analogs tethered in the
epidermis. The change in fluorescence of the substrate when bound
to the enzyme would be indicative of the enzyme concentration.
[0250] Thus, the metabolic differences in the cancer cell can be
targeted using metabolic monitoring technology. Those parts of
metabolism that are altered when a cell becomes cancerous can be
monitored using the methods and compositions of the invention. Such
a system can be used to monitor high-risk individuals or
populations for a particular cancer and to determine the progress
of the disease in a subject undergoing treatment.
Suitable Small Molecule Metabolic Reporters of the Invention
[0251] Suitable small molecule metabolic reporters of the invention
include, but are not limited to: fluorophores, protein labeled
fluorophores, proteins comprising a photooxidizable cofactor, and
proteins comprising another intercalated fluorophore as described
herein.
[0252] It is well known that specific dyes bind to cellular
structures and allow imaging and anatomical/histological studies of
intracellular structures. See, e.g., the information available from
companies such as Molecular Probes or Sigma-Aldrich. It is also
well known that some signals from these dyes can be used to
characterize cellular metabolism in vitro. Fluorescent chemical
sensors have been reported to play a critical role in the
elucidation of cellular mechanisms by giving real-time information
about the environment of a cell in a non-destructive manner, See,
e.g., Glass, J. Am. Chem. Soc. 2000, 122: 4522-4523. For these
applications, sensor affinity and selectivity are of utmost
concern. Thus, a useful sensor must recognize its analyte with high
specificity and possess an affinity that is commensurate with the
average concentration of the analyte in solution. Id. However, no
specific fluorophores have been named, and no fluorophore design
requirements have been published for in vivo, non-invasive
elucidation of metabolic pathways for any medical applications in
general, and, specifically, for those described by this invention
(e.g., in a preferred embodiment for measurement of blood glucose).
A surprising discovery has been made that the detailed and specific
absorption and emission spectral characteristics of a select set of
dyes, when introduced into living cells of organisms (in vivo),
change as a qualitative and quantitative indication of
extracellular and intracellular metabolism. One or more dyes of the
select set presented herein are specifically used to report
metabolite concentration, which are then used to further define the
quantity or quality of metabolic activities within living
organisms, such as glycolysis.
[0253] This invention relates to SMMRs that indicate the rate and
quantity of glycolysis occurring within the living cell loci. The
detailed spectral changes noted as direct and indirect metabolic
reporters include: variation in fluorescence emission intensity and
lifetime, variation in wavelength position for absorption and
emission maxima, and variation in bandwidth and spectral shapes of
absorption and emission spectra. These measurable changes vary in
direct proportion to the changes in concentrations of metabolite
molecules within the physical proximity of associated extracellular
and intracellular structures. The information provided by measuring
the changes in specific reporter dye spectra following introduction
into living organisms has utility as part of a low-cost method and
apparatus for the detailed, real-time measurement and delineation
of metabolic pathways and processes in living organisms.
[0254] In vivo small molecule metabolic reporter measurements
require the in situ interaction of living cells with the reporter
molecules to give an accurate and real-time indication of the
metabolic state for a whole organism, an organ, a tissue type, or
individual cells. The measurements of the metabolic state for
living organisms can thus be made non-destructively and
non-invasively using spectroscopic measurements on living tissues
and cells. Furthermore, custom molecules can be synthesized based
on detailed understanding of reporter interactions with in vivo
metabolic processes. See, e.g., FIGS. 17A through 17D. This
discovery has allowed optimization of these dye molecules in their
active role as SMMR, for reduced toxicity, selective residence time
in targeted tissues, cellular binding site specificity, analyte
selectivity and sensitivity, photostability, and fluorescence
spectral characteristics. These fluorescence spectral
characteristics can be selected based on molecular structures for
SMMR, and include: emission intensity and lifetime, location of
excitation/absorption and emission maxima, Stokes shift, bandwidth,
spectral shape changes due to the presence of metabolites, quantum
yield, and quantum efficiency.
[0255] Therefore, the methods and devices disclosed herein
represent an improvement over current techniques such as
antibody:antigen labeling, because they relates explicitly to a
unique use of small molecules capable of penetrating the stratum
corneum, that when placed in living tissue allow a measurable
fluorescence response proportional to metabolic changes in living
cells, tissues, and whole organisms (e.g., animals and humans),
without initiating an immune response. These measured metabolite
signals provide delineation of metabolic pathways by measuring the
spectra of certain dye molecules when the molecules are used in
precise ways, under exacting conditions, and when placed in
specific structures within living cells and tissues.
[0256] A dye that is classified as a reporter according to the
invention must meet several minimum criteria: low toxicity; ability
to be delivered precisely to target tissue; report quantitative
information with respect to the concentration of specific
metabolites when measured in vivo; and detectable using wavelength
emission-related technology. Preferably the dyes are fluorescent.
Mechanisms for identifying and/or constructing exemplary reporters
of the invention are described below. Mathematical models are
provided based on the metabolite or metabolic pathway to be
analyzed.
[0257] In order to qualify as a SMMR according to this invention,
dyes require one or more of the following criteria: [0258] 1.
Enhancement of signal-to-noise ratio of native autofluorescence
measurements through the process of: [0259] a. ENERGY TRANSFER from
NADH, NAD(P)H, or FAD.sup.+ to SMMR (which boosts signal by 5 to 50
fold) that is an indirect indication of redox transfer coenzyme
activity within cells and tissues due to glycolysis (see FIG. 12;
Mechanism 1); [0260] 2. Enhancement of Specific Metabolite and
Precursor Signals such as: [0261] a. Lactate SMMRs that indicate
lactate/hydrogen ion formation from anaerobic glycolysis activity
(measurement sites include intracellular, extracellular, and
organelle loci) (see FIG. 13; Mechanism 2); [0262] b. Mitochondrial
Membrane Potential SMMRs that indicate overall changes in
mitochondrial membrane redox-potential that corresponds to changes
in glucose (see FIG. 14; Mechanism 3); [0263] c. Calcium ion
(Ca.sup.2+) tracking SMMRs that indicate available adenosine
triphosphate (ATP) and ion pump transport activity fueled by
glycolytic activity (see FIG. 13; Mechanism 2); [0264] d. Glycogen
SMMRs using glycogen-staining molecules that indicate the
occurrence of glycolysis and resultant storage of glycogen
molecules (see FIG. 13; Mechanism 2). [0265] 3. Direct measurement
of glucose molecules in vivo using: [0266] a. Protein-labeled
fluorophores such as proteins that are specifically bound to
glucose and have enhanced fluorescence quantum efficiency. When
placed into the skin, the resulting fluorescence is indicative of
the amount of glucose present (see FIG. 15; Mechanism 4); [0267] b.
Proteins comprising a photoredox active cofactor (such as flavin
adenine dinucleotide, i.e., FAD) that are used to observe excited
state lifetime fluorescence by monitoring the triplet state of FAD
(.sup.3FAD*) (see FIG. 16; Mechanism 5).
[0268] These mechanisms are referred to as Mechanisms 1-5 and are
depicted schematically in FIGS. 11-16.
[0269] Some suitable reporters according to the invention are
available commercially, and include, but are not limited to, the
following: (1) Rh123 for measuring NAD(P)H (nicotinamide adenine
dinucleotide (phosphate), reduced form) using energy transfer, or
FAD.sup.+ (flavin adenine dinucleotide, oxidized form) using energy
transfer; (2) membrane localizing dyes such as diphenylhexatriene,
xanthenes, cyanines as well as diphenyl hexatriene and its
derivatives, for measurement of energy and glucose transport by
membrane receptors such as GluT1; (3) pH (i.e., lactate/H.sup.+)
indicating dyes such as phenolphthalein, xanthene dyes such as
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, (BCECF),
benzenedicarboxylic acid, 2(or
4)-[10-(dimethylamino)-3-oxo-3H-benzo[c]xanthene-7-yl]-(SNARF-1)
for calculations of lactate/H+ ratios, cytosolic NAD/NADH ratios or
pyruvate/lactate ratios; (4) dyes known to have altered emission
properties depending on the redox potential, ATP/ADP ratio,
Ca.sup.2+-pumping rate, Mg.sup.2+-pumping rate, Na.sup.+-pumping
rate, or K.sup.+-pumping rate of its surroundings, as these
processes are ATP regulated and ATP formation in keratinocytes is a
direct result of glycolysis fueled by glucose; (5) vital
mitochondrial membrane stains or mitochondrial membrane dyes,
especially those that produce a fluorescence response to changes in
mitochondrial membrane potential; (6) reactive molecules that
directly or inversely correlate to glucose concentration, such as
nitric oxide (NO); and (7) molecules that directly bind to
D-glucose producing a fluorescence response. Additional exemplary
SMMRs are provided throughout the disclosure. Other appropriate
SMMRs of the invention will be apparent to those skilled in the
art.
1. Energy Transfer Measurements
[0270] The use of energy transfer as a mechanism to measure the
presence and quantity of coenzymes in the cellular environment has
been demonstrated in vitro after removing cells from organisms.
Singlet bimolecular electronic energy transfer reactions, which can
be designated as B*+A.fwdarw.B+A*, where the energy is transferred
from molecule B to A, proceed by at least four different
mechanisms: (1) long-range resonance energy transfer ("fluorescence
resonance energy transfer (FRET)" or Forster transfer), which
occurs between dipole-dipole interactions over a molecule distance
of up to 5 nm; (2) short-range collisional energy transfer (CET),
requiring electron-exchange interactions between the donor and
acceptor molecular orbitals (that is the main mechanism of transfer
in the majority of SMMRs); (3) static quenching, in which the donor
and acceptor molecules are in close proximity in the ground state
and; (4) radiative energy transfer (RET), involving donor emission
and reabsorption of the photon by the acceptor.
[0271] A number of SMMRs (e.g., Rh123) provide excellent energy
transfer capacity wherein the metabolite of interest is excited.
SMMRs report an enhanced signal at its characteristic emission
wavelength. This energy transfer mechanism provides signal
enhancement for normally very weak autofluorescence. What is
normally a very weak signal with about 10 percent relative
discrimination (i.e., a signal to noise of 10:1 to 50:1), can be
discriminated at 0.2 to 1 percent signal (i.e., signal-to-noise of
100:1 and higher to about 500:1). This signal enhancement allows
the use of low-cost diode-based instruments or sensors for making
accurate measurements of fluorescence signal.
[0272] The specific application of energy transfer is for the
measurement of metabolic coenzymes essential in reduction-oxidation
(redox) molecular biosyntheses, wherein the molecule has a
stoichiometric or highly correlated relationship with glucose
concentration. Coenzymes directly involved in redox mediated
reactions include NADH, NAD(P)H, and FADH.sub.2. A measurement of
the change in fluorescence signal brought about by using a reporter
of the invention in vivo to track the formation of NAD(P)H
(nicotinamide adenine dinucleotide (phosphate), reduced form) for
energy transfer, FAD.sup.+ (flavin adenine dinucleotide, oxidized
form) for energy transfer, can be used as an indirect measurement
of the quantity of glucose entering a cell. Enhancement of these
signals allows accurate tracking of glucose metabolism and other
biosynthetic processes within the living cell. Low-cost tracking of
the activity for these specific coenzymes enables oxidative
phosphorylation and anaerobic glycolysis to be monitored in
real-time.
2. Enhancement of Specific Metabolite and Precursor Signals
[0273] The sensor composition used in these methods for monitoring
and detecting the concentration of one or more metabolite(s) or
analyte(s) can include, for example, a reporter that is a
mitochondrial stain that is sensitive to membrane potential or
chemical gradient. Examples of suitable mitochondrial stains
include a polycyclic aromatic hydrocarbon dye, such as, for
example, rhodamine 123; di-4-ANEPPS; di-8-ANEPPS; DiBAC.sub.4(3);
RH421; tetramethylrhodamine ethyl ester, perchlorate;
tetramethylrhodamine methyl ester, perchlorate;
3,3'-dihexyloxacarbocyanine,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride; 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
iodide; nonylacridine orange; dihydrorhodamine 123 dihydrorhodamine
123, dihydrochloride salt; xanthene; benzenedicarboxylic acid;
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; 2(or
4)-[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; or iodine
dissolved in potassium iodide. The reporter dye can also be
coumarin; derivatives of coumarin, anthraquinones; cyanine dyes,
azo dyes; xanthene dyes; arylmethine dyes; pyrene derivatives; or
ruthenium bipyridyl complexes.
[0274] Reporters useful for illustrating this mechanism include
pH:lacate/H.sup.+ indicating molecules where two or more
wavelengths change directly in proportion to a change in
pH:lacate/H.sup.+ concentration. A two-photon fluorescence lifetime
imaging within the dead uppermost layers of the epidermis (i.e.,
the stratum corneum) has been described, where the fluorophores are
introduced into the tissue to measure the pH gradient across human
skin. See, e.g., Hanson et al. Hanson et al., 2002, Biophysical
Journal 83: 1682-1690, incorporated herein by reference. However,
the skin tissue was removed from the animal prior to analysis, such
that their in vitro technique was performed on dying tissue.
[0275] Proteins acting as reporters, as described herein, can be
used in vivo for direct measurement of intracellular or
extracellular glucose. Fluorescence emission and lifetime intensity
response is proportional to the glucose concentration within the
cell or external to the cell in interstitial tissue fluid or
blood.
[0276] The essential characteristic in identifying a member of the
class of SMMR dyes includes those compounds that report
fluorescence changes in proportion to changes in glucose
concentration for in vivo measurements. These dyes may be
discovered empirically by screening large numbers of compounds for
signal efficacy, or they may be designed using a basic
understanding of photochemistry. The spectroscopic properties of
SMMRs useful for routine analysis, especially when using low-cost
instruments include, but are not limited to, one or more of the
following: molecules that exhibit a large molar absorption
coefficient (10,000 L mol.sup.-1 cm.sup.-1 and above), molecules
that exhibit a high Stokes shift (e.g., 20 to 150 nm), long (e.g.,
2 hours to 4 weeks) residence time at target site, molecules that
are highly photostable (e.g., less than 5 percent signal loss at
use excitation power), molecules that exhibit little or no excited
state chemistry (i.e., inert or non-reactive in excited state), and
molecules that exhibit large fluorescence quantum yield (e.g.,
Quantum Yield [.phi..sub.F] greater than 0.4).
[0277] Examples of suitable SMMRs include, but are not limited to,
modifications of fluorescence dyes to include molecular size
attachments as: acetoxy methyl esters, chloro-methyl derivatives,
alkyl chain adducts, highly charged moieties, enzyme substrate
mimic, enzyme cofactor tethers, and membrane binding tethers.
[0278] The basic starting dyes used to develop these reporters are
polycyclic aromatic hydrocarbon dyes, including, but not limited
to: rhodamine 123; di-4-ANEPPS, di-8-ANEPPS; DiBAC.sub.4(3); RH421;
tetramethylrhodamine ethyl ester, perchlorate; tetramethylrhodamine
methyl ester, perchlorate; 3,3'-dihexyloxacarbocyanine,
2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
chloride;
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarboc-
yanine iodide; nonylacridine orange; xanthene dyes especially
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein;
dihydrorhodamine 123 dihydrorhodamine 123, dihydrochloride salt;
and benzenedicarboxylic acid, 2(or
4)-[10-(dimethylamino)-3-oxo-3-H-benzo[c]xanthene-7-yl]; and iodine
dissolved in potassium iodide. Other dyes or stains that are useful
as SMMRs include, but are not limited to: fluorescein derivatives,
modified coumarin; derivatives of coumarin, anthraquinones; cyanine
dyes, azo dyes; xanthene dyes; arylmethine dyes; pyrene
derivatives; and ruthenium bipyridyl complexes. An exemplary
backbone of a contemplated SMMR is shown in FIG. 17C and described
further in the Examples, infra.
[0279] However, the use of other dyes exhibiting similar
characteristics and chemical structure for the in vivo
determination of glucose is an extension of this concept, and an
important aspect of this invention. Dyes that exhibit the provided
characteristics and chemical structures would be known to one
skilled in the art. Alternative embodiments would differ only in
the dye selected and in the optimization of the techniques shown
herein. This invention includes methodologies to develop optimum
conditions for the use of other dyes expressly for the purpose of
extending or refining this application.
[0280] Mitochondrial stains have been used in vitro for measuring
glucose concentration in immortal cell lines by fluorescence. See,
e.g., N. Borth, G. Kral, H. Katinger, Cytometry 14:70-73 (1993).
However, no known previous work determines the glucose
concentration in blood for a living organism by non-invasive, in
vivo measurement of the glucose level in skin by means of
fluorescence measurements of metabolic indicators/reporters (such
as SMMR) of glucose metabolism.
[0281] One specific redox potential indicating dye, Rhodamine 123
(Rh123), provides an illustrated working example of the present
invention. Rh123 dye has the systematic name
(2-(6-Amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester),
given CAS No. 62669-70-9. Membrane reporting redox potential
indicating dyes such as Rh123 have been used in concentrations of
10-150 .mu.M for multiple applications, many related to
intracellular mitochondrial activity, specifically for measurement
of fluorescence response proportional to changes in transmembrane
redox potential in order to research the mechanics of cell
metabolism.
[0282] Rh123 is commonly known as green fluorescent mitochondrial
dye and is widely applied in cytometry studies involving
mitochondrial membrane potential. Its spectral properties include
an excitation maximum wavelength of 485 to 505 nm with an emission
wavelength of 525 to 534 nm. It exhibits an absorption maximum from
485 to 505 nm and has a molar absorption coefficient of 97,000
Lmol.sup.-1 cm.sup.-1. This dye is an orange-red solid that is
soluble in methanol (MeOH), dimethyl sulfoxide (DMSO) and
dimethylformamide (DMF). These dyes are light sensitive. Once in
solution they should be kept at less than 5.degree. C. and
protected from direct illumination for long-term storage and
optimum efficacy. Rh123 has a molecular formula of
C.sub.21H.sub.17ClN.sub.2O.sub.3 and a molecular weight of 381
daltons. The molecule has low toxicity and has a reported
intravenous lethal dose for animals (LD10) of approximately 20
mg/kg of body weight; and a fifty percent lethality (LD50) of 89.5
mg/kg (i.v.) in rats (Merck).
[0283] Rh123, and other dyes exhibiting similar molecular
structures, have a specific set of chemical properties whereby the
molecule is fluorescent, cationic (i.e., positively charged), of
low molecular weight, lipophilic, and configurable as a
water-soluble salt. Having these molecular properties, dyes such as
Rh123 exhibit preferential binding to negatively charged
mitochondrial membrane lipids. The final quantity of dye that
collects within the mitochondrial membrane is dependent on the
molar concentration of the dye within the surrounding medium (i.e.,
intercellular and cytosol concentrations) and, more importantly,
the mitochondrial membrane potential. The dye is distributed into
the membrane by means of general diffusion such that the molecules
move into the cell and then to the mitochondrial membrane at a rate
that is dependent on chemical kinetics and metabolic rate. Thus,
increases in temperature and thereby metabolic rate, will increase
the rate of random motion that is driving the concentration of
Rh123 molecules in solution to equilibrium.
[0284] Accurate in vivo and in vitro measurements can be made over
nominal temperature ranges from 75 to 105.degree. F., or wider.
Variations in the subject temperature wider than approximately
.+-.5.degree. F. of the target tissue require re-calibration, as
noted elsewhere. The method used to recalibrate for any temperature
range is provided to make certain that the temperature is measured
while the calibration is performed using equations 1-5 and 13-16 of
the invention. Any subsequent measurement of the test sample may be
performed within .+-.5.degree. F. without concern for temperature
variation.
[0285] In the case of mitochondrial dyes, each cationic molecule of
dye accumulates stoichiometrically as negatively charged moieties
within the inner mitochondrial membrane of healthy metabolizing
cells at a concentration dependent rate. The final concentration of
dye uptake for each cell is dependent upon the number of
mitochondria present within the treated cells as well as the
changes in the mitochondrial membrane potential within each
cell.
[0286] Under conditions where glucose is the major metabolic
substrate for the cell, oxidative phosphorylation is fueled by the
products of glycolysis. See, e.g., Johnson, L. V., et al. J. Cell
Biol. 83, 526 (1981). Additional discussions describing research
applications of membrane potential-indicating dyes are found in,
e.g., R. C. Scaduto, and L. W. Grotyohann, Biophysical Journal 76,
469 (1999) and related references. For most of the reducing
reactions that occur in cells, the reducing power is provided by
NAD(P)H. The pH gradient that generates the mitochondrial membrane
potential is fueled by NADH. This NADH may be derived from the
Krebs citric acid cycle as well as from glycolysis.
[0287] Illumination using energy at approximately 490 nm excites
Rh123 directly and its fluorescence emission can be detected at
approximately 530 nm. The final baseline intensity of the dye is
proportional to the concentration of dye present at the
mitochondria, and to the mitochondrial density. The changes in the
net fluorescence intensity of Rh123-like dyes are directly
proportional to the changes in the membrane potential of the cell.
Molecules behaving in this manner are referred to as transmembrane
redox potential indicating SMMRs (FIG. 14). Metabolic activity
fueled by glucose is indicated by Rh123-like dye fluorescence
intensity.
[0288] Specific chemical agents are known to disrupt oxidative
phosphorylation and glucose metabolism. Any such agent causing
decreased cellular respiration, cellular energy balance, and cell
viability will affect the fluorescence intensity of the dye bound
to the mitochondria. A decrease in the glucose concentration
available to the cell causes a reduction in ATP production due to
depletion in metabolism from lowered oxidative phosphorylation.
Such a decrease in glucose concentration is indicated by a
corresponding decrease in fluorescent intensity. The demonstration
of a linear increase in mitochondrial-bound Rh123 fluorescence with
changes in respective glucose concentration for immortal cell lines
has been shown. See, e.g. Borth, et al. Cytochemistry 14, 70
(1993), incorporated herein by reference. Borth demonstrated that
for isolated 3D6-LC4 human-mouse heterohybridoma cells in
suspension the mean fluorescence intensity was dependent upon
glucose availability (i.e., concentration) rather than to either
increased growth rate or metabolic rate.
[0289] One embodiment of the invention utilizing a sensor that acts
as a reporter and marker channel detector is provided in FIG. 4.
The signal detected by the sensor is derived from the relaxation of
a metastable excited state generated by the absorption of energy
from a lamp, light-emitting diode, or laser source. The
fluorescence process is repetitive, meaning the fluorescence
response to glucose can be measured repetitively or continuously,
as long as the molecular tag molecules are not destroyed or
removed. The same molecular tag can be repeatedly excited and its
emitted energy detected. In the methods and compositions of this
invention, the emission intensity, given from a known concentration
of at least one molecular tag, is proportional to the number of
energy transfer events from NAD(P)H to Rh-123 where Rh-123 acts as
the SMMR. Compounds exhibiting such a mechanism of action are
representative of a family of compounds referred to as "energy
transfer from reducing equivalents indicating SMMRs" (FIG. 12). The
number of these energy transfer events is proportional to the rate
of glycolysis modulated by the glucose concentration of the
intercellular environment and ultimately to the blood glucose
concentration.
[0290] The chemical structure of Rh123 is shown below as Structure
A. This dye belongs to a broad range of compounds referred to as
xanthene dyes. The general structure of xanthene dyes is shown in
Structure B. Substitution of these dyes at any of the positions
marked "R" on the xanthene moiety influences the wavelengths of
absorption and emission while substitution of the phenyl ring at
position 9, shown in Structure B, influences the solubility of the
molecule. As drawn, the molecule absorbs light in the ultraviolet
region of the spectrum. Substitution at the positions marked R with
a heteroatom that readily exchanges hydrogen causes extended
conjugation across the ring, wherein the molecule absorbs in the
visible region of the spectrum. In the case of Rh123, the
heteroatom is nitrogen and the R group may exist as an amino group
or an imino group. Many xanthene dyes are amphipathic, that is,
they have both polar and non-polar regions on the molecule. This
property gives the molecule a high affinity for binding to the
surface of biological membranes.
##STR00001##
[0291] Molecular structures of some of the SMMRs of the invention
include those shown as Structures A-F. Other mitochondrial or
membrane potential dyes useful for this invention include any
molecules exhibiting properties as defined for Rh123 above
(Structure A) including those mentioned here, and general compounds
falling within these molecular structures, activity, solubility,
toxicity, and overall action as described. Specific dyes meeting
some or all of these requirements include, but are not limited to,
the following.
Xanthene Type Dyes (Structure B):
[0292] Examples of Xanthene type dyes include: TMRE as
tetramethylrhodamine ethyl ester, perchlorate
(C.sub.26H.sub.27ClIN.sub.2O.sub.7. Molecular Weight 515), TMRM as
tetramethylrhodamine methyl ester, perchlorate
(C.sub.25H.sub.25ClIN.sub.2O.sub.7. Molecular Weight 501),
Dihydrorhodamine 123 (C.sub.20H.sub.18N.sub.2O.sub.3. Mwt: 346),
Dihydrorhodamine 123, dihydrochloride salt
(C.sub.20H.sub.20Cl.sub.2N.sub.2O.sub.3. Mwt: 419).
Cyanine Type Dyes (Structure C):
[0293] Examples of cyanine type dyes include:
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine
both the chloride and the iodide salts.
Bis-Oxonol Dyes
[0294] Examples of bis-oxonol type dyes include: DiBAC4(3) as
bis-(1,3-dibarbituric acid)-trimethine oxanol
(C.sub.27H.sub.39N.sub.4O.sub.6, Molecular Weight 519).
Styryl Pyridinium Dyes:
[0295] Examples of styryl pyridinium type dyes include: RH421,
N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)-pyridinium
(C.sub.29H.sub.42N.sub.2O.sub.3S, Molecular Weight 498.72), DASPEI
as 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide) with a
molecular formula of C.sub.17H.sub.21IN.sub.2 and molecular weight
of 380 daltons, Pyridinium,
4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-,
hydroxide Di-4-ANEPPS(C.sub.28H.sub.36N.sub.2O.sub.3S; Molecular
Weight 481).
Carbocyanine Dyes (Structure D):
[0296] T-3168 is a cationic carbocyanine dye that yields green
fluorescence. It accumulates in mitochondria and is a sensitive
marker for mitochondrial membrane potential. It exists as a monomer
at low concentrations and forms J-aggregates at higher
concentrations that exhibit a broad excitation spectrum and an
emission maximum at .about.590 nm.
Glucose Analog (Structure E):
[0297]
2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose
(2-NBDG). The 2-NBDG fluorophore typically displays excitation and
emission maxima at around 465 nm and around 540 nm, respectively.
It is visualizable using optical filters designed for fluorescein
and is sensitive to its environment. A fluorescent nonhydrolyzable
glucose analog 6-NBD-deoxyglucose (6-NBDG) is also available
commercially to track glucose diffusion rates in cells. (Molecular
Probes cat. no. N-23106).
Viability and Toxicity Dyes (Structure F):
[0298] The cell-impermeant Ethidium Bromide is excited by an
argon-ion laser and is useful for detecting and sorting dead cells
by flow cytometry. It is also used in combination with
fluorescein-based probes (such as calcein, CellTracker Green CMFDA
or BCECF) for two-color applications, and as a marker when a
reporter dye responds at only one emission wavelength.
[0299] The dyes mentioned above are available commercially in
relatively pure forms from suppliers of custom molecules as well as
from Biotium, Inc., 3423 Investment Blvd. Suite 8, Hayward, Calif.
94545. The preceding dyes are commonly described in the scientific
literature as molecules "that stain mitochondria in living cells in
a membrane potential-dependent fashion [with varying excitation and
emission wavelengths]." See the Merck Index (The Merck Index: An
Encyclopedia of Chemicals, Drugs, and Biologicals, Thirteenth
Edition, Maryadele J. O'Neil, Ann Smith, Patricia E. Heckelman,
John R. Obenchain Jr., Eds., Merck & Co., Inc., Whitehouse
Station, N.J., USA, 2001), and other comprehensive collections of
properties for organic compounds. Such references provide
information regarding details of chemical and physical properties
of molecules, including availability, solubility, and synthesis for
each class of molecule described herein. Additional information is
available from commercial suppliers, e.g., Aldrich Chemical
Company, Inc., 1001 West St. Paul Avenue, Milwaukee, Wis. 53233;
Sigma Chemical Co., Inc., 3050 Spruce Street, St. Louis, Mo. 63103;
Fluka-RdH, P.O. Box 2060, Milwaukee, Wis. 53201 USA; Molecular
Probes, Inc., 29851 Willow Creek Rd., Eugene, Oreg. 97402 USA; and
other manufacturers.
[0300] Preferred dyes, acting as SMMRs according to the invention,
emit fluorescence signals at wavelengths above 350 nm.
[0301] The design of specific SMMRs for particular locations and
mechanisms within tissue takes into consideration the specific
molecular properties of the SMMRs. Under conditions where
intracellular and extracellular pH measurements are to be made
simultaneously using redox potential measurements, for example, it
is important that the dyes emit in different wavelength regions of
the electromagnetic spectrum. The spectral regions that are
preferentially selected for emission bands are bands at or about
100 nm wide and centered at or about 600 nm, 700 nm and 800 nm.
These regions contain little or no autofluorescence and a
relatively small and stable absorption background. These properties
allow a relatively interference-free measurement of the SMMR
fluorescence.
[0302] The use of known mitochondrial specific redox potential
fluorescing dyes, or energy transfer fluorescing dyes for the
purpose of sensing live human keratinocyte glucose metabolism in
situ has not been previously described. The fluorescence intensity
of mitochondrial-bound redox potential sensing dyes, as well as
dyes reporting on reducing equivalents via energy transfer, are
indicative of the reduction potential for the sum of oxidative
phosphorylation, fatty acid metabolism, and NADH shuttling. The use
of redox potential fluorescing dyes has not previously been applied
in situ for direct determination of in vivo skin glucose
concentration. The ability to use these dyes for such an
application is based upon an understanding of skin glucose
metabolism, the mechanism and importance of reducing equivalents in
glycolytic metabolism of skin, an understanding of the fluorescent
properties of the selected dyes, and an understanding of the
optical properties of skin. In addition, these methods and
compositions require a detailed understanding and optimization of
dye introduction to the human keratinocyte cells; of the derivation
of the appropriate conditions for temperature, pH, concentration,
purity, lag times, reaction times, response times, quantum yields,
optical properties of the various skin layers for the appropriate
excitation and emission wavelengths, of the
concentration/fluorescence response model, metabolic models for
skin; and of the lag time between blood glucose and skin glucose
concentrations.
[0303] One skilled in the art will recognize that the SMMR
compositions and methods of the invention have both in vitro and in
vivo applications. However, a unique advantage of using SMMR in
clinical diagnostic and treatment applications is that their
spectral response measurements are made in vivo, a distinct
improvement over current in vitro analysis techniques.
[0304] Measurement of these specific analytes and metabolites,
individually or combined with ancillary measurements, provides
detailed information describing glucose metabolism in living
tissue. The specific invention delineated here relates to the
determination of blood glucose levels based upon skin glucose
levels for use in the monitoring and control of diseases related
to, but not limited to, diabetes mellitus, heart disease,
autoimmune disease, kidney disease, memory dysfunction, cancer,
stress and organ transplantation. A description of the metabolic
pathways for glucose in dermis and epidermis is helpful to provide
a basis for this present invention. Mechanisms operating in skin
metabolism are shown in Scheme 1 of FIG. 17A. An additional
overview scheme is provided in FIG. 17B. This present invention
models systemic blood glucose levels based upon the application of
specific first principle mathematical models to direct non-invasive
fluorescence measurements made using SMMR placed within the
skin.
[0305] This invention targets in vivo measurement of
analyte/metabolites that provide detailed information for epidermal
glycolytic pathways that are driven specifically by D-glucose,
fructose, galactose and other simple sugars, but are unaffected by
molecules similar to D-glucose that are not metabolically active.
Such non-active metabolites include L-glucose and other
levorotatory optical isomers, or enantiomeric forms of simple or
complex sugars. This in fact is used as an efficacy test for the
action of glycolytic reporting SMMRs. For complex glycolytic
processes such as the biosynthesis of NAD(P)H, or for glycolytic
processes that are distinctly non-linear, more than one pathway can
be combined to enhance analytical information content to model
glucose concentration. The additional information provided by
monitoring more than one metabolite is used to improve analytical
performance for monitoring glucose. In this way, a final
measurement system provides for a wide dynamic range for glucose
and is less prone to measurement errors caused by potential
interferences.
[0306] Although Scheme 1 (FIG. 17A) shows that the substrate for
oxidative phosphorylation is glucose-derived, this pathway may also
be fueled by lipid metabolism. This is not a concern when
monitoring glycolysis fueled by glucose for human or mammalian
epidermal keratinocytes, since this metabolic pathway is not
relevant to glucose measurement in keratinocytes, as only two
percent of skin metabolism comprises this alternative lipid
pathway, whereas 70% of assimilated glucose is metabolized by
glycolysis, which is a metabolic process that derives energy for
the cell exclusively from the metabolism of glucose. This and other
details of skin metabolism can be found, e.g., in Johnson and
Fusaro. The Role of the Skin in Carbohydrate Metabolism in:
Advances in Metabolic Disorders, R. Levine (Ed.), Academic Press,
1972, 60, 1-55.
[0307] The supply of glucose in the blood both diffuses and is
actively transported into the cytosol of epidermal cells. The rate
of transport into the epidermis is indicative of the differential
concentration of skin glucose levels and blood glucose levels. The
rate of transport into skin allows for an accurate first principles
mathematical extrapolation of blood glucose levels.
[0308] Once modeled, the kinetics of blood glucose transport to the
skin from the blood supply of subcutaneous blood vessels enables
the determination of the precise first principles mathematical
relationship between the rate of change of skin glucose and the
rate of change of blood glucose. Thus, rapid up or down changes in
blood glucose concentration can be accurately tracked by knowing
the skin glucose mean concentration levels and the rate of change
of skin glucose levels. First principles mathematical models can be
developed, preferably for individual patients, more preferably for
small local populations, and most preferably for the universal
patient case.
[0309] The invention provides at least one sensor composition that
includes endogenous chromophores and exogenous
fluorophore/reporters (i.e., SMMR as molecules that fluoresce as an
indication of metabolic rate or by an increase in metabolite
levels). By convention, factors routinely affecting the glycolytic
velocity assumption set for quantitative analysis of metabolites,
including lactate/H.sup.+, are as follows: (1) pH generally has a
small effect at less than 5% relative change between pH 7 and 8;
(2) temperature has a small metabolic effect at semi-controlled
temperatures (e.g., 25.degree. C. to 27.degree. C.); (3)
enzyme/coenzyme concentration is normally in excess to allow
glycolysis over all physiological ranges of glucose; (4) cellular
substrate concentrations are normally in excess to allow glycolysis
over all physiological ranges of glucose; (5) anaerobic/aerobic
ratio for target cells of interest (e.g., epidermal keratinocytes)
is assumed constant per individual; and (6) cell maturity is
relatively constant and assumed to be constant over the gradient of
the epidermis.
[0310] For human keratinocytes in situ, a specific layer of the
epidermis (above the dermal papillae and within or above the
stratum basale) is in a comparatively homeostatic condition and the
major metabolic biosynthetic process is anaerobic glycolysis. This
layer of cells is referred to as the stratum germinativum.
Therefore, cells in the stratum germinativum make an ideal location
for the introduction of SMMR into the skin. See FIGS. 1-3 and 10.
Other tissues favorable for use in the methods and compositions of
the invention include all those having predominantly anaerobic
glycolysis as the main biosynthetic process for glucose
utilization. Thus, the epidermis throughout the human skin and at
all locations becomes a prospective target site for the invention.
Other epithelial tissues lining cavities within the body are also
target cells for the invention. These tissues include: simple
epithelium, e.g., squamous, cuboidal, and columnar; stratified
epithelium, e.g., squamous, cuboidal, columnar, and transitional;
and pseudostratified epithelium. Preferred sites for measurement
application include, but are not limited to, the fingertip, the
volar forearm, the upper arm, the foot, or any location where easy
access to the skin is obtained without the need to disrobe.
[0311] The transport of glucose into the cell is non-insulin
regulated, and the stoichiometry of anaerobic glycolysis provides
two lactate/H+ molecules per one glucose molecule. Thus,
intracellular lactate/H+ measurement provides the basis for
inferring interstitial fluid glucose concentration in normal
keratinocytes. The direct in vivo intracellular measurements of
intermediate or end-product metabolites (analytes) resulting from
glycolysis within keratinocytes are thus used to infer glucose
substrate concentrations within the cell in real-time without the
use of invasive techniques. Endogenous, native fluorophores are not
considered useful reporters of metabolic state due to low
signal-to-noise and to background interferences, but they do
provide information about the optical properties of the tissue and
the integrated history of premature tissue glycosylation that
occurs over time due to the diabetic condition.
[0312] In contrast, the exogenous molecules described herein that
are added as SMMRs to the skin result in fluorescent signals that
directly report on the type and level of metabolite present in the
cell. The SMMRs described herein provide unique fluorescence
signals that are of sufficient magnitude to be measured using
standard, low-cost, commercial photonic components. By using SMMRs,
the extracellular, intracellular, and organelle microenvironments
can be accurately and specifically assessed for glycolytic function
within a tissue or for an organism. The exogenous
fluorophore/reporters are added to the skin and are used to locate
and measure metabolites located within the epidermal layer of the
living skin in situ, thereby indicating the metabolic state of the
organism. In alternative embodiments, these dyes are applied
through oral ingestion, or more preferably by passive or active
topical administration.
[0313] Effective concentrations of SMMRs to be applied are in the
range of at least 1 to 500 .mu.g/ml, e.g. 5 to 150 .mu.g/ml or 10
to 100 .mu.g/ml. The concentration of SMMRs used is preferably from
10 to 500 .mu.M, more preferably from 100 to 300 .mu.M and most
preferably from 150 to 250 .mu.M.
[0314] The methods and compositions of the invention employ the
measurement of the fluorescence of SMMRs added to the skin to
monitor glycolytic metabolic processes in the skin. These processes
respond to blood analyte levels and to disease states affected by
glycolytic activity. Autofluorescence by itself is insufficient to
monitor many analytes, particularly glucose, because it does not
have the necessary signal-to-noise ratio and dynamic range to be
useful (i.e., accurately measured at low cost). Instead, the
instant methods and compositions replace or supplement
autofluorescence measurements with measurements of exogenous
molecules that act as metabolite reporters localized within the
epidermis.
[0315] Each of the following aspects of the SMMR system was
optimized in order to derive the methods for utilizing exogenous
molecule fluorescent signals in the keratinocytes for deriving
blood glucose levels. The key informational requirements and
assumptions include: [0316] 1. Diffusion following the laws of mass
transport is the main mechanism of transport for small molecules
(including D-glucose) from blood in the dermis to the keratinocytes
of the epidermal layers; [0317] 2. Human keratinocytes utilize
GluT1 (GenBank Accession Number: K03195) at the cell membrane
(i.e., glucose transport is not insulin or GluT4 (GenBank Accession
Number: M91463) regulated); [0318] 3. Glucose transport at the
keratinocytes is constant relative to the maximum velocity of
molecular transport and the number of active transporters within
the keratinocyte cell membrane. If these are not constant, they
must be modeled based upon a first principles understanding of the
events that bring about changes in the transport rate. The overall
effect must allow modeling of extracellular glucose levels based
upon intracellular glucose levels. Thus, the intracellular glucose
concentration must be based upon a known relationship to the
concentration of glucose within the interstitial fluid; [0319] 4.
Keratinocytes are relatively simple cells utilizing as much
D-glucose as is available at any time without changing metabolic
mechanisms (they remain essentially glycolytic);
[0320] and they process glucose in real-time into metabolites that
are directly measurable using SMMRs; [0321] 5. There is a net
NAD(P)H production via the pentose shunt from glycolysis, thereby
providing a mechanism for glucose measurement by using an amplified
NAD(P)H signal; [0322] 6. SMMR compounds can be synthesized to
demonstrate desired performance properties based upon known
characteristics of molecular structure; [0323] 7. All proposed
techniques using the SMMR compounds described in this invention are
adaptable to small, inexpensive measurements, such as using a
handheld device; [0324] 8. pH (as lactate/H.sup.+), NAD(P)H,
Ca.sup.2+, FAD.sup.+, ATP/ADP ratio, and redox potential can be
used to directly track D-glucose concentration present in the fluid
surrounding human skin keratinocyte cells; [0325] 9. For anaerobic
glycolysis (i.e., the metabolism of target human skin cells or
keratinocytes), pH (as lactate/H.sup.+), NAD(P)H energy transfer,
and redox potential provide the most rapid and trackable responses
to glucose. The shortest response times are from 15 seconds to 2
minutes. SMMRs utilize three separate reporting mechanisms to
report for these three glycolytic metabolites, including direct
reporting, energy transfer, and redox potential, respectively;
[0326] 10. There is a lag time for diffusion of glucose from the
capillary fields of the dermis to the cells of the epidermis of no
more than approximately 5-10 minutes for highly vascularized
regions of the body, such as the fingertip; [0327] 11.
Intracellular, extracellular and organelle lactate/H.sup.+ is
measured as a direct indication of D-glucose concentration of
surrounding fluid, where lactate/H.sup.+ is an indicator of
keratinocyte glycolysis; [0328] 12. Measurable D-Glucose response
range for these parameters is 5 to 500-plus mg/dL; [0329] 13. Human
skin cells are scavenger cells, which utilize as much D-glucose as
is available at any time without changing glycolytic or transport
mechanisms; [0330] 14. Commercially available dye probes are useful
but not optimal. Thus, strategies for independent new molecules in
this regard have been developed; [0331] 15. Reporters passively
transported to the skin can last up to 4 days or more using
currently known methods; [0332] 16. Direct glucose measurements are
possible for small treated areas of the skin but require the use of
larger SMMR compounds (i.e., 100-160 kDa or more), indicating the
possible requirement for electroporation schemes; [0333] 17. Small
quantities of larger SMMR compounds can be optimized for signal
intensity and, thus, are useful for making glucose measurements
without toxicity or irritation issues in mammals, including humans;
[0334] 18. A very small reaction site (i.e., 200 to 300 microns in
diameter) can be used, thereby minimizing toxicity issues; [0335]
19. SMMRs as proteins, reporters and markers are placed at desired
locations at the skin surface or below, namely from 10 to 500
microns in depth from the tissue surface; [0336] 20. Reporters are
easy to get into the skin using passive mechanisms, but
electroporation gives enhanced signal magnitudes by factor of 2 to
3 times. Electroporation is inexpensive, but adds a degree of
complexity to the method; [0337] 21. None of the tested mechanisms
respond to L-glucose, thereby making the tests specific for
D-glucose only. (This is the `gold standard` for testing the
efficacy and veracity of any glycolytic and physiologically active
glucose-concentration measuring technique); [0338] 22. Simple
sugars, such as D-glucose, fructose, and galactose, are the sugars
of interest relative to fueling glycolysis, and all cause
glycolytic activity in keratinocytes. Techniques for Placement of
SMMRs into the Epidermis
[0339] For any of the embodiments described herein, a series of
techniques exist that allow the placement of specialized
fluorescent or absorptive molecules (SMMRs) into the epidermis,
epithelial cells, or peripheral cells (for organs or muscle tissue
during invasive surgery). Penetration of the sensor composition can
be accomplished using an active transport technique, such as, for
example, electroporation, laser poration, sonic poration,
ultrasonic poration, iontophoresis, mechanical poration, solvent
transport, direct application by painting, tattooing methods
involving application by needle, an equivalent electrical tattooing
technique; or most preferably by using passive transport using
special solvent and reporter molecule mixtures. Passive transport
may be used to allow small molecules of typically 100 Daltons (Da)
to 1000 Da to enter tissues and cells.
[0340] Exemplary methods for passive transport are pressurized
delivery and wicking. The method is comprised of a direct
measurement of the fluorescence of SMMRs placed within epidermal
cells, i.e., keratinocytes. This fluorescence is measured using
molecules with specific properties for defining glucose metabolism
in epidermis and for inferring the magnitude of the change in
fluorescence signal to blood glucose concentrations.
[0341] Incorporation of a reporter into the tissue without use of
an external device is preferred, due to the reduced cost,
convenience, and ease of use. Such a passive transdermal delivery
solvent system must be accurate and safe. Thus, a more elaborate
solvent regime must be applied than that used for the active
mechanisms such as tattooing, electroporation, and ultrasonic
poration. Suitable solvent systems useful for passive transdermal
delivery include creams, emulsions, and oils. These solvent systems
provide passive transdermal stain delivery into the tissue at a
depth of less than 50 microns. The following additives aid the
process of tissue penetration for SMMR and create a diffusion rate
enhancing solvent system: Soybean Oil, Hazelnut Oil, Jojoba Oil,
Sweet Almond Oil, Olive Oil, Calendula Oil, Apricot Kernel Oil,
Grapeseed Oil, Wheat Germ Oil, refined Light Mineral Oil,
Triundecanoin (Akomed C), Undecanoic acid, Caprylic/Capric
Glycerides (Akoline MCM), Caprylic/Capric Triglycerides, Propylene
glycoldiester of caprylic-/capric acid, Emu oil, all as low
viscosity mixtures, preferably less than 35 cSt at 35.degree. C. In
addition, mixtures of one or more of the above oils in combination
with a non-polar dilution solvent can also be used. The solvent
system is allowed to passively penetrate the tissue for from about
1 minute, about 5 minutes, about 10 minutes, about 30 minutes to
about 2 hours to allow diffusion of the SMMR into the appropriate
tissue layer(s).
[0342] In addition, penetration of the sensor composition to the
desired depth can be accomplished by combining the composition with
various molecular size attachments.
[0343] After the reporters are injected into, or applied to the
surface of the tissue, they are allowed to penetrate in proximity
to superficial cells of tissues and organs at a depth from the
surface of the cells of from about 10 .mu.m to about 1500 .mu.m.
For measurement of specific metabolites, the preferred placement of
the reporters should be near the surface of the tissue (i.e., about
10 to about 175 .mu.m) yet be representative of the overall
metabolic state of the tissue in which the reporters are placed.
The reporters may also be placed at a greater depth into the
tissue. The precise placement of the reporters is controlled by the
combination of its molecular properties, including: specific
molecular size (i.e., 100 daltons to 100 kilodaltons), polarity,
charge, structure, pKa, solubility, the size and type of molecular
attachments or anchors, the solvent system used, as well as the
specific conditions used for poration (if required). A combination
of these factors provides the ability to control the location,
diffusion rate, and duration or lifetime of the SMMR within the
tissue or organ layers.
[0344] The dyes may be introduced into the skin by passive
diffusion over a period of 2448 hours, more preferably over a
period of 2-6 hours, and most preferably in 10 seconds to 5
minutes. Contemplated diffusion times include periods less than 48
hrs, 24 hrs, 10 hrs, 6 hrs, 2 hrs, 1 hr, 30 min, 15 min, 10 min, 5
min, 1 min, 30 sec, 10 sec, or 1 sec. With passive absorption, a
molecule is placed on the surface of the skin and allowed to
penetrate in proximity to the epidermal cells (keratinocytes)
directly above the basal layer (stratum basale) at a depth from the
surface of skin from 10 .mu.m to 50 .mu.m and up to 175 .mu.m in
the pits of the stratum basale extending into the dermis between
the dermal papillae. For measurement of glucose, the placement of
the SMMR is below the stratum corneum yet above the dermis, more
specifically in the stratum spinosum or stratum basale immediately
above the upward extensions of the dermal papillae. This SMMR
placement is accomplished by varying the combination of the
polarity and charge on the SMMR, the size of molecular attachments
or anchors, as well as by the polarity and hydrophilicity
characteristics of the solvent system. The specific conditions for
poration or passive diffusion for placement of the SMMR in the skin
are controllable factors. Using any combination of these factors,
it is possible to control the localization of the dye within the
skin layers and target cells.
[0345] Another embodiment of the reporter application involves the
use of a reservoir containing reporter, which is used to
automatically or manually dispense a dose of the reporter mixture
topically prior to poration or passive transport. For measurement
of metabolites and precursors the reporter is placed in the tissue
at a depth of up to 300 .mu.m. A solution of 10-400 .mu.L volume
made from 1-50 .mu.M SMMR in a solvent system penetrates into the
tissue for some period of time to allow activation following
passive diffusion kinetics. Once activated the change in
fluorescence or absorption response of the tissue cells to changes
in extracellular and intracellular metabolite or precursor
concentrations is monitored directly using an optical reader.
Irritant chemicals such as salicylic acid can be used to facilitate
the penetration of reporters into skin or peripheral tissue.
[0346] In another embodiment, a small disposable film patch
composed of polyolefin, polyester, or polyacrylate and having an
SMMR dispersed into a transfer gel applied to the transfer side of
the film patch, is used for SMMR application. The patch is applied
with the gel side toward the skin and the gel contacts the external
surface of the skin. Following the gel application, a poration or
passive transfer technique is used to introduce the mixture into
the appropriate skin layer(s) (as described above). Another
embodiment of the SIAM application involves the use of a reservoir
containing molecular tag or SMMR. This reservoir is used to either
automatically or manually dispense a dose of the SMMR mixture
topically prior to poration or passive transport. A non-limiting
example of a topical dose is a small dot or spot from 100 .mu.m to
5 mm. A smaller area is preferred in most embodiments, but a larger
area is also contemplated. For measurement of glucose, the SMMR is
placed in the keratinocytes at 30 .mu.m to 50 .mu.m and up to 175
.mu.m so that placement is precisely in the specific layer of the
epidermis (e.g., above the dermal papillae and within or above the
stratum basale), within a comparatively homeostatic keratinocyte
stratum. The molecular tag or SMMR penetrates into the skin for
some period of time (depending upon molecular size and solvent
mixture used) to allow activation following passive diffusion
kinetics (i.e., mass transport). Once activated, the change in
fluorescence response of the skin cells to changes of extracellular
and intracellular glucose is monitored directly using an optical
reader.
[0347] An active mechanism utilizing tissue permeation,
electroporation, laser poration, or ultrasonic poration is another
procedure for introducing SMMRs into the skin. Pulse lengths for
poration technologies are provided below. An example of an
ultrasonic poration device includes those manufactured by Sontra
Medical Corporation, Cambridge Mass. Sontra and other commercial
manufacturers of devices useful for this application have
previously described a method for sensing glucose directly in the
interstitial fluid surrounding the skin cells by removing fluid or
gaining access to removed fluid for analysis. See e.g., J A Tamada,
M Lesho and M J Tierney, "Weekly Feature: Keeping Watch on
Glucose--new monitors help fight the long-term complications of
diabetes." IEEE Spectrum Online, Jun. 10, 2003 at website:
<http://www.spectrum.ieee.org/WEBONLY/publicfeature/apr02/glu.html>
(last visited Jun. 26, 2003). The methods and compositions of the
invention do not remove fluid but, rather, place small quantities
of solution containing low concentrations of SMMRs into the skin
for direct reading of the SMMR fluorescence spectral
characteristics as an indication of both epidermal skin and blood
glucose levels.
[0348] For some reporters above 1,000 daltons in size,
electroporation may be used to introduce reporter into tissue.
Electroporation has been utilized for introducing chemotherapy
treatments, for introduction of DNA into living cells and tissues,
and broadly recommended for introducing materials into tissues for
cosmetic or medical treatment applications. If poration schemes are
used, the optimized settings for an electroporation device are
achieved by commercially available or by a customizable device
having settings that provide conditions as described within this
invention. Commercial systems utilizing a square wave voltage pulse
have been described within the literature, such as those available
from Genetronics Biomedical Corporation, 11199 Sorrento Valley
Road, San Diego, Calif. 92121. Such a small device can be
inexpensively made to have one or more constant settings for the
optimized conditions disclosed for this invention.
[0349] Electroporation uses a short pulse electrical field to alter
cell membrane permeability. Micro-pores form in the membrane of
skin cells allowing the introduction of various molecular size
mixtures into the cells at an appropriate depth of penetration for
this specific inventive application. When the electric field is
discontinued, the cells return to normal and one or more SMMRs
introduced into the cell using the technique remains at the
cellular site specifically within the epidermal cell until either
the dye is chemically degraded and disposed of within the tissue or
is sloughed off in a normal desquamating cycle. The process of
sloughing off (or desquamation) follows a normal ten-day to
twenty-day (typically fourteen-day) cycle as the residence time of
epidermal keratinocytes moving from the basal layer (stratum
basale) to the desquamating layer of the stratum corneum.
[0350] When employed, electroporation is optimized for use in this
invention by selection of voltage range (from about 40 to 90
Volts), gap distance (from about 0 to 2 mm), pulse length (from
about 150 to 250 ms), number or pulses (from about 1-10), pulse
interval (from about 5 to 60 s), specific electrode design, and
desired field strength (from about 40 to 60 V/cm). In addition, the
selection of molecular tag molecules, solvent molecules,
concentration, and lag times relative to measurement onset is
determined as precisely as possible. In certain embodiments,
specific parameters are determined empirically using specific
solvent and SMMR selection. For example, optimization of
electroporation involves the following specifications: [0351] 1.
Output voltage range: 0 to +200 VDC; [0352] 2. Discharge capacitor
(Cdis) values in microfarads are on or about: 200, 500, 700, 1000,
1200, 1500, 1700 .mu.F; [0353] 3. Pulse type: exponential decay;
[0354] 4. Pulse RtCdis decay time constant where Rt (total)=5+Rskin
in parallel with 50 ohms. If Rskin>>50 ohms then Rt=55 ohms
and Rt.times.Cdis=11, 27.5, 38.5, 55, 66, 82.5, 93.5 milliseconds
(ms).
[0355] Electroporation also facilitates the delivery of dyes bound
to large molecules that serve as anchors such as polymer beads,
large polysaccharides, or colloidal particles. These approaches are
contemplated as being within the invention, but are less
advantageous in that the particles are often too massive to pass
through the stratum corneum without active poration or mechanical
injection. Once in the skin, they do not readily dissolve or
organically reabsorb into the body. Such less desirable approaches
would create undesirable particles that would either remain in
place indefinitely or accumulate in lymph nodes, in other
circulatory cavities and/or in other organ sites.
[0356] Reporters of the invention can be made with specific
properties such that they are retained only within skin cells
(keratinocytes) where they report on glycolytic activity and do not
harm or affect cellular metabolism. These reporter compounds are
sloughed off after a few days, even when permanently integrated
into, or attached to, keratinocyte cells. The small quantity of
reporter(s) that diffuse away from the epidermis are rapidly
degraded within the body and are completely eliminated within a few
days. In preferred embodiments, reapplication of the reporter(s) is
relatively easy to perform. The process of sloughing off (or
desquamating) follows a normal ten-day to twenty-day (typically
fourteen-day) cycle as the residence time of epidermal
keratinocytes moves from the basal layer (stratum basale) to the
desquamating layer of the stratum corneum. Thus, reporters are
developed to be applied once every 2 to 3 days, preferably every 3
to 4 days, and more preferably every 5 or more days.
[0357] The present invention introduces one or more SMMRs into the
skin and then measures the fluorescence of the SMMR as an indicator
of the skin glucose concentration. Electroporation can be used to
introduce SMMRs into a specific skin site for measurement of SMMRs
to report glucose concentration. Specifically, electroporation or
passive transport via diffusion and wicking is used explicitly to
introduce one or more specific molecular compounds (SMMR) and a
solvent system into the appropriate skin layer in order to more
rapidly introduce the SMMR for subsequent fluorometric
analysis.
[0358] In another preferred embodiment, the passive transdermal
delivery solvent system employed is efficacious and safe. A more
elaborate solvent regime may be applied than for the active
mechanisms of traditional tattooing procedures, where dyes and inks
are placed into the dermis for permanent marking; or poration
schemes such as electroporation, laser-poration, iontophoresis,
mechanical-poration, pressurized delivery and ultrasonic
poration.
[0359] The more advanced solvent systems useful for passive
transdermal delivery include, but are not limited to, e.g., creams,
emulsions (both oil-in-water and water-in-oil), oils (ointments),
gel film patches, a reservoir device, paints, polar solvents and
non-polar solvents. Non-polar solvents are preferred, as these are
most miscible with the SMMRs of the invention and the stratum
corneum lipids cementing the keratinocyte lamellae in place. "Lipid
solvent systems" have been reported in the literature for use in
transdermal drug delivery, and are composed to resemble the
chemistry of stratum corneum lipids. Such a mixture may also be
used to place the SMMRs into the appropriate point within the
epidermis. Such a suggested mixture includes: (w/w): ceramide
(50%), cholesterol (28%), palmitic acid (17%) and cholesteryl
sulfate (5%). See, e.g., Downing et al.: Partition of dodecyl
sulfate into stratum corneum lipid liposomes. Arch. Dermatol. Res.
1993, 285:151-157.
[0360] The objective of each of these solvent systems is to provide
passive transdermal SMMR delivery into the skin at a preferred
depth of from about 10 to 175 .mu.m (microns), more preferred from
about 20 to 100 microns, and most preferred from about 20 to 50
microns. For example, the following solvents as additives to the
final SMMR mixtures are added to the skin to initiate passive
transport of the SMMR to the target cellular site. The materials
listed aid the process of skin penetration for SMMRs and create a
diffusion rate enhancing solvent system for transdermal delivery:
dimethyl sulfoxide, ethanol, isopropanol, chloroform, acetic acid,
saturated hydrocarbon solvent (with from 10 to 40 carbons as linear
or branched chained molecules), soybean oil, hazelnut oil, jojoba
oil, sweet almond oil, olive oil, calendula oil, apricot kernel
oil, grapeseed oil, wheat germ oil, refined light mineral oil and
mineral oil spirits, triundecanoin (akomed C), undecanoic acid,
caprylic/capric glycerides (akoline MCM), caprylic/capric
triglycerides, propylene glycoldiester of caprylic-/capric acid,
and emu oil. All are low viscosity mixtures, preferably less than
35 cSt at 35.degree. C. In certain embodiments, mixtures of one or
more of the above oils are used in combination with a non-polar
dilution solvent.
[0361] Factors that control the depth of penetration of the SMMR
and its compartmentalization into the cells and domains of the
epidermis include the polarity and partition coefficient of the
SMMR as well as the solvent and the molecular size. The SMMR
compound may also be derivatized so that it is readily taken up by
the cell and then acted upon by enzymes that chemically alter the
SMMR to prevent it from leaking out of the cell. One advantage of
this type of approach is that the SMMR is only taken up in its
active form by viable cells. Predictive schemes for determining
appropriate derivatization of SMMR compounds are provided below.
Alternative methods of derivatization well known to those skilled
in the art are also contemplated as part of the invention.
[0362] The physical properties of the solvent system that strongly
influence permeability in the skin include the molecular size, the
vapor pressure, the water solubility, and the octanol water
coefficient. Smaller molecular size increases the diffusion
coefficient. The vapor pressure controls the balance between
diffusion into the skin and evaporation from the surface. The water
solubility and the octanol water partition coefficient determine
the miscibility of the SMMR solution between aqueous interstitial
fluid and hydrophobic core of the cell membrane.
[0363] For a passive solvent delivery system, the depth of
penetration of the SMMR is strongly dependent on the volume of
solvent added. Typically, the volume of SMMR used is from 10 .mu.L
to less than about 100 .mu.L. Preferably, the concentration of SMMR
is from 10 to 500 .mu.M, more preferably from 100 to 300 .mu.M, and
most preferably from 150 to 250 .mu.M. Target cells are exposed to
extracellular concentrations in the range of 1 to 10 .mu.M.
Dilution of the SMMR concentration arises because of the diffusion
properties from the surface of the tissue to the target cell
site.
[0364] The proposed volume range added to the skin or other tissue
is preferably from 1 to 50 .mu.L, more preferably from 5 to 20
.mu.L, and most preferably from 5 to 15 .mu.L. Alternatively, a gel
patch is used containing an SMMR coated surface of approximately 6
mm in diameter consisting of a concentration of SMMR preferably
from 10 to 500 .mu.M, more preferably from 100 to 300 .mu.M, and
most preferably from 150 to 250 .mu.M.
[0365] Solvent systems used for SMMRs may be adjusted depending
upon their molecular properties and compatibility with the specific
SMMR being delivered. For example, solvent hydrophobicity and
polarity are noted along with the solubility properties of the
SMMR, Which will all have an effect on the movement of the SMMR
into the tissue. Each SMMR has a certain affinity for the solvent
and the tissue. The solvent's activity for delivering the SMMR
directly to target tissue is a matter for empirical testing. One
preferred embodiment of the invention uses an SMMR dissolved in
DMSO (dimethyl sulfoxide) and further diluted in a saturated
hydrocarbon solvent (with from 10 to 40 carbons as linear or
branched chained molecules), or an alcohol (with from 2 to 4
carbons) at a volume ratio of 5:95 to 20:80, respectively. The
optimum volume of DMSO in the delivery solvent is less than 20
percent, as the DMSO is used to facilitate dissolution of the SMMR
into the carrier hydrocarbon mixture. The mixture is added to the
tissue in the concentrations and volumes described above.
[0366] A gel patch may be used to apply the SMMR. In one
embodiment, a gel contains the SMMR in a volatile hydrocarbon
solvent in suspension with a polymer such as PVA (polyvinyl
alcohol). When placed against the skin or other living tissue, the
heat of the skin causes the SMMR (dissolved in the PVA-hydrocarbon
solvent) to diffuse into the skin. The final diffusion depth is
controlled by length of application time. Volumes below 100 .mu.L
minimize extraneous transdermal delivery and maximize delivery into
the epidermis target area. Optimum passive solvent delivery is
attained by using a solvent mixture or emulsion that facilitates
the movement of SMMR across the stratum corneum into the epidermis,
but then dissipates rapidly to limit movement of the SMMR away from
the target area. Solvent systems that have the lowest toxicity
include water, saturated hydrocarbon oils, polyethylene glycols and
glycerol. Solvents systems that include alcohols and dimethyl
sulfoxide are less favored in this application since these solvents
are less biologically inert.
[0367] The SMMRs are applied directly to the surface of the skin
and then passively allowed to penetrate the skin for a period of 1
minute to 5 hours, more preferably less than 4 hours, and most
preferably less than 1 hour. Ideally, a solvent delivery system
would be developed to provide SMMR delivery to the target tissue in
less than 1 hour, more preferably less than 30 minutes, and most
preferably in less than 5 minutes. This time period allows the
passive diffusion of the SMMRs into the appropriate epidermal
cells.
[0368] Once the one or more SMMRs are activated as a result of
placement within the skin, measuring fluorescence monitors the
response of the skin cells to glucose. As described herein, the
fluorescence mechanism used is either a direct or indirect
indication of the glucose concentration in the target cell
environment. Fluorescence is typically measured using an optical
reader. The optical reader calculates the skin response to glucose,
applies first principles mathematical models to the response (as
described below and shown in FIG. 7), and provides a determination
of the blood glucose levels (See FIGS. 3, 8-9). The choice of the
particular commercially available or custom designed optical reader
that is compatible for use with the methods and compositions of
this invention is within the ability of one skilled in the art of
the invention.
[0369] The application of reporters to the glucose glycolytic
pathway is illustrated in FIGS. 22 and 23. FIG. 22 illustrates the
glycolytic pathway and FIG. 23 highlights the positions within this
metabolic pathway that are measured using reporters (e.g., the
bold, underlined, italicized molecules*). In corresponding pathways
for fructose metabolism (FIGS. 24 and 25), and galactose metabolism
(FIGS. 26 and 27), the reporters technique measures portions of
each metabolic pathway that are highlighted similarly.
[0370] FIG. 7 is a flow chart showing signal processing logic for
determining final corrected blood glucose levels from a
fluorescence measurement of an SMMR in the skin or other tissue.
The Detector signal (as fluorescence or diffuse reflectance) is
pre-amplified and the initial fluorescence ratio calculation is
made. The signal is corrected using a diffuse reflection or
empirical correction scheme (*Corr.) to produce G.sub.C. Next, one
or more of a series of Demographic functions are applied to the
initial glucose (G.sub.C) calculation to obtain a demographically
corrected glucose level (G.sub.D). This correction takes into
account optical and minor physiological differences derived for
demographic groups. The G.sub.D value is then corrected for
physiological differences such as exercise level, diabetes state,
and health conditions resulting in a glucose value corrected for
unique physiological conditions (G.sub.P). The G.sub.P value is
subjected to a final correction for the lag component of
translating diffusion of blood glucose to skin glucose levels
resulting in the final blood glucose estimation (G.sub.BG). This
result is reported as the physiological blood glucose for an
individual and is considered the analytical result.
[0371] Simultaneously, a quality value is calculated telling the
user the quality of the measurement taken and of the resultant
glucose value reported. Based on this quality value, the user may
be instructed to make one or more additional measurements until the
quality value is indicative of an accurately reported glucose
result.
[0372] An extension of this embodiment is the addition of SMMR
molecules that are allowed to penetrate more deeply into the skin.
In some embodiments, SMMR penetrate as far as the papillary layer
of the dermis (upper corium), and into the reticular layer of the
dermis (lower corium), up to about 300 .mu.m. In other embodiments,
SMMR are applied into the subcutaneous layers of the skin. In
further embodiments, injection or ingestion of reporter molecules
into the bloodstream, or into specific organs or tissues, is
utilized. The resultant fluorescence response is measured at the
site of application, e.g., by using an optical reader with remote
optics (i.e., optical waveguides).
[0373] In alternative embodiments, the tissue being monitored is
exposed, as in surgery or injury, or viewed remotely using invasive
fiber optics, light pipes, or camera-based remote optics.
[0374] The SMMRs may be applied directly to the tissue of a subject
or is integrated into paint or gel. The paint or gel containing the
SMMR is placed directly on the stratum corneum (outer skin) at one
or more of several recommended sites. These sites include, but are
not restricted to, e.g., the fingertip, the volar forearm, the
upper arm, the foot, or any location where easy access to the skin
is obtained without the need to disrobe. An added advantage for
such a choice of location is to not induce embarrassment when in
public display. A preferable location is on the side of the finger,
where SMMR are tagged just above the first knuckle. This avoids
both inconvenience to the user and contamination brought about by
prevalent use of the fingertip for routine activities. The volar
forearm may also be used but is less preferred due to a decrease in
vascularization at this skin site. The fingertip area is most
preferred due to its increased relative vascularization as compared
to the volar forearm and due to its convenience as a personal
monitor site for both public and private use.
[0375] Specialized methods using painless jet injection for
placement of reporter into the tissue are also encompassed by the
invention. However, such techniques are not preferred, due to the
high initial cost of the device. Multiple versions of jet injectors
are commercially available (e.g., the INJEX.TM. developed and made
available to the European market by ROSCH .DELTA.G Medizintechnik
i.G.)
[0376] SMMR may be packaged and sold in any clinically appropriate
manner known to those skilled in the art, including in individual
containers or in kits, and with or without disposable or
non-disposable applicators. A disposable applicator containing a
solvent mixture, including but not limited, e.g., to a liquid or
gel, and containing one or more SMMR, can be placed directly onto
the outer skin and allowed to remain in place for a period of time.
The time required for the SMMR to become activated is typically
from 1 sec to 3 hrs, preferably less than 2 hrs, less than 1 hr,
less than 30 min, less than 10 min or less than 5 minutes.
[0377] The mechanism of action of the SMMR is to act as an in vivo
fluorescence reporter for metabolites that are stoichiometrically
proportional to other non-fluorescent metabolites that are part of
well characterized metabolic pathways (such as glycolysis). These
stoichiometric relationships are most applicable when operative in
living systems. These mechanisms and the techniques used to target
these pathways are summarized in Scheme 1, FIGS. 10-17.
Mitochondrial Membrane Redox Potential
[0378] Once introduced to the epidermal intercellular fluid and
keratinocytes, the SMMRs will migrate preferentially to the target
cells and cellular structures of the live epidermal cells
(keratinocytes), which are directly above the basal layer (stratum
basale). For effective measurement of glucose, the placement of the
SMMR is preferably below the stratum corneum yet above the dermis,
more specifically above the dermal papillae, because the device
measures the glycolytic/metabolic activity of living cells, and the
cells in the stratum corneum are essentially dead. The use of dyes
that require activation by metabolic processes within the cell
limits background interferences from a SMMR that has penetrated
into dead tissue. Therefore, the methods and compositions of this
invention are also useful for distinguishing between live and dead
tissue using the principle of activation by metabolic processes.
For example, SMMRs used as metabolic reporters will report the
level of metabolic activity in target cells whether dead, normal,
hypo-metabolic, or hyper-metabolic. This is due to the surprising
discovery that SMMR-treated tissue provides unique spectral
responses for metabolically active living tissue that are
significantly different from the spectral responses from dead
tissue. SMMRs that provide the most useful spectral responses
include, but are not limited to, molecules providing fluorescence
reporting of reducing equivalents, reduction-oxidation potential,
and the presence of metabolites actively produced during
biosynthetic processes such as glycolysis.
[0379] The SMMRs selected for use in the methods and compositions
of the invention should have an affinity for the keratinocyte
target cells and cellular structures located within the stratum
spinosum. In some embodiments, the SMMR remains in place for
several hours and/or throughout the life cycle of the epidermal
keratinocyte cells and is eventually sloughed off as part of the
desquamating layer of the stratum corneum. Epidermal keratinocytes
have an average lifespan of 14 days, ranging from 4 to 20 days, and
even up to 30 days, depending upon the individual subject skin
health conditions, physical abrasion, or unprotected use of caustic
or acidic chemicals on the skin. In most embodiments, the SMMR is
introduced in low concentration, typically from 10 .mu.M to 500
.mu.M, at nominal volumes from 200 .mu.L to 5 .mu.L, respectively.
The insertion of the SMMR within the epidermis provides an added
safety feature, such that only short-term exposure to the SMMR
occurs at any potential measurement site. In certain embodiments,
the final interstitial fluid concentration of SMMR used is from
0.01 to 500 .mu.gml.sub.-1, more preferably 1 to 100
.mu.gml.sup.-1, and most preferably 5 to 20 .mu.gml.sup.-1, based
upon a molecular weight of approximately 380 Daltons for the SMMR.
These concentrations apply irrespective of the molecular weights of
the SMMR, which range from approximately 100 Da to approximately
250 kDa. For the invention, the SMMR for these molecular weight
ranges can be placed into target tissues for reporting of
glycolytic activity or other metabolic processes. Dosage of the
SMMR solution involves adding 1 to 100 .mu.L to a spot that is
0.1-5 mm in diameter, preferably less than 2 mm in diameter. One
skilled in the art will be able to modify these dosage requirements
based on empirical test results for specific metabolic reporting
applications and signal intensity requirements.
Direct Measurement of Glucose Using the Sensor Compositions of the
Invention
[0380] In another embodiment of the invention, glucose levels are
measured using a direct mechanism for in vivo fluorescence
measurement of glucose. Direct measurement technologies utilize a
mechanism for fluorescent spectra that responds directly to the
glucose molecule itself, rather than ones that respond to changes
in a related metabolite or analyte. The methods involve applying
the sensor composition of the invention to a surface of the skin
for a predetermined period of time, causing penetration of the
sensor composition to a depth of about 10 .mu.m to about 175 .mu.m,
monitoring a change in glucose concentration in the skin by
detecting changes in the fluorescence or absorption, and
correlating the glucose concentration within the skin with blood
glucose levels, thereby determining the concentration of glucose in
the blood. However, depths up to about 300 .mu.m are also
contemplated as part of the invention.
[0381] Accordingly, the present invention provides materials,
apparatuses, and methods for several non-invasive techniques for
determining in vivo blood glucose levels based upon the direct
measurement of glucose levels present in the skin. These methods
use reporters of the invention to determine glucose levels in the
skin, which may then be correlated to blood glucose levels as
described herein.
[0382] Sensor compositions are disclosed, wherein one or more
reporters are deposited at a depth from the surface of the skin of
from about 10 .mu.m to about 175 .mu.m in the epidermis at an
effective concentration. However, depths up to about 300 .mu.m are
also contemplated as part of the invention. When the reporter
contacts a molecule of glucose, a change in fluorescence or
absorption of the one or more reporters occurs, thereby allowing
quantification of the change in fluorescence. The measured change
in fluorescence is indicative of the total glucose concentration
within the skin. The quantification of the change in fluorescence
is performed using fluorescence or absorption spectroscopy, or an
equivalent wavelength emission detection technology.
[0383] As one or more reporters are deposited at the epidermis, the
fluorescence response is measured using a handheld sensor,
preferable a low cost handheld sensor. Reporters are preferably
relatively small entities of specific molecular size, polarity,
charge, and structure, which undergo a change in fluorescence or
absorption when brought in contact with an analyte molecule.
[0384] These methods utilize glucose oxidase or modified glucose
oxidase to measure skin glucose in vivo by reacting SMMRs directly
with glucose within the skin to form a colored or fluorescent
product. The quantity of color change or fluorescence is indicative
of the total glucose concentration within the skin. The skin
glucose thus determined is used to infer blood glucose levels as
calibrated and described herein. A SMMR fluorophore can be
intercalated into glucose oxidase at the FAD site or secondarily
attached to the periphery of the molecule where it fluoresces when
brought in contact with a specific analyte molecule or a byproduct
of a reaction of the fluorophore-attached enzyme with the analyte
molecule.
[0385] The one or more SMMRs used in this aspect of the invention
include, for example, Glucose Oxidase-Labeled Fluorophore (GO-LF)
and Glucose Oxidase-having FAD in the triplet state
(GOx-.sup.3FAD*).
[0386] The use of a Glucose Oxidase--Labeled Fluorophore (GO-LF) or
Glucose Oxidase--with a photooxidizable cofactor (such as FAD), or
another intercalated fluorophore, provides detailed information
regarding in vivo glucose levels in the picomolar through
millimolar range in living skin tissue, interstitial fluid, or
blood. Measurement and determination of blood glucose levels, based
upon skin glucose levels, is a valuable tool in the monitoring and
control of diabetes mellitus. According to the present invention,
specific first principle mathematical models are applied to the
direct non-invasive determination of skin glucose levels in order
to model the blood glucose levels.
[0387] In another embodiment of this invention, methods for
monitoring blood glucose levels using photo-induced electron
transfer are described. These methods, as an exemplary
fluorescence-labeled protein SMMR is used, such that the reaction
of glucose with the triplet excited state of the FAD moiety
contained with the glucose oxidase protein (GOx-.sup.3FAD*) may be
monitored kinetically by a reduction in the lifetime of the triplet
state and under steady state conditions by a decrease in the
triplet absorption. This measurement of the triplet state of FAD
for glucose monitoring is provided. The skin glucose thus
determined is used to infer blood glucose levels.
[0388] In yet another embodiment, protein-labeled fluorophores and
proteins comprising a photooxidizable cofactor (such as FAD), or
proteins comprising another intercalated fluorophore are provided
as direct reporters of glucose or glucose metabolic products
throughout the anaerobic or aerobic glycolytic pathways.
Preferrably, these reporters would indicate quantitative levels of
D-glucose (FIG. 7).
[0389] The supply of glucose at the epidermis is provided by mass
transport from the blood vessels and capillary fields located
within the dermis, immediately beneath the epidermis. The movement
of glucose from the blood stream to the epidermis is
concentration-dependent, rather than insulin-regulated, thereby
allowing the skin glucose levels to provide the basis for
measurement of blood glucose as a direct inference from skin
glucose measurement. The rate of glucose transport into the
epidermis is indicative of the differential between skin glucose
and blood glucose levels. Thus, the rate of transport into skin
allows an accurate extrapolation of blood glucose levels using
first principles mathematical extrapolation techniques. Once
modeled, the kinetics of blood glucose transport to the skin from
the blood enables the determination of the precise first principles
mathematical relationship between the rate of change of skin
glucose concentration and the rate of change of blood glucose
concentration. Thus, rapid blood glucose concentration changes up
or down are accurately tracked by determining the skin glucose mean
concentration levels and the rate of change of skin glucose levels.
First principles mathematical models can be developed for the
individual case, preferably for small local populations, and most
preferably for a universal patient case.
[0390] The use of fluorescence and absorption of endogenous and
exogenous chromophores and fluorophores is directed by known
metabolic pathways that operate in living tissue. The
interpretation of these data and the application of the invention
to the monitoring of in vivo analytes, particularly glucose, is
simplified by the use of mathematical models of these metabolic
processes. A number of computer models of these processes which
vary in complexity and include: glucose transport, glycogen
synthesis, lactate formation and transport, oxidative
phosphorylation and the generation of reducing equivalents in
tissue have been reported. These models are used to identify the
optimum experimental conditions to measure an analyte concentration
in particular the blood glucose concentration.
[0391] The glucose oxidase is reacted with glucose containing
flavin adenine dinucleotide (FAD) to generate the triplet state of
FAD denoted as .sup.3FAD*. The .sup.3FAD* reacts with molecular
oxygen (O.sub.2) and glucose. The reaction of the .sup.3FAD* with
glucose may be monitored kinetically using low-cost instrumentation
by measuring a reduction in the lifetime of the triplet state.
Under steady-state conditions the reaction of glucose and
.sup.3FAD* can be monitored by a decrease in .sup.3FAD*
absorption.
[0392] It has been shown that fluorophores, or colored dyes
utilizing absorption spectroscopy, can be used to measure glucose
in solution or serum by using a series of separate reagents. These
generic reagents include glucose oxidase (which oxidizes glucose
forming hydrogen peroxide); peroxidase (generally horseradish
peroxidase: HRP) used to create an oxidizing reaction in the
presence of hydrogen peroxide with the dye or fluorophore and a dye
reagent or fluorophore, which changes its color or fluorescence
spectrum when brought in contact with hydrogen peroxide, and
peroxidase. The resultant colored or fluorescent species is
measured with a colorimeter or fluorometer, and the amount of
glucose in solution is calculated. In addition, other analytical
techniques have been shown to be commercially useful for measuring
hydrogen peroxide generated from the reaction of glucose oxidase
and glucose.
[0393] In one embodiment, a small molecule metabolic reporter
(i.e., Glucose Oxidase-Labeled Fluorophore: GO-LF), when brought in
contact with glucose, forms a fluorescent species. The GO-LF
molecule is in the form of glucose oxidase protein whereby a
fluorescent cofactor analog is incorporated as a substituent
molecule (SubMol) to the enzyme cofactor flavin adenine
dinucleotide (FAD). One advantage of the present invention is the
increased sensitivity of 10-100 times the former visible color
reaction, smaller analyte concentration requirements (pM vs. .mu.M
or mM of glucose), and greater simplicity of the chemical strip
system (i.e., a single reagent versus multiple reagents as
described in detail herein). This sensor or reader measuring the
strip response requires less sample volume, less sensitivity, and
less power than previous strips while yielding improved
accuracy.
[0394] The basic science required to add dyes to protein molecules
has been previously described and are well known to those skilled
in the art. See, E. Katz et al. Glutathione reductase was
transformed into a `photoenzyme` by tethering to the protein
photoactive eosin dye units (Eo.sup.-2) with the resulting
mechanism of the enzyme photoactivation summarized.
[0395] One embodiment of this invention employs a specific enzyme
(i.e., glucose oxidase), whereby a specific fluorescent cofactor
analog is incorporated as a substituent to the enzyme cofactor
flavin adenine dinucleotide (FAD). This molecule is then deposited
into the skin of a living individual, and is used for the purpose
of detecting glucose in the skin fork predicting blood glucose
levels. The concept of creating this specific molecule for
incorporation into as living organism for routine monitoring of
glucose levels is unique.
[0396] The reaction between glucose and the excited triplet state
of the cofactor within the protein is detected in a method for in
vivo, non-invasive glucose or glucose-pathway derived metabolite
detection in living organisms.
Glucose Monitoring Using Glucose Oxidase-Labeled Fluorophore
(GO-LF)
[0397] Many current commercially available blood glucose test strip
products utilize a well-known color reaction caused by the presence
of glucose in a body fluid sample drawn from interstitial tissue
fluid or from blood. This reaction is described as the formation of
hydrogen peroxide from the reaction of dissolved or suspended serum
glucose with a test strip containing glucose oxidase. Glucose
oxidase, a flavoenzyme, catalyzes the following reactions as shown
in M9, M10 and M11 below:
##STR00002##
[0398] The stoichiometric formation of hydrogen peroxide in
proportion with molar serum glucose concentration is detected by
the addition of peroxidase (generally horseradish peroxidase: HRP)
to form a colored component when further reacted with an indicator
(i.e., an oxidizable dye). The additional extraneous oxidation of
the colored dye is inhibited using a color stabilizing reaction.
Thus, the peroxidase catalyzes the oxidation of an indicator in the
presence of hydrogen peroxide while the final color-changing
reaction is stabilized.
[0399] The present invention eliminates the color reaction step by
reacting a Glucose Oxidase-Labeled Fluorophore (GO-LF) directly
with glucose yielding a fluorescence response. One advantage of the
present invention is the increased sensitivity of 10-100 times the
former visible color reaction, smaller analyte concentration
requirements pM versus .mu.M or mM of glucose, and greater
simplicity of the chemical strip system (i.e., a single reagent
versus multiple reagents). The sensor or reader measuring the strip
response requires less sample volume, less sensitivity, and less
power than previous strips, while yielding improved accuracy. The
invention is described using descriptive text equation form as: 1
Glucose plus 1 Glucose oxidase labeled fluorophore (GO-LF) yields 1
Hydrogen peroxide; 1 Hydrogen peroxide plus 1 GO-LF yields 1 GO-LF*
(Fluorescence signal); and by using the more detailed chemical
symbols as reactions M12, M13 and M14:
.beta.-D-glucose+(GO-LF)(GO-LF)+.delta.-D-gluconolactone (M12)
(GO-LF)+O.sub.2.fwdarw.(GO-LF)+H.sub.2O.sub.2 (M13)
H.sub.2O.sub.2+(GO-LF).fwdarw.(GO-LF*fluorescent)+O.sub.2 (M14)
[0400] Hydrogen peroxide is measured using the enzyme catalase
combined with an oxygen sensing fluorophore (FL) such that a
fluorescence signal, molecular oxygen, and water are generated from
the reaction shown as M15:
##STR00003##
[0401] Multiple fluorescence sensors or molecules can be used for
detection of hydrogen peroxide or oxygen once formed from the
reactions of glucose and GO or hydrogen peroxide and catalase,
respectively. Addition of glucose to a solution of glucose oxidase
causes an increase in fluorescence after a lag time. The lag period
can be related to the concentration of the glucose oxidase, the
oxygen concentration and the glucose concentration. Assuming that
the rate constant for the reoxidation of the reduced enzyme is
significantly greater than the binding and oxidation of glucose,
and that the concentration of the free oxidized enzyme is higher
than that of other forms before the time at which the fluorescence
changes, then the following expression shown as M16 has been
derived. See for example, Q. H. Gibson et al., Biol. Chem.
239:3927, 1964; and J. F. Sierra et al., Anal. Chem. 69:1471, 1997.
It should be noted that the measurement of oxygen within the tissue
is required for optimum utilization of equation M16 for
determination of glucose concentration.
t m - t 0 = 1 k 1 [ GO x ] 0 ln ( [ G ] 0 [ G ] 0 - 2 [ O 2 ] 0 ) (
M16 ) ##EQU00007##
where
[0402] t.sub.m Time between the change in glucose and the time at
which the fluorescence changes;
[0403] t.sub.0 Time at which glucose changes;
[0404] k.sub.1 Rate constant for the reduction of GO.sub.x by
glucose;
[0405] [GO.sub.x].sub.0 Initial concentration of glucose
oxidase;
[0406] [G].sub.0 Initial concentration of glucose; and
[0407] 2[O.sub.2].sub.0 Initial concentration of oxygen.
[0408] The application of this analysis to the in vitro measurement
of glucose is shown in FIG. 38
[0409] With the concentration of glucose oxidase and oxygen used in
this experiment, the experiment is most sensitive over a glucose
concentration range of about 18-216 mg/dL, i.e., about 1 mM-12 mM.
This range can be adjusted by changing the glucose oxidase
concentration. The actual concentration of glucose oxidase is
determined from diffuse reflectance. It can be seen from this data
that the intensity of the fluorescence is also indicative of the
glucose concentration and this measurement may be combined with the
dynamic measurements to determine glucose concentration.
[0410] Energy transfer within the GO-LF molecule mediates the GO-LF
fluorescence intensity. Because of this, it is proposed that the
fluorescence of GO-LF reports the in vivo reaction of glucose with
glucose oxidase in the presence of molecular oxygen (occurring
within skin tissue or the surrounding tissue fluid).
[0411] The specific fluorophores useful in the methods and
compositions of this aspect of the invention include any number of
a group of fluorophores having a three-ring structure similar to
that demonstrated by the fluorescent moiety of FAD as an enzyme
cofactor substituent molecule. This fluorophore is inserted into
the FAD location within the glucose oxidase molecule (protein)
structure and is here termed the substituent molecule (SubMol).
These principles for glucose determination are described herein and
are illustrated in FIGS. 18 through 21.
[0412] Examples of SMMR suitable for direct glucose measurement
include Glucose Oxidase-Labeled Fluorophore (GO-LF), Glucose
Oxidase-Intercalated Fluorophore (GO-IF), Glucose Oxidase--having
FAD in the triplet state (GOx-.sup.3FAD*), or another protein
designed to act as a molecular sensor by substitution of a
fluorophore into the protein so as to produce and optimize an
optical signal when an analyte molecule is inserted into a protein
specific binding site.
[0413] The types and groups of molecules useful as fluorescent
SubMols are illustrated in structures G through J. It should be
noted that one skilled in the art could employ these methods to
similar biomolecules and protein variants to achieve similar
results.
##STR00004##
[0414] It should also be noted that a variety of proteins could be
used to create small molecule metabolic reporters for many other
analytes, including, but not limited to the following examples.
Thus, one skilled in the art could utilize anyone of these
molecular sensors to create in vivo, low-cost, non-invasive
metabolite sensors.
[0415] Reporters designed for fluorescence detection of reactive
species (RS) such as hydrogen peroxide (H.sub.2O.sub.2), molecular
oxygen (O.sub.2), hydroxyl radical (HO*), peroxyl radical (HOO*)
singlet oxygen (.sup.1O.sub.2) and superoxide anion
(*O.sub.2.sup.-) can all be used to measure glucose concentration
based on stoichiometric formation of colored or fluorescent
species. The colored or fluorescent compounds result from the
reaction of the fluorophore or colored compound with hydrogen
peroxide, which is formed from the reaction of glucose with the
glucose oxidase portion of the GO-LF molecule. The reporter will
yield a fluorescence signal when placed in near proximity to the
reactive species. Molecular structures useful for SubMol insertion
for specific detection of the reactive species described herein can
be obtained commercially from suppliers such as Molecular Probes,
Inc., 29851 Willow Creek Rd., Eugene, Oreg. 97402 USA. Other
suppliers may provide similar molecules also useful for detection
of RS.
[0416] Electrochemical sensors can also be used to detect this
hydrogen peroxide after a reaction of glucose with glucose oxidase
in ex vivo body fluids that were removed from the skin. See, e.g.,
S. Gebhart et al., Glucose Sensing in Transdermal Body Fluid
Collected under continuous vacuum pressure via Micropores in the
Stratum Corneum.
[0417] Many enzymes, especially redox active enzymes, utilize
cofactors to facilitate their catalytic activity. These factors are
not amino acids but are often redox active species. Common
cofactors and the enzymes they are found in include, but are not
limited to:
1. NADH
[0418] Alcohol dehydrogenase, Lactate dehydrogenase
2. FAD
[0419] Glucose oxidase, Malate dehydrogenase, Cholesterol
oxidase
3. Thiamine pyrophosphate
[0420] Pyruvate dehydrogenase
4. Herne
[0421] Cytochrome c peroxidase, Chloride peroxidase, Hemoglobin
(Oxygen).
5. Metals [metals given after the enzyme]
[0422] Glutathione peroxidase [selenium], L-Ascorbate oxidase
[Copper], Superoxide dismutase [copper, zinc].
[0423] The potential analytes are denoted with italics. The
cofactors listed here may be replaced with a number of fluorescent
analogs that report the activity of the enzyme by their
fluorescence or absorption properties. For example, NADH may be
replaced by substituted benzoquinones, which are highly colored
molecules in the oxidized form and fluoresce in the visible part of
the spectrum. They are also redox active. A range of xanthene dyes
or analogs of methylene blue may replace FAD. Xanthene dyes are
redox active and highly fluorescent. Heme cofactors may be replaced
by porphyrins. Porphyrins are fluorescent, have very high molar
absorption coefficients and generate excited triplet states in high
yield when photoexcited. They are commonly used to determine oxygen
concentrations from the lifetime of the triplet state. Metal ions
are not easily replaced with a fluorescent species, however the
oxidation state of the metal ion may be determined by electron
transfer with dyes bound to other parts of the enzyme. Metal ions
are readily oxidized and reduced and the oxidation state may be
determined from their reactivity with an exogenous photoexcited
dye. The oxidation state of the metal reports the reactivity of the
enzyme with the relevant substrate.
[0424] Those skilled in the art will recognize that there are
specific commercially available molecules useful for detecting the
RS listed within this invention. For example, for hydrogen peroxide
detection, commercial reagents available from Molecular Probes,
Inc. include: Carbioxyl-H.sub.2DCFDA, CM-H.sub.2DCFDA,
Dihydrocalcein AM, Dihydrorhodamine 123, Dihydrorhodamine 6G,
H.sub.2DCFDA, Lucigenin, Luminol, and RedoxSensor Red CC-1.
Reagents, which respond to peroxidase introduction or which undergo
fluorescence change when oxidized, are also useful for this
detection.
[0425] Molecular oxygen can be detected using one of several
regimes. However, these techniques use a region of the ultraviolet
spectrum that is not practical for living organisms, and this
mechanism is described here to note that the method could be used
for ex vivo analysis. However, multiple dyes used as reagents can
be applied for determination of molecular oxygen as shown in M17
and M18 below:
O.sub.2+h.nu..fwdarw.O+O (M17)
and
O.sub.2+h.nu..fwdarw.O.sub.2.sup.++e.sup.- (M18)
[0426] The free electron is then sensed via
reduction-induced-fluorescence (RIF) detection. In RIF detection,
fluorophores such as fluorescein, rhodamine, and others are reduced
from a highly colored fluorescent state to a colorless,
nonfluorescent leuco dye state. These dyes are available
commercially and their actions are described in various literature
sources and in commercial fluorophore and reagent catalogs. See for
example, Arch Toxicol 68:582, 1994; Brain Res 635:113, 1994; Chem
Res Toxicol 5, 227, 1992.
[0427] The hydroxyl radical can be measured using CM-H2DCFFDA,
Proxyl fluorescamine, and TEMPO-9-AC. Peroxyl radical detection is
performed using BODIPY FL EDA, BODIPY 665/676, H.sub.2DCFDA,
Carboxyl-H.sub.2DCFDA, CM-H.sub.2DCFDA, DPPP, Luminol,
cis-Parinariuc Acid, RedoxSensor Red CC-1. Singlet oxygen is
detected using commercial reagents as trans-1-(2'-methoxyvinyl)
pyrene. One skilled in the art of synthetic organic chemistry and
photochemistry could synthesize additional molecules with similar
structures, which would also respond in a likewise manner.
Glucose Monitoring Using Flavin Adenine Dinucleotide Triplet State
(.sup.3FAD*)
[0428] One embodiment of this invention describes a method for
monitoring blood glucose levels in live tissue, such as skin, solid
tissue, tissue fluids, and plasma, using photo-induced electron
transfer as described herein. The direct oxidation of glucose in
vivo is facilitated by the enzyme glucose oxidase, which catalyses
the oxidation of glucose to gluconolactone. Gluconolactone
spontaneously hydrolyzes to gluconic acid. The cofactor in this
reaction is flavin adenine dinucleotide (FAD) and the reaction
involves the reduction of the FAD moiety within the glucose
oxidase. FAD is eventually re-oxidized by molecular oxygen with the
resultant production of hydrogen peroxide.
[0429] The reduction of FAD to FADH.sub.2 is a two-electron
reduction process. In vivo the kinetics of this reaction are
facilitated by the enzyme matrix, which orients the reactants in an
optimum conformation. One electron transfer to generate the
semi-reduced FAD radical and the semi-oxidized glucose radical may
be induced by the absorption of a photon. Electron transfer then
proceeds from the first excited singlet state or, more likely from
the longer-lived first excited triplet state. FAD or another
specific photo-oxidant may be used to generate this reaction
[0430] The reaction scheme as written for FAD may be summarized as
shown in M19:
##STR00005##
[0431] FAD absorbs a quantum of light, represented by h.nu. to form
the first excited singlet state, designated as .sup.1FAD*. The most
likely fate of this species is decay to the ground state with
concomitant fluorescence emission. The singlet state may also react
with some quencher, Q, which may be molecular oxygen, glucose or
some other reactive species. The fraction of species that decay by
this route is relatively small since the intrinsic lifetime of this
species is short (i.e., nanoseconds). Approximately thirty percent
of .sup.1FAD* may also form the triplet state, designated as
.sup.3FAD*. This .sup.3FAD* species has an intrinsic lifetime of
about 30 .mu.s and may decay in a radiationless transition to the
ground state. In vivo, .sup.3FAD* may react with molecular oxygen
and glucose. The reaction of the glucose with the triplet-excited
state of FAD may be monitored kinetically by a reduction in the
lifetime of the triplet state and under steady state conditions by
a decrease in the triplet absorption.
[0432] For measurement of glucose, the reporter protein is placed
in the keratinocyte layer at 30 .mu.m to 50 .mu.m and up to 175
.mu.m in the pits of the papillae. The reporter protein penetrates
into the skin for some period of time to allow activation following
passive diffusion kinetics. Once activated, the change in
fluorescence or absorption response of the skin cells to changes in
inter- and intra-cellular glucose is monitored directly using an
optical reader. Chemicals such as salicylic acid can be used to
facilitate the penetration of reporter into the skin.
[0433] The reporter protein may be introduced into the skin by
passive diffusion over 24-48 hours, more preferably within 2-6
hours, and most preferably within about 30 seconds to 5 minutes. An
active mechanism utilizing skin permeation, electroporation, or
ultrasonic poration (see for example Sontra Medical Corporation,
Cambridge, Mass.) is another procedure for introducing reporter
protein into the skin. Devices useful for this application sense
glucose directly in the interstitial fluid surrounding the skin
cells by removing fluid or gaining access to fluid for analysis.
This present invention can be used for introducing a small quantity
of low concentration reporter protein solution into the skin for
direct reading of the reporter protein as an indication of both
skin and inferred blood glucose levels.
Instrumentation Required for Reporter Monitoring
[0434] The instrumentation required to detect changes in reporter
signal may consist of simple light emitting diode sources combined
with low-cost solid-state detectors. The mechanism of signal
extraction relating to a biochemical or physiological process is
derived from the elucidation and measurement of key metabolic
pathways. The reporters are excited, and the remitted energy
detected over the wavelength region of 190 nm to 850 nm (See FIG.
38). The three mechanisms of measurement for metabolites or
precursors using the reporters of the invention include (1) using
reporters to increase the signal-to-noise of native
autofluorescence signals indicative of human reductive metabolism
[FADH.sub.2, NADH, and NAD(P)H], (2) using reporters for selection
and enhancement of specific metabolite and precursor signals in
tissue that are indicative of metabolic state and allow
determination of changes in metabolism [Ca.sup.2+, lactate,
oxygen], and (3) using reporters to directly measure the presence
of intracellular or extracellular molecular metabolites
[protein-FL, and protein-.sup.-3FAD*].
[0435] All three mechanisms of signal identification and
enhancement allow utilization of low-cost, hand held
spectrophotometric equipment (e.g., LED excitation and diode
detectors) that is simple in design and does not require advanced
or complicated computational algorithms. Such equipment is not
harmful to subjects and requires just an additional disposable
component (other than a calibration strip) to prepare the subject
for metabolite monitoring. A measurement device approximately the
size of a personal cell phone having quality features, such as
those which allow the user to determine whether a specific
measurement is valid, or whether a repeat measurement is required,
can be used. Such a hand-held, battery powered device is intended
to be used either occasionally, or on a continuous, real-time
monitoring basis for subjects requiring serious health management
regimes. A single calibration allows continuous monitoring for up
to several hours. A calibration technology that utilizes a
calibration strip, which mimics the optical response of the subject
and allows freedom from continuous correction using primary
analysis devices, can be used. Other calibration technologies
contemplated by the invention will be readily discerned by those
skilled in the art.
[0436] As an example, to use the device, the subject or physician
prepares the area to be measured using the enhancement technology,
which is painless and requires a patch (similar in appearance to a
Band-Aid.RTM.), paint, or spray to be applied to the targeted
tissue area. This treatment conditions the tissue area for from a
few minutes up to 30 days, depending upon the SMMR properties
selected and the depth at which it has been deposited in the
subject tissue. The device is then calibrated using a calibration
strip and is ready to make measurements for up to 2 hours or more,
without requiring additional calibration. The subject or physician
examines the conditioned area with the sensor and makes a
measurement. Typically, the measurement takes less than about 5
seconds, and the sensor provides the appropriate metabolite
concentration or reports that a repeat measurement is required.
[0437] In another embodiment, if the photophysics of fluorescent
dyes are considered, the fluorescence changes associated with the
SMMR and the analyte may also be monitored using fluorescence
lifetime technology. One preferred embodiment for such a hand held
device capable of measuring lifetime changes is to use a phase and
modulation spectrometer, which is a device constructed from a radio
frequency modulated light emitting diode and a miniature
photomultiplier or photodiode, whose signal is amplified by a phase
sensitive amplifier. Such devices have been well characterized in
the literature and are commercially available in a variety of
forms. Manufacturers of such devices include: Photon Technology
International, Inc., 1009 Lenox Drive, Lawrenceville, N.J. 08648;
PicoQuant GmbH, Rudower Chaussee 29 (IGZ) 12489 Berlin, Germany;
Tecan Systems Inc., 2450 Zanker Road, San Jose, Calif. 95131;
Thermo Oriel, 150 Long Beach Blvd., Stratford, Conn. 06615. These
devices measure both the degree of modulation of the fluorophore
and the phase shift of the emission relative to the excitation
light, and these two parameters are then related to the lifetime of
the dye. Determination of these parameters at a number of
frequencies increases the accuracy of the device.
[0438] The instrumentation suitable for monitoring glucose via
.sup.3FAD* determination includes a device that is capable of
monitoring transient absorption changes induced by an excitation
source. Two examples of such instrumentation are described
here.
[0439] First, an excitation wavelength is chosen to match an
absorption band of the electron acceptor. For FAD this wavelength
is either 380 nm or 450 nm. It is also an option to use both
wavelengths. The excitation source is modulated at a frequency that
is different from any sources of electrical or optical
interference. Any harmonic of 60 Hz in the United States would not
be used because this is the frequency of the AC electric supply.
The triplet state absorption is monitored from its absorption at
650 nm. A suitable 650 nm source (e.g., a laser diode) irradiates
the sample volume irradiated by the excitation source and the light
backscattered from the sample is detected with a suitable detector
(e.g., photodiode). Triplet state generation results in a fraction
of the 650 nm light being absorbed, and provided the modulation
frequency is sufficiently short compared to the lifetime of the
triplet state (typically 30 .mu.s in oxygen free solution) then the
backscattered light will be modulated at the same frequency. The
signal seen by the detector appears as a modulated signal
superimposed on a constant background. AC coupling of this signal
to a lock-in amplifier allows rejection of interfering light
sources. In the presence of glucose, the triplet state reacts with
the electron acceptor (FAD) and the triplet state absorption is
reduced. The amount of FAD in the skin is quantified by a ground
state absorption measurement at the excitation wavelength.
[0440] Second, an instrument that operates in the time domain may
also quantify the triplet state. The apparatus is similar to that
described above, with some important differences. The excitation
source is intensity modulated. If the frequency of this modulation
is chosen so the lifetime of the triplet state is relatively long
compared to one cycle of the excitation source oscillation then a
phase shift is introduced between the excitation and the detected
modulated monitoring beam. The magnitude of the phase shift is
given by the expression shown in M20:
tan .phi.=.omega..tau. (M20)
where .phi. is the phase shift, .omega. is the circular modulation
frequency and .tau. is the lifetime of the transient species. The
phase shift as a function of transient lifetime at a modulation
frequency of 20 kHz is shown in FIG. 39.
[0441] As shown in FIG. 39, at the designated frequency, the phase
shift is most sensitive to changes of lifetime in the 2-10 .mu.s
timescale. The monitoring source is a laser diode or LED operating
at a wavelength where the electron acceptor triplet state absorbs.
The detector is a photodetector, AC-coupled to a lock-in amplifier
that returns the magnitude and the phase shift of the signal. The
phase shift is related to the lifetime of the acceptor by the
expression given above and the lifetime is related to the glucose
concentration by the expression in M21:
1 .tau. = k d + k ox [ O 2 ] + k q [ G ] ( M21 ) ##EQU00008##
where k.sub.d is rate constant for decay by all means other than
reaction with oxygen or reaction with glucose, k.sub.ox is the rate
constant for reaction with oxygen, [O.sub.2] is the oxygen
concentration, k.sub.q is the rate constant for reaction with
glucose and [G] is the glucose concentration.
[0442] The specificity of the instrumentation used for the
measurement of glucose concentration is brought about by attaching
the electron acceptor (SubMol) to a protein that has a high
affinity for glucose. One example of such a protein is glucose
oxidase, which already contains the electron acceptor FAD. FAD may
be exchanged for other xanthene dyes that have similar size and
charge. For specific determination of glucose concentration,
selectivity to glucose is improved by linking the electron acceptor
to a glucose specific binding protein such as glucose oxidase
Calibration of Instrumentation Using Solutions or Strips with Known
Metabolite Concentrations
[0443] One advantage of the present invention is the use of small
reagent strips for calibrating the instrumentation required for
measuring metabolites (i.e., glucose). The reagent strips are
polymer strips with wicking capacity that contain an exact molar
concentration of SMMR and metabolite to elicit a specific optical
response corresponding to the known metabolite (i.e., glucose)
levels for human tissue, tissue fluid, and blood. This technique
allows precise optical calibration of metabolite measuring
instruments from 0 to 2000 mg/dL in fluids (i.e., approximately 0
to 100 mM for molecules of approximately 350 to 400 daltons); or
from 0 to 10 percent by weight or volume of metabolite comprising
the range found in human tissue. This technique allows precise
optical calibration of glucose measuring instruments from 0 to 650
mg/dL (i.e., approximately 0 to 35 mM) glucose comprising the range
of glucose found in human tissue.
[0444] Instruments may also be designed to image specific tissue
areas where an enhanced signal for metabolites and precursors could
allow easy tissue discrimination for damages, circulation poor,
necrotic or cancerous tissue versus normal. This tissue having
enhanced signal content can be used for physiological studies
related to genome, pharmacogenomic, and proteomic studies where
genetic code is related to metabolic factors.
[0445] To calibrate an instrument, the calibration strip is
activated by mixing a known concentration of metabolite into a
known concentration of reporter protein. The resultant optical
response is used to set the reported metabolite measurement or
reading levels on the measuring instrument. This calibration
procedure can be conducted for any level of metabolite and is most
often completed for two levels to bracket the normal concentration
levels. Calibration strips can be made at any metabolite level,
however it is preferred that metabolite concentrations of from 0 to
150 percent of the highest expected or theoretical levels be used
for calibration, most preferably from 50 to 150 percent of expected
levels be used for calibration.
[0446] For example, for glucose, it is preferred that glucose
concentrations of from about 0 mg/dL to about 500 mg/dL be used for
calibration, and most preferred from about 50 mg/dL to about 350
mg/dL be used for calibration. The combined SMMR and detection
system can also be used for qualitative analysis of metabolites
wherein the purpose of the technique is to identify the presence of
a metabolite or precursor (analyte) or to discriminate tissues
having high and low levels of a metabolite or precursor, rather
than to quantify it. These methods and compositions are useful for
identifying the condition of tissue metabolic health during injury,
surgery, or cancer detection.
[0447] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Relating Fluorescence of Mitochondrial Membrane Probes to D-Glucose
Concentration
[0448] Described herein is a technique for establishing the
dose-response relationship for tracking skin and blood glucose
concentrations using mitochondrial membrane potential. The SMMR
used in this embodiment have the demonstrated property of being
mitochondrial-specific vital stains that respond in a direct
relationship to the rate of glycolysis, which is directly related
to intracellular glucose concentration. The fluorescence response
of one specific embodiment of this invention uses SMMR exhibiting
an excitation wavelength of from 290 to 790 nm, more preferably 400
to 550 nm, and most preferably from 440 to 490 nm, i.e., the
wavelengths used to excite a fluorescence response of the SMMR. The
fluorescence is monitored at above 480 nm, preferably above 490 nm
and most preferably at 501 nm. The upper range for monitoring is at
or below 790 nm. Excitation and emission wavelengths are selected
to minimize absorption and fluorescence by endogenous chromophores
and fluorophores.
[0449] Mitochondrial activity as monitored by oxidative
phosphorylation is directly correlated to the number of reducing
equivalents derived from NADH, which is generated by aerobic
glycolysis or from the conversion of pyruvate to CO.sub.2 within
the mitochondrial organelles. For aerobic metabolism the number of
reducing equivalents is directly and quantitatively (i.e.,
stoichiometrically) equal to ten times the number of glucose
molecules entering the metabolizing cell. The glucose glycolysis
pathway is depicted in FIG. 23.
[0450] For anaerobic glycolysis, the metabolism of glucose to
pyruvate generates two NADH molecules in the cytoplasm of the cell
per glucose molecule. NADH is available to the mitochondria by a
NADH shuttle system in the mitochondrial membrane. The
stoichiometry of this process is such that for every glucose
molecule metabolized, two pyruvate molecules are generated. The
conversion of pyruvate to acetyl CoA and subsequently to carbon
dioxide in the Krebs citric acid cycle is accompanied by the
generation of an additional four NADH molecules per pyruvate
metabolized. Therefore, the overall yield of NADH per glucose
metabolized is ten molecules. The final product of glucose
metabolism is carbon dioxide and water.
[0451] Under conditions where the most important metabolic
substrate is glucose to drive glycolysis, as occurs in the skin,
the fluorescence response is linear and in direct proportion to the
intracellular glucose concentration. Once the SMMR is introduced to
the appropriate cell layers (specifically live epidermal cells
(keratinocytes) directly above the basal layer (stratum basale)),
the SMMR enters the keratinocyte cell membrane and accumulates in
the cell mitochondria. When the SMMR is in place for living cells,
the fluorescence response may be fully sufficient for in vivo
noninvasive determination of the rate of oxidative phosphorylation
(i.e., the Kreb's cycle) for living human epidermal cells.
[0452] The fluorescence response of these dyes is then related to
blood glucose level by the relationships shown in equations 1 and
2. The action of any SMMR or other dye meeting the requirements
outlined above include those molecules that are
mitochondrion-selective vital SMMRs, which act to indicate the
NAD(P)H activity within the mitochondria and, in some cases, the
cytosol. The dyes, when used singly or in combination, have an
affinity for the mitochondria and accumulate within this organelle
in a quantity that is directly proportional to the living cell
membrane potential. Changes in membrane potential are reflected in
changes in dye levels, thus providing real time monitoring of
metabolic state. In other preferred embodiments, all such dyes
useful for this invention are nontoxic, non-carcinogenic,
non-teratogenic, and do not deleteriously affect the skin when
exposed to ultraviolet light or natural sunlight. In preferred
embodiments, such dyes included in the present invention are highly
fluorescent, are evenly dispersible in the cell and interstitial
cell fluid, cannot aggregate or agglomerate, and do not exhibit
binding-dependent fluorescent efficiency and quantum yields. In
most embodiments, these dyes do not inhibit or restrict normal cell
metabolism nor adversely affect cell viability or health in the
concentrations and manner used.
[0453] Indirect measurement of blood glucose concentration is made
as follows. A two-dye measurement regime is provided wherein a non
redox indicating dye, which exhibits stable fluorescence with a
change in glucose or other metabolites (i.e., the marker dye); and
a dye that exhibits direct changes in fluorescence intensity with a
change in glucose (i.e., the reporter dye) are measured
individually. Optimized dyes are safe, relatively permanent, and
non-absorbing into the dermal tissue. The individual dye
fluorescence intensity measurements are made using an ultraviolet
or visible light emitting diode (LED) or laser diode for an
excitation source. The emission detector (i.e., the sensor)
collects the light from the emission of the dye signal within the
skin. In most embodiments, the sensor device also calculates the
ratio of reporter dye fluorescent (following a predetermined lag
time as lagt) to the marker dye fluorescence (following the same
lag period lagt). A linear univariate computational formula for
calibrating such an analyzer for blood glucose is given in equation
(1) as:
[ Glucose Blood ] = k 1 .times. Reporter Fluorescence lagt Marker
Fluorescence lagt + k o ( 1 ) ##EQU00009##
where k.sub.1 is the regression coefficient (slope for the line)
describing a change in fluorescence for the Reporter to Marker
ratio versus glucose concentration in the blood, and k.sub.0 is the
calibration line intercept. Additionally, a change in glucose
concentration over a time interval from T.sub.1 to T.sub.2 involves
the relationship given in equation (2) as:
.DELTA. [ Glucose Blood ] = k 1 .times. Reporter Fluorescence lagt
( T 2 - T 1 ) Marker Fluorescence lagt ( T 2 - T 1 ) + k o ( 2 )
##EQU00010##
where .DELTA.[Glucose.sub.Blood] represents the change in blood
glucose concentration and the terms (T.sub.2-T.sub.1) represent the
change in reporter or marker dye fluorescence over the time
interval,
[0454] The dyes described within this invention may also exhibit an
exponential, logarithmic, power, or other non-linear relationship
between fluorescence intensity and glucose concentration such that
the computational formula for calibrating such an analyzer for
blood glucose using an exponential relationship is given in
equation (3) as:
[Glucose.sub.Blood]=k.sub.0e.sup.k.sub.1.sup.R (3)
where R is the ratio of Reporter Fluorescence.sub.lagt to Marker
Fluorescence.sub.lagt.
[0455] The computational formula for calibrating such an analyzer
for blood glucose using a logarithmic relationship is given in
equation (4) as:
[Glucose.sub.blood]=k.sub.0+k.sub.1 In R (4)
where R is the ratio of Reporter Fluorescence.sub.lagt to Marker
Fluorescence.sub.lagt.
[0456] The computational formula for calibrating such an analyzer
for blood glucose using a power relationship is given in equation
(5) as:
[Glucose.sub.blood]=k.sub.0x.sup.k.sub.1 (5)
where R is the ratio of Reporter Fluorescence.sub.lagt to Marker
Fluorescence.sub.lagt.
[0457] R can represent the intensity at either a measure wavelength
referenced to a baseline wavelength, or as described above as the
ratio of Reporter Fluorescence.sub.lagt to Marker
Fluorescence.sub.lagt.
[0458] Specific methods and compositions of the invention relate to
the measurement of glucose using the mitochondrial membrane
potential as the metabolic marker. However, as described in Scheme
1 (FIG. 17A), other pathways may also be used to make this
measurement and/or to give additional or validation information
about the measurement. As noted above, the invention also provides
compositions and methods for monitoring and calibrating
concentrations of metabolites and small molecules other than
glucose.
Example 2
Relating Fluorescence of Energy Transfer to a Reporter Dye to
D-Glucose Concentration
[0459] SMMRs can also be used to report the metabolic state of
cells, by using such dyes to monitor NAD(P)H concentration. NAD(P)H
can be excited at wavelengths of 340 to 360 nm. Over this
wavelength range, the molar absorption coefficient of SMMRs such as
Rh123 is low (Rh123.di-elect cons.<500 LM.sup.-1 cm.sup.-1 from
345 nm to 425 nm compared with 6.3.times.10.sup.3 LM.sup.-1
cm.sup.-1 for NADH. (NADH and NAD(P)H are indistinguishable by
their absorption or emission spectra.) Excitation at 350 nm of
tissue that has been incubated with Rh123 shows a distinct
fluorescence signal at 530 nm. This fluorescence arises because of
collisional energy transfer from NAD(P)H to the Rh123. Under
conditions where the energy transfer is efficient this process
leads to an enhancement of the sensitivity with which NAD(P)H can
be detected, shown in equation (6) as:
##STR00006##
[0460] The excited state of Rh123 (Rh-123*) relaxes to the ground
state by fluorescence with almost unit efficiency. As a result, the
sensitivity of the fluorescence technique to monitor NAD(P)H is
increased by at least an order of magnitude or more over
autofluorescence. One skilled in the art of photochemistry can
easily identify similar conjugated molecules to be used for
collisional energy transfer reporting for reducing equivalent
molecules, including predominantly NAD(P)H and FADH.
Example 3
Relating Fluorescence of Membrane Localizing Reporter Dyes to
D-Glucose Concentration
[0461] Membrane localizing dyes are used to detect activity of
membrane bound proteins. Dyes such as diphenylhexatriene have been
used in the past to monitor membrane fluidity. However many dyes
may be used to monitor membrane activity by energy transfer
mechanisms. Dyes that are useful in this role include molecules
that have lower singlet energy levels than amino acid residues such
as tryptophan, that is, they absorb light at longer wavelengths
than 320 nm. Suitable dyes include, but are not limited to
xanthenes, cyanines as well as diphenyl hexatriene and its
derivatives. The efficiency of energy transfer is determined by the
separation of the donor and acceptor pair and is given by the
expression in equation (7):
E = R o 6 R o 6 + r 6 ( 7 ) ##EQU00011##
where E is the efficiency, R.sub.o is the Forster radius and r is
the donor acceptor separation. The Forster radius is defined as the
donor acceptor separation that gives an energy transfer efficiency
of 50% and is dependent on the donor and acceptor used. This
mechanism is particularly useful for proteins that physically move
during activity such as glucose transporter (GluT) proteins. The
distance between excited state amino acid residues and an acceptor
molecule, usually located in the membrane, will change as the
protein carries out its function and hence the efficiency with
which the acceptor fluoresces will vary with the activity of the
protein. GluT undergoes conformational changes as it transports
glucose across the membrane. Excitation of tryptophan residues in a
GluT molecule leads to energy transfer to the membrane bound
acceptor and the overall fluorescence is then dependent on the
concentration of glucose transported across the membrane.
Example 4
Relating Fluorescence of pH Indicating Reporter Dyes to D-Glucose
Concentration
[0462] Determination of the cytosolic intracellular pH relates the
ratio of the cytosolic NAD/NADH ratio to the pyruvate/lactate ratio
by the expression as can be derived from textbook information such
as that provided by L. Stryer, Biochemistry, W.H. Freeman and Co.,
New York, 1988 (3.sup.rd Ed.), pp. 363-364, Chapter 18. An example
calculation of intracellular pH is given in equation (8):
[ NAD ] cyt [ NADH ] cyt .varies. [ pyruvate ] .times. 10 - pH [
lactate ] ( 8 ) ##EQU00012##
[0463] The measurement of pH as a direct indicator of
lactate/H.sup.+ concentration in skin yields direct information on
skin and blood glucose concentrations. The parameter of pH as
-log.sub.10[H.sup.+] can be measured using calibrated pH sensitive
dyes or with a variety of known microprobe electrodes specifically
designed for pH determination. One embodiment involves a series of
techniques that allow the placement of a specialized "tattoo", or
more precisely the "active viewing window" comprised of one of a
choice of specific pH indicating SMMRs, into the epidermis using
methods including, but not limited to, electroporation, direct
application by painting with specific transporter solvent mixtures,
tattooing methodologies, laser poration, sonic poration,
iontophoresis, mechanical-poration, solvent transport, wicking,
microneedle, pressurized delivery or by an equivalent active or
passive application technique.
[0464] In another embodiment of the SMMR application techniques a
small disposable polymer patch containing an SMMR dispersed into a
transfer gel is applied to the skin using a pre-specified
protocol.
[0465] Another embodiment is to have a small dispenser with a
specialized tip for placing a measured dose of the SMMR, with or
without a solvent mixture, onto the skin. The molecular tag or SMMR
is allowed to penetrate the skin for some period of time to allow
activation (from 1 minute to 3 hours, depending upon the mixture
used). Once activated, the response of the skin cells to glucose is
monitored directly using an optical reader on the SMMR-treated
viewing window. The optical reader calculates the skin fluorescence
response to glucose, applies first principles mathematical models
to the response, and provides a determination of the blood glucose
levels.
[0466] These concepts and results are demonstrated in FIGS. 1-9,
especially in FIGS. 3-5, 8, 9. A quality value may be
simultaneously calculated in the optical reader/sensor telling the
user the quality of the glucose value reported. Based on this
quality value, the user may be instructed to make one or more
additional measurements until the quality value is indicative of an
accurate result.
[0467] These features provide a technique for establishing the
dose-response relationship for tracking glucose. See, e.g., FIGS.
3, 8, 9. Specific SMMRs to be used have demonstrated properties of
being pH:lactate/H.sup.+-indicating SMMRs that respond in a direct
linear, exponential, or sigmoid relationship to intracellular
glucose concentration. An increase in intracellular glucose causes
a direct increase in intracellular lactate/H.sup.+ via glycolysis,
thereby decreasing the intracellular pH in real-time by a
stoichiometric inverse proportionality, relative to the increase in
glucose concentration. The visible light response of these SMMRs is
such that a diffuse reflection or fluorescent emission spectrum or
signal obtained after excitation at one or more optimum
wavelength(s), e.g., between 300 nm and 750 nm, and more preferably
at least 450 nm, is directly correlated to the quantity of glucose
available to fuel metabolic (glycolytic) activity and is unaffected
by cellular metabolic rate. Therefore the absorption/diffuse
reflection or fluorescence spectrum measured is in direct
proportion to the intracellular glucose concentration. The reaction
velocity assumption set for quantitative analysis of metabolites,
including pH, is described above.
[0468] When the SMMR is comprised of a mixture including a
fluorescent indicator dye, then fluorescence spectroscopy may be
used to determine the pH/lactate/H.sup.+ in the microenvironment of
the cell. Once the tattoo or SMMR mark is produced, these methods
and compositions can be used for in vivo noninvasive determination
of the rate of glucose utilization, whether occurring by
glycolysis, oxidative phosphorylation (i.e., the Kreb's cycle), or
a combination of these metabolic processes. In the case of
mammalian or human keratinocytes, anaerobic glycolysis is the
pathway defined using this technique. The determination of glucose
is accurate for living mammal or human epidermal cells as long as
the SMMR remains within the area of the stratum spinosum. SMMRs
meeting the requirements for this embodiment include but are not
limited to phenolphthalein, which is useful for absorption
measurements of pH. Fluorescent SMMRs include but are not limited
to molecules that are xanthene dyes, especially
2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, (BCECF),
and other standard pH indicating fluorescent dyes available from,
e.g., Aldrich, Sigma, Molecular Probes, and other manufacturers.
Alternatively, as the structures are known, those skilled in the
art may be able to synthesize these materials.
[0469] Other SMMRs meeting these requirements include BCECF, which
can be used at 439 nm and 490 nm excitation. pH is calculated from
the emission detected at 520 nm. Measurements may also be made of
the lifetime of BCECF, and such measurements have been made in the
stratum corneum. See, e.g., Hanson, K. M., et al., Two-photon
fluorescence lifetime imaging of the skin stratum corneum pH
gradient. Biophysical Journal, Vol. 83, pp. 1682-1690. An
alternative molecule is benzenedicarboxylic acid, 2(or
4)-[10-(dimethylamino)-3-oxo-3H-benzo[c]xanthene-7-yl]-(SNARF-1)
using 514 nm excitation and fluorescence detection at 640 nm and
587 nm, respectively. The fluorescence ratio at these emission
wavelengths allows the determination of the ratio of the protonated
and unprotonated forms of the dye. This ratio allows the
determination of the pH of the dye environment using the
Henderson-Hasselbalch equation (9).
pH = pKa - log HA A - ( 9 ) ##EQU00013##
As an illustrative example, for BCECF this relationship becomes
equation (10):
pH = pK a + log ( ( F 490 / F 439 ) - ( F 490 a / F 439 a ) ( F 490
b / F 439 b ) - ( F 490 / F 439 ) ) ( 10 ) ##EQU00014##
In this expression the fluorescence is monitored at a wavelength of
535 nm, the terms F.sub.490 and F.sub.439 refer to the fluorescence
intensity monitored at excitation wavelengths of 490 nm and 439 nm
respectively and the terms with superscripts a and b represent the
limiting values of the fluorescence ratio in acid (a) and base (b)
respectively.
[0470] Use of the dye to measure absolute values of pH requires a
small correction of the fluorescence ratio since the two
fluorescence emission bands are not completely separated.
[0471] A difference comparison of both intracellular and
extracellular pH measurements allows measurements to be made of
lactate synthesis, transport and diffusion out of the interstitial
fluid. The difference between intracellular and extracellular pH is
indicative of the hydrogen ion produced within the cell (due to
glycolysis) and those hydrogen ions that are produced
systemically.
Estimation of the Effect of Glucose Metabolism on Changes in
Intracellular pH
[0472] Numerous prior studies measured intracellular pH in a
variety of organisms and cell types. See, e.g., Roos, A. and Boron,
W. F. (1981) Intracellular pH. Physiological Reviews, vol. 61, pp.
297-434. Of interest are experiments that examine the effect of
weak acids and bases on the pH of cell extracts and homogenates.
Using a simple equation (11) from Michaelis to describe the
buffering capacity of a solution, the physicochemical buffering of
these samples can be expressed as "intracellular buffering power"
as follows:
.beta. = ( A or B ) pH ( 11 ) ##EQU00015##
[0473] .beta.: total buffering power of intracellular fluid
[0474] A: amount of added acid
[0475] B: amount of added base
See, e.g., Roos and Boron, 1981, pp. 389-400.
[0476] The intracellular buffering power of different tissues and
cell types are summarized in Roos and Boron (1981), supra, Table
13, at p. 399. Table 1 (below) uses equation (11) and the
intracellular buffering power of rat tissues to calculate the
potential effect of 5 mM glucose (undergoing glycolysis) on
intracellular pH. These calculations are based on studies reporting
that a net of two protons (two lactate) are produced for every
molecule of glucose that is metabolized. See e.g., Busa, W. B. and
Nuccitelli, R. (1984) Am. J. Physiol., vol. 246, R409-R438; and
Robergs, R. (2001), Professionalization of Exercise
Physiology-Online. vol. 4, no. 11. Thus, by deriving this
information for a specific cell type and for the conditions of an
individual subject, the glucose available to a cell for glycolysis
can be calculated from the measured pH.
TABLE-US-00001 TABLE 1 Buffering pH change Tissue Power (5 mM
glucose or 10 mM H.sup.+) Rat Brain (whole) 18.5 -0.54 Rat
Diaphragm Muscle 67 -0.15 Rat Skeletal Muscle 66-68 -0.15 to -0.16
Rat Cardiac Muscle 51 -0.19 Rat Ventricular Muscle 77 -0.13
Measurement Protocol
[0477] The rationale for making measurements of D-glucose and other
simple sugars using pH (i.e., lactate/H.sup.+) sensitive
intracellular dyes is described. The specific rationale is based
upon the concept that glycolytic mechanisms may be monitored via
metabolite concentration to give an estimation of the total
D-glucose available to the cell (Scheme 1, FIG. 17A). In this
embodiment of the invention, the fluorescence of a pH-sensitive dye
is used to determine blood glucose concentrations. This measurement
is possible because for every glucose molecule undergoing
glycolysis, two lactate/H.sup.+ molecules result. Thus, depending
upon the buffering capacity for any specialized cell types, the pH
is indicative of the quantity of glucose available. During
glycolysis, the glucose is immediately converted to lactate.
[0478] In most embodiments, two steps are required for the glucose
measurement. The measurements to be made are the intensity of
fluorescence at about 580 nm and 640 nm with 532 nm excitation. The
bandwidth of these measurements is typically 10 nm wide.
Intracellular pH is monitored using an intracellular dye that is
equivalent or superior in efficacy to SNARF 5 AM; i.e.,
extracellular pH is monitored using an extracellular dye equivalent
or superior in efficacy to SNARF 5 (SNARF.RTM.-5F
5-(and-6)-carboxylic acid). The dyes are typically applied in two
different places. A third spot is applied using an intracellular
dye equivalent or superior in efficacy to SNARF 5 AM to be used to
determine the spectra of the acidic and basic forms of the dye. All
dyes are applied in a 10 .mu.L volume having a final concentration
of 200 .mu.M.
[0479] The protocol used for application of the dye requires that a
skin temperature between 30.degree. C. and 37.degree. C. be
maintained. The area of skin to be measured is rinsed with
approximately 1 mL of distilled water and wiped dry with an
uncoated tissue or Kimwipe. It is preferable to wipe the area clean
with a clinical alcohol wipe. Once a clean area of skin has been
prepared, 10 .mu.L of dye is applied to the skin with an automatic
dispenser or pipette. The dye spots are protected from room light
and all manipulations are carried out under dimmed room light.
After one hour, any dye that remains on the surface of the skin is
blotted off with a Kimwipe. The uptake of dye was monitored using a
two-photon microscope and by measuring spectra after the dye is
applied. It was determined by observation that measurement should
begin three hours following application of the SMMR.
[0480] To test the efficacy of the sensor measurement during the
measurement period, sensor readings were recorded every time a
blood sample was withdrawn for reference measurements. The test
measurement was designed so that the autofluorescence, and the
fluorescence from SMMRs located in the intra and extracellular
spaces can be acquired ideally at the same time. The only way to do
this at present is to move the sensor to different sites between
measurements. However, other methods may be used as they become
available to provide equivalent information.
[0481] Reference blood samples drawn were analyzed for blood
glucose, lactate and hematocrit. Spectra were acquired from the
skin, to obtain autofluorescence, from the spot where an
extracellular dye equivalent or superior in efficacy to SNARF 5 was
applied to obtain the extracellular pH, and from the spot where an
intracellular dye equivalent or superior in efficacy to SNARF 5 AM
form (available from Molecular Probes, Inc. Eugene, Oreg.) was
applied to obtain the intracellular pH. Finally, acid and base were
applied to the control spot to obtain the spectra from the fully
protonated and fully deprotonated dyes.
[0482] For normal prandial studies, this measurement protocol lead
to a smaller range of glucose values than those obtained using
clamp studies. Typically, fasted mammals have a blood glucose
concentration of anywhere between 50 mg/dL to 100 mg/dL, whereas
fed mammals have a glucose concentration range of 100 mg/dL to 150
mg/dL.
[0483] The data were analyzed according to equation (12):
pH = pK A + log [ R - R B R A - R .times. F B ( .lamda. 2 ) F A (
.lamda. 2 ) ] ( 12 ) ##EQU00016##
where R is the ratio of the fluorescence intensity at 580 nm and
640 nm, R.sub.B is the same ratio when the dye has been made
alkaline and R.sub.A is when the dye has been acidified. The terms
F.sub.A and F.sub.B are the intensity measurements at 640 nm in
acid and base respectively.
[0484] Equation (12) is a modified version of the
Henderson-Hasselback equation that describes the fraction of
molecules that are protonated in an acid-base system at a certain
pH. The term in parentheses is inversely proportional to the
hydrogen ion concentration. The ability to relate glucose
concentration to pH is based on the stoichiometry of glycolysis.
For every equivalent of glucose that is metabolized, two
equivalents of hydrogen ions are generated. pH is simply the
negative log of the hydrogen ion concentration.
[0485] The fluorescence ratio values were obtained after the
intensity of the autofluorescence has been subtracted. Although
this expression actually gives the pH value in these measurements,
it should be realized that the glucose concentration is only a
function of the pH. If the oxidation of glucose results in the
formation of hydrogen ions then it is the corrected fluorescence
ratio that is important in the determination of glucose
concentration. As far as the influence of external pH, it is
hypothesized that the changes in intracellular pH are dependent on
the difference between intracellular and extracellular pH. The
basis for this assumption is that the monocarboxylate transporter
is a facilitated diffusion pump. As a result, hydrogen ions can be
pumped out if the external pH is high compared to the intracellular
pH. It is more difficult to pump hydrogen ions out if the pH is
low.
Example 5
Empirical Calibration Scheme
General Case
[0486] An empirical correction scheme for obtaining quantitative
fluorescence spectra from molecules embedded within the skin of
individual human subjects is required due to the unique scattering
and absorptive properties of individuals. The effects on
fluorescent spectra brought about by these individualistic optical
properties include changes in bandshape and relative fluorescence
intensity. A general equation (13) for obtaining quantitative
fluorescence calibration spectra, which will accommodate for unique
tissue matrix effects, is written as:
C ^ i = ( - B ) c 1 - c 2 ( f 1 - B ) - ( f 2 - B ) ( 13 )
##EQU00017##
where C.sub.i is the estimated concentration for a test sample I; I
is the fluorescence response of the test sample I; I.sub.B is the
fluorescence response of the test sample site with solvent
treatment only; f.sub.1 is the fluorescence response of the sample
site at concentration c.sub.1 (a concentration higher than the
expected concentration of the test sample I); f.sub.2 is the
fluorescence response of the sample site at concentration c.sub.2
(a concentration lower than the expected concentration of the test
sample I). See, e.g., Harrison, G. R., Lord, R. C., and Loofbourow,
J. R. Practical Spectroscopy, Prentice-Hall, Inc., New York, N.Y.,
1948, pp. 412-414.
Example 6
Empirical Calibration Scheme
Special Case of Lactate/H+: pH Measurements
[0487] Specifically for the case involving quantitative
determination of lactate/H.sup.+ using intracellular or
extracellular pH measurements, one would work with hydrogen ion
concentration directly as [H.sup.+]. In the case where an indicator
dye exhibits a fluorescence response due to a change in [H.sup.+]
following the relationship as shown in equation (14):
[ H + ] = k a ( f ( .lamda. 1 ) f ( .lamda. 2 ) - f B ( .lamda. 1 )
f B ( .lamda. 2 ) f A ( .lamda. 1 ) f A ( .lamda. 2 ) - f ( .lamda.
1 ) f ( .lamda. 2 ) ) ( f B ( .lamda. 1 ) f A ( .lamda. 2 ) ) ( 14
) ##EQU00018##
where f(.lamda..sub.i) is the fluorescence measurement at
wavelength i and the subscripts A and B represent the respective
acidic and basic endpoints using a titrimetric approach. See, e.g.,
Molecular Probes Product Information Sheet #MP 01270, SNARF pH
Indicators, Molecular Probes, Eugene, Oreg., Oct. 22, 2002). This
relationship, shown in equation (15), holds noting that background
correction is applied to each fluorescent signal prior to ratio
calculation. If .lamda..sub.2 is selected as the isosbestic point,
then the relationship below holds. For a dye such as SNARF-1:
.lamda..sub.1=580 nm, .lamda..sub.2=640 nm, and .lamda..sub.EX=514
nm, .lamda..sub.Isosbestic=608 nm.
[ H + ] = k a ( f ( .lamda. 1 ) f ( .lamda. 2 ) - f B ( .lamda. 1 )
f B ( .lamda. 2 ) f A ( .lamda. 1 ) f A ( .lamda. 2 ) - f ( .lamda.
1 ) f ( .lamda. 2 ) ) ( 15 ) ##EQU00019##
Then the corrected equation 13 for measurement of hydrogen ion
concentration accounting for matrix effects should be as equation
(16):
[ H ^ + ] i = ( - B ) [ H + ] 1 - [ H + ] 2 ( f 1 - B ) - ( f 2 - B
) ( 16 ) ##EQU00020##
where [H.sup.+] is the estimated concentration for a test sample i;
I is the fluorescence response of the test sample i; I.sub.B is the
fluorescence response of the test sample site with solvent
treatment only; f.sub.1 is the fluorescence response of the sample
site at concentration [H.sup.+].sub.1 (a concentration higher than
the expected concentration of the test sample i); f.sub.2 is the
fluorescence response of the sample site at concentration [H].sub.2
(a concentration lower than the expected concentration of the test
sample i).
Example 7
Use of External Calibration Standards for General Case
[0488] The use of external calibration standards (i.e., standard
addition) is essential in providing a bloodless method for
calibrating in vivo measurements. In theory, a set of two or more
calibration standards comprised of known concentrations of analytes
(e.g., glucose) can be externally added to tissue and delivered to
the specific analysis target site(s). Such a practice does not rely
on a purely theoretical approach dependent on some fixed assumption
set. Thus a more broadly applicable method would involve reliance
on an empirical measurement approach. Such an approach must be
applied across idiosyncratic physiological properties of specific
tissue sites including: perfusion rate, interstitial fluid volume,
rates of diffusion into and out of the tissue, glucose transport,
and the like. Such phenomena can be illustrated generally as in
Scheme 4 (FIG. 17D) illustrating fluid issues related to in vivo
skin calibration.
[0489] As an example, an in vitro experiment using such standard
addition can be reviewed. The experiment is to determine the final
concentration of a cuvet initially containing a liquid of 100
volume units (Vi) and a concentration of 100 w/v (Ci). In this
case, neither the initial volume nor the initial concentration is
known. To begin, a known Standard liquid (A1) is added to the cuvet
having a volume of 100 volume units (Va1) and a concentration of
0.0 w/v (Ca1). A fluorescence measurement is made of the solution
plus Standard A1 and the result recorded as Ia1. The final
concentration of the cuvet at this point may be determined using
the general equation (17):
C f a 1 = ( C i V i ) + ( C a 1 V a 1 ) V i + V a 1 = ( 100 100 ) +
( 0.0 + 100 ) 100 + 100 = 50 w v ( 17 ) ##EQU00021##
[0490] A second Standard liquid (A2) is then added to the cuvet
having a volume of 100 volume units (Va2) and a concentration of
500 w/v (Ca2). A fluorescence measurement is made of the solution
plus Standard A2 and the result recorded as Ia2. Following the
addition of Standard A2 the cuvet now contains a concentration
calculated using equation (18):
C f a 2 = ( C i V i ) + ( C a 2 V a 2 ) V i + V a 2 = ( 50 200 ) +
( 500 100 ) 200 + 100 = 200 w v ( 18 ) ##EQU00022##
[0491] If a fluorescence method has been developed capable of
measuring the concentration of analyte defined as a linear
relationship over a concentration range of between 50 and 400 w/v
(see Table 2), then equation (19) holds as:
a 2 a 1 = C f a 2 C f a 1 ( 19 ) ##EQU00023##
[0492] Thus a ratio measurement of Ia2 and Ia1 yields a value of
200/50=4.0 and provides sufficient information to compute absolute
concentration of the initial fluid as well as the final fluid
levels. The examples below consider two examples of the in vivo
case.
Example 8
[0493] Equivalent volumes of Standards A1 and A2 are added to the
tissue as volumes Va1 and Va2; these volumes approximate the
current interstitial volume. The concentration of the added
Standards A1 and A2 are 0.0 w/v (Ca1) and 300 w/v (Ca2). The
interstitial volumes are assumed to remain approximately the same
as the liquid from the Standards mix with the interstitial fluid,
i.e., after a period, there is a mixing of liquids causing an
equilibrium of the analyte levels, but no overall interstitial
fluid volume change. The equivalent relationships can be calculated
for any set of assumptions. Since in this case the equivalent
volume assumption is made then equation (20) holds:
C f a i = ( C i V i ) + ( C a i V a i ) V i + V ai ( 20 )
##EQU00024##
[0494] Equation (20) reduces to a simple relationship where the
volumes Vi, Va1 and Va2 are equivalent and there is assumed
diffusion of the analyte from the Standards to the interstitial
fluid equilibrating the concentration given sufficient time. Thus,
equation (21) is used:
C f a 1 = C i + C ai 2 ( 21 ) ##EQU00025##
This scenario takes into consideration that the addition of the
first Standard A1 at 0.0 w/v concentration reduces the
concentration; and then the second Standard A2 at 300 w/v
concentration is added. As such the following Table 2 holds.
TABLE-US-00002 TABLE 2 Application of Standards A1 and A2 as 100
unit volume and 0.0 w/v and 300 w/v concentration. Initial Volume
(Vi) Initial Conc. (Ci) C.sub.f.sub.a1 C.sub.f.sub.a2 C f a 2 C f a
1 = ##EQU00026## 100 units 50 25 162.5 6.5 100 units 100 50 175 3.5
100 units 150 75 187.5 2.5 100 units 200 100 200 2.0 100 units 250
125 212.5 1.7 100 units 300 150 225 1.5 100 units 350 175 237.5
1.36 100 units 400 200 250 1.25
Example 9
[0495] Equivalent volumes of Standards A1 and A2 are added to the
tissue as volumes Va1 and Va2; these volumes approximate the
current interstitial volume. The concentration of the added
Standards A1 and A2 are 0.0 w/v (Ca1) and 400 w/v (Ca2). As in
Example 8, the interstitial volumes are assumed to remain
approximately the same as the liquid from the Standards mix with
the interstitial fluid, i.e., there is a mixing of liquids causing
an equilibrium of the analyte levels, but no overall interstitial
fluid volume change. Since this assumption is made, then equations
20 and 21 are used.
[0496] This scenario takes into consideration that the addition of
the first Standard A1 at 0.0 w/v concentration reduces the
concentration; and then the second Standard A2 at 400 w/v
concentration is added. As such the following Table 3 holds as.
TABLE-US-00003 TABLE 3 For the application of Standards A1 and A2
as 100 unit volume and 0.0 w/v and 400 w/v concentration. Initial
Volume (Vi) Initial Conc. (Ci) C.sub.f.sub.a1 C.sub.f.sub.a2 C f a
2 C f a 1 = ##EQU00027## 100 units 50 25 212.5 8.5 100 units 100 50
225 4.5 100 units 150 75 237.5 3.17 100 units 200 100 250 2.5 100
units 250 125 262.5 2.1 100 units 300 150 275 1.83 100 units 350
175 287.5 1.64 100 units 400 200 300 1.5
Example 10
Screening and Optimizing Organic Dyes for SMMR Activity
[0497] In some embodiments of the invention, a dye is added into a
tissue with an anticipated SMMR-response activity, and spectra are
collected for a set of predetermined excitation and emission
wavelengths. The excitation wavelength set selected corresponds to
the maximum absorption spectrum of the dye being used. The optimal
measurement wavelength for excitation and emission is then
determined empirically for each SMMR application such that the
selected excitation wavelength results in a combined effect where
maximum emission intensity and response is achieved for each
metabolite of interest. Metabolites useful for tracking glucose
were derived from an understanding of the glycolytic pathway for
the cells of interest and an understanding of which dyes may
actually behave as SMMRs for quantitative reporting of these
metabolites. By selecting the optimum wavelengths for SMMR
measurement in an empirical fashion, the precise method for
quantitative detection of each metabolite was achieved, thereby
yielding maximum analytical selectivity, repeatability, and
reproducibility.
Empirical Procedure for the Development of Calibration
Protocols
[0498] The following procedure can be used to develop the
calibration protocol for a blood glucose analysis method combining.
SMMRs with a low-cost, handheld sensor: (1) the glucose (or another
blood or tissue analyte) is measured for the test subject (or
series of test subjects) by withdrawing blood from a subject and by
analysis via a reference blood glucose measurement (the glucose may
be intentionally varied within the test subject for the test
evaluation period); (2) the metabolic reporter signal and a marker
(or reference) wavelength signal are measured at a time-stamped
interval corresponding to the blood glucose reference measurement
(this is completed for a series of excitation and emission
wavelengths); (3) the ratio of the metabolic reporter and reference
or marker wavelengths is calculated for each set of excitation and
emission wavelengths; (4) the series of ratio measurements of the
reporter/reference is compared to the reference blood glucose
measurements; (5) the optimum wavelength sets are derived and the
absolute ratios determined that best correspond to specific blood
glucose levels, taking into account the lag times and best
mathematical model; (6) a small handheld device is provided,
preferably where the device has the capability to measure the
signal at the optimized specific wavelengths using exact excitation
sources and emission detection schemes (with defined intensity and
bandshape characteristics); (7) the ratio measurements of the
device when coupled with specific SMMRs produces a metabolite
profile that is used to directly predict blood glucose
concentration using algorithms described herein. Those skilled in
the art will recognize that when the metabolic pathways for
multiple biosyntheses are defined, that this empirical testing
method can be used to screen multiple dyes for their efficacy as
SMMRs for a variety of metabolic measurements. Thus, without much
knowledge about specific pharma-kinetic activity or dyes, a series
of compounds can be screened and optimized for SMMR activity.
[0499] Ideally, all dye candidates to be tested for SMMR activity
in humans are first screened properly to ensure safety.
Example 11
Factors Affecting the Molecular Structure and Action of Organic
Dyes Suitable for Use as SMMRs
[0500] Molecular Design
[0501] There are six main characteristics of a dye molecule that
determine its efficacy as an SMMR according to the methods and
devices of the invention. These include: (1) its affinity and
specificity for target cells and cell structures; (2) its binding
properties and residence time in skin; (3) its safety to cells and
organisms; (4) its speed of delivery; (5) its specificity for the
metabolite of interest; and (6) its spectral properties. Properties
that control the affinity and specificity for target cells and cell
structures for SMMR molecules into skin cells include: [0502] 1.
The partition coefficient in octanol/water together with the
solubility in aqueous solution, which determines how the molecule
is distributed between the aqueous and lipid phases in the tissue;
[0503] 2. The charge, which affects electrostatic interactions of
the compound; [0504] 3. The vapor pressure at 25.degree. C., which
determines the evaporation rate at the skin surface; [0505] 4. The
molecular size, which controls the diffusion of the material
through a porous interface or a viscous liquid.
[0506] Factors affecting the functionality of the molecule include
the reactivity and reduction potential of the molecule, the pKa and
the energy level of the first excited state.
[0507] Spectral properties that are important for SMMRs include the
absorption spectrum of the chromophore, the fluorescence spectrum
and the emission quantum yield. Properties that moderate the
absorption characteristics include the degree of conjugation in the
molecule, the number of electrons in the conjugated system and the
electro-negativities of substituents attached to the molecule.
Factors that affect the fluorescence emission spectrum are similar
to those that affect the absorption spectrum. The fluorescence
quantum yield, which determines the intensity of the fluorescence,
is influenced by the flexibility of the molecule and the
intramolecular reactivity.
[0508] An example of how a xanthene dye may be modified to act as a
long wavelength pH sensitive dye for specific action as a
lactate/H.sup.+ SMMR is described herein. One possible structure is
shown in Scheme 3 (FIG. 17C). As shown in FIG. 17C, the xanthene
ring is substituted with hetero atoms (A) that extend the
conjugation of the molecule across the fused ring system. Electron
density in the ring system is increased by the lone pair of
electrons on the heteroatom that are partly delocalized into the
ring system. Typical groups at these positions would include an
amine and an amide, such as rhodamine.
[0509] The pKa of the molecule is controlled by the substitution of
acidic and basic groups (B) and the nature of the heteroatoms (A).
Small changes to the pKa may be made by substitution of electron
donating or withdrawing groups to the ring (D). The quantum yield
of fluorescence (.phi..sub.F) and hence the intensity of the
fluorescence is determined by the balance between the rate
constants for radiative (k.sub.r) and non-radiative (k.sub.nr)
decay as shown in equation (22):
.phi. F = k r ( k r + k nr ) ( 22 ) ##EQU00028##
The radiative rate constant is determined by the probability of a
transition whereas the non-radiative rate constant is affected by
the number of modes of vibration that a molecule has and any
intramolecular reactivity that can quench the excited state.
[0510] The absorption spectrum of the molecule is determined by the
extent of the conjugation as well as substitution on the ring (C).
Substitution of both electron withdrawing and electron donating
groups in a push-pull type of system extends the overall
conjugation of the system and causes a bathochromic shift (to
longer wavelengths) of the spectrum. A number of empirical rules
have been put forward to predict spectra. The well-known Woodward
rules, for example, predict that for a simple conjugated system the
addition of a double bond adds about 30 nm to the wavelength
maximum.
[0511] The polarity of the molecule can be altered, without grossly
affecting other properties of the molecule, by substitution of
non-conjugated groups to the ring system (E). Many xanthene dyes
are synthesized with a substituted phenyl ring at R.sub.2. It is by
specific modification of this dye and the measurement of its
fluorescence signature that allows the dye to function as an SMMR
to relate lactate/H.sup.+ to D-glucose concentration (as noted in
FIGS. 1-17).
Example 12
Use of Glycogen Particle Density
[0512] The measurement of glycogen particle numbers indicates a
direct proportionality to the amount of glucose in the metabolic
pathway of the cell (for an individual metabolic rate). As Scheme 2
(FIG. 17B) illustrates, a measurement of glycogen synthesis
provides an indicator of glucose concentration because the only
biochemical route to glycogen is directly from glucose. The use of
iodine-based SMMRs within the skin can be measured using an optical
reader as a direct indicator of glycogen particle concentration in
the skin. Skin glycogen concentration can be related to skin
glucose levels, which in turn are mathematically related to blood
glucose levels. Thus, skin glycogen concentration yields direct
information on skin and blood glucose concentrations. The parameter
of glycogen particles can be measured using a variety of known
techniques. Glycogen particles are known to have a mean particle
size diameter of approximately 30 nanometers (nm). Thus, an ideal
wavelength for characterizing the presence of these particles in an
absorptive-scattering media such as skin would be at 2.5 to 3.5
times the diameter or approximately 75 to 105 nm ultraviolet light.
This invention contemplates utilizing such a wavelength to
characterize the number of glycogen particles within the skin, as
well as utilizing other potential methodologies for measuring the
particle density for glycogen include scattering measurements in
the 290 nm to 750 nm spectral regions, and includes optical
coherent tomography. Mathematical manipulations of the data derived
from these techniques can provide correlative information allowing
prediction of glycogen particle numbers.
[0513] In one specific embodiment, a series of techniques are
described in the invention which allow the placement of a
specialized tattoo, comprised of at least one of a choice of
specific glycogen indicating SMMR, into the epidermis for analysis
of mitochondrial membrane potential and pH indicating signals.
Measurement of glycogen particles, which preferentially absorb
SMMR, is monitored directly using an optical scattering reader. The
optical reader calculates the total absorption of the SMMR into the
glycogen particles. Once determined, the glycogen content of the
skin is empirically related (by first principles mathematical
models) to reference skin and blood glucose levels. Simultaneously,
a quality value is calculated, which tells the user the quality of
the glucose value reported. Based on this quality value, the user
may be instructed to make one or more additional measurements until
the quality value is indicative of an accurate result.
[0514] Once the tattoo or mark is produced, this invention may be
fully sufficient for in vivo noninvasive determination of the rate
of glucose utilization within living human epidermal cells as long
as the SMMR remains within the stratum spinosum. SMMR meeting the
requirements for this embodiment are described above, and include,
e.g., iodine dissolved in potassium iodide. Iodine forms a
blue-black complex with glycogen, the intensity of which is
directly related to the number of particles of glycogen present in
the tissue. The visible response of these SMMR is then related to
blood glucose level by the relationship given in equation (23):
[ G ] .varies. # glycogen particles . .times. NAD ( P ) H FAD
.times. NO .times. pH .times. O 2 ( 23 ) ##EQU00029##
[0515] Equation (23) is based on measuring a cell function and
normalizing this function for the relative metabolic rate of the
tissue. The number of glycogen particles is directly related to the
glucose concentration. This relationship will break down when
metabolism is high and all the glycogen reserves have been
utilized. The concentration of glycogen particles can be obtained
from measurements using optical coherent tomography, light
scattering, or differential staining of glycogen particles using
iodine stains.
Example 13
An Example of a Targeted Pathway
Mathematical Modeling Applications to Glucose Concentration
[0516] FIG. 23 is a proportionality-qualitative description of how
the glycolytic pathway (e.g., glycolysis) relates to glucose
concentration in cellular metabolism. The quantitative description
of these pathways is developed dependent upon accurate, selective,
and responsive measurement parameters yielding indirect or direct
information for glucose concentration. An example of the
quantitative treatment for fluorescence changes associated with the
activity of glucose oxidase is given in Equation (24).
[0517] Several examples of the mathematical models required for
fitting the reported glucose to the measured blood glucose for this
invention are given in Equations 1-5. The addition of glucose to a
solution of glucose oxidase causes an increase in fluorescence
after a lag time. The lag period can be related to the
concentration of the glucose oxidase, the oxygen concentration and
the glucose concentration. Assuming that the rate constant for the
reoxidation of the reduced enzyme is significantly greater than the
binding and oxidation of glucose, and that the concentration of the
free oxidized enzyme is higher than that of other forms before the
time at which the fluorescence changes, then the following
expression in equation (24) has been derived.
t m - t 0 = 1 k 1 [ GO x ] 0 ln ( [ G ] 0 [ G ] 0 - 2 [ O 2 ] 0 ) (
24 ) ##EQU00030##
where
[0518] t.sub.m Time at which the fluorescence changes
[0519] t.sub.0 Time at which glucose is introduced
[0520] k.sub.1 Rate constant for the reduction of GO.sub.X by
glucose
[0521] [GO.sub.X].sub.0 Initial concentration of glucose
oxidase
[0522] [G].sub.0 Initial concentration of glucose
[0523] 2[O.sub.2].sub.0 Initial concentration of oxygen
See, e.g., Sierra J. F., Galban J., Castillo, J. R. "Determination
of Glucose in Blood Based on the Intrinsic Fluorescence of Glucose
Oxidase." Anal. Chem. 1997 69(8), 1471-1476).
Example 14
Other Monitoring Techniques and Metabolites
Lactate Transport
[0524] Lactate transport is monitored by measuring intracellular
and extracellular pH using fluorescent SMMRs, as previously
described. The xanthene dye BCECF has been used to monitor lactate
transport in a number of tissues (see e.g., Carpenter, L. and
Halestrap, A. P. 1994 Biochem. J. 304, 751-760). In the present
invention this dye is used to monitor both intracellular and
extracellular pH. The extracellular pH is monitored to measure
variations in physiology within the body that are unrelated to
glucose metabolism in the epidermis, but are related to metabolic
pH changes in the body. The intracellular pH, as measured, is then
corrected using the value of the measured extracellular pH.
Oxidative Phosphorylation
[0525] Oxidative phosphorylation can be monitored by NADH
fluorescence. This fluorescence is measured in the presence and
absence of oxygen. These two measurements yield the rate of
oxidative phosphorylation and a measure of the overall metabolism
of the cell. The rate of oxidative phosphorylation is dependent on
the overall substrate availability to the cell, which requires
oxygen. In the absence of oxygen, the overall metabolism is
dependent on glycolysis alone.
[0526] The oxidative phosphorylation pathway for glucose is
determined by measuring oxygen consumption along with the NADH/FAD
fluorescence ratio. This ratio has been used in the past to
determine the overall reduction potential of the cell. The
measurement of the oxygen consumption rate determines the rate of
oxidative metabolism in the tissue. The sensitivity of the NADH/FAD
fluorescence ratio can be increased by the use of an energy
transfer or redox potential measuring dye to amplify overall signal
intensity. An example of such a dye suitable for use as an SMMR is
rhodamine 123, although other compounds containing conjugated
aromatic systems can also be used.
[0527] In a preferred embodiment, the amplifying SMMR molecule is
positively charged at pH 7 and has a high quantum yield of
fluorescence. In a further embodiment, the SMMR molecule has little
absorption in the region where NADH absorbs. Excitation of NADH
results in energy transfer to the SMMR dye that then fluoresces
with efficiency at least ten times greater than that of NADH
alone.
Photobleaching
[0528] Photobleaching is a process that occurs with virtually all
fluorescent dyes. The term is something of a misnomer since it
literally means the loss of color as a result of irradiation by
light. The loss of color is the result of a photochemical reaction
that results in a new chemically distinct compound being formed
that does not exhibit the same fluorescence properties as the
parent SMMR compound. This new photo-degraded compound will have
altered photophysics compared to the parent molecule but its
properties are not necessarily loss of color. Photobleaching is a
hindrance to continuous fluorescence-based monitoring and is
exacerbated by the presence of oxygen, high concentrations of
reactive species and high light levels. For this present invention,
SMMRs are used that have high quantum yields of fluorescence (which
implies that the main process for deactivation of the excited state
is fluorescence), and they are excited with the minimum amount of
excitation light. Photoreactivity is also reduced by the low oxygen
tension in the skin.
Differential Monitoring
[0529] The mechanism presented in Scheme 1 (FIG. 17A) for the
measurement of glucose requires that the majority of glucose be
metabolized by glycolysis because oxidative phosphorylation may
also utilize fatty acid metabolites as substrates instead of
glucose. Oxidative phosphorylation in skin comprises only .about.2%
of metabolism and this fraction may be controlled by reducing the
oxygen available to the cells, although experimental data suggests
that there is little or no effect of oxygen concentration on
glycolysis. By performing a differential measurement with and
without oxygen, the fraction of glycolytic and oxidative metabolism
is determined.
Glycolysis
[0530] In tissues that undergoes primarily anaerobic metabolism
(i.e., glycolysis) the products of the glycolysis reaction pathway
are lactate and adenosine triphosphate (ATP). ATP is synthesized
from ADP, the diphosphate analog, and a phosphate. Lactate is
generated as a waste product of the pathway. The lactate
concentration within the cell is dependent on lactate transport out
of the cell and on the rate of glycolysis. The extracellular
lactate concentration is dependent on lactate transport and
diffusion of lactate into the blood stream. The production of
lactate correlates with the intracellular pH. The pH of epidermal
tissue, using intra- and extracellular pH sensitive SMMRs, can be
used to specifically relate intracellular pH changes to glucose
utilization via glycolysis. The use of NMR techniques using
phosphorous (.sup.31P) and proton (.sup.1H) probes allows the
measurement of ATP, phosphate, pH and lactate simultaneously. This
technique alone can be used to determine the relationship between
glucose concentration and glycolysis. The use of .sup.31P NMR is
described specifically for measuring the effect of exercise on the
levels of ATP, phosphocreatine, and orthophosphate in human forearm
muscle. See e.g., G. K. Radda. Science 233: 641 (1986). pH can also
be measured in vivo and directly using .sup.31P NMR. See e.g.,
citations in D. G. Gadian et al. IN: Biological Applications of
Magnetic Resonance, R. G. Shulman, ed., (Academic Press, 1979), p.
475. The .sup.31P magnetic resonance technique also provides
information on the orthophosphate concentration for glucose
metabolism in the Kreb's cycle and/or oxidative phosphorylation
pathway. The lactate/pyruvate ratio and the
.beta.-hydroxybutyrate/acetoacetate ratios have been used to
estimate cytosolic and mitochondrial NADH/NAD(P)H ratios
respectively. See e.g., Tischler, M. E., et al., Arch Biochem
Biophys, 1977. 184(1): p. 222-36; Poole, R. C. and A. P. Halestrap,
Am J Physiol, 1993. 264(4 Pt 1): p. C761-82; Groen, A. K., et al.,
J Biol Chem, 1983. 258(23): p. 14346-53.
Nitric Oxide (NO):
[0531] NO has been shown to correlate inversely with glucose
concentration. This reactive molecule acts as a vasodilator and
interacts with thiol groups. The reaction of NO with hemoglobin has
also been monitored in the past using absorption spectroscopy. NO
may also be measured using an NO meter using a probe head that is
as small as 30 .mu.m.
[0532] Scheme 1 (see FIG. 17A) points to the measurement sites
required to define the glucose metabolism in epidermis thereby
providing complete information for the fate of glucose metabolized
in the skin. NO causes physiological effects such as vasodilation
and is a reactive material that interacts with thiols and the
basement membrane of the dermal/epidermal junction. Direct
measurement of NO is possible using commercially available
technology. The measurement of NO will be used, if necessary, for
final correction of the glucose concentration. The determination of
the NO correction follows initial comparisons of blood glucose
estimated from fluorescence measurements when compared to blood
glucose measured using a reference technique (e.g., YSI
Incorporated, PO Box 279, Yellow Springs, Ohio 45387 USA). The
change in glucose concentration as affected by NO concentration is
described in the equation (23). The use of NO concentration
information for final blood glucose correction is also described
herein. When required, equations (1-5) of the invention are
modified by the addition of an NO term as shown in equation (25).
This adjustment accounts for the cases where NO alters the
perfusion rate significantly.
[ Glucose blood ] = f ( reporter Reference ) k i ( 1 [ NO ] ) Where
, f ( Reporter Reference ) ( 25 ) ##EQU00031##
is the in vivo fluorescence signal ratio of reporter fluorescence
to reference (or marker) fluorescence varying with respect to
changes in glucose concentration within the measured target tissue;
and k; is the computed weighting factor attributing the effect of
NO concentration on the perfusion rate. The factor k.sub.i is
computed empirically following comparisons of blood glucose
optically determined versus reference values using standard
regression methods (see for example H. Mark and J. Workman,
Statistics in Spectroscopy, 1.sup.st Ed., Academic Press, 1991; and
2.sup.nd Ed., Elsevier Publishers, 2003)
Example 15
Consideration of Blood Glucose Concentration and Fluorescence
[0533] Previous work has demonstrated that the lag time between
blood glucose levels and non-perturbed epidermis is 2.9 to 4
percent per minute for the differential concentrations (vis-a-vis
blood and epidermal glucose concentrations). See, e.g., J. M.
Ellison et al. Diabetes Care, June 2002, 25(6), 961-964; B. M.
Jensen et al. Scandinavian Journal of Clinical Laboratory
Investigation, 1995, 55, 427-432; P. J. Stout, Diabetes Technology
& Therapeutics 2001, 3(1), 81-90; C. P. Quinn, Publication
0193-1849/95 The American Physiological Society, E155-E161). In
practice, a 5 to 15 minute lag is most often experienced between
real-time measured blood glucose levels and glucose levels
determined at the keratinocyte/epidermal layers. The fingertip area
keratinocyte/epidermal layers are considered ideal due to their
high vascularization. The time required for the epidermis to reach
an equilibrium with blood glucose at steady-state, dependent on the
measurement site, has been reported to be from 25 to 35 minutes.
See, e.g. K. Jungheim and T. Koschinsky Diabetes Care, 25(6), 956,
2002; and J. Ellison et al. Diabetes Care, 25(6), 961, 2002.
[0534] When blood glucose is rapidly increasing (hyperglycemia) or
decreasing (hypoglycemia), the lag time becomes a critical issue
for determining the response time for any external, non-invasive
blood glucose monitor. Rapid response is required for identifying
important health related changes in glucose levels and to avoid
critical blood glucose scenarios (i.e., clinically important high
or low blood glucose levels). Issues of rapid response are
addressed by using elevated temperatures at the measurement site to
increase blood flow to these regions. Therefore, in various
embodiments, the sensor unit is combined with a regulatable heating
element and/or temperature gauge. The sensors are calibrated by
comparing actual blood glucose to the sensor output. The
temperature is either controlled at the measurement site or
compensated for in the final blood glucose estimation. K.sub.a and
.phi..sub.F are only slightly temperature dependent. The zero and
slope of the sensor calibration are determined by measuring an
initial baseline glucose level, and a second glucose level at
higher concentration. The sensor calibration is then measured as
shown in equation (26):
[G]=K.sub.1(sensor response)+K.sub.0 (26)
The K.sub.1 and K.sub.o values are entered into the sensor and the
calibration is checked against a reference standard material. The
reference standard material is comprised of a matrix that responds
to glucose concentration in such a way as to provide primary
standard concentration and fluorescence response data. Their
relationship is given in equation (27), where A, B, C, and D are
comprised of one or more individual analyte measurements or ratios
of measurements. The method shown in equations (26) and (27) can be
used either for calibration using YSI-determined blood reference
data, or without blood reference data via use of equations (13)
through (21).
[0535] Algorithm:
[G]=f([A],[B],[C],[D])* (27)
*Where [A], [B], [C], and [D] are directly measured using one or
more measurement techniques for one or more metabolite signals.
Each metabolite signal represents a mechanism for quantitatively
measuring intracellular or extracellular glycolysis. The primary
fuel for glycolysis is D-glucose combined with low concentrations
of other simple sugars, such as galactose and fructose.
EQUIVALENTS
[0536] From the foregoing detailed description of the specific
embodiments of the invention, it should be apparent that particular
novel compositions and methods involving utilizing SMMRs for direct
or indirect measurements of metabolic analyte concentrations have
been described. Although these particular embodiments have been
disclosed herein in detail, this has been done by way of example
for purposes of illustration only, and is not intended to be
limiting with respect to the scope of the appended claims that
follow. In particular, it is contemplated by the inventors that
various substitutions, alterations, and modifications may be made
as a matter of routine for a person of ordinary skill in the art to
the invention without departing from the spirit and scope of the
invention as defined by the claims. Indeed, various adaptations and
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within and be incorporated into the scope of
the appended claims.
* * * * *
References