U.S. patent number RE40,198 [Application Number 10/671,436] was granted by the patent office on 2008-04-01 for method and device for electrochemical immunoassay of multiple analytes.
This patent grant is currently assigned to Roche Diagnostics Operations, Inc.. Invention is credited to Harvey B. Buck, Jr., Zhi David Deng, Eric R. Diebold.
United States Patent |
RE40,198 |
Buck, Jr. , et al. |
April 1, 2008 |
**Please see images for:
( Certificate of Correction ) ** |
Method and device for electrochemical immunoassay of multiple
analytes
Abstract
A method and device for detection and quantification of
biologically significant analytes in a liquid sample is described.
The method includes contacting a volume of a liquid sample with
predetermined amounts of at least a first and second redox
reversible species having redox potentials differing by at least 50
millivolts. At least one of the redox reversible species comprises
a liquid sample diffusible conjugate of a ligand analog of an
analyte in the liquid sample and a redox reversible label. A
predetermined amount of at least one specific binding partner for
each analyte to be measured is combined with the sample and current
flow is measured at first and second anodic and cathodic potentials
and correlated with current flows for known concentrations of the
respective diffusible redox reversible species. Diagnostic devices
and kits, including such devices and the specified specific binding
partner(s) and redox reversible species are also described.
Inventors: |
Buck, Jr.; Harvey B.
(Indianapolis, IN), Deng; Zhi David (Weston, FL),
Diebold; Eric R. (Fishers, IN) |
Assignee: |
Roche Diagnostics Operations,
Inc. (Indianapolis, IN)
|
Family
ID: |
22205995 |
Appl.
No.: |
10/671,436 |
Filed: |
September 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60087576 |
Jun 1, 1998 |
|
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Reissue of: |
09330422 |
May 28, 1999 |
06294062 |
Sep 25, 2001 |
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Current U.S.
Class: |
205/777.5;
204/403.1 |
Current CPC
Class: |
C07F
15/0026 (20130101); C07K 5/101 (20130101); C07K
7/02 (20130101); C07K 7/06 (20130101); C07K
9/001 (20130101); C07K 14/805 (20130101); G01N
33/532 (20130101); G01N 33/536 (20130101); G01N
33/58 (20130101); G01N 33/723 (20130101); G01N
27/3277 (20130101); G01N 2333/805 (20130101); G01N
2458/30 (20130101); Y10S 436/805 (20130101); Y10S
436/806 (20130101); Y10T 436/105831 (20150115) |
Current International
Class: |
G01N
27/327 (20060101) |
Field of
Search: |
;204/403.01-403.15
;205/777.5,778 |
References Cited
[Referenced By]
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Primary Examiner: Noguerola; Alex
Attorney, Agent or Firm: Barnes & Thornburg LLP
Parent Case Text
This application claims benefit of provisional application
60/087,576 Jun. 1, 1998.
Claims
What is claimed is:
1. A method for measuring the concentration of one or more analytes
in a liquid sample, said method comprising contacting a volume of
said liquid sample with 1) predetermined amounts of at least a
first and second redox reversible species, each perspective species
having a redox potential differing by at least 50 millivolts from
that of each other species, at least one species comprising a
liquid sample diffusible .Iadd.covalent .Iaddend.conjugate of a
ligand analog of an analyte in the liquid sample and a redox
reversible label, said conjugate capable of competitive binding
with a specific binding partner for said analyte, and 2) a
predetermined amount of at least one specific binding partner for
each analyte to be measured; and electrochemically determining the
concentration of each of said diffusible redox-reversible species
in the liquid sample by contacting said sample with an electrode
structure including a reference electrode and at least first and
second working electrodes dimensioned to allow diffusional
recycling of the diffusible redox reversible species in the sample
when a predetermined redox-reversible-species-dependent cathodic
potential is applied to one working electrode and a predetermined
redox-reversible-species-dependent anodic potential is applied to a
second working electrode, said diffusional recycling of said
species being sufficient to sustain a measurable current through
said sample, applying a first cathodic potential to the first
working electrode and a first anodic potential to the second
working electrode, said first cathodic and anodic potentials
corresponding to those respective potentials necessary to establish
current flow through the sample due to diffusional recycling of the
first redox reversible species without significant interference
from said second redox reversible species, measuring current flow
at said first anodic and cathodic potentials, applying a second
cathodic potential to said first or second working electrode and a
second anodic potential to the other working electrode, said second
cathodic and anodic potential corresponding to those respective
potentials necessary to establish current flow through the sample
due to diffusional recycling of the second redox-reversible-species
without significant interference from the first redox reversible
species, measuring current flow at said second anodic and cathodic
potentials, and correlating the respective measured current flows
to that for known concentrations of the respective diffusible redox
reversible species.
2. The method of claim 1 wherein the cathodic and anodic potentials
are applied to the working electrodes using a bipotentiostat.
3. The method of claim 1 wherein the redox reversible label is a
metal ion complex selected from ferrocene and nitrogen-coordinate
complexes of transition metal ions.
4. The method of claim 1 wherein the redox reversible label is a
redox reversible organic group.
5. The method of claim 1 for measuring the concentration of two
analytes in a liquid sample wherein the respective redox potentials
of the first and second redox-reversible-species differ by at least
100 millivolts.
6. The method of claim 1 for measuring the concentration of one or
more analytes in a liquid sample wherein current flow is measured
as at least one of the anodic or cathodic potentials is held at the
predetermined value and the potential of the other is swept through
its predetermined value.
7. The method of claim 1 for measuring two proteinaceous analytes
in a liquid sample wherein the ligand analog component of the first
redox-reversible-species is a peptide comprising an epitope of a
first analyte and the ligand analog component of a second
redox-reversible-species is a peptide comprising an epitope of a
second analyte.
8. The method of claim 7 wherein one specific binding partner is an
antibody recognizing the epitope of the first analyte and the other
specific binding partner is an antibody recognizing the epitope of
the second analyte.
9. .[.The.]. .Iadd.A .Iaddend.method .[.of claim 1.]. for measuring
.[.one.]. .Iadd.the concentration of an .Iaddend.analyte in a
liquid sample.Iadd., said method comprising contacting a volume of
said liquid sample with 1) predetermined amounts of at least a
first and second redox reversible species, each respective species
having a redox potential differing by at least 50 millivolts from
that of each other species, said first and second redox reversible
species comprising a liquid sample diffusible conjugate of a ligand
analog of an analyte in the liquid sample and a redox reversible
label, said conjugate capable of competitive binding with a
specific binding partner for said analyte, wherein the respective
ligand analog component of the first and second
redox-reversible-species are different ligand analogs of a single
analyte and 2) a predetermined amount of at least one specific
binding partner for each analyte to be measured; and
electrochemically determining the concentration of each of said
diffusible redox-reversible species in the liquid sample by
contacting said sample with an electrode structure including a
reference electrode and at least first and second working
electrodes dimensioned to allow diffusional recycling of the
diffusible redox reversible species in the sample when a
predetermined redox-reversible-species-dependent cathodic potential
is applied to one working electrode and a predetermined
redox-reversible-species-dependent anodic potential is applied to a
second working electrode, said diffusional recycling of said
species being sufficient to sustain a measurable current through
said sample, applying a first cathodic potential to the first
working electrode and a first anodic potential to the second
working electrode, said first cathodic and anodic potentials
corresponding to those respective potentials necessary to establish
current flow through the sample due to diffusional recycling of the
first redox reversible species without significant interference
from said second redox reversible species, measuring current flow
at said first anodic and cathodic potentials, applying a second
cathodic potential to said first or second working electrode and a
second anodic potential to the other working electrode, said second
cathodic and anodic potential corresponding to those respective
potentials necessary to establish current flow through the sample
due to diffusional recycling of the second redox-reversible-species
without significant interference from the first redox reversible
species, measuring current flow at said second anodic and cathodic
potentials, and correlating the respective measured current flows
to that for known concentrations of the respective diffusible redox
reversible species.Iaddend..
10. The method of claim 9 wherein the ligand analog component of
the first redox reversible species is a peptide comprising a first
epitope of the analyte, and the ligand analog component of the
second redox-reversible-species is a peptide comprising a second
epitope of the analyte, and the specific binding partners are first
and second antibodies each recognizing the respective first and
second epitopes.
11. A device for detecting or quantifying one or more analytes in a
liquid sample, said device comprising a sample chamber for holding
the liquid sample, at least two redox reversible species located
for contact with the liquid sample in the chamber, each redox
reversible species capable of diffusion in said liquid sample at
least in the presence of a respective predetermined analyte, said
redox reversible species having respective redox potentials
differing by at least 50 millivolts, and at least one of said redox
reversible species comprising a ligand capable of blinding to a
specific binding partner for the analyte, an electrode structure
for contact with the liquid sample, said electrode structure
including a reference electrode and working electrodes dimensioned
to allow diffusional recycling of a diffusible redox reversible
species in the liquid sample in contact with the electrode
structure when a predetermined redox-reversible-species-dependent
cathodic potential is applied to one working electrode and a
predetermined redox-reversible-species-dependent anodic potential
is applied to a second working electrode, said diffusional
recycling of said species being sufficient to sustain a measurable
current through each working electrode, and conductors
communicating with the respective electrodes for applying said
anodic potential and said cathodic potential and for carrying the
current conducted by the electrode.
12. The device of claim 11 wherein said chamber has a sample
receiving port and is dimensioned so that it fills by capillary
flow when the liquid sample is contacted with the sample receiving
port.
13. The device of claim 12 wherein the redox reversible species are
located for contact with the liquid sample as it flows into the
chamber.
14. The device of claim 11 wherein the electrode structure
comprises microarray electrodes selected from the group consisting
of arrays of microdiscs, microbands or microholes.
15. The device of claim 11 wherein the electrode structure
comprises interdigitated microarray electrodes.
16. The device of any of claims 11 wherein at least one redox
reversible species includes an osmium complex.
17. The device of claim 11 wherein at least one of the redox
reversible species comprises ferrocene or a redox reversible
derivative thereof.
18. The device of claim 11 wherein two redox reversible species are
positioned for contact with the liquid sample as it is delivered to
the chamber and each species is an osmium complex.
19. The device of claim 11 including at least one redox reversible
species comprising ferrocene or a redox reversible derivative
thereof and at least one redox reversible species comprising an
osmium complex.
20. The device of claim 11 wherein at least one of the
redox-reversible species is an electrochemically detectable
compound of the formula ##STR00006## wherein R and R.sub.1 are the
same or different and are 2,2'-bipyridyl,
4,4'-disubstituted-2,2'-bipyridyl, 5-5'-disubstituted,
-2,2'-bipyridyl, 1,10-phenanthrolinyl, 4,7-disubstituted-1,
10-phenanthrolinyl, or 5,6-disubstituted-1,10-phenanthrolinyl,
wherein each substituent is a methyl, ethyl, or phenyl group, R and
R.sub.1 are coordinated to Os through their nitrogen atoms; q is 1
or 0; R.sub.7 is B--(L).sub.k--Q(CH.sub.2).sub.i--; R.sub.2 is
hydrogen, methyl, or ethyl when q is 1, and R.sub.2 is
B--(L).sub.k--Q(CH.sub.2).sub.i--when q is 0; wherein in the group
B--(L).sub.k--Q(CH.sub.2).sub.i--Q is O, S, or NR.sub.4 is
hydrogen, methyl or ethyl; --L-- is a divalent linker; k is 1 or 0;
i is 1, 2, 3, 4, 5 or 6; and B is a group comprising a ligand
capable of binding to a specific binding partner; Z is chloro or
bromo; m is+1 or+2; X is mono or divalent anion; Y is a monovalent
anion; and n is 1 or zero, provided that when X is a divalent
anion, n is zero, and when m is 1, n is zero and X is not a
divalent anion.
21. The device of claim 11 wherein at least one of the redox
reversible species is an electrochemically detectable compound of
the formula ##STR00007## wherein R and R.sub.1 are the same or
different and are 2,2'-bipyridyl,
4,4'-disubstituted-2,2'-bipyridyl,
5-5'-disubstituted,-2,2'-bipyridyl, 1,1 0-phenanthrolinyl,
4,7-disubstituted-1, 10-phenanthrolinyl, or
5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is
a methyl, ethyl, or phenyl group, R.sub.5 is
4-substituted-2,2'-bipyridyl or 4,4'-disubstituted-2,2'-bipyridyl
wherein the substituent is the group
B--(L).sub.k--Q(CH.sub.2).sub.i--and the 4'-substituent is a
methyl, ethyl or phenyl group; R, R.sub.1 and R.sub.5 are
coordinated to Os through their nitrogen atoms; Q is O, S, or
NR.sub.4 wherein R.sub.4 is hydrogen, methyl or ethyl; --L--is a
divalent linker; k is 1 or 0; i is 1, 2, 3, 4, 5 or 6; B is a group
comprising a ligand capable of binding to a specific binding
partner; d is+2 or+3; X and Y are anions selected from monovalent
anions and divalent anions sulfate, carbonate or sulfite wherein x
and y are independently 0, 1, 2, or 3 so that the net charge of
X.sub.xY.sub.y is-2 or-3.
22. The device of claim 11 wherein the redox reversible species
have respective redox potentials differing by at least 100
millivolts.
23. The device of claim 11 wherein the redox reversible species
have respective redox potentials differing by at least 200
millivolts.
24. The device of claim 11 wherein the device comprises at least
two electrode structures, each in the form of microarray electrodes
dimensioned to enable diffusible recycling of a diffusible redox
reversible species.
25. The device of claim 11 for quantifying a first analyte and a
second analyte in a liquid sample, said device comprising two redox
reversible species, a first redox reversible species comprising a
conjugate of a ligand analog of the first analyte and a second
redox reversible species comprising a conjugate of a ligand analog
of the second analyte, each of said analyte analog conjugates being
capable of binding competitively with its respective analyte to a
specific binding partner.
26. The device of claim 25 further comprising a binding partner
specific for both the first analyte and the redox reversible
conjugate of the ligand analog of the first analyte and a binding
partner specific for both the second analyte and the redox
reversible conjugate of the ligand analog of the second analyte
said specific binding partners located for contact with the liquid
sample in the chamber.
27. The device of claim 11 further comprising a bipotentiostat in
electrical communication with the conductors for applying a
redox-reversible-species-dependent-cathodic potential to one
working electrode and a redox-reversible-species-dependent-anodic
potential to a second working electrode.
28. The device of claim 27 for quantifying one or more analytes in
a liquid sample, said device including first and second redox
reversible species, wherein the bipotentiostat is programmable, and
it is programmed to apply a first cathodic potential to a first
working electrode and a first anodic potential to a second working
electrode, said first anodic and cathodic potentials corresponding
to those potentials necessary to establish current flow through the
sample due to diffusional recycling of the first redox reversible
species, and wherein the bipotentiostat is programmed to apply a
second cathodic potential to said first working electrode and a
second anodic potential to the second working electrode, said
second cathodic and anodic potentials corresponding to those
potentials necessary to establish current flow through the sample
due to diffusional recycling of the second redox reversible
species, and means for measuring current flow through the sample at
each of the first and second potentials.
29. The device of claim 27 for quantifying one or more analytes in
a ligand sample, said device including first and second redox
reversible species, and at least first and second electrode
structures for contact with the liquid sample in the chamber, each
of said electrode structures comprising a microarray of working
electrodes, and a switch for changing the electrical communication
of the bipotentiostat between the first and second electrode
structures.
30. The device of claim 29 wherein the bipotentiostat is
programmable, and it is programmed to apply a first cathodic
potential to a working electrode of the first electrode structure
and a first anodic potential to a second working electrode of the
first electrode structure, said first anodic and cathodic
potentials corresponding to those necessary to establish current
flow through the sample due to diffusional recycling of the first
redox reversible species, and wherein the bipotentiostat is
programmed to apply a second cathodic potential to a working
electrode of the second electrode structure and a second anodic
potential to a second electrode of the second electrode structure,
said second cathodic and anodic potential corresponding to those
potentials necessary to establish current flow through the sample
due to diffusional recycling of the second redox reversible
species, and means for measuring current flow through the sample at
each electrode structure.
31. The device of claim 11 wherein the first and second reversible
species each comprise a conjugate of different ligand analogs of
one analyte, each of said conjugates capable of binding
competitively with said analyte to one of two independent specific
binding partners for said analyte.
32. The device of claim 11 for quantifying glycosylated hemoglobin
wherein at least one of the two redox reversible species comprises
a conjugate of the formula Gluc-Val-His-Leu-Thr - L - M.sub.1
wherein M.sub.1 is a redox reversible label, L is a linker and
Gluc-Val-His-Leu-Thr- is the N-terminal sequence of the
.beta.-chain of hemoglobin Al c.
33. The device of claim 32 wherein the redox reversible label is a
metal ion complex.
34. The device of claim 32 wherein M.sub.1 is an osmium ion complex
or ferrocene.
35. The device of claim 32 wherein the other reversible redox
species comprises a redox reversible conjugate of the formula
Val-His-Leu-Thr- L -M.sub.2 wherein M.sub.2 is a redox reversible
label and L is a linker.
36. The device of claim 35 wherein the redox reversible label is a
metal ion complex.
37. The device of claim 35 wherein the redox potential of M.sub.1
and M.sub.2 differ by at least 100 millivolts.
38. The device of claim 35 wherein the redox potential of M.sub.1
and M.sub.2 differ by at least 200 millivolts.
39. The device of claim 35 further comprising a specific binding
partner for both hemoglobin Alc and the redox reversible conjugate
Gluc-Val-His-Leu-Thr-L -M.sub.1, said specific binding partner
located for contact with the sample in the chamber.
40. The device of claim 39 further comprising a specific binding
partner for both hemoglobin and the redox reversible conjugate
Val-His-Leu-Thr -L M.sub.2, said specific binding partner located
for contact with the sample in the chamber.
41. A kit for measuring the concentration of one or more analytes
in a liquid sample, said kit comprising at least two redox
reversible species for contact with the liquid sample, each capable
of diffusion in the liquid sample at least in the presence of a
predetermined analyte, at least one species comprising a
.Iadd.covalent .Iaddend.conjugate of a ligand analog of an analyte
and a redox reversible label, said redox reversible species having
respective redox potentials differing by at least 50 millivolts; a
specific binding partner for each analyte; an electrode structure
for contact with the liquid sample, said electrode structure
including a reference electrode and working electrodes dimensioned
to allow diffusional recycling of diffusible redox reversible
species in the sample when a predetermined
redox-reversible-species-dependent-cathodic potential is applied to
one working electrode and a predetermined
redox-reversible-species-dependent-anodic potential is applied to
the second working electrode, said diffusional recycling of said
.Iadd.diffusible redox reversible .Iaddend.species .[.means.].
sufficient to sustain a measurable current through the sample; and
conductors communicating with the respective electrodes for
applying said anodic potential and said cathodic potential and for
carrying the current conducted by the electrodes.
42. The kit of claim 41 wherein the electrode structure comprises
microarray electrodes selected from the group consisting of arrays
of microdiscs, microbands, or microholes.
43. The kit of claim 41 wherein the electrode structure comprises
interdigitated microarray electrodes.
44. The kit of claim 41 wherein the redox reversible species are
mixed as a composition for contact with the liquid sample.
45. The kit of claim 41 wherein the redox reversible label of at
least one redox reversible species comprises an osmium complex.
46. The kit of claim 41 wherein the redox reversible species have
respective redox potentials differing by at least 100
millivolts.
47. The kit of claim 41 wherein the redox reversible species have
respective redox potentials differing by at least 200
millivolts.
.Iadd.48. A method of determining the amount or concentration of a
plurality of diffusible redox-reversible species in a solution,
comprising: providing an electrochemical measurement cell
comprising at least two working electrodes and a reference
electrode, said working electrodes so configured and arranged that
redox recycling of diffusible redox-reversible species takes place
between the working electrodes when appropriate potentials are
applied, contacting the solution with the electrodes in the
measurement cell, applying potentials to the working electrodes
such that a current through the cell is generated as a result of
redox recycling of at least one diffusible redox-reversible
species, applying potentials to the working electrodes such that a
current through the cell is generated as a result of redox
recycling of a second diffusible redox-reversible species wherein
said diffusible redox-reversible species have equilibrium
potentials that differ by more than about 50 mV and the measured
concentration of one species is corrected by the response of
another species. .Iaddend.
.Iadd.49. The method of claimed 48 where the current generated
correlates to the concentration of one or more diffusible redox
recycling species. .Iaddend.
.Iadd.50. The method of claim 48 where at least one of the
responses are correlated with at least one analyte concentration.
.Iaddend.
.Iadd.51. A method of determining the relative diffusion
coefficients of a plurality of diffusible redox-reversible species
in a solution, comprising: providing an electrochemical measurement
cell comprising at least two working electrodes and a reference
electrode, said working electrodes so configured and arranged that
redox-recycling of diffusible redox-reversible species takes place
between the working electrodes when appropriate potentials are
applied, contacting the solution with the electrodes in the
measurement cell, applying potentials to the working electrodes
such that a current through the cell is generated as a result of
redox recycling of at least one of the diffusible redox-reversible
species, applying potentials to the working electrodes such that a
current through the cell is generated as a result of redox
recycling of a second diffusible redox-reversible species wherein
said diffusible redox-reversible species have equilibrium
potentials that differ by more than about 50 mV. .Iaddend.
.Iadd.52. A method for measuring the concentration of an analyte in
a liquid sample, said method comprising: reaching a compound with
said analyte to generate a first redox reversible species in said
liquid in the presence of a second redox reversible species,
wherein said first and second redox reversible species have redox
potentials differing by at least 50 millivolts, wherein at least
one of said first and second redox reversible species comprises a
liquid sample diffusible covalent conjugate of a ligand analog of
said analyte and a redox reversible label, electrochemically
determining the concentration of each of said redox-reversible
species in the liquid sample by containing said sample with an
electrode structure including a reference electrode and at least
first and second working electrodes dimensioned to allow
diffusional recycling of the redox reversible species in the sample
when a predetermined redox-reversible-species-dependent cathodic
potential is applied to one working electrode and a predetermined
redox-reversible-species-dependent anodic potential is applied to a
second working electrode, said diffusional recycling of said
species being sufficient to sustain a measurable current through
said sample, applying a first cathodic potential to the first
working electrode and a first anodic potential to the second
working electrode, said first cathodic and anodic potentials
corresponding to those respective potentials necessary to establish
current flow through the sample due to diffusional recycling of the
first redox reversible species without significant interference
from said second redox reversible species, measuring current flow
at said first anodic and cathodic potentials, applying a second
cathodic potential to said first or second working electrode and a
second anodic potential to the other working electrode, said second
cathodic and anodic potential corresponding to those respective
potentials necessary to establish current flow through the sample
due to diffusional recycling of the second redox-reversible-species
without significant interference from the first redox reversible
species, measuring current flow at said second anodic and cathodic
potentials, and correlating the respective measured current flows
to that for known concentrations of the respective diffusible redox
reversible species. .Iaddend.
Description
FIELD OF THE INVENTION
This invention relates to a method and device for detection and
quantification of biologically significant analytes in a liquid
sample. More particularly the invention is directed to a biosensor
and method of using same for electrochemical immunoassays of
multiple analyte species in a single liquid sample.
BACKGROUND AND SUMMARY OF THE INVENTION
Therapeutic protocols used today by medical practitioners in
treatment of their patient population requires accurate and
convenient methods of monitoring patient disease states. Much
effort has been directed to research and development of methods for
measuring the presence and/or concentration of biologically
significant substances indicative of a clinical condition or
disease state, particularly in body fluids such as blood, urine or
saliva. Such methods have been developed to detect the existence or
severity of a wide variety of disease states such as diabetes,
metabolic disorders, hormonal disorders, and for monitoring the
presence and/or concentration of ethical or illegal drugs. More
recently there have been significant advancements in the use of
affinity-based electrochemical detection/measurement techniques
which rely, at least in part, on the formation of a complex between
the chemical species being assayed (the "analyte") and another
species to which it will bind specifically (a "specific binding
partner"). Such methods typically employ a labeled ligand analog of
the target analyte, the ligand analog selected so that it binds
competitively with the analyte to the specific binding partner. The
ligand analog is labeled so that the extent of binding of the
labeled ligand analog with the specific binding partner can be
measured and correlated with the presence and/or concentration of
the target analyte in the biological sample.
Numerous labels have been employed in such affinity based sample
analysis techniques, including enzyme labeling, radioisotopic
labeling, fluorescent labeling, and labeling with chemical species
subject to electrochemical oxidation and/or reduction. The use of
redox reversible species, sometimes referred to as electron
transfer agents or electron mediators as labels for ligand analogs,
have proven to provide a practical and dependable results in
affinity-based electrochemical assays. However, the use of
electrochemical techniques in detecting and quantifying
concentrations of such redox reversible species (correlating with
analyte concentrations) is not without problem. Electrochemical
measurements are subject to many influences that affect the
accuracy of the measurements, including those relating to
variations in the electrode structure itself and/or matrix effects
deriving from variability in liquid samples.
The present invention relates to immunosensors based on direct
electrochemical measurement of detectable species with microarray
electrodes under bipotentiostatic control. An electrochemical
label, for example as Oe mediator, is covalently attached to a
peptide which has amino acid sequence of the binding epitope for
the antibody. When indicator/peptide conjugate is bound to
antibody, the indicator does not function electrochemically or it
is said to be "inhibited". The analyte present in sample will
compete with indicator/peptide conjugate for the limited number of
binding sites on the antibody. When more analyte is present, more
free indicator/peptide conjugate will be left producing higher
current at a sensor electrode, i.e., one of the working electrodes
where measured events (oxidation or reduction) are taking place. In
the opposite case, when less analyte is present, more
indicator/peptide conjugate will be bound to antibody resulting
less free conjugates and producing lower current levels at the
working electrodes. Therefore the current detected at either one of
the working electrodes will be a function of analyte
concentration.
It is frequently desired to measure more than one analyte species
in a liquid sample. Measurement of multiple species in a mixture
has been achieved with photometry and fluoroescence, via selection
of the appropriate wavelengths. Electrochemical measurements of a
single species in a complex mixture are routinely made by selecting
a potential at which only the desire species is oxidized or reduced
(amperometry) or by stepping or varying the potential over a range
in which only the desired species changes its electrochemical
properties (AC and pulse methods). These methods suffer from
disadvantages including lack of sensitivity and lack of
specificity, interference by charging and matrix polarization
currents (pulse methods) and electrode fouling due to the inability
to apply an adequate overpotential. Moreover, electrochemical
measurements are complicated by interference between the
multiplicity of electroactive species commonly extant in biological
samples.
Electrode structures which generate steady state current via
diffusional feedback, including interdigitated array electrodes
(IDAs) (FIGS. 1 and 2) and parallel plate arrangements with
bipotentiostatic control are known. They have been used to measure
reversible species based on the steady state current achieved by
cycling of the reversible species. A reversible mediator (redox
reversible species) is alternately oxidized and reduced on the
interdigitated electrode fingers. The steady state current is
proportionate to mediator concentration (FIG. 3) and limited by
mediator diffusion. A steady state current is achieved within
seconds of applying the predetermined anodic (more positive) and
cathodic (less positive or negative) potentials (FIG. 6) to the
microelectrode array. The slope of a plot of the IDA current vs.
mediator concentration is dependent on IDA dimensions, and the
slope increases with narrower electrode spacings (FIG. 7).
One embodiment of the present invention provides a method for
measuring multiple analyte species in the same sample, and
optimally on the same electrode structure, thus improving the
accuracy of the relative measurements. This invention also provides
an electrochemical biosensor with capacity to provide improved
accuracy through the use of self-compensation. Analyte
concentration can be measured/calculated from electrometric data
obtained on the same liquid sample with the same electrode
structure (the working electrodes), thereby minimizing
perturbations due to variability in sample or electrode
structure.
The various embodiments of this invention utilize the principle of
diffusional recycling, where a diffusible redox reversible species
is alternately oxidized and reduced at nearby electrodes, thereby
generating a measurable current. As alternate oxidation and
reduction is required for measurement, only electroactive species
which are electrochemically reversible are measured thereby
eliminating, or at least reducing, the impact or interference from
non-reversible electroactive species in the sample. Redox
reversible species having different oxidation potentials can be
independently measured in a mixture by selecting and
bipotentiostatically controlling the oxidizing and reducing
potentials for neighboring electrode pairs so that only the species
of interest is oxidized at the anode (the electrode with the more
positive potential) and reduced at the cathode (the electrode with
the less positive or negative potential). When the working
electrodes (the anode/cathode arrays) are dimensioned to allow
diffusional recycling of the redox-reversible-species at the
selected oxidizing and reducing potentials appropriate for that
species, a steady state current at the working electrodes where the
measurable oxidative and reductive events are taking place, is
quickly established through the sample and the electrode structure.
The magnitude of the current is proportional to the concentration
of the diffusible redox reversible species in the sample. When two
or more redox reversible species are utilized, they are selected to
have redox potentials differing by at least 50 millivolts, most
preferably at least 200 millivolts, to minimize interference
between one species and the other in measurements of the respective
steady state currents.
Any electrode structure which allows for diffusional recycling to
achieve steady state current in response to application of
pre-selected species-specific anodic and cathodic potentials can be
utilized in carrying out the invention. Suitable electrode
structures include interdigitated array microelectrodes and
parallel plate electrodes separated by distances within the
diffusion distance of the respective redox reversible species. The
electrode structures typically include a reference electrode (e.g.,
Ag/AgCl), at least two working electrodes (one at positive
potential and another at a less positive or negative potential
relative to the reference electrode), and optionally an auxiliary
electrode for current control. In use, a programmable
bipotentiostat is placed in electrical communication with the
electrode structure for applying the respective anoidic and
cathodic potentials specific for each of the respective redox
reversible species utilized in the method/biosensor. Several novel
osmium complexes have been developed for use as labels for
preparing ligand analog conjugates having potential differences
sufficient to allow the use of two osmium complexes (as opposed to
an osmium complex and a ferrocene or other redox reversible label)
in this invention.
Accordingly, one embodiment of the invention provides a device for
detecting or quantifying one or more analytes in a liquid sample.
The device comprises at least two redox reversible species having
respective redox potentials differing by at least 50 millivolts,
and an electrode structure for contact with the liquid sample. In
one embodiment the device further comprises a chamber for
containing the liquid sample, optionally dimensioned for capillary
fill. The electrode structure includes a reference electrode and an
anode and a cathode (working electrodes) dimensioned to allow
diffusional recycling of the redox reversible species in the sample
when a redox-reversible-species-dependent cathodic potential is
applied to one working electrode and a
redox-reversible-species-dependent anodic potential is applied to a
second electrode to enable and sustain a measurable current through
the sample. The device also includes conductors communicating with
the respective electrodes for applying potentials and for carrying
current conducted between the sample and the respective
electrodes.
The device in accordance with this invention is typically utilized
in combination with a meter which includes a power source, for
example a battery, a microprocessor, a register for storing
measured current values, and a display for reporting calculated
analyte concentrations based on measured current values. The
construction and configuration of such meters are well known in the
art. Meters for use in accordance with the present device further
comprise a bipotentiostat under control of the microprocessor or
separately programmable to apply predetermined potentials to the
device component microelectrode arrays during liquid sample
analysis. Improvements in meter construction and design for
biosensor systems are described in U.S. Pat. Nos. 4,999,632;
5,243,516; 5,366,609; 5,352,351; 5,405,511; and 5,48,271, the
disclosures of which are hereby incorporated by reference.
In another embodiment of the invention there is provided a method
for measuring the concentration of one or more analytes in a liquid
sample. The method includes contacting a portion of the sample with
pre-determined amounts of at least a first and second redox
reversible species having a redox potential differing by at least
50 millivolts from that of each other species. Each respective
species comprises a liquid sample diffusible conjugate of a ligand
analog of an analyte in the liquid sample and a redox reversible
label. The liquid sample is also contacted with a predetermined
amount of at least one specific binding partner for each analyte to
be measured. The diffusible conjugate is selected so that it is
capable of competitive binding with the specific binding partner
for said analyte.
The concentration of diffusible redox-reversible-species in the
liquid sample is then determined electrochemically. The sample is
contacted with an electrode structure, including a reference
electrode and at least first and second working electrodes
dimensioned to allow diffusional recycling of at least one of the
diffusible redox-reversible-species in the sample, when a
predetermined redox-reversible-species-dependent cathodic potential
is applied to one working electrode and a predetermined
redox-reversible-species-dependent anodic potential is applied to
the second working electrode. Typically, a first cathodic potential
is applied to the first working electrode and a first anodic
potential is applied to the second working electrode to establish
current flow through the sample due to diffusional recycling of the
first redox-reversible-species without significant interference
from the second redox-reversible-species. Current flow through one
or more of the electrodes at the first anodic and cathodic
potentials is measured. Similarly current flow responsive to
application of second cathodic and anodic potentials to electrodes
in contact with the sample is measured and correlated with measured
current flows for known concentrations of the respective
redox-reversible-species, said concentrations being proportionate
to the respective analyte concentrations at a predetermined
redox-reversible species-dependent potential (anoidic or cathodic).
Alternatively, the potential of one of the working electrodes can
be held constant and current flow is monitored as the potential of
the other working electrode is varied and swept through the other
redox-reversible species-dependent potential.
The reagent components for the invention, including the redox
reversible species and the specific binding partners, can be
provided in the form of a test kit for measuring the targeted
analyte(s) in a liquid sample, either as separate reagents or, more
preferably, combined as a multi-reagent composition, e.g. combined
redox reversible species, combined specific binding partners, or
combined redox reversible species and specific binding partners.
The kit optionally, but preferably, includes an electrode structure
dimensioned to allow diffusional redox recycling of diffusable
redox reversible species in the liquid sample. The electrode
structure includes conductors for connecting the structure
bipotentiostat programmed to apply
redox-reversible-species-dependent-anodic and cathodic potentials
to the electrode structure and to sense and measure current flow,
typically at one or both of the working electrodes, responsive to
such applied potentials.
Also described herein is the preparation and use of
electrochemically detectable osmium complexes and covalent
conjugates of said complexes having oxidation potentials differing
sufficiently to enable their use together in the respective method
and device embodiments of the invention. Osmium labeled ligand
analogs capable of binding to a specific binding partner of a
biologically significant analyte are prepared. One group of
electrochemically detectable conjugates comprise a bis(bipyridyl)
imidazolyl chloroosmonium complex characterized by fast mediation
kinetics and low redox potential (+15 mV vs. Ag/AgCl). Another
group of osmium complex labeled, electrochemically detectable
conjugates include tris(biphenyl) osmium complexes, which, like the
bis(bipyridyl) imidazolyl chloroosmium complexes are characterized
by fast mediation kinetics, but the tris(bipyridyl) complexes have
a redox potential sufficiently different from the bis(pyridyl)
imidazolyl chloroosmium complexes to allow their use together in
the various embodiments of this invention to enable use of
microelectrode arrays for measuring more than one analyte in a
single liquid sample by concentration dependent currents amplified
by diffusional redox recycling.
In one preferred embodiment of the invention at least one osmium
complex conjugates is used in combination with another conjugated
redox-reversible-species for the measurement of both glycosylated
hemoglobin and hemoglobin in a lysed blood sample. One
redox-reversible-species preferably comprises an osmium complex
covalently linked to a ligand analog of either hemoglobin or
glycosylated hemoglobin, and the second redox-reversible-species
comprises a second redox reversible label covalently bound to a
ligand analog of the other of the two target analytes. The method
enables measurement of the concentration of both the glycosylated
hemoglobin (HbAlc) and the concentration of either total hemoglobin
or that of unglycosylated hemoglobin (HbA.sub.0) thereby enabling
calculation of the results as a ratio of the two measurements (%
HbAlc). It is advantageous to assay both HbAlc and total hemoglobin
(or HbA.sub.0) using the same principle in a single sample.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an enlarged plan view of an interdigitated array
electrode for reversible mediator measurement in accordance with
the present invention.
FIG. 2 is a partial cross-sectional view of the electrode of FIG. 1
illustrating the conditions of steady state current limited by
diffusion of reversible mediator (M) which is alternately oxidized
and reduced on the interdigitated electrode fingers.
FIG. 3 is a graphic presentation of dose response currents for a
bis-(bipyridyl) imidazolyl chloroosmonium mediator in a peptide
conjugate of that mediator.
FIG. 4 is a graphic illustration of current flow vs. concentration
of glycosylated hemoglobin (HbAlc) in blood samples using an osmium
conjugate and enzyme amplified DC amperometry.
FIG. 5 is a graphic illustration of the inhibition of current flow
due to free conjugate as a function of antibody concentration
(C.sub.n) as measured using enzyme amplified DC amperometry
[C.sub.1>C.sub.2>C.sub.3].
FIG. 6 is a graphic illustration of current flow vs. time using an
interdigitated array electrode.
FIG. 7 is a graphic illustration of the effect of the dimensions of
the interdigitated array electrode structure on current flow as a
function of concentration of an osmium conjugate (Os-DSG-Alc).
FIG. 8 is a graphic illustration of current flow as a function of
applied potential for a liquid sample containing equimolar (50
.mu.M) of a bis-(bipyridyl) imidazolyl chloroosmium complex and a
tris(bipyridyl) osmium complex.
FIG. 9 is a graphic presentation of current flow vs. concentration
of a ferrocene-biotin conjugate in the presence of varying amounts
of an osmium complex conjugate on interdigitated array electrodes
with bipotentiostatic control.
FIG. 10 is a graphic illustration of the effect of concentration of
an unlabeled conjugate (BSA-Alc) on current flow in a solution
containing osmium labeled conjugate (osmium-DSG-Alc)) in the
presence of three separate Alc-recognizing antibody
compositions.
FIG. 11 illustrates the structure of a tris(bipyridyl) osmium
labeled conjugate for use in accordance with this invention.
FIGS. 12-14 are similar and each depict the chemical structure of a
bis(bipyridyl) imidazolyl chloroosmium labeled peptide conjugate
for use in accordance with this invention.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the invention is a method for measuring the
concentration of one or more analytes in a liquid sample. The
method enables two or more independent amperometric measurements of
the sample on a single electrode structure. The method comprises
contacting a volume of said liquid sample with 1) predetermined
amounts of at least a first and second redox reversible species,
each respective species having a redox potential differing by at
least 50 millivolts from that of each other species, at least one
species comprising a liquid sample diffusible conjugate of a ligand
analog of an analyte in the liquid sample and a redox reversible
label, said conjugate capable of competitive binding with a
specific binding partner for said analyte, and 2) a predetermined
amount of at least one specific binding partner for each analyte to
be measured; and electrochemically determining the concentration of
each of said diffusible redox-reversible species in the liquid
sample by contacting said sample with an electrode structure
including a reference electrode and at least first and second
working electrodes dimensioned to allow diffusional recycling of
the diffusible redox reversible species in the sample when a
predetermine redox-reversible-species-dependent cathodic potential
is applied to one working electrode and a predetermined
redox-reversible-species-dependent anodic potential is applied to a
second working electrode, said diffusional recycling of said
species being sufficient to sustain a measurable current through
said sample, applying a first cathodic potential to the first
working electrode and a first anodic potential to the second
working electrode, said first cathodic and anodic potentials
corresponding to those respective potentials necessary to establish
current flow through the sample due to diffusional recycling of the
first redox reversible species without significant interference
from said second redox reversible species, measuring current flow
at said first anodic and cathodic potentials, applying a second
cathodic potential to said first or second working electrode and a
second anodic potential to the other working electrode, said second
cathodic and anodic potential corresponding to those respective
potentials necessary to establish current flow through the sample
due to diffusional recycling of the second redox-reversible-species
without significant interference from the first redox reversible
species, measuring current flow at said second anodic and cathodic
potentials, and correlating the respective measured current flows
to that for known concentrations of the respective diffusible redox
reversible species.
The method of the invention has very broad applicability but in
particular may be used to assay: drugs, hormones, including peptide
hormones (e.g., thyroid stimulating hormone (TSH), luteinizing
hormone (LH), follicle stimulating hormone (FSH), insulin and
prolactin) or non-peptide hormones (e.g., steroid hormones such as
cortisol, estradiol, progesterone and testosterone, or thyroid
hormones such as thyroxine (T4) and triiodothyronine), proteins
(e.g., human chorionic gonadotropin (hCG), carcino-embryonic
antigen (CEA) and alphafetoprotein (AFP)), drugs (e.g., digoxin),
sugars, toxins or vitamins.
The method can be performed on liquid samples comprising biological
fluids such as saliva, urine, or blood, or the liquid sample can be
derived from environmental sources. The liquid samples can be
analyzed "as is," or they can be diluted, buffered or otherwise
processed to optimize detection of the targeted analyte(s). Thus,
for example, blood samples can be lysed and/or otherwise denatured
to solubilize cellular components.
The method can be performed using widely variant sampling handling
techniques. Thus, the sample can be premixed with either or both of
the specific binding partner for the targeted analytes and the
redox reversible species prior to contacting the sample with the
electrode structure, or the liquid sample, either neat or
pre-processed, can be delivered to a vessel containing
predetermined amounts of the redox reversible species and the
specific binding partner for subsequent or simultaneous contact
with the electrode structure. The order of introduction of the
components into the sample is not critical; however, in one
embodiment of the invention the predetermined amounts of the
specific binding partners are first added to the sample, and
thereafter, there is added the predetermined amounts of the redox
reversible species. It is also possible to combine the
predetermined amounts of the specific binding partners with the
redox reversible species to form the respective complexes prior to
combining those components with the liquid sample. In that latter
case the redox reversible species will be displayed from its
respective specific binding partner by the corresponding analyte to
provide a concentration of the redox reversible species
proportionate to the concentration of analyte in the liquid sample.
The reagents, that is, the predetermined amounts of the specific
binding partner of each analyte and the predetermined amounts of
the corresponding redox reversible species can, for example, be
deposited in a vessel for receiving a predetermined volume of the
liquid sample. The liquid sample is added to the vessel, and
thereafter, or simultaneously, the liquid sample is contacted with
the electrode structure.
The electrode structure includes a reference electrode and at least
first and second working electrodes dimensioned to allow
diffusional recycling of the diffusible redox reversible species in
the sample when predetermined
redox-reversible-species-dependent-cathodic and anodic potential is
applied to the working electrodes. The term "working electrode" as
used herein refers to an electrode where measured events (i.e.
oxidation and/or reduction) take place and resultant current flow
can be measured as an indicator of analyte concentration. "Anodic
potential" refers to the more positive potential (applied to the
anode) and "cathodic potential" refers to the less positive or
negative potential applied to the cathode (vs. a reference
electrode). Electrodes dimensioned to allow diffusional recycling
are well known in the art and are typically in the form of arrays
of microdiscs, microholes, or microbands. In one embodiment the
electrodes are in the form of an interdigitated arrangement of
microband electrodes with micron or submicron spacing. Short
average diffusional length and a large number of electrodes are
desirable for effective current amplification by recycling of
reversible redox species. The microelectrode arrays can be
fabricated, for example, as pairs of interdigitated thin film metal
electrodes in micron and submicron geometry arranged on an
insulator substrate, for example, oxidized silicon. Each of the
electrode fingers (FIG. 1) are spaced from its neighboring finger
in the nanometer to low micrometer (1-10 microns) range.
Microelectrode arrays can be fabricated using photolithography,
electron bean lithography, and so-called lift-off technique. Thus,
an interdigitated electrode array (IDA) can be deposited on glass,
silicon or polyamide utilizing the following general procedure: 1.
Grow thermal oxide layer on silicon substrate; 2. Sputter 400 .ANG.
chromium seed layer, 2000 .ANG.gold; 3. Spin-coat and soft-bake
photo resist; 4. Expose and develop photo resist with IDA pattern;
5. Pattern gold and chromium with ion beam milling; 6. Strip photo
resist; and 7. Cut electrodes into chips by first coating with a
protective layer, cutting into strips, stripping the protective
layer, and cleaning electrode surfaces in oxygen plasma.
The electrode structure can be formed on an inner surface of a
chamber for receiving the liquid sample, e.g., a cuvette, a
capillary fill chamber, or other sample receiving vessel wherein
the electrode structure can be contacted with the liquid sample.
Alternatively, the electrode structure can form part of a probe for
dipping into the liquid sample after the sample has been contacted
with the predetermined amounts of the redox reversible species and
the specific binding partners. The electrode structure is in
contact with conductors that enable application of the respective
cathodic and anodic potentials for carrying out the present method.
The anodic and cathodic potentials are applied relative to a
reference electrode component of the electrode structure using a
bipotentiostat. The electrode structure can optionally include an
auxiliary electrode for current control. The bipotentiostat is
utilized to apply a first cathodic potential to a first working
electrode and a first anodic potential to a second working
electrode, the first cathodic and anodic potentials corresponding
to those respective potentials necessary to establish current flow
through the sample due to diffusional recycling of the first redox
reversible species. Optionally the potential on one working
electrode can be set at a first diffusible species dependent,
anodic potential and current flow is measured as the potential of
the other working electrode is swept through a potential
corresponding to the predetermined diffusible species dependent
cathodic potential (or vice versa).
The cathodic and anodic potentials appropriate for each reversible
redox species can be readily determined by empirical measurement.
The multiple redox reversible species used in performance of the
method of this invention are selected to have redox potentials
differing by at least 50 millivolts, more preferably at least 100
millivolts, more preferably at least 200 millivolts, from that of
each other redox reversible species utilized in the method. The
difference in redox potentials of the redox reversible species
being used allow each species to be detected without significant
interference from the second or any other redox reversible species
in the liquid sample. A steady state current flow is rapidly
established at each of the working electrodes following application
of the anodic and cathodic potentials. Current flow can be measured
at either or both working electrodes, and it is proportionate to
the concentration of the recycling redox reversible species.
Second cathodic and anodic potentials are applied to the working
electrodes wherein said second potentials correspond to those
respective potentials necessary to establish current flow through
the sample due to diffusional recycling of the second redox
reversible species without significant interference from the first
redox reversible species, and the resulting steady state current
flow is measured. This step is repeated for each redox reversible
species utilized in the method. The measured current flows are then
correlated to known concentrations of the respective diffusible
redox reversible species. Those concentrations are proportionate to
the respective analyte concentrations.
The method steps can be conducted using a programed bipotentiostat
to control potentials on the electrode structure in contact with
the sample. The bipotentiostat can be included either in a desktop
or hand-held meter further including means for reading values for
steady state current, storing said values, and calculating analyte
concentrations using a microprocessor programmed for making such
calculations.
The redox reversible species utilized in the method comprise a
liquid-sample-diffusible conjugate of a ligand analog of an analyte
in the liquid sample and a redox reversible label. The term "ligand
analog" as used in defining the present invention refers to a
chemical species capable of complexing with the same specific
binding partner as the analyte being measured and can include the
analyte itself, provided that the molecular weight of the conjugate
is less than about 50,000, more preferably less than about 10,000
Daltons. Most preferably the molecular weight of the conjugate of
the ligand analog and the redox reversible label is between about
500 and about 5,000 Daltons. Low molecular weight redox reversible
species are most desirable in view of the diffusion-based
electrochemical detection technique utilized in carrying out the
present method.
The term "redox reversible label" as used herein refers to a
chemical species capable of reversible oxidation and reduction in a
liquid sample. It can be in the form of an organic moiety, for
example, a chemical group comprising a nitrosoaniline, a catechol,
hydroquinone, or an aminophenol group. Alternatively, the redox
reversible label can be an inorganic or organometallic species
capable of undergoing reversible oxidation and reduction in a
liquid sample. Such species may be, for example, complete
molecules, portions of molecules, atoms, ions, or more
particularly, ion complexes. Redox reversible labels are well-known
in the art and include ligand complexes of transition metal ions,
for example iron (ferrocene and ferrocene derivatives), ruthenium
and osmium.
The relative amounts of the first and second redox reversible
species and the respective specific binding partners for the
targeted analytes to be measured in the method can be determined
empirically. They are dependent on the concentration ranges of the
targeted analyte, and the binding stoichiometry of the specific
binding partner, the binding constant, the analyte and the
corresponding redox reversible species. The amounts of each reagent
appropriate for each analyte being measured can be determined by
empirical methods.
The redox reversible species typically comprises a conjugate of a
ligand analog of an analyte in a liquid sample and a redox
reversible label. The conjugate is prepared by linking the ligand
analog to the label either covalently through bifunctional linking
agents or by combination of covalent linkages and art-recognized
specific binding entities (for example, biotin-avidin).
In one embodiment of the invention the specific binding partner for
each analyte is an antibody and the ligand analog is selected so
that it binds competitively with the analyte to the antibody. There
are, however, other examples of ligand-specific binding partner
interactions that can be utilized in developing applications of the
present method. Examples of ligands and specific binding partners
for said ligands are listed below.
TABLE-US-00001 Ligand Specific Binding Partner Antigen (e.g., a
drug substance) Specific antibody Antibody Antigen Hormone Hormone
receptor Hormone receptor Hormone Polynucleotide Complementary
polynucleotide strand Avidin Biotin Biotin Avidin Protein A
Immunoglobulin Immunoglobulin Protein A Enzyme Enzyme cofactor
(substrate) Enzyme cofactor (substrate) Enzyme Lectins Specific
carbohydrate Specific carbohydrate Lectins of lectins
The term "antibody" refers to (a) any of the various classes or
subclasses of immunoglobulin, e.g., IgG, IgM, derived from any of
the animals conventionally used, e.g., sheep, rabbits, goats or
mice; (b) monoclonal antibodies; (c)intact molecules or "fragments"
of antibodies, monoclonal or polyclonal, the fragments being those
which contain the binding region of the antibody, i.e., fragments
devoid of the Fc portion (e.g., Fab, Fab.sup.1, F(ab').sub.2) or
the so-called "half molecule" fragments obtained by reductive
cleavage of the disulfide bonds connecting the heavy chain
components in the intact antibody. The preparation of such
antibodies are well-known in the art.
The term "antigen" used in describing and defining the present
invention includes both permanently antigenic species (for example,
proteins, peptides, bacteria, bacteria fragments, cells, cell
fragments, drug substances, and viruses) and haptans which may be
rendered antigenic under suitable conditions.
In one embodiment of the invention there is provided a method for
measuring two proteinaceous analytes in a liquid sample wherein the
ligand analog component of the first redox reversible species is a
peptide comprising an epitope of a first analyte and the ligand
analog component of a second redox reversible species is a peptide
comprising an epitope of a second analyte. One specific binding
partner utilized in the method is an antibody recognizing the
epitope of the first analyte, and the other specific binding
partner is an antibody recognizing the epitope of the second
analyte. In another application of the present method two
independent measurements are performed on a single analyte in a
liquid sample. In that embodiment the respective ligand analog
component of the first and second redox reversible species are
different ligand analogs of the targeted analyte. Where the
targeted analyte is a proteinaceous compound, the ligand analog
component of the first redox reversible species is a peptide
comprising a first epitope of the analyte, and the ligand analog of
the second redox reversible species is a peptide comprising a
second epitope of the analyte, and the specific binding partners
are first and second antibodies, each recognizing respective first
and second analyte epitopes.
In one preferred embodiment of the invention, at least one of the
redox reversible labels is an osmium complex. In another
embodiment, both redox reversible labels are osmium complexes, each
having an oxidizing potential difference of at least 50, most
preferably at least 200 millivolts. Illustrative of the redox
reversible osmium complexes for use in this invention are complexes
of the formula ##STR00001## wherein
R and R.sub.1 are the same or different and are 2,2'-bipyridyl,
4,4'-disubstituted-2,2'-bipyridyl,
5-5'-disubstituted,-2,2'-bipyridyl, 1,10-phenanthrolinyl,
4,7-disubstituted-1,10-phenanthrolinyl, or 5,6-disubstituted-1,1
0-phenanthrolinyl, wherein each substituent is a methyl, ethyl, or
phenyl group,
R and R.sub.1 are coordinated to Os through their nitrogen
atoms;
q is 1 or 0;
R.sub.7 is B-(L).sub.k--Q(CH.sub.2).sub.i--;
R.sub.2 is hydrogen, methyl, or ethyl when q is 1, and R.sub.2 is
B--(L).sub.k--Q(CH.sub.2).sub.i--when q is 0;
wherein in the group B--(L).sub.k--Q(CH.sub.2).sub.i--Q is O,S, or
NR.sub.4 wherein R.sub.4 is hydrogen, methyl or ethyl;
--L--is a divalent linker;
k is 1 or 0;
i is 1, 2, 3, 4, 5 or 6; and
B is hydrogen or a group comprising a ligand capable of binding to
a specific binding partner;
Z is chloro or bromo;
m is+1 or+2;
X is a mono- or divalent anion, e.g., chloride, bromide, iodide,
fluoride, tetrafluoroborate, perchlorate, nitrate, sulfate,
carbonate, or sulfite;
Y is monovalent anion, e.g., chloride, bromide, iodide, fluoride,
tetrafluoroborate, perchlorate or nitrate; and
n is 1 or zero,
provided that when X is a divalent anion, n is zero,
and when m is 1, n is zero and X is not a divalent anion.
Another redox reversible osmium complex for use in the present
method is a compound of the formula ##STR00002## wherein
R and R.sub.1 are the same or different and are 2,2'-bipyridyl,
4,4'-disubstituted-2,2'-bipyridyl,
5-5'disubstituted,-2,2'-bipyridyl, 1,10-phenanthrolinyl,
4,7-disubstituted-1,10-phenanthrolinyl, or
5,6-disubstituted-1,10-phenanthrolinyl, wherein each substituent is
a methyl, ethyl, or phenyl group,
R.sub.5 is 4-substituted-2,2'-bipyridyl or
4,4'-disubstituted-2,2'-bipyridyl wherein the substituent is the
group B--(L).sub.k--Q(CH.sub.2).sub.i--and the 4'-substituent is a
methyl, ethyl or phenyl group;
R, R.sub.1 and R.sub.5 are coordinated to Os through their nitrogen
atoms;
Q is O, S, or NR.sub.4 wherein R.sub.4 is hydrogen, methyl or
ethyl;
--L--is a divalent linker;
k is 1 or 0;
i is 1, 2, 3, 4, 5 or 6;
B is hydrogen or a group comprising a ligand capable of binding to
a specific binding partner;
d is+2or+3;
X and Y are anions selected from monovalent anions, e.g., chloride,
bromide, iodide, fluoride, tetrafluoroborate, perchlorate, and
nitrate and divalent anions, e.g., sulfate, carbonate or sulfite
wherein x and y are independently 0, 1, 2, or 3 so that the net
charge of X.sub.xY.sub.yis-2 or-3.
Redox reversible conjugate species of each of those formulas are
prepared from the corresponding compounds wherein K is O and B is
hydrogen by reacting such compounds with either a hetero functional
crosslinker of the formula S--L'--T wherein L' is a divalent linker
and S and T are different electrophilic groups capable of reacting
with a nucleophilic group to form a covalent bond, or with a
homofunctional crosslinker of the formula S--L'--T wherein L' is a
divalent linker and S and T are the same electrophilic groups
capable of reacting with a nucleophilic group to form a covalent
bond. The resulting products are then reacted with ligand analogs
using classical coupling reacting conditions to product the
conjugate species. The oxidizing potentials of the respective
bis(pyridyl) and tris(bipyridyl)osmium complexes defined above is
such that the respective complexes can be used as reversible redox
labels for the respective redox reversible species in performance
of the method. FIG. 8 illustrates a cyclic voltammogram for a
liquid sample containing equimolar (50 .mu.M) amounts of a
bis(pyridyl) imidazolyl chloroosmium complex and a tris(bipyridyl)
osmium complex.
In another embodiment of the invention there is provided a device
for detecting or quantifying one or more analytes in a liquid
sample. The device comprises at least two redox reversible species,
each capable of diffusion in said liquid sample at least in the
presence of a respective predetermined analyte, said redox
reversible species having respective redox potentials differing by
at least 50 millivolts. an electrode structure for contact with the
liquid sample in said chamber, said electrode structure including a
reference electrode and working electrodes dimensioned to allow
diffusional recycling of a diffusible redox reversible species in a
liquid sample in contact with the electrode structure when a
predetermined redox-reversible-species-dependent cathodic potential
is applied to one working electrode and a predetermined
redox-reversible-species-dependent anodic potential is applied to a
second working electrode, said diffusional recycling of said
species being sufficient to sustain measurable current through each
working electrode, and conductors communicating with the respective
electrodes for applying said anodic potential and said cathodic
potential and for carrying the current conducted by the
electrodes.
The device can be constructed using procedures and techniques that
have been previously described in the art for construction of
biosensors employing electrometric detection techniques. Thus, for
example, the device can include a chamber that has a receiving
port, and the chamber is dimensioned so that it fills by capillary
flow when the liquid sample is contacted with the sample receiving
port. The electrode structure can be formed on a plate that defines
a wall of the chamber so that the electrode structure will contact
a liquid sample in the chamber. Thus, for example, the device can
be constructed using the general procedures and designs described
in U.S. Pat. No. 5,141.868, the disclosure of which is expressly
incorporated herein by reference. The features of the present
invention can also be incorporated into other electrochemical
biosensors or test strips, such as those disclosed in U.S. Pat.
Nos. 5,120,420; 5,437,999; 5,192,415; 5,264,103; and 5,575,895, the
disclosures of which U.S. Pat. are expressly incorporated herein by
reference. The device can be constructed to include the
predetermined amounts of the redox reversible species and the
specific binding partners. For example, a mixture of such reagents
can be coated onto a wall of the sample chamber in said device
during device construction, so that the liquid sample is contacted
with the reagent mixture as it is delivered into the chamber for
containing the sample. In one embodiment the device is constructed
for quantifying a first analyte and a second analyte in liquid
sample. The device comprises two redox reversible species, a first
redox reversible species comprising a conjugate of a ligand analog
of the first analyte and a second redox reversible species
comprising a conjugate of a ligand analog of the second analyte,
and a specific binding partner for each analyte so that each of
said analyte analog conjugates are capable of binding competitively
with its respective analyte to a specific binding partner.
In one device embodiment of this invention the device further
comprises a bipotentiostat in electrical communication with the
conductors for applying a
redox-reversible-species-dependent-cathodic potential to one
working electrode and a redox-reversible-species-dependent-anodic
potential to a second working electrode. The biopotentiostat can be
programmed to apply a sequence of potentials to the respective
working electrodes. More particularly, the bipotentiostat can be
programmed to apply first cathodic potential to a first working
electrode and a first anodic potential to a second working
electrode, said first anodic and cathodic potentials corresponding
to those potentials necessary to establish current flow to the
sample due to diffusional recycling of the first redox reversible
species. The bipotentiostat is also programmed to apply a second
cathodic potential to said first working electrode and a second
potential to the second anodic electrode, said second cathodic and
anodic potentials corresponding to those potentials necessary to
establish current flow through the sample due to diffusional
recycling of the second redox reversible species. In an alternate
embodiment the device includes first and second redox reversible
species, and at least first and second electrode structures for
contact with the liquid sample in the chamber, each of said
electrode structures comprising a microarray of working electrodes,
and means for switching the bipotentiostat between the first and
second electrode structures. In preferred device embodiments there
is provided means for measuring current flow through the sample at
each of the first and second potentials and preferably storing
values for said current flows in a register coupled to a
microprocessor programmed to calculate analyte concentrations based
on said values.
In still another embodiment of the present invention there is
provided a kit for measuring the concentration of one or more
analytes in liquid sample. The kit comprises at least two redox
reversible species for contact with the liquid sample, each capable
of diffusion in the liquid sample at least in the presence of a
predetermined analyte, at least one of such species being a
conjugate of a ligand analog of an analyte and a redox reversible
label, said redox reversible species having respective redox
potentials differing by at least 50 millivolts; a specific binding
partner for each analyte; an electrode structure for contact with
the liquid sample, said electrode structure including a reference
electrode and working electrodes dimensioned to allow diffusional
recycling of diffusible redox reversible species in the sample when
a predetermined redox-reversible-species-dependent-cathodic
potential is applied to one working electrode and a predetermined
redox-reversible-species-dependent-anodic potential is applied to
the second working electrode, said diffusional recycling of said
species means sufficient to sustain a measurable current through
the sample; and conductors communicating with the respective
electrodes for applying said anodic potential and said cathodic
potential and for carrying the current conducted by the
electrodes.
In one embodiment, the redox reversible species are mixed as a
novel composition for contact with the liquid sample. In another
embodiment each of the redox reversible species and the specific
binding partner for each analyte is mixed as a novel composition
for contact with the liquid sample. Preferably, the redox
reversible label of at least one of the redox reversible species
comprises an osmium complex.
Preparation of Os Mediator Labels
The Os mediator bis(bipyridyl) imidazolyl chloroosmium has been
shown to be an excellent electron mediator for many oxido-reductase
enzymes (U.S. Pat. No. 5,589,326). It has fast mediation kinetics
(about 500 times faster than ferricyanide with glucose oxidase) and
a relatively low redox potential (+150 mV vs. Ag/AgCl). It has also
very fast electron transfer rate at electrode surface. More
importantly, the organic ligands on Os mediator can be
functionalized so that it can be covalently linked to other
molecules without detrimental effects on redox properties of the Os
center. These unique properties of Os mediator make it an ideal
electrochemical indicator for sensors based on immunoaffinity.
Os mediators with these new ligands were synthesized using the same
procedure used for Os free mediator. Their synthesis consists of
two major process steps as outlined below. Details of these
processing steps are described below.
The first process step involves the synthesis of Os intermediate,
cis-bis(2,2'-bipyridyl) dichloroosmium(II), from commercially
available osmium salt using the following scheme. The intermediate
product is isolated through recrystallization in an ice bath.
K.sub.2Os.sup.IVCl.sub.6+2bpy.sup.DMF[Os.sup.III(bpy).sub.2Cl.sub.2]Cl+2K-
Cl
2[Os.sup.III(bpy).sub.2Cl.sub.2]+Na.sub.2S.sub.2O.sub.4+2H.sub.2O.sup.-
0.degree. C
2Os.sup.II(bpy).sub.2Cl.sub.2.dwnarw.+2Na.sup.++2SO.sub.3.sup.-+4H.sup.++-
2Cl
The second process step involves the reaction between Os
intermediate and histamine or 4-imidazoleacetic acid (or a
substituted bipyridine for preparation of the tris(bipyridyl)
complexes) to produce Os mediators with the appropriate "handle".
The desired product is then precipitated out from solution by
addition of ammonium tetrafluoroborate.
.function..times..times..times..DELTA..function..function..times..times..-
times..function..times..times..times..times. >
.function..times..times..times..dwnarw..times. ##EQU00001##
These Os mediators can also be easily converted to oxidized form,
i.e. Os(III) using nitrosonium tetrafluoroborate. However, this is
unnecessary since the Os revert back to reduced form anyway at
alkaline conditions during conjugation reactions. And it does not
require oxidized form of Os(III) for the detection on the
biosensor.
A. Simple Mixed Mediator Measurement
1. Interdigitated array microelectrodes (IDA) are produced through
photolithographic means by art-recognized methods, (See W 97/34140;
EP 0299,780; D. G. Sanderson and L. B. Anderson, Analytical
Chemistry, 57 (1985), 2388; Koichi Aoki et al., J.
Electroanalytical Chemistry, 256 91988) 269; and D. Niwa et la., J.
Electroanalytical Chemistry, 167 (1989) 291. Other means which are
standard in lithographic processing may also be used to produce the
desired patterns of a conductor on insulator substrate.
2. Reversible mediators are selected from those described herein
and those described references (U.S. Pat. Nos.4,945,045 and
5,589,325, the disclosures of which are incorporated herein by
reference). Preferably two different mediators are selected with
potentials which differ by at least 100 mV, more preferably at
least 200 mV. Examples of suitable mediators include the
Os(bipy)ImCl described herein and in U.S. Pat. No. 5,589,326, the
disclosure of which is incorporated herein by reference, and
ferrocene, described in U.S. Pat. No. 4,945,045 and EP 0142301, the
disclosures of which are incorporated herein by reference. Mixtures
of these mediators are made in aqueous solution, for example
phosphate-buffered saline (PBS). Concentrations between about 1 uM
and 1000 uM may conveniently be measured.
3. The IDA is connected to a bipotentiostat, an instrument capable
of controlling the potential of two separate electrodes. Also
provided is a reference electrode. This nonpolarizable electrode
serves as the reference for the two applied potentials and may also
serve as the counter electrode. Any non-polarizable electrode may
be used, for example Ag/AgCl, such as may be obtained from
ABI/Abtech. An auxiliary electrode can also be used for controlling
current flow through the working electrodes. The mixtures are
placed on the IDA electrode and the reference electrode also
contacted with the mixture, or the IDA along with the reference
electrode may be dipped into the mixture.
4. To measure Mediator 1 (Os(bipy)21mCl) A cathodic potential is
applied to one set of fingers of the IDA which is capable of
reducing mediator 1 (ca-50 mV vs. Ag/AgCl.) An anodic potential is
applied to the other set of finger of the IDA which is capable of
oxidizing mediator 1 but not mediator 2 (or any other mediators)
(ca 250 mV vs Ag/AgCl). After a short time (msec to sec), a steady
state current will be measurable which is dependent only on the
concentration of mediator 1.
5. To measure Mediator 2 (Ferrocene) A cathodic potential is
applied to one set of fingers of the IDA which is capable of
reducing mediator 2 but not mediator 1 (ca 250 mV vs. Ag/AgCl.) An
anodic potential is applied to the other set of fingers of the IDA
which is capable of oxidizing mediator 2 (ca 550 mV vs Ag/AgCl).
After a short time (msec to sec), a steady state current will be
measurable which is dependent only on the concentration of mediator
2.
Specific Binding Assay with Mixed Mediator Measurement
Specific Assay of HbAlc in a Blood sample
1. IDA electrodes are provided as in Paragraph A above.
2. Conjugates of mediators 1 and 2 and haptens or specific binding
members are provided using art-recognized procedures for covalent
coupling using either a homo-functional or hetero-functional
linker. Specifically, a synthetic peptide corresponding to the
N-terminal sequence of the .beta.-chain of HbAlc is conjugated to
the osmium complex. Similarly, a synthetic peptide corresponding to
the N-terminal sequence of HbA0 is conjugated to a second mediator,
for example ferrocene.
3. Antibodies for the analytes (HbAlc and HbAO) which react
specifically with the N-terminal peptides which have been
incorporated into the conjugate are provided by standard methods
for producing polyclonal antibodies. In this case, sheep were
immunized with carrier proteins to which were conjugated the
synthetic peptide sequences for HbAlc and HbA0. Following the
appropriate immunization schedule, the sheep were bled, and the
antibody isolated from the blood via ion exchange chromatography,
followed by immunosorbent purification on a column of the same
N-terminal peptide with a different linker.
4. Appropriate stoichiometry of the reaction was determined for the
two reactions independently by methods standard for immunoassay
development. A solution containing a fixed amount of labeled
conjugate was mixed with a solution with varying amounts of
antibody, and, following an appropriate incubation period, the
amount of free conjugate remaining was measured on the IDA
electrode using the procedure described above. The amount of
antibody just sufficient to achieve maximum inhibition of the
conjugate (ca>80%) was selected.
5. Reagent solution 1 was made containing a mixture of the two
conjugates in the appropriate concentrations. Reagent solution 2
was made containing a mixture of the two antibodies in the amounts
determined above. A blood sample was diluted ca 20-fold in a
solution of 25mM citric acid/0.5% Brij-35. Following a 30 second
incubation to allow for lysis and denaturation of the hemoglobin,
to 66 uL of this diluted sample was added 33 uL of 1 M phosphate
buffer, to adjust the pH back to neutral. 30 uL of antibody
solution 2 was added, and the mixture allowed to incubate 30 sec.
Then 30 uL of conjugate solution 1 was added, and the mixture
measured on the IDA electrode. The concentration of HbAlc in the
sample is related to the current measured from Mediator 1, and the
concentration of HbA0 is related to the current from Mediator 2.
The %HbAlc in the sample is related to the ratio of the measured
amounts of Mediator 1 and Mediator 2.
Application to HbAlc Assay
Hemoglobin Alc is a specific glycohemoglobin in which the
glycosylation takes place at the N-terminal of hemoglobin
.beta.-chain. The antibody binds specifically to HbAlc has an
epitope sequence of Gluc-Val-His-Leu-Thr. To facilitate conjugation
to other molecules, a nonnative amino acid has been added to the
sequence e.g., Cys, Lys, or Lys-MH, to produce Alc peptides
including: 1) Gluc-Val-His-Leu-Thr-Lys-MH; 2)
Gluc-Val-His-Leu-Thr-Lys; 2) Gluc-Val-His-Leu-Thr-Cys.
HbAlc assay requires measuring both Al c concentration and total
hemoglobin concentration and reports the results as a ratio of
these two measurements (% HbAlc). It is advantageous to assay both
Alc and total hemoglobin using same principle because ratioing
would minimize biases due to environmental effects. Thus antibody
has been raised to bind specifically to hemoglobins with
unglycosylated N-terminus, i. e. with an epitope sequence of
Val-His-Leu-Thr. Similarly, nonnative amino acid is added to the
sequence to facilitate conjugation. The peptides used for total
hemoglobin measurement is termed as AO peptide. AO peptides that
have been used in the preparation of Os mediator-peptide conjugates
include Val-His-Leu-Thr-Cys and Val-His-Leu-Thr-Lys.
Conjugation Chemistry and Conjugates
There are many types of conjugation chemistry that can be employed
to link Os mediator to a peptide. The following two conjugation
chemistries employed for the preparation of Os mediator-peptide
conjugates have also been commonly used for preparing protein
conjugates: 1) formation of amide bond by reactive ester with
primary amine; 2) formation of thioether bond by maleimide with
sulfhydryl group. Amide bond is preferred over thioether bond
because amide bond is generally more stable. Based the preferred
conjugation chemistry, the ligand on Os mediator can be
functionalized with either a primary amine group or a carboxylic
acid group. The best location for these functional groups is
believed to be the C-4 or C-5 positions on the imidazole ligand of
Os mediator, however, functionalization through the
non-Os-complexed imidazole ring nitrogen atom can also be carried
out. Two different functionalized Os mediators were synthesized as
described above. ##STR00003##
Os mediator (a) was formed with histamine while Os mediator (b) was
formed with imidazolacetic acid. However, it was found that the
imino nitrogen of the imidazole ring interferes with the activation
of carboxylic acid group to reactive ester (i.e.,
N-hydroxysuccinimide ester) using carbodiimide. Thus, use of
carboxylic acid functionalized Os mediator in the synthesis of Os
mediator-peptide conjugates gave much less favorable results.
The amine group on histamine ligand of Os mediator readily reacts
with N-hydroxysuccinimide (NHS) ester to form amide bond. Two types
of crosslinkers have been employed to link Os mediator to peptides,
(a) heterofunctional crosslinker, having a NHS ester at one end and
the other end has a maleimide or a sulthydryl group; and (b)
homofunctional crosslinker, e.g. both ends have NHS esters.
In the case of heterofunctional crosslinker, the crosslinker is
first reacted with Os mediator with histamine ligand (Os histamine)
at 1:1 molar ratio. One particular point needs to be noted here. Os
mediator in oxidized form, i.e. Os(III), can instantly oxidize
sulfhydryl group to form disulfide bond. It is importnat to keep Os
center in the reduced form by addition of a small amount of
reductant such as sodium dithionite during the conjugation
processes. The reaction progress can be monitored by analytical
reverse-phase HPLC on a C 18 column. Then the Os
mediator-crosslinker adduct is isolated via preparative HPLC and
the collected fraction is subsequently freeze-dried. Finally, the
Os mediator-crosslinker adduct is reacted with the appropriate
peptide to form Os mediator-peptide conjugate. Again, the product
is isolated by collecting appropriate fraction in preparative HPLC
and the collected fraction is then freeze-dried.
Two different heterofunctional crosslinkers have been used for the
synthesis of Os mediator-peptide conjugates. SMCC (succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate) is used for
cystein-containing peptide, while SATA (N-succinimidyl
S-acetylthioacetate) is used for maleimide-containing peptide.
Three Os mediator-peptide conjugates (two with Alc peptide and one
with A0 peptide) have been made using heterofunctional crosslinkers
and their structure are shown below: (a) Os-SMMC-A1c; (b)
Os-SATA-A1c, and (c) Os-SATA-AO. ##STR00004##
However, it has been found that these conjugates were not stable
when they were stored as solutions. Analytical HPLC results
indicated that these conjugates degrade. Mass spectroscopy
confirmed that the instability is due to splitting of thioether
bond present in these conjugates.
In order to avoid thioether bond in the conjugate, homofunctional
crosslinker containing two NHS esters was used instead to prepare
the conjugates. The crosslinker used was DSG (disuccinimidyl
glutarate). In order to prevent the formation of crosslinked Os
mediator, i.e. Os-crosslinker-Os, a large excess of homofunctional
crosslinker was used in the reaction with Os histamine at 4:1 molar
ratio. Under this condition, only the desired product, i.e.
Os-crosslinker, was formed. The Os-DSG-Alc conjugate was similarly
prepared using the procedure described earlier. ##STR00005##
The preparation of analogous Os-DSG-A.sub.o conjugate requires and
extra step since the unglycated N-terminal amine of HbAA.sub.o
peptide is also reactive toward NHS ester. In this case, the
N-terminal amine of HbA.sub.o peptide is first protected by either
a base-labile Fmoc.sup.1 or an acid-labile Boc.sup.2 group. After
reacting with Os-DSG adduct to form Os-DSG-A.sub.o conjugate, the
protecting group is then cleaved using appropriate deprotection
method (adding base for Fmoc or acid for Boc). The peptides
prepared by solid-phase peptide synthesis already have N-terminal
Fmoc protecting groups. The protecting groups are usually removed
prior to cleavage of peptide from the resin beads, but they can
also be left on if so desired. The HbA.sub.o peptide from Zymed has
an intact Fmoc protecting group at N-terminus. Using this strategy
the Os-DSG-A.sub.o conjugate was successfully synthesized.
.sup.1Fmoc=9 flourenylmethyloxycarbonyl
.sup.2Boc =t-butyloxycarbonyl
Many analytes cannot be assayed using enzyme-based sensors. They
require the development of affinity biosensors or immunosensors
which are based on the selective binding of antigens to antibodies.
The key to the detection of this binding event on electrochemical
sensors is the inclusion of antigens labeled with redox labels.
Bis(2,2'-bipyridyl) imidazolyl chloroosmium, a.k.a. Os mediator,
possesses many properties that make it an excellent redox label for
this purpose. In addition, a "handle" for linking it covalently to
antigens can be added on without affecting its redox
properties.
Several assay schemes can be used in affinity biosensors including,
i) competitive binding assay (labeled antigen is competing for a
limited number of binding sites); ii) sequential binding assays
(labeled antigen is bound to excess binding sites); iii)
heterogeneous assay (uses a separation step to separate bound and
free labeled antigens); and iv) homogeneous assay (no separation
step). The steps involved in a homogeneous sequential binding assay
include binding the analyte to an antibody. The labeled antigen
(analyte analog) binds to the remaining binding sites on the
antibody. Finally the leftover free labeled antigen is detected at
electrode surface. The resulting current will be a function of the
amount of analyte present.
The detection of free labeled antigens can be achieved using either
direct detection or amplified detection methods. Direct detection
requires the use of advanced electrochemical techniques such as ac
voltammetry, differential pulse voltammetry, square wave
voltammetry or ac impedance in order to reach a sensitivity of 5
.mu.M or less. Amplified detection methods use dc amperometry with
amplification through reaction with enzyme or chemical reductants
or by using interdigital array (IDA) microelectrodes. The preferred
detection method is amplified amperometry through cycling of free
Os mediator label by using IDA microelectrodes in accordance with
this invention.
General Analytical HPLC Method For Osmium Conjugates
All HPLC analysis were performed using a Beckman System Gold HPLC
system consists of a 126 pump module and a 168 diode array detector
module.
Stationary phase is a Vydac analytical reverse-phase C18 analytical
column. Other parameters are listed below. Mobile Phase:
A=0.1%TFA.sup.3 in H.sub.2O B=0.1% TFA in CH.sub.3CN Flow rate: 1
mL/min Gradient: 0-5 min: 10% B 5-45 min: 10% B->50% B at 1%/min
45-50 min: 100% B Detector: Channel A at 384 nm Channel B at 220
nm.
.sup.3TFA=trifluoroacetic acid.
Synthesis of Bis(2,2'-bipyridyl) dichloroosmium
1. Charge a 1 L one-neck RB flask with 10.335 grams
K.sub.2OsCl.sub.6 (0.0419 mole) and 13.295 grams 2,2'-dipyridyl
(0.08512 mole). Add 400 mL DMF to dissolve all reactants. 2. Heat
the solution to reflux and then maintain reflux for 1 hour. Then
turn off the heat and let solution cool to 30-40.degree. C. at
ambient. 3. Filter the reaction mixture using a medium grade
glass-frit filter. Rinse the flask with additional 20 mL DMF and
wash the filter. 4. Transfer the filtrate into a 3-L beaker. Charge
another 2-L beaker with 22.799 grams of NaS.sub.2O.sub.4 and
dissolve in 2 L deionized water. Add this solution to the beaker
containing Os/DMF filtrate dropwise using a dropping funnel. Keep
the solution stirring at all time. 5. Then cool the mixture in an
ice bath for at least 3 hours. Add ice as necessary, 6. Filter the
mixture "cold" using a ceramic filter with filter paper. Wash the
content on the filter with 50 mL, water twice and 50 mL ether
twice. 7. Dry the product under high vacuum at 50.degree. C.
overnight (at least 15 hours). Weigh the product and transfer into
a brown bottle. Store in a desiccator at room temperature. Typical
yield=16 gram or 70%. Product is analyzed by UV-Visible
spectroscopy and elemental analysis.
TABLE-US-00002 UV-Vis: Peak .lamda. (nm) .epsilon.
(M.sup.-1cm.sup.-1) 382 10,745 465 9,914 558 11,560 836 3,873 EA: C
% H % N % Cl % Os % H.sub.2O % Theoretical 41.89 2.81 9.77 12.36
33.17 0 Actual 40.74 2.92 9.87 11.91 0.41
1. Charge a 2L one-neck RB flask with 11.3959 gram
Os(bpy).sub.2Cl.sub.2 (0.0198 mole) and 4.9836 gram histamine
(0.0448 mole). Add 500 mL ethanol to dissolve the reactants. Then
add 250 mL deionized water. 2. Heat the solution to reflux and
maintain reflux for 6 hours. Let solution cool to RT at ambient. 3.
Remove all ethanol using rotary evaporation. The transfer the
solution into a 500 mL beaker. Dissolve 2.136 gram NH .sub.4BF 4 in
20 mL water. Add dropwise to Os solution. Precipitate forms. Cool
in an ice bath for 30 min. Filter the mixture using a ceramic
filter with filter paper. Wash the content on filter with -20 mL
water twice. 4. Dry under high-vacuum at 50.degree. C. overnight
(at least 15 hour). 5. Weigh the product and transfer to a brown
bottle. Store in a desiccator at room temperature. Typical
yield=7.6 gram or 52%. Product is analyzed by UV-visible
spectroscopy and HPLC.
TABLE-US-00003 UV-Vis: Peak .lamda. (nm) .epsilon.
(M.sup.-1cm.sup.-1) 355 7,538 424 7,334 514 7,334 722 2,775
HPLC: Elutiontime=18.0 min Purity by HPLC range from 65-85%
Preparation of [Os(bpy).sub.2(histamine)Cl]heterofunctional
crosslinker adduct
1. Weigh 0.1167 g [Os(bpy).sub.2(histamine)Cl] BF.sub.4 (0.162
mmol) and transfer to a 5 mL Reacti-Vial. Add 1.0 mL DMF to
dissolve the reactant. Add 25 .mu.L triethylamine. 2. Add 0.0508 g
SMCC(0.150 mmol)or 0.0390 g SATA (0.168 mmol). Stir the reactants
at RT for 2 hours. Inject a sample into HPLC to monitor reaction
progress. 3. If reaction is complete, dilute the solution with 0.1%
TFA buffer to a final volume of 4.5 mL. Inject into preparative
BPLC and collect the product peak. 4. Freeze dry the collected
fraction overnight. 5. Weigh the product and transfer to a brown
bottle. Store in a desiccated bag at-20.degree. C. Typical yield=40
mg or 25%. Product is analyzed by HPLC and ES/MS.
TABLE-US-00004 HPLC ES/MS Os-SMCC: Elution time = 32.1 min
m.sup.+/e = 434.2 Os-SATA: Elution time = 27.5 min m.sup.+/e =
382.8
Preparation of [OC(bpy).sub.2(histamine)Cl ]homofunctional
crosslinker adduct
1. Weigh 0.2042 g DSG (0.626 mmol) ) and transfer to a 5 mL.
Reacti-Vial. Add 0.75 mL DMF to dissolve the reactant. 2. Weigh
0.1023 g [Os(bpy).sub.2(histamine)Cl]BF.sub.4 (0.142 mmol) and
transfer to a separate 5 mL Reacti-Vial. Add 1.0 mL DMF to dissolve
the reactant. Add 25 .mu.L triethylamine. Then add Os/DNF solution
dropwise to DSG/DW solution with constant stirring. After reacting
for 2 hours at RT, inject a sample into HPLC to monitor reaction
progress. 3. If reaction is complete, dilute the solution with 0.1%
TFA buffer to a final volume of 4.5 mL. Inject the preparative HPLC
and collect the product peak. 4. Freeze dry the collected fraction
overnight. 5. Weigh the product and transfer to a brown bottle.
Store in a desiccated bag at-20.degree. C. Typical yield=45 mg or
35%. Product is analyzed by HPLC and ES/MS.
TABLE-US-00005 HPLC ES/MS Os-DSG: Elution time = 27.1 min m.sup.+/e
= 429.2 and 859.6
Preparation of Os-SATA-Alc Conjugate
1. Weigh 40.5 mg OS-SATA (0.0529 mmol) and transfer to a 5 mL
Reacti-Vial with stir bar. Add 1.0 mL PBS (pH 7.5) to dissolve. Add
20 mg Na.sub.2SO.sub.4 in order to keep Os in reduced form. 2. Add
1.0 mL deacetylation buffer (PBS pH7.5+0.5 M hydroxylamine and 25
mM EDTA) to deprotect the sulfhydryl group. Inject a sample into
analytical HPLC to determine whether deprotection is complete by
appearance of a new peak at 25.8 min. 3. Add 45 ig HbAlc-MH peptide
(0.474 mmol) and let react at RT for 1 hour. Inject a sample into
analytical HPLC to monitor reaction progress. 4. If reaction is
complete, dilute the mixture with 0.1%TFA buffer to a final volume
of 4.5 mL. Inject into preparative HPLC to collect product peak. 5.
Freeze dry the collected fraction overnight (at least 15 hour). 6.
Weigh the product and transfer to a brown bottle. Store in a
desiccated bag at-20.degree. C. Typical yield=12 mg Product is
analyzed by HPLC and ES/MS.
TABLE-US-00006 HPLC ES/MS Os-SATA-Alc: elution time = 27.6 min
m.sup.+/e = 559.1 and 838.5
Preparation of OS-SMCC-Alc Conjugate
1. Weigh 39.0 mg Os-SMCC (0.0452 mmol) and transfer to a 5 mL
Reacti-Vial with stir bar. Add 1.0 mL PBS (pH 6.0) to dissolve. 2.
Add 30.0 mg Hblc-Cys peptide (0.0450 mmol). Let reaction proceed at
RT for 2 hours. Inject a sample into analytical HPLC to monitor
reaction progress. 3. If reaction is complete, dilute the mixture
with 0.1% TFA buffer to a final volume of 4.5 mL. Inject into
preparative HPLC to collect product peak. 4. Freeze dry the
collected fraction overnight (at least 15 hour). 5. Weigh the
product and transfer to a brown bottle. Store in a desiccated bag
at-20.degree. C. Typical yield=12 mg Product is analyzed by HPLC
and ES/MS.
TABLE-US-00007 HPLC ES/MS Os-SMCC-Alc: elution time = 27.6 min
m.sup.+/e = 480.5 and 534.4
Preparation of Os-SMCC-Ao Conjugate
1. Weigh 37.0 mg OS-SMCC (0.0426 mmol) and transfer to a 5 mL
Reacti-Vial with stir bar. Add 1.0 mL PBS (pH=6.0) to dissolve. 2.
Add 24.3 mg HbAo-Cys peptide (0.0425 mmol). Let reaction proceed at
RT for 2 hours. Inject a sample into analytical HPLC to monitor
reaction progress. 3. If reaction is complete, dilute the mixture
with 0.1 %TFA buffer to a final volume of 4.5 mL. Inject into
preparative HPLC and collect the product peak. 4. Freeze dry the
collected fraction overnight (at least 15 hour). 5. Weigh the
product and transfer to a brown bottle. Store in a desiccated bag
at-20.degree. C. Typical yield=15 mg Product is analyzed by HPLC
and ES/MS.
TABLE-US-00008 HPLC ES/MS Os-SMCC-A.sub.o: elution time = 27.9 min
m.sup.+/e = 360.7 and 720.5
Preparation of Os-DSG-Alc Conjugate
1. Weigh 32.0 mg Os-DSG (0.037 mmol) and transfer to a 5 mL
Reacti-Vial with stir bar. Add 0.75 mL DMF to dissolve. Add 25
.mu.L triethylamine. 2. Add 26.5 mg HbAlc-Lys peptide (0.0349
mmol). Let reaction proceed at RT for 2 hours. Inject a sample into
analytical BPLC to monitor reaction progress. 3. If reaction is
complete, dilute the mixture with 0.1%TFA buffer to a final volume
of 4.5 mL. Inject into preparative HPLC and collect the product
peak. 4. Freeze dry the collected fraction overnight (at least 15
hour). 5. Weigh the product and transfer to a brown bottle. Store
in a desiccated bag at-20.degree. C. Typical yield=16 mg Product is
analyzed by HPLC and ES/MS.
TABLE-US-00009 HPLC ES/MS Os-DSG-Alc: elution time = 23.5 min
m.sup.+/e = 501.8 and 752.8
Preparation of OS-DSG-Ao Conjugate
1. Weigh 52.0 mg Os-DSG (0.0605 mmol) and transfer to a 5 mL
Reacti-Vial with stir bar. Add 1.0 mL DMF to dissolve. Add 25 .mu.L
triethylamine. 2. Add 49.1 mg Fmoc-HbA.sub.0 peptide (0.0606 mmol).
Let reaction proceed at RT for 2 hours. Inject a sample into
analytical HPLC to monitor reaction progress by the appearance of
peak at 40.3 min for Os-DSG-A.sub.o(Fmoc). 3. If reaction is
complete, inject additional 100 .mu.L triethylamine. After I hour,
inject sample into analytical HPLC to determine whether all Fmoc
protection group is removed by disappearance of the peak at 40.3
min. 4. If removal of Fmoc is complete, dilute the mixture with 0.
1%TFA buffer to a final volume of 4.5 mL. Inject into preparative
HPLC to collect product peak. 5. Freeze dry the collected fraction
overnight (at least 15 hour). 6. Weigh the product and transfer to
a brown bottle. Store in a desiccated bat at-20.degree. C. Typical
yield=16 mg Product is analyzed by HPLC and ES/MS.
TABLE-US-00010 HPLC ES/MS Os-DSG-A.sub.o: elution time = 23.2 min
m.sup.+/e = 447.4 and 670.3
Synthesis of bis (4,4'-dimethyl-2,2'-biphenyl) 4-methyl
-4'-carboxylpropyl-2,2'-bipyridyl osmium
[Os(dm-bpy)2(mcp-bpy)]Cl2
Potassium hexachloroosmium was reacted with
4,4'-dimethyl-2,2'-dipyridyl at 1:2 molar ratio by refluxing in
DMF. The potassium chloride preparative was filtered and the
dimethyl-bipyridyl dichloroosmium complex was reduced from+3
oxidation state to+2 oxidation state using excess sodium
dithionite. The product was recrystallized in DMF/water mixture at
0.degree. C. and recovered by filtration.
4,4'-Dimethyl-2,2'-bipyridyl dichloroosmium was reacted with
4-methyl-4'-carboxylpropyl-2,2'-dipyridyl by refluxing in ethylene
glycol. The solvent was removed by rotary evaporation. The product
was dissolved in DMF and precipitate in ethyl ether. The product
was dried in a vacuum oven overnight.
Analyticals: Product and intermediate product were analyzed by HPLC
and mass spectroscopy for purity and identity of the compound.
Os(dm-bpy)2Cl2: Theoretical MW=629.6, MS showed 8 isotope peaks
with most abundant peak at 630. HLPC elution time at 29.94 min with
a purity of 90%+
Os(dm-bpy)2(mcp-bpy): MS confirmed the MW at 814.5 and HPLC showed
a purity greater than 85%.
Synthesis of biotin-Os complex conjugate
Biotin-Os(dm-bpy)2(mcp-bpy)]Cl2
The carbonyl group was activated by reacting the above Os complex
with dicyclohexylcarbodiimide in the presence of
N-hydroxysuccinimide. The active ester Os complex was isolated
using preparative HPLC method and then reacted with
amine-containing biotin to form the final conjugate.
Experiment to Independently Measure the Concentration of Two
Electroactive Conjugate Species
Os(bipy)HisCl-DSG-HbAlc was prepared as described above.
Ferrocene-AMCHA-DADOO-biotin was prepared from ferrocene
monocarboxylic acid, the crosslinker aminomethylcyclohexylic acid,
the chain extender 1,8-diamino-3,6-DiOxoOctane and biotin as
described elsewhere.
Mixtures of the two conjugates were prepared to evaluate the
ability of the method of the invention to independently measure the
concentration of the conjugates, and make corrections for
variations in reagent amounts, electrode response, and
environmental conditions.
Part 1: Simple Mixed Conjugate Response
The following matrix of solutions was prepared in 10 mM phosphate
buffer with 150 mM NaCl and 0.5 % Brij-35, a non-ionic
surfactant.
TABLE-US-00011 Os-DSG- Ferrocene- Os-DSG- Ferrocene- HbAlc Biotin
HbAlc Biotin uM/l uM/l uM/l uM/l 0 0 0 12.5 6.25 0 6.25 12.5 12.5 0
12.5 12.5 25 0 25 12.5 0 25 0 50 6.25 25 6.25 50 12.5 25 12.5 50 25
25 25 50
An Interdigitated array (IDA) microelectrode was fabricated
according to the procedures described. In addition to the IDA, the
chip had a silver/silver chloride electrode on the surface to
function as the reference electrode and counter electrode. This
electrode was produced with the same lithographic process, and then
electroplated with silver and silver chloride according to standard
techniques. The IDA was connected to a bipotentiostat capable of
controlling the potential relative to the reference and measuring
the current at each of the electrodes of the IDA. Aliquots of the
solutions were placed onto the surface of the chip, such that the
IDA and the reference electrodes were covered.
Measurements were made by first applying-100 mV (vs. Ag/AgCl) to
one electrode of the IDA, and 200 mV to the other electrode for a
period of 30 seconds. At this time, current was measured at each
electrode. Then 200 mV was applied to one electrode, and 550 mV to
the other. After 30 seconds, current was again measured. See FIG. 9
for a summary of the results, which clearly demonstrate that the
concentrations of the two mediators can be independently measured
with this method.
Part 2: Concentration Co-variance of Dual Mediators on IDA
Electrodes
In this experiment, it was demonstrated that by making a mixture of
known concentrations of two different mediators, and measuring
different dilutions of that mixture by the method of the invention,
the ratio of the concentrations of the mediators remains constant.
(Internal standard application).
The same two mediator conjugates wee used as in Part 1. (
Os-DSG-Alc and Fc-Bi).
From a solution containing 40 uM of each conjugate, solutions
containing 27 uM, 32 uM, and 36 uM of each conjugate were prepared
in the same buffer ( PBS/Brij).
The solutions were measured as in the previous example. Each
solution was measured on 5 different IDA electrodes. The results
are presented as the means, standard deviations, and coefficient of
variation for each solution separately, and for all solutions
pooled over all electrodes.
TABLE-US-00012 Individual Os-DSG- concentrations Alc Fc-Bi Os/FC 28
uM Mean 156 85 1.84 S.D. 17.5 13.2 0.08 % C.V. 11.2 15.5 4.4 32 uM
Mean 165 88 1.88 S.D. 11 7.2 0.04 % C.V. 6.7 8.2 2.0 36 uM Mean 189
102 1.85 S.D. 12.5 6.9 0.04 % C.V. 6.6 6.7 2.3 40 uM Mean 208 110
1.90 S.D. 9.5 5.5 0.06 % C.V. 4.6 5.0 3.4 Pooled Concentrations
Mean 182 97 1.87 S.D. 24.4 13.2 0.06 % C.V. 13.4 13.5 3.4
This example clearly demonstrates that the internal standard effect
of measuring two conjugates or mediators and calculating the ratio
gives significantly improved precision of measurement, not only
within each solution (compensation for variation between
electrodes) but over all solutions (compensation for variation in
sample dilution or amount).
Part 3: Temperature Compensation
It was desired to show the effectiveness of the method in
compensating for environmental influences such as Temperature
variation on the accuracy or the measurement.
The same two conjugates were prepared in solution at 40 uM as
before. They were measured as before on IDA electrodes, either at
room temperature or warmed to 35-40.degree. C. on a heated metal
plate prior to the measurement. The solutions were also warmed to
37.degree. C. prior to application to the electrodes.
TABLE-US-00013 Room Temperature Warmed Ratio of response
(23.degree. C.) (35-40.degree.) C. Warm/RT Os-DSG-Alc 261 387 1.45
Fc-Bi 179 268 1.5 Ratio Os/Fc 1.46 1.44 0.99
As demonstrated by the results, the measured values increase by
almost 50 % in the case of the warmed samples, which would lead to
a large measurement error. However the use of the internal standard
and ratio calculation effectively eliminates the temperature
dependent of the result.
Immunoassay Detection of HbAic with Osmium Mediator Conjugates
The goal of all diabetic therapy is to maintain a near normal level
of glucose in the blood. Home blood glucose kits are available to
monitor the current glucose values and are valuable for diabetics
to adjust day to day insulin doses and eating habits.
Unfortunately, since the tests only measures a point in time
result, it does not tell them the overall effectiveness of their
actions in maintaining glycemic control. Measurement of
glycosylated hemoglobin is now becoming widely accepted as an index
of mean blood glucose concentrations over the preceding 6-8 weeks
and thus provides a measure of the effectiveness of a diabetic's
total therapy during periods of stable control. Since monitoring a
diabetic's glycated hemoglobin can lead to improved glycemic
control, the ADA recommends routine measurements of four times a
year up to once a month for less stable type I diabetics.
Several technologies are available for the measurement of glycated
hemoglobin. They include immunoassays for HbAlc (TinaQuant, BMC;
DCA2000, Ames; and Unimate, Roche), icon exchange (Variant, BioRad;
Eagle Diagnostics), and affinity chromatography (ColumrMate,
Helena; GlyHb, Pierce).
One objective of this project is to develop a simple to use
disposable strip for electrochemical detection of HbAlc for use in
both physician offices and the home.
The most significant parameter for assessing patient condition is
ratio of HbAlc to HbA.sub.o, and thus the measurement of both
glycated (HbAlc) and nonglycated (HbA0) values is required to
calculate the ratio. This requires two separate measurements. It is
preferable to use the same technology to measure both the glycated
and nonglycated fractions, thus removing some sample and
environmental interferences. Measurement of HbAlc via
electrochemical immunoassay is described below. Electrochemical
HbA0 immunoassay measurements are carried out using the same
methods as that for HbAlc. The concentrations of HbA.sub.0 are
significantly higher. One alternative to A.sub.0 measurements using
immunoaffinity would be to measure total hemoglobin directly using
biamperometry or differential pulse voltammetry. This can be easily
accomplished since hemoglobin is readily oxidized by
[Os(bpy).sub.2(im)Cl].sup.2+Cl .sub.2.
The N-terminal valine of the .beta.-chain of hemoglobin A is the
site of glycosylation in HbAlc, and serves as a recognition site
for the antibody. In whole blood the N-terminal valine is not
accessible for the antibody to bind. Access is gained by lysing the
red cells to release the hemoglobin followed by a conformation
change (denaturing or unraveling) to adequately expose the HbAlc
epitope. Dilution of the sample may occur as part of the
lysing/denaturing process or may be required post denaturing to
prepare the sample for the antibody (adjust pH, other) or bring the
sample into a range suitable for electrochemical immunoassay. In
one embodiment, a fixed amount of antibody is incubated with the
prepared sample and it binds to the HbAlc epitopes of the sample.
The free antibody and the antibody bound sample is then combined
with the osmium peptide conjugate (Alc or A.sub.0) to allow the
remaining unbound antibody to bind to the mediator label. When the
mediators is bound to the antibody (a macromolecule), it can not
freely diffuse to interact with the electrode and thus currents
generated are significantly reduced. The remaining unbound mediator
label is therefore proportional to the concentration of the HbAlc
in the sample. The unbound mediator can be measured
electrochemically preferably using an interdigitated array
electrode with bipotentiostatic control.
Blood lysis is necessary to release the hemoglobin followed by
denaturing to expose the HbAlc epitope. Lysis can easily be
accomplished via surfactants, osmotic effects of dilution with
water, and directly by many denaturants. Blood lysed through a
freeze/thaw cycle was shown not to significantly interfere with the
biamperometric measurement ("open rate" with and without lysed
blood was almost identical). Conversely, denaturing the lysed blood
with a variety of known denaturants to expose the HbAlc epitope has
shown significant suppression of the electrochemical response,
inhibiting measurement of an HbAlc dose response. Only LiSCN and
citric acid from the list of evaluated denaturants shown in Table 1
was able to expose the Alc epitope and minimize protein fouling
enough to measure an HbAlc dose response.
Denaturing the sample for antibody recognition without severely
fouling the electrode surface is important for successful
development of an HbAlc immunoassay. Although LiSCN has been used
almost exclusively to show feasibility, it has many limitations
that would hinder its use in the disposable. Citric acid, a solid
at RT may offer benefits as a denaturant if it could be dried onto
a strip followed by a diluent to adjust the pH to neutral. Acid or
base blood denaturing followed by a final pH adjustment with a
buffered diluent is an area worth further evaluation. One problem
that was initially encountered was precipitation in adjusting the
pH back to neutral, which can be overcome by using a different
buffer or with the addition of surfactants.
TABLE-US-00014 TABLE 1 Blood Denaturants Method Comments KSCN
Initial work did not show a dose response with KSCN denatured
blood. KSCN has a larger negative effect on the electrochemical
response than LiSCN. Literature shows that LiSCN is more effective
than KSCN (concentrated efforts on LiSCN). DCA2000 Denaturing of
blood not evident with the higher blood Buffer concentrations
required for this assay. Higher concen- trations of LiSCN are shown
below. LiSCN Method used by Ames DCA2000 HbAlc immunoassay. Citric,
Blood HbAlc dose response (high/low) was seen with Sulfuric, Hy-
citric acid and was comparable to the response drochloric, with
LiSCN & Evidence of blood denaturing was seen by Perchloric
all: "solution turned brown." Citric acid is preferred. Acid
Adjustment of pH to neutral after denaturing also saw problems of
precipitation. Enzyme mediated responses with Gluc-Dot at pH 5.7
reduces response 50% compared to pH 7-8. Citric acid blood
denaturing method is shown in FIG. 5. Pepsin/ Roche HbAlc
immunoassay uses pepsin/citric acid to Citric Acid hemolyze and
proteolytically degrade hemoglobin in glycoproteins accessible by
the antibody. Denaturation was apparent by the color change to a
brown- ish red solution. Hemoglobin Alc dose response (high/low)
was obtained comparable to LiSCN and citric acid denaturants. The
procedure was identical to that of citric acid used above with the
exception of pepsin added to the acid. Results were identical to
that of citric acid. TTAB (Tetra Method of denaturing used in the
TinaQuant HbAlc decyl- turbidimetric immunoassay. trimethyl
Evidence of denaturing: "solution turned green" ammonium TTAB
concentrations 0.0125-0.2% severely suppressed bromide) enzyme
mediated (Glucdor/PQQ/Glucose) biamperometric measurements. Open
rates were 16-50 nA compared to 140 nA without TTAB. NaOH Evidence
of denaturing: "solution turned brown." NaOH does not adversely
effect the enzyme mediated electochemistry. Even at high pH the
open rates do not change, although pH adjustment will probably be
required to bring it within an optimal range for the antibody. NaOH
denatured blood suppresses the open rates probably due to protein
fouling. Lowering the pH to neutral tends to cause some
precipitation.
TABLE-US-00015 TABLE 2 Effective Blood Denaturing Procedure for 2%
Blood LiSCN (One Step) LiSCN (Two Step) Citric Acid (2 Step) 40
.mu.L 6 M LiSCN 960 .mu.L 1.5 M LiSCN 200 .mu.L 0.2 M Citric Acid
20 .mu.L 5% Tween in 20 .mu.L 5% Tween in PBS 20 .mu.L 5% Tween in
PBS PBS 20 .mu.L Blood 20 .mu.L Blood 20 .mu.L Blood Mix (vortex)
and Mix (vortex) and allow Mix (vortex) and allow to denature to
denature for 10 allow to denature for 10 minutes. minutes. for 10
minutes. Dilute with 920 .mu.L Dilute with 760 .mu.L DI H.sub.2O.
8X PBS (0.1% Tween) Denaturing time Denaturing time was not No
optimization was not optimized. optimized. studies were Limited
data supports Data indicates shorter performed. longer times for
better times may be adequate. precision using this method. PBS = 10
mM Phosphate Buffer, 2.7 mM KCl, 137 mM NaCl pH = 7.4 Increased
level of surfactant (5% Tween) reduces or eliminates
precipitate.
Electrode fouling caused by denatured blood proteins adsorbing to
the electrode surface can impede electron transfer and thus
decrease electrode sensitivity.
Electrode fouling or passivation occurs more or less immediately
following sample contact with the surface thus minimizing the
severity of denaturing in the sample should be the first approach.
Surface conditions that are hydrophobic will favor adhesion of the
proteins and thus fouling may be minimized with electrode surfaces
of higher surface energies. This explains why gold electrodes shows
less fouling with denatured blood than palladium. Reduction of
protein fouling may be achieved by changing or protecting the
electrode surface. Modifications that make the surface more
hydrophilic should reduce the amount of fouling and can be
accomplished by argon or oxygen plasma treatment or corona
treatment. Selective coatings that could block the proteins from
reaching the electrode surface can usually partially circumvent the
problem have been used in the field to reduce fouling.
Unfortunately, dramatic decreases in responses greater than seen
with the denatured blood proteins are normally noted with their
use. Hydrophilic coatings such as PEO were also evaluated and
showed some improvement, but have similar problems of decreased
magnitude and precision caused by forcing reagents to diffuse
through the polymers. Reagents dispensed and dried over the
electrodes may help reduce the magnitude of protein fouling with
less negative effects.
Mediator concentration dose response, inhibition with antibody and
reversal with a BSA-Alc polyhaptan were evaluated and summarized in
Table 3. The Os-DSG-Alc is stable in a lyophilized form and when
frozen in solution at-20.degree. C. (40 and 80 .mu.M).
TABLE-US-00016 TABLE 3 Osotium Mediator Labels Mediator
Concentration Inhibition with Label Response Antibody Reversal
Comments Os-SMCC- Linear PAB IS (.ltoreq.92%) Yes with BSA-Alc % =
Inhibition values ranged Alc PAB DE (.ltoreq.97%) polyhaptan from
16% to 97% depending on MAB (.ltoreq.50%) age of Os-SMCC stock
solution. Degrades in solution. Os-SATA- Linear PAB IS
(.ltoreq.44%) Yes with BSA-Alc Stability similar to SMCC. Alc
polyhaptan Os-DSG-Alc Linear PAB IS (.ltoreq.91%) Yes with BSA-Alc
More stable than conjugate PAB DE (<87%) polyhaptan made with
SATA and SMCC MAB (<78%) crosslinker but still degrades in
solution. Os-SATA-A.sub.o Linear Yes with Sheep No with A.sub.oHB-
A.sub.o conjugate was found to be B<HbA.sub.o> peptide #1
unstable in solution. Conjugate (.ltoreq.84%). No was not
lyophilized. with Zymed rabbit antibodies
Polyclonal DE (ion-exchange) purified sheep antibody is used in the
TinaQuant HbAlc assay. IS (immunosorbent) antibody is prepared
using standard IS purification methodology. Samples of a monoclonal
antibody were also obtained for evaluation. Inhibition curves were
performed in solution with all mixing occurring in microcentrifuge
tubes. Assays were measured by applying 20 .mu.L onto 6 mm.sup.2
palladium electrodes with the conditions shown in Table 3.
Inhibition curves with the three hemoglobin Alc antibodies (PAB IS,
PAB DE, and MAB) were generated by fixing OS-DSG-Alc at 5.mu.M and
varying the antibody concentration. Both PAB IS and MAB showed the
expected stoichiometric relationship for inhibition with the osmium
peptide conjugate indicating efficient and fast binding of the
antibody to the Alc peptide. The polyclonal IS and monoclonal
bother showed steep inhibition curves with maximum change being
reached close to 5 .mu.M. Additional antibody above 5 .mu.M showed
little effect on increasing the inhibition. The less purified PAB
DE antibody had a much smaller slope and as expected required more
than 3 times the amount to get close to maximum inhibition. FIG. 6
shows the inhibition curves for each of the HbAlc antibodies
tested. From the inhibition curves we were able to select
reasonable concentrations of antibody for maximum reversal with Alc
samples.
Inhibition curves were also performed for the Os-SMCC-Alc (Max=97%)
and OS-SATA-Als (Max=44%) mediator labels. Stability of the
mediator labels were also evaluated by monitoring % inhibition
values over time. All of the mediator labels showed some
degradation when stored in dilute solutions (40 .mu.M) at RT.
Samplers frozen at-20.degree. C. appear to be stable.
For demonstrating inhibition reversal, antibody concentrations of 4
.mu.M for both PAB IS and MAB and 15 .mu.M for PAB DE were chosen
from the inhibition curves shown above. Reversal curves were then
generated using a series of dilutions ofBAS-Alc-polyhaptan with
a-1:1 Alc:BAS. The BAS-Alc acts as our sample and binds to the
antibody. FIG. 10 shows the reversal curves for the three
antibodies.
While these feasibility studies for a HbAlc immunoassay used an
enzyme mediated amplification method (Glucdor/PQQ/glucose was used
to regenerate reduced mediator after oxidation at the electrode
surface providing a higher diffusion controlled current is given by
the cottrell equation), they are considered to be indicative of
results attainable with the use of IDA electrodes with
bipotentiostatic control is most preferred for measuring mediator
labeled conjugates in accordance with this invention.
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