U.S. patent application number 13/686760 was filed with the patent office on 2013-12-26 for glucose sensor.
This patent application is currently assigned to LIGHTSHIP MEDICAL LIMITED. The applicant listed for this patent is LIGHTSHIP MEDICAL LIMITED. Invention is credited to Nicholas Paul Barwell, Barry Colin Crane, Peter Edgley, William Paterson.
Application Number | 20130344619 13/686760 |
Document ID | / |
Family ID | 48741413 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344619 |
Kind Code |
A1 |
Crane; Barry Colin ; et
al. |
December 26, 2013 |
GLUCOSE SENSOR
Abstract
A method of quantifying the amount of glucose in a sample is
provided herein that may further comprise an interferent such as
mannitol. At least two measurements are obtained using measurement
methods that differ in their sensitivity to the amount of
interferent in the sample, thus enabling the results to be compared
to determine whether any interferent is present in the sample. A
glucose sensor for carrying out a method described herein is also
provided
Inventors: |
Crane; Barry Colin;
(Oxfordshire, GB) ; Paterson; William;
(Oxfordshire, GB) ; Barwell; Nicholas Paul;
(Coventry, GB) ; Edgley; Peter; (Oxfordshire,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIGHTSHIP MEDICAL LIMITED |
London |
|
GB |
|
|
Assignee: |
LIGHTSHIP MEDICAL LIMITED
London
GB
|
Family ID: |
48741413 |
Appl. No.: |
13/686760 |
Filed: |
November 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61662560 |
Jun 21, 2012 |
|
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Current U.S.
Class: |
436/501 ; 422/69;
600/316 |
Current CPC
Class: |
G01N 2021/6434 20130101;
G01N 2021/773 20130101; G01N 21/7703 20130101; G01N 2021/772
20130101; A61B 5/14532 20130101; G01N 2021/6484 20130101; G01N
2021/6441 20130101; G01N 27/3271 20130101; G01N 33/66 20130101;
G01N 2021/7786 20130101; G01N 21/6428 20130101; A61B 5/1459
20130101; G01N 2021/7793 20130101; G01N 21/6408 20130101; G01N
2021/6432 20130101; A61B 5/1473 20130101 |
Class at
Publication: |
436/501 ;
600/316; 422/69 |
International
Class: |
G01N 33/66 20060101
G01N033/66; A61B 5/1459 20060101 A61B005/1459; A61B 5/1473 20060101
A61B005/1473; A61B 5/145 20060101 A61B005/145 |
Claims
1. A glucose sensor for quantifying the amount of glucose in a
sample that may further comprise an interferent, the sensor
comprising: a sensing region comprising: a first indicator system
comprising a first receptor for binding to glucose and a first
fluorophore associated with the first receptor, wherein said first
receptor has an association constant K.sub.G1 with glucose and an
association constant K.sub.M1 with said interferent, and a second
indicator system comprising a second receptor for binding to
glucose and a second fluorophore associated with the second
receptor, wherein said second receptor has an association constant
K.sub.G2 with glucose and an association constant K.sub.M2 with
said interferent, and wherein K.sub.G2/K.sub.M2 is different from
K.sub.G1/K.sub.M1; and an optical waveguide for directing light
onto the sensing region.
2. The glucose sensor according to claim 1, wherein said first
fluorophore and said second fluorophore have peak emission
wavelengths that differ by at least 5 nm.
3. The glucose sensor according to claim 1, wherein
[K.sub.G2/K.sub.M2]/[K.sub.G1/K.sub.M1] is greater than 2 or less
than 0.5.
4. The glucose sensor according claim 1, wherein said first
receptor and said second receptor are boronic acid receptors.
5. The glucose sensor according to claim 4, wherein said first
receptor contains two boronic acid groups and said second receptor
contains one boronic acid group.
6. The glucose sensor according claim, wherein said interferent is
selected from: (a) an interferent that comprises a cis-diol group;
and (b) an amine.
7. The glucose sensor according to claim 6, wherein the interferent
that comprises a cis-diol group is selected from a sugar alcohol
and a saccharide.
8. The glucose sensor according claim 1, wherein the interferent is
selected from the group consisting of mannitol, sorbitol,
galactitol, inositol, fructose, galactose, arabinose, glutamine and
catechol amines.
9. The glucose sensor according to claim 8, wherein the interferent
is mannitol.
10. A method of quantifying the amount of glucose in a sample that
may further comprise an interferent, the method comprising:
inserting into the sample a glucose sensor comprising: a sensing
region comprising at least a first indicator system comprising a
first receptor for binding to glucose and a first fluorophore
associated with the first receptor; and an optical waveguide for
directing light onto the sensing region; providing incident light
to the sensing region of the sensor and obtaining a first
measurement of the amount of glucose by a first measurement method
comprising detecting the emission of said first fluorophore;
obtaining a second measurement of the amount of glucose by a second
measurement method, wherein the second measurement method differs
in its sensitivity to the amount of said interferent in the sample
from the first measurement method; and comparing said first
measurement and said second measurement and thereby determining
whether any of said interferent is present in the sample; wherein
said interferent is a substance that is capable of interfering with
the binding of glucose to said first receptor.
11. A method according to claim 10, comprising: inserting into the
sample a glucose sensor comprising: a sensing region comprising: a
first indicator system comprising a first receptor for binding to
glucose and a first fluorophore associated with the first receptor,
wherein said first receptor has an association constant K.sub.G1
with glucose and an association constant K.sub.M1 with said
interferent, and a second indicator system comprising a second
receptor for binding to glucose and a second fluorophore associated
with the second receptor, wherein said second receptor has an
association constant K.sub.G2 with glucose and an association
constant K.sub.M2 with said interferent, and wherein
K.sub.G2/K.sub.M2 is different from K.sub.G1/K.sub.M1; and an
optical waveguide for directing light onto the sensing region;
providing incident light to the sensing region of the sensor;
obtaining a first measurement of the amount of glucose by a first
measurement method comprising detecting the emission of said first
fluorophore; obtaining a second measurement of the amount of
glucose by a second measurement method comprising detecting the
emission of said second fluorophore; and comparing said first
measurement and said second measurement, thereby determining
whether any of said interferent is present in the sample, and
correcting for the presence of any said interferent in the
sample.
12. A method according to claim 11, wherein said first measurement
and said second measurement are obtained in a single emission
detection step.
13. A method according to claim 11, wherein said first fluorophore
and said second fluorophore have peak emission wavelengths that
differ by at least 5 nm.
14. A method according to claim 11, wherein
[K.sub.G2/K.sub.M2]/[K.sub.G1/K.sub.M1] is greater than 2 or less
than 0.5.
15. A method according to claim 11, wherein said first receptor and
said second receptor are boronic acid receptors.
16. A method according to claim 15, wherein said first receptor
contains two boronic acid groups and said second receptor contains
one boronic acid group
17. A method according to claim 10, wherein said sample that may
further comprise an interferent is an in vivo sample.
18. A method according to claim 10, wherein said sample that may
further comprise an interferent is an in vivo sample and wherein
said method comprises: (i) obtaining said first measurement at a
time point t.sub.test; (ii) obtaining said second measurement by
said second measurement method, wherein said second measurement
method comprises electrochemically measuring the amount of glucose,
or of said interferent, in the sample at said time point
t.sub.test; and (iii) comparing said first measurement and said
second measurement, thereby determining whether said first
measurement contains a contribution from said interferent in the
sample.
19. A method according to claim 18, wherein the method is carried
out on a human or animal subject and the sample is an in vivo
bodily fluid of said subject, and wherein step (i) comprises
carrying out an in vivo measurement on said bodily fluid at time
t.sub.test, and wherein step (ii) comprises extracting a portion of
said bodily fluid from said subject at time t.sub.test and carrying
out an in vitro electrochemical measurement thereon.
20. A method according to claim 18, wherein said method comprises
carrying out the sequence of steps (i) to (iii) one or more times,
each at different time points, until in said step (iii) of
comparing said first measurement and said second measurement it is
determined that said first measurement contains substantially no or
a reduced contribution from said interferent.
21. A method according to claim 18, wherein in said step (iii) of
comparing said first measurement and said second measurement an
estimate of the amount of said interferent in the sample at
t.sub.test is obtained and wherein said method further comprises:
(iv) allowing a sufficient time delay for any said interferent
present in the sample at t.sub.test to substantially clear the
sample; and (v) providing incident light to the sensing region of
the sensor and detecting the emission of said first fluorophore,
thereby obtaining a third measurement of the amount of glucose in
the sample, said third measurement having substantially no or a
reduced contribution from said interferent.
22. A method according to claim 10, wherein the interferent is
selected from: (a) an interferent that comprises a cis-diol group;
and (b) an amine.
23. A method according to claim 22, wherein the interferent that
comprises a cis-diol group is selected from a sugar alcohol and a
saccharide.
24. A method according to claim 10, wherein the interferent is
selected from the group consisting of mannitol, sorbitol,
galactitol, inositol, fructose, galactose, arabinose, glutamine and
catechol amines.
25. A method according to claim 24, wherein the interferent is
mannitol.
26. A glucose sensor for quantifying the amount of glucose in a
sample that may further comprise an interferent, the sensor
comprising: a sensing region comprising: a first indicator system
comprising a first receptor comprising two boronic acid groups for
binding to glucose and a first fluorophore associated with the
first receptor, and a second indicator system comprising a second
receptor comprising one boronic acid group for binding to glucose
and a second fluorophore associated with the second receptor; and
an optical waveguide for directing light onto the sensing
region.
27. The glucose sensor according to claim 26, wherein said
interferent comprises a cis-diol group.
28. The glucose sensor according to claim 26, wherein said
interferent is selected from the group consisting of mannitol,
sorbitol, galactitol, inositol, fructose, galactose and
arabinose.
29. The glucose sensor according to claim 26, wherein said
interferent is mannitol.
30. The glucose sensor according to claim 26, wherein the first
receptor has an association constant K.sub.G1 with glucose and an
association constant K.sub.M1 with said interferent and the second
receptor has an association constant K.sub.G2 with glucose and an
association constant K.sub.M2 with said interferent, and wherein
K.sub.G2/K.sub.M2 is different from K.sub.G1/K.sub.M1.
Description
CLAIM OF PRIORITY
[0001] This application claims priority under 35 USC .sctn.119(e)
to U.S. Patent Application Ser. No. 61/662,560, filed on Jun. 21,
2012, the entire contents of which are hereby incorporated by
reference.
FIELD
[0002] The document relates to glucose sensors for detecting
glucose in a sample that may contain an interferent species, for
example mannitol. Methods of quantifying the amount of glucose in
such a sample are also provided.
BACKGROUND
[0003] Boronic acids are known to form reversible adducts with 1,2-
and 1,3-diols. This phenomenon has been utilised by attaching a
boronic acid receptor to chromophores or fluorophores in attempts
to design sensors for the continuous measurement of glucose.
Diboronic acid fluorophore chemistries have also been designed that
produce 1:1 adducts with 1,2-diols. Furthermore, if the diboronic
acid is linked to the fluorophores via a short linker, then the
chemistry operates by a Photo-induced Electron Transfer ("PET")
mechanism.
[0004] There has been an enormous drive to utilise these
chemistries in the development of sensors to measure physiological
glucose. The focus has been to improve the accuracy and reliability
for calculating a glucose concentration.
SUMMARY
[0005] Interferent species can sometimes be present in significant
quantities in the blood. For example, mannitol can be present in a
clinical setting. Interferent species, including mannitol, can be
capable of competing with glucose for binding to the boronic acid
receptor in a glucose sensor.
[0006] Interferent species, such as mannitol, can lead to errors
during glucose sensing methods. Some potential interferents, such
as saccharides (e.g., mannitol), have a similar molecular weight
and chemical structure to glucose. Thus, although protective
barrier layers can be used to block some potential interferents,
certain potential interferents, including mannitol, can permeate
glucose permeable barrier layers. Where significant errors arise
during monitoring of a patient's glucose levels (e.g., due to the
presence of an interferent such as mannitol in the blood),
potentially dangerous mis-diagnoses and/or patterns of medical
treatment can arise.
[0007] Glucose sensors and methods provided herein therefore
address the problem of inaccuracies arising during glucose sensor
indicating chemistry, in particular where these inaccuracies arise
due to the presence of an interferent in the sample.
[0008] Methods provided herein can include obtaining two
measurements of the amount of glucose using both a first and a
second measurement method, which methods differ in their
sensitivity to the amount of interferent in the sample. Comparison
between the two measurements can be used to determine whether any
interferent is present in the sample and, if so, for suitable steps
to be taken to address this issue, e.g., for an accurate assessment
of the glucose concentration in the sample to be obtained.
[0009] Thus, a method of quantifying the amount of glucose in a
sample that may further include an interferent can include: [0010]
inserting into the sample a glucose sensor comprising: [0011] a
sensing region comprising at least a first indicator system
including a first receptor for binding to glucose and a first
fluorophore associated with the first receptor; and [0012] an
optical waveguide for directing light onto the sensing region;
[0013] providing incident light to the sensing region of the sensor
and obtaining a first measurement of the amount of glucose by a
first measurement method including detecting the emission of said
first fluorophore; [0014] obtaining a second measurement of the
amount of glucose by a second measurement method, wherein the
second measurement method differs in its sensitivity to the amount
of said interferent in the sample from the first measurement
method; and [0015] comparing said first measurement and said second
measurement and thereby determining whether any said interferent is
present in the sample; wherein said interferent is a substance that
is capable of interfering with the binding of glucose to said first
receptor.
[0016] In some cases, methods provided herein include: [0017]
inserting into the sample a glucose sensor comprising: [0018] a
sensing region including: [0019] a first indicator system including
a first receptor for binding to glucose and a first fluorophore
associated with the first receptor, wherein said first receptor has
an association constant K.sub.G1 with glucose and an association
constant K.sub.M1 with said interferent, and [0020] a second
indicator system including a second receptor for binding to glucose
and a second fluorophore associated with the second receptor,
wherein said second receptor has an association constant K.sub.G2
with glucose and an association constant K.sub.M2 with said
interferent, and wherein K.sub.G2/K.sub.M2 is different from
K.sub.G1/K.sub.M1; and [0021] an optical waveguide for directing
light onto the sensing region; [0022] providing incident light to
the sensing region of the sensor; [0023] obtaining a first
measurement of the amount of glucose by a first measurement method
including detecting the emission of said first fluorophore; [0024]
obtaining a second measurement of the amount of glucose by a second
measurement method including detecting the emission of said second
fluorophore; and [0025] comparing said first measurement and said
second measurement, thereby determining whether any of said
interferent is present in the sample, and correcting for the
presence of any said interferent in the sample.
[0026] This preferred method is particularly advantageous because a
single experimental protocol can be used simultaneously to obtain
the first and the second measurements, namely an emission
fluorescence technique which involves providing incident light to
the sensing region of the sensor (in a single step) and detecting
(in a single emission spectrum/single emission measurement) the
emission of both the first and the second fluorophore. Comparison
between the fluorescence of the two fluorophores can permit for
correction (elimination) of any interferent contribution to the
fluorescence of the system, as further described herein.
[0027] Thus, in this preferred method there is no need for a
separate experimental verification of the interferent content of
the sample, for example by taking a blood sample and subjecting it
to laboratory analysis (e.g., by electrochemical methods).
Consequently, the preferred method of the document is particularly
convenient, rapid and easy to operate (e.g., without the presence
of specially trained medical practitioners).
[0028] The present document also provides a related a glucose
sensor, specifically a glucose sensor for quantifying the amount of
glucose in a sample that may further include an interferent, the
sensor including:
[0029] a sensing region including: [0030] a first indicator system
including a first receptor for binding to glucose and a first
fluorophore associated with the first receptor, wherein said first
receptor has an association constant K.sub.G1 with glucose and an
association constant K.sub.M1 with said interferent, and [0031] a
second indicator system including a second receptor for binding to
glucose and a second fluorophore associated with the second
receptor, wherein said second receptor has an association constant
K.sub.G2 with glucose and an association constant K.sub.M2 with
said interferent, and wherein K.sub.G2/K.sub.M2 is different from
K.sub.G1/K.sub.M1; and
[0032] an optical waveguide for directing light onto the sensing
region.
[0033] This glucose sensor can be used to carry out the preferred
method of the document.
[0034] Further preferred features and embodiments are described in
the accompanying description and the appended claims.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIGS. 1 and 1a depict a sensor incorporating an optical
fibre and a monitor for such a sensor.
[0036] FIGS. 2, 3 and 3a depict various embodiments of a sensing
region of a sensor.
[0037] FIG. 4 depicts a flow chart that schematically illustrates a
method provided herein.
[0038] FIG. 5 depicts a flow chart that schematically illustrates
one embodiment of the a method provided herein.
[0039] FIG. 6 depicts a flow chart that schematically illustrates
another embodiment of a method provided herein.
[0040] FIG. 7 depicts the results of Example 2 as follows: A)
Fluorescence emission spectra recorded in the binding study of
hydrogel 4 vs. D-glucose in PBS at excitation wavelengths of 350
and 405 nm and at [D-glucose].sub.titrant of 214 mM (note that
lower lines to upper lines follow in order of lower concentrations
to high concentrations); B) Fluorescence emission spectra recorded
in the binding study of hydrogel 4 vs. D-mannitol in PBS at
excitation wavelengths of 350 and 405 nm and at
[mannitol].sub.titrant of 184 mM (note that lower lines to upper
lines follow in order of lower concentrations to high
concentrations); C) Relative fluorescence intensity versus
carbohydrate concentration profile of hydrogel 4 vs. D-glucose,
.lamda.ex=350 nm, .lamda.em=380 nm and .lamda.ex=405 nm,
.lamda.em=487 nm; and D) Relative fluorescence intensity versus
carbohydrate concentration profile of hydrogel 4 vs. D-mannitol,
.lamda.ex=350 nm, .lamda.em=380 nm and .lamda.ex=405 nm,
.lamda.em=487 nm.
DETAILED DESCRIPTION
[0041] A method described herein can include the use of a glucose
sensor including a sensing region including at least a first
indicator system including a first receptor for binding to glucose
and a first fluorophore associated with the first receptor; and an
optical waveguide for directing light onto the sensing region.
[0042] A method described herein can use fibre optic glucose
sensors (i.e., a glucose sensor where the optical waveguide is an
optical fibre), but the presently described methods can also be
carried out with sensors having different types of optical
waveguides.
[0043] Glucose sensing methods described herein can be carried out
in an aqueous solution. In some cases, glucoses sensing methods
described herein can be carried out in bodily fluids such as
interstitial tissue or blood. For example, the glucose sensors
described herein can be used as invasive sensors and inserted into
a blood vessel. Glucose sensing methods provided herein can use
non-invasive sensors for in vitro use, implantable sensors, and/or
subcutaneous sensors.
[0044] An example a sensor incorporating an optical fibre is
depicted in FIGS. 1 and 1a. The sensor 1 includes an optical fibre
2 including a sensing region 3 at its distal end. In some cases,
sensor 1 can be an invasive sensor. Sensor 1 includes a fibre 2.
Fibre 2 can be adapted for insertion into a patient, for example
insertion into a blood vessel through a cannula. The sensing region
3 (depicted in more detail in FIGS. 2, 3 and 3a) contains a cell or
chamber 7 in which the, or each, indicator system is contained. The
optical fibre extends through cable 4 to connector 5 which is
adapted to mate with an appropriate monitor 8. The monitor can
include a further optical cable 4a that mates with the connector at
5a and at the other bifurcates to connect to (a) an appropriate
source of incident light for the optical sensor 9 and (b) a
detector for the return signal 10.
[0045] In some embodiments, the sensor is a disposable sensor. A
disposable sensor can be adapted to be connected to a
non-disposable monitor including a light source 9 and detector
10.
[0046] As depicted in FIG. 2, the sensing region 3 incorporates a
cell 7 in the form of a chamber within the fibre. The cell may take
any form, as long as the one or more indicator systems are
contained in the path of the incident light directed by the
waveguide (e.g., a fibre). Thus, the cell may be attached to the
distal end of the fibre or waveguide or may be in the form of a
chamber within the fibre having any desired shape.
[0047] Optionally, a reactive oxygen species ("ROS") quenching
agent may be present in the sensor, for example within the sensing
region or within an optional barrier layer as further described
herein. An example of a reactive oxygen species is hydrogen
peroxide (H.sub.2O.sub.2). Suitable ROS-quenching agents are as
described in U.S. patent application No. 61/524,525 and
PCT/GB2012/051921, the contents of which are incorporated by
reference herein. Suitable means for incorporating the
ROS-quenching agent into a glucose sensor are also described in
U.S. patent application No. 61/524,525 and PCT/GB2012/051921 and
again are incorporated by reference herein.
[0048] The sensing region 3 of the glucose sensor has one or more
openings 6a, 6b to enable glucose to enter the cell. A glucose
permeable barrier layer "BL" may optionally be provided across
these openings so that glucose enters the cell through the barrier
layer. A glucose permeable barrier layer used in a glucose sensor
provided herein can be capable of at least partially restricting
the passage of high molecular weight materials such as
proteins.
[0049] In FIGS. 2, 3 and 3a, the optional barrier layer BL is
provided over the entire sensing region 3. Alternatively, however,
the optional barrier layer BL may be provided on only part of the
sensing region, for example only across openings 6a and 6b.
[0050] Briefly, FIG. 2 shows an embodiment in which the optional
barrier layer BL is applied directly onto the sensing region 3,
here onto the tip of the optical fibre, via joints (e.g.,
thermoformed attachment points) Ja and Jb. In FIG. 3, the sensing
region 3 is provided within a separate support 11 and the barrier
layer BL is provided on the support 11 (again via joints Ja and
Jb). In FIG. 3a, the barrier layer itself forms a support structure
"BL/11". Further details on suitable sensor constructions when a
barrier layer is incorporated, and on suitable materials for such
barrier layers, are described in U.S. patent application No.
61/524,525, PCT/GB2012/051921 and WO2011/101626, the contents of
which are incorporated by reference herein.
[0051] Glucose sensors having design features in addition to or
different from those shown in the attached Figures are of course
possible, provided that these include both of the required: (i)
sensing region including at least a first indicator system
including a first receptor for binding to glucose and a first
fluorophore associated with the first receptor; and (ii) optical
waveguide for directing light onto the sensing region. For example,
glucose sensors such as those described and illustrated in
WO2008/141241, WO2008/098087 and WO2011/113020 can be used.
[0052] By "indicator system" is meant a combination of a receptor
for binding to glucose and a fluorophore associated therewith, such
that the emission pattern (e.g. the wavelength, intensity,
lifetime) of the fluorophore is altered when glucose is bound to
the receptor (thus enabling the emission behaviour of the
fluorophore to act as an "indicator" for the presence of
glucose).
[0053] The first receptor and first fluorophore may be directly
bonded to one another as a first receptor-first fluorophore
construct. Examples of suitable first fluorophores include
anthracene, pyrene and derivatives thereof. Examples of suitable
first receptors are boronic acid receptors, such as compounds
having at least one (for example one or two), or at least two (for
example two or three) boronic acid groups.
[0054] Suitable receptors also include arsenic-containing
receptors, tellurium-containing receptors and germanium-containing
receptors, for example receptors comprising one or more groups of
formula H.sub.3AsO.sub.3, H.sub.2AsO.sub.3.sup.-, H.sub.6TeO.sub.6,
H.sub.5TeO.sub.6.sup.-, Ge(OH).sub.6 or GeO(OH).sub.3.sup.-, or
derivatives thereof. Examples of such receptors include the
receptors disclosed in US 2005/0158245, the content of which is
herein incorporated by reference in its entirety. Still further
suitable receptors include synthetic lectins that are capable of
binding to glucose, for example via non-covalent interactions such
as hydrogen bonding, CH-.pi. interactions and/or hydrophobic
interactions. Examples of such synthetic lectins are described in
Nat Chem. 2012 4(9) 718-23, the content of which is herein
incorporated by reference in its entirety.
[0055] In a preferred embodiment, the first receptor is a group of
formula (I)
##STR00001##
wherein m and n are the same or different and are typically one or
two, preferably one; Sp is an aliphatic spacer, typically an
alkylene moiety, for example a C.sub.1-C.sub.12 alkylene moiety,
e.g. a C.sub.6 alkylene moiety; and L.sub.1 and L.sub.2 represent
possible points of attachment to other moieties, for example to a
fluorophore or to a hydrogel. For example, L.sub.1 and L.sub.2 may
represent an alkylene, alkylene-arylene or
alkylene-arylene-alkylene moiety, linked to a functional group.
Where no attachment to another moiety is envisaged, the functional
group is protected or replaced by a hydrogen atom. Typical alkylene
groups for L.sub.1 and L.sub.2 are C.sub.1-C.sub.4 alkylene groups,
e.g. methylene and ethylene. Typical arylene groups are phenylene
groups. The functional group is typically any group which can react
to form a bond with, for example, the fluorophore or hydrogel, e.g.
ester, amide, aldehyde or azide. Varying the length of the spacer
Sp alters the selectivity of the receptor. A C.sub.6-alkylene chain
can provide a receptor which has good selectivity for glucose.
Further details of such receptors are found in U.S. Pat. No.
6,387,672, the contents of which are incorporated herein by
reference in their entirety.
[0056] Further examples of first receptors suitable for the
presently described sensors include those of formula (II):
##STR00002##
wherein X represents O, S, NR.sub.2 or CHR.sub.3; n is from 1 to 4;
m is from 1 to 4, and n+m is 5; R.sub.2 represents hydrogen or
C.sub.1-4 alkyl; each R.sub.1 is the same or different and
represents hydrogen, C.sub.1-4 alkyl or C.sub.3-7 cycloalkyl; or
R.sub.1, together with an adjacent R.sub.1, R.sub.2 or R.sub.3
group and the carbon or nitrogen atoms to which they are attached,
form a C.sub.3-7 cycloalkyl or a 5- or 6-membered heterocyclyl
group, wherein when X represents CHR.sub.3, R.sub.3 together with
an adjacent R.sub.1 group and the carbon atoms to which they are
attached form a C.sub.3-7 cycloalkyl group. Further details of
receptors of this type are found in U.S. 61/431,756, the contents
of which are incorporated herein by reference.
[0057] An example of a boronic acid receptor having one boronic
acid group is a compound of the formula (III) below
##STR00003##
wherein: n is from one to three, preferably one or two, and more
preferably one; and L.sub.1 and L.sub.2 represent possible points
of attachment to other moieties, for example to a fluorophore or to
a hydrogel. For example, L.sub.1 and L.sub.2 may represent an
alkylene, alkylene-arylene or alkylene-arylene-alkylene moiety,
linked to a functional group. Where no attachment to another moiety
is envisaged, the functional group is protected or replaced by a
hydrogen atom. Typical alkylene groups for L.sub.1 and L.sub.2 are
C.sub.1-C.sub.4 alkylene groups, e.g. methylene and ethylene.
Typical arylene groups are phenylene groups. The functional group
is typically any group which can react to form a bond with, for
example, the fluorophore or hydrogel, e.g. ester, amide, aldehyde
or azide.
[0058] As explained above, the first receptor may be a compound
having one boronic acid group such as that of formula (III).
However, more typically the first receptor includes two boronic
acid groups. As explained in further detail below, where a second
receptor is additionally present, this second receptor may be a
compound having one boronic acid group, such as a compound of
formula (III).
[0059] As used herein the term alkyl or alkylene is a linear or
branched alkyl group or moiety. An alkylene moiety may, for
example, contain from 1 to 15 carbon atoms such as a C.sub.1-12
alkylene moiety, C.sub.1-6 alkylene moiety or a C.sub.1-4 alkylene
moiety, e.g. methylene, ethylene, n-propylene, i-propylene,
n-butylene, i-butylene and t-butylene. C.sub.1-4 alkyl is typically
methyl, ethyl, n-propyl, i-propyl, n-butyl or t-butyl. For the
avoidance of doubt, where two alkyl groups or alkylene moieties are
present, the alkyl groups or alkylene moieties may be the same or
different.
[0060] An alkyl group or alkylene moiety may be unsubstituted or
substituted, for example it may carry one, two or three
substituents selected from halogen, hydroxyl, amine, (C.sub.1-4
alkyl)amine, di(C.sub.1-4 alkyl)amine and C.sub.1-4 alkoxy.
Preferably an alkyl group or alkylene moiety is unsubstituted.
[0061] As used herein an arylene group is an unsaturated group
which may be monocyclic, bicyclic, or which may contain three or
four fused rings. Typically, an arylene group is phenylene. Arylene
groups may be unsubstituted or substituted. Suitable substituents
are C.sub.1-4 alkyl groups, for example methyl and ethyl.
Preferably, an arylene group is unsubstituted.
[0062] As used herein a C.sub.3-7 cycloalkyl group is typically a
cyclopentyl or cyclohexyl group. C.sub.3-7 cycloalkyl groups may be
unsubstituted or substituted. Suitable substituents are C.sub.1-4
alkyl groups, for example methyl and ethyl. Preferably, a C.sub.3-7
cycloalkyl group is unsubstituted. As used herein a 5- or
6-membered heterocyclyl group is a 5- or 6-membered saturated ring
containing one or more, typically one or two, e.g. one, heteroatom
selected from N, O and S. Preferred heterocyclyl groups are those
containing a nitrogen atom, for example piperidinyl and
pyrrolidinyl. Heterocyclyl groups may be unsubstituted or
substituted. Suitable substituents are C.sub.1-4 alkyl groups, for
example methyl and ethyl. Preferably, a heterocyclyl group is
unsubstituted.
[0063] The first receptor and first fluorophore are typically bound
to one another and may further be bound to a polymeric matrix such
as a hydrogel, or to a dendrimer. Examples of suitable hydrogels
and dendrimers are those described in WO 2011/101624, the content
of which is incorporated herein by reference.
[0064] Alternatively, the first receptor and first fluorophore may
be not directly bonded to one another (for example, they may be not
bonded to one another or they may be bonded only via a polymeric
chain such as a polymeric chain contained within a hydrogel
matrix). It will be clear that when the first receptor and first
fluorophore are not directly bonded to one another, they must still
be capable of interacting in such a way that the fluorescence
behaviour of the first fluorophore changes when the indicator
system is exposed to glucose. For example, the first fluorophore
and the second fluorophore may be capable of binding
electrostatically (e.g., as a charge pair), which binding is
capable of being at least partly disrupted by the presence of
glucose. Examples of suitable first fluorophores include pyranine
(HPTS) and its derivatives, such as HPTS itself and the derivatives
HPTS-PEG, HPTS-MA, HPTS-CO.sub.2, HPTS-TriCys-MA and HPTS-LysMA
disclosed in US 2009/0177143, the content of which is herein
incorporated by reference in its entirety. Further suitable first
fluorophores may include the SNAF and SNAFL dyes commercially
available from Molecular Probes. Examples of suitable first
receptors include aromatic boronic acids covalently bonded to a
conjugated nitrogen-containing heterocyclic aromatic bis-onium
structure (e.g. a viologen). Examples of such first receptors are
provided in US 2009/0177143, the content of which is herein
incorporated by reference in its entirety. One particularly
suitable first receptor is 3,3'-oBBV, as described in US
2009/0177143.
[0065] As used herein, the phrase "in vivo sample" means that the
sample is a sample that is present in the human or animal
(preferably human) body. For example, such a sample may be a bodily
fluid such as the blood that is present in the human or animal
(preferably human) body. An in vivo sample can be contrasted with
an ex vivo sample, for example an ex vivo blood sample, wherein the
sample has been removed (isolated) from the human or animal body.
Consequently, references throughout this specification to methods
carried out on an "in vivo sample" mean that the method is a method
of quantifying the amount of glucose in the human or animal
(preferably human) body. It should be clarified, however, that an
in vivo sample can be subject to glucose (or interferent, e.g.
mannitol interferent) measurement either by directly measuring the
glucose concentration in vivo, for example using an invasive method
as described herein, or by extracting the sample from the body and
measuring the glucose concentration in vitro. In the latter case,
the glucose (or interferent, e.g. mannitol interferent)
concentration of the sample as measured corresponds to the
concentration of the in vivo sample at the time point at which it
was extracted from the body.
[0066] It follows from the above that an in vivo sample as used
herein will vary in terms of its glucose and interferent (e.g.,
mannitol) concentration over time due to, for example, metabolism
of the interferent (e.g., mannitol) in the body. Accordingly, where
the presently described methods take two or more measurements of an
in vivo sample at different points in time, the sample will not
necessarily have the same composition at those different time
points.
[0067] To the extent that the methods of the document are carried
out on in vivo samples, all of the procedures carried out in
accordance with the methods provided herein (including inserting
into the sample a glucose sensor and electrochemically measuring
the amount of glucose or interferent in the sample) are routine
procedures that do not involve any substantial health risk to the
human or animal patient and which moreover can be carried out
without substantial medical expertise. It will be appreciated that
steps such as insertion of a cannula (for example, in the context
of inserting an exemplary glucose sensor of the present document)
and extraction of blood samples for the purposes of obtaining
electrochemical measurements (e.g., by needle extraction) are
routine in the art and can be carried out for example by a nurse
rather than requiring the involvement of doctors. Thus, the methods
provided herein do not constitute surgical methods carried out on
the human or animal body.
[0068] The methods provided herein can provide incident light to a
sensing region of a sensor and a first measurement of the amount of
glucose is obtained by a first measurement method including
detecting the emission of said first fluorophore.
[0069] The first measurement method may comprise making an
equilibrium measurement of the emission of the first fluorophore
after the glucose sensor has been contacted with the sample.
[0070] In one aspect, the first indicator system may comprise a
first receptor that is capable of at least partially quenching the
fluorescence emission of the first fluorophore when the said first
fluorophore is exposed to excitation light (e.g., by association of
the receptor with a quencher). In this aspect, when the glucose
sensor is contacted with the sample, glucose in the sample binds
with the first receptor and thereby at least partially reduces the
quenching efficiency of the first receptor, thus leading to an
enhancement in emission from the first fluorophore.
[0071] The first measurement method may use a dye displacement
technique.
[0072] In the light of the discussion herein, it will be
appreciated that this first measurement may contain a contribution
arising from binding of an interferent (one example of which is
mannitol), rather than the desired glucose, to the receptor when
there is an interferent present in the sample. In other words, the
first measurement may not of itself be capable of providing an
accurate quantitative determination of the glucose concentration in
a sample that contains interferent (i.e., without taking further
steps to account for the presence of interferent).
[0073] Accordingly, the methods provided herein further include
obtaining a second measurement of the amount of glucose by a second
measurement method, as shown schematically in the flow chart of
FIG. 4. The nature of the second measurement method is not
critical. The methods provided herein are capable of quantitatively
determining the quantity of glucose in a sample.
[0074] The second measurement method differs in its sensitivity to
the amount of interferent in the sample from the first measurement
method. By "differs in its sensitivity to the amount of
interferent" it is meant that for a sample that does contain some
interferent, the contribution made by that interferent to the
second measurement, relative to the contribution made by the
glucose, is different from the corresponding contribution in the
first measurement.
[0075] It can easily be established by one of ordinary skill in the
art whether two measurement methods "differ in sensitivity to the
amount of interferent in the sample". For example, when the second
measurement method is an electrochemical method, then typically
that second measurement method will be sensitive only to the
analyte (i.e., usually glucose, though possibly the interferent)
that it is probing, thereby rendering it necessarily different in
sensitivity to the amount of interferent in the sample from the
first measurement method. When the second measurement method
includes detecting the emission of a second fluorophore in a second
indicator system, typically both the first and second indicator
systems are pre-calibrated to assess their detection sensitivities
to glucose and the interferent, and the first indicator system's
detection sensitivities to glucose and interferent are different
from the second indicator detection sensitivities to glucose and
interferent.
[0076] Usually both the first and second measurement methods are
capable of "direct" measurement of the amount of glucose in a
sample, i.e. both the first and second measurement methods are
sensitive to the amount of glucose in the sample and therefore can,
in the absence of interferent, be used to determine quantitatively
the concentration of glucose in a sample.
[0077] However, it will be appreciated that the methods provided
herein can also be carried out when the second measurement method
is sensitive to the interferent concentration, but is actually
substantially or completely insensitive to glucose concentration.
For example, the second measurement method in certain cases
directly indicates only the concentration of the interferent, but
clearly still allows for comparison with the first measurement so
as to determine whether any said interferent is present. In other
words, the second measurement method can provide an "indirect",
rather than a "direct" second measurement of the amount of glucose.
For the avoidance of doubt, it is emphasised that the methods
provided herein can use a second measurement of an amount of
glucose that is direct (the second measurement including a glucose
contribution) or a second measurement of an amount of glucose that
is indirect (the second measurement including no glucose
contribution).
[0078] The second measurement method used in the methods provided
herein may for example include electrochemically measuring the
amount of glucose (or interferent) in the sample (as shown
schematically in the flow chart of FIG. 6). In some cases, the
second measurement method may for example include detecting the
emission of a second fluorophore included in a second indicator
system including a second receptor for binding to glucose and a
second fluorophore (as shown schematically in the flow chart of
FIG. 5). These exemplary second measurement methods will now be
discussed in turn.
Electrochemical Second Measurement Methods
[0079] Electrochemically measuring the amount of glucose in the
sample can be achieved using well known techniques in the art for
electrochemical detection of glucose. Commercially available
glucose sensors working on electrochemical principles can be used
to carry out the methods provided herein. Exemplary devices for
electrochemical glucose detection are the analyzers provided by
YSI.TM. Life Sciences. In some cases, these devices have a small
amount of (ex vivo) sample inputted, from which glucose
concentration can then accurately be determined For example, when
carrying out a real-time in vivo analysis of glucose concentration
using the glucose sensor described herein, a blood sample may be
extracted from the patient and used for the electrochemical
analysis.
In some cases, methods provided herein use a second measurement
method that electrochemically measures an amount of the interferent
in the sample. It will be appreciated this corresponds to the
"indirect" second measurement of glucose described herein.
[0080] In some cases, an amount of glucose in an in vivo sample is
determined using the methods described herein using a second
measurement method that includes electrochemically measuring the
amount of glucose (or interferent) in the sample. Methods described
herein can rely on in vivo metabolic clearance of any of the
interferent initially present in the sample over time, such methods
of course therefore being suitable specifically for interferents
that are capable of being metabolically cleared (i.e.,
metabolised). Methods described herein can obtain the final,
quantified glucose concentration once the interferent has been at
least substantially metabolised.
[0081] In some cases, a second measurement method used in a method
provided herein includes electrochemically measuring the amount of
glucose (or interferent) in the sample, the method including:
[0082] (i) obtaining said first measurement at a time point
t.sub.test; [0083] (ii) obtaining said second measurement by said
second measurement method, wherein said second measurement method
includes electrochemically measuring the amount of glucose, or
interferent, in the sample at said time point t.sub.test; and
[0084] (iii) comparing said first measurement and said second
measurement, thereby determining whether said first measurement
contains a contribution from the interferent in the sample.
[0085] Methods provided herein can be carried out on a human or
animal subject and the sample is an in vivo bodily fluid of said
subject. For example, step (i) can include carrying out an in vivo
measurement on said bodily fluid at time t.sub.test (e.g. an
invasive measurement as described herein, or any other method
carried out directly on the body without extracting the sample).
Step (ii) can include extracting a portion of said bodily fluid
from said subject at time t.sub.test and carrying out an in vitro
electrochemical measurement thereon.
[0086] In some cases, results obtained at t.sub.test can be used to
establish whether the interferent is having a significant adverse
effect on (i.e., making a significant interfering contribution to)
the glucose concentration as measured by the (fluorescence
emission) glucose sensor.
[0087] The reference to "electrochemically measuring the amount of
glucose, or interferent, in the sample at said time point
t.sub.test" means obtaining an electrochemical measurement of the
amount of glucose, or interferent, that was present in the sample
at the time point t.sub.test. The electrochemical measurement can
be obtained by obtaining a quantity of the sample, e.g. by needle
extraction in the case of an in vivo sample, and then by
electrochemically measuring the amount of glucose, or interferent,
therein.
[0088] If the comparison step (iii) indicates that there is no
significant interferent contribution to the first measurement, then
there is no need for further steps to occur (it having been
confirmed that the first measurement is accurate). It will of
course be appreciated that during practical use of the glucose
sensor, this means that ongoing (continuous) measurement of glucose
concentration over a prolonged period can be carried out using the
first indicator system, it now having been established that there
is no danger of a false reading due to unmetabolised interferent in
the sample.
[0089] If the comparison step (iii) indicates that there is a
significant interferent contribution to the first measurement, a
skilled person (e.g., a nurse) can identify that the accuracy of
the glucose concentration measurement from the glucose sensor is
compromised by the interferent. The skilled person may determine
that an accurate measurement using the glucose sensor should be
taken at a later time when the interferent has (at least partially)
been metabolised and therefore been eliminated from the in vivo
system. In some cases, an estimated time for interferent
elimination from the in vivo system can be calculated by the
skilled person (e.g., a nurse) and/or by a sensor provided herein.
For example, the sensor provided herein can be connected to a
monitor that displays a proposed time or countdown timer to show
when an accurate measurement can be taken. By "said first
measurement contains a contribution from interferent in the sample"
may be meant that the first measurement is at least 1%, for example
at least 2%, at least 5% or at least 10% different from the "true"
(i.e., actual) concentration of glucose in the sample (as measured,
for example, by an electrochemical measurement).
[0090] A suitable later time for establishing when the interferent
has (at least partially) been metabolised involves carrying out the
sequence of steps (i) to (iii) one or more times, each at different
time points, until in said step (iii) of comparing said first
measurement and said second measurement it is determined that said
first measurement contains substantially no or a reduced
contribution from the interferent. In other words, one carries one
or more measurements, and comparisons, until the interferent has
been metabolised to an acceptable extent. By "substantially no or a
reduced contribution from interferent" may be meant that the first
measurement is at most 10%, preferably at most 5%, more preferably
at most 2% and more preferably still at most 1% higher or lower
than the "true" (i.e., actual) concentration of glucose in the
sample (as measured, for example, by an electrochemical
measurement). For example, the method may include carrying out the
sequence of steps (i) to (iii) at least twice, at least three
times, at least four times or at least five times. In some cases,
the method limits the carrying out the sequence of steps (i) to
(iii) to no more than twenty times, for example not more than ten
times or not more than five times. In some cases, suitable later
time for when the interferent has (at least partially) been
metabolised can be established by comparing, in said step (iii),
the first measurement and the second measurement and thereby
obtaining an estimate of (e.g., calculating) the amount of
interferent in the sample at t.sub.test. The rate of metabolization
of many interferents in vivo is a known quantity and therefore the
method may then include: [0091] (iv) allowing a sufficient time
delay for any interferent present in the sample at t.sub.test to
substantially clear the sample; and [0092] (v) providing incident
light to the sensing region of the sensor and detecting the
emission of said first fluorophore, thereby obtaining a third
measurement of the amount of glucose in the sample, said third
measurement having substantially no or a reduced contribution from
said interferent.
[0093] By allowing "any interferent present in the sample at
t.sub.test to substantially clear the sample" may be meant that
there is sufficient time delay such that the third measurement is
at most 10%, preferably at most 5%, more preferably at most 2% and
more preferably still at most 1% higher or lower than the "true"
(i.e., actual) concentration of glucose in the sample (as measured,
for example, by an electrochemical measurement).
[0094] As an illustrative example, consider the embodiment in which
said interferent is mannitol. The half life, t.sub.1/2, of mannitol
in vivo is approximately 100 minutes and it would be a matter of
routine from this for a skilled person to determine, once the
amount of mannitol initially present in the sample has been
estimated, how long a time delay would be required to bring the
concentration of mannitol interferent down to an acceptable level.
For example, if after the initial estimate it is established that
the amount of mannitol interferent would need to be approximately
halved for an acceptable result to be obtained, then one would
allow a time delay of approximately 100 minutes. In some cases, a
sensor can calculate an estimated time for meeting a certain
accuracy and the estimated time can be displayed on a monitor
(e.g., as a countdown timer).
Emission Fluorescence as the Second Measurement Method
[0095] A method provided herein can include a second measurement
method that includes emission fluorescence detection. In some
cases, a second measurement can include detecting the emission of a
second fluorophore included in a second indicator system including
a second receptor for binding to glucose and the second
fluorophore. This second indicator system can be included in the
sensing region of the glucose sensor that includes the first
indicator system.
[0096] In some cases, a glucose sensor provided herein can include
a first indicator system including a first receptor for binding to
glucose and a first fluorophore associated with the first receptor,
wherein said first receptor has an association constant K.sub.G1
with glucose and an association constant K.sub.M1 with the
interferent. This glucose sensor can include a second indicator
system including a second receptor for binding to glucose and a
second fluorophore associated with the second receptor, wherein
said second receptor has an association constant K.sub.G2 with
glucose and an association constant K.sub.M2 with the interferent,
and wherein K.sub.G2/K.sub.M2 is different from
K.sub.G1/K.sub.M1.
[0097] Methods for measuring the association constant for a
particular receptor with a particular guest substance (e.g.,
glucose or the interferent, for example mannitol) are well known in
the art. One such means involves calibrating the changes in
fluorescence behaviour (e.g., intensity) of a fluorophore
associated with the receptor as the concentration of the guest
substance in a control sample containing no other guest substances
is varied.
[0098] A method provided herein using this glucose sensor can then
be carried out by providing incident light to the sensing region of
the sensor and obtaining: (a) a first measurement of the amount of
glucose by a first measurement method including detecting the
emission of said first fluorophore; and (b) a second measurement of
the amount of glucose by a second measurement method including
detecting the emission of said second fluorophore. The incident
light provided to the sensing region can have a suitable wavelength
profile to trigger the fluorescence emission of both the first
fluorophore and the second fluorophore, i.e. the fluorescence
emission of the fluorophores occurs at the same time and can be
detected in a single experiment. In some cases, incident light of a
first wavelength profile is provided to trigger the fluorescence
emission of the first fluorophore and incident light of a second
wavelength profile is provided to trigger the fluorescence emission
of the second fluorophore with a temporal delay between each
wavelength profile, e.g. with a delay of up to ten seconds, for
example a delay of up to five seconds.
[0099] The second receptor and the second fluorophore are chosen
such that K.sub.G2/K.sub.M2 is different from K.sub.G1/K.sub.M1.
The second receptor can, for example, be chosen from the receptors
described herein as being suitable for the first receptor (e.g.,
those compounds of formulae (I) and/or (II) described herein).
Similarly, the second fluorophore can, for example, be chosen from
the fluorophores described herein as being suitable for the first
fluorophore. The second indicator system (i.e., the second
fluorophore/second receptor system) is different from the first
indicator system (i.e., the first fluorophore/first receptor
system). In some cases, the second fluorophore is different from
the first fluorophore and the second receptor is different from the
first receptor. Typically the second receptor is different from the
first receptor. Typically both the first receptor and the second
receptor have a non-zero association constant with glucose (i.e.,
both the first receptor and the second receptor are capable of
binding to glucose and therefore are sensitive to changes in
glucose concentration in the sample).
[0100] In some cases, the first fluorophore and second fluorophore
are chosen such that their respective peak fluorescence emission
wavelengths differ by at least 5 nm, preferably at least 10 nm,
more preferably at least 20 nm (when associated with their
respective receptors and said receptors are bound to glucose and/or
the interferent). A separation of peak fluorescence emission
wavelengths can simplify analysis of a fluorescence emission
spectrum containing the fluorescence emission of both the first
fluorophore and the second fluorophore.
[0101] In some cases, when the steps of detecting the emission of
the first fluorophore and the second fluorophore, respectively,
include detecting the emission lifetime of the first fluorophore
and the second fluorophore, respectively, the first and second
fluorophores may be chosen such that they have a substantially
different fluorescence lifetime (for example, differing by at least
0.5 ns or 1 ns or 2 ns).
[0102] The second receptor and the first receptor can have
different relative abilities to bind to glucose and the
interferent. In some cases, this difference in relative abilities
to bind to glucose and the interferent can be significant. For
example, it may be preferable that the quotient
[K.sub.G2/K.sub.M2]/[K.sub.G1/K.sub.M1] is greater than 2 or less
than 0.5 (it being appreciated that these values give rise to the
same effective difference, depending on whether it is the first or
the second receptor that has the higher relative interferent
sensitivity). It may be more preferable that the quotient
[K.sub.G2/K.sub.M2]/[K.sub.G1/K.sub.M1] is greater than 10 or less
than 0.1, and even more preferable that the quotient
[K.sub.G2/K.sub.M2]/[K.sub.G1/K.sub.M1] is greater than 50 or less
than 0.02. As the difference in the relative ability of the two
receptors to bind to glucose and the interferent increases, a
correction for the presence of interferent can become more
accurate, even for smaller concentrations of interferent.
[0103] In some cases, the two receptors that to bind to glucose and
the interferent can have substantially different chemical and/or
stereochemical properties. For example, a first receptor and a
second receptor can both be boronic acid receptors, but the first
receptor can have two boronic acid groups (for example, it is a
receptor of the formula (I) or the formula (II) as defined herein)
whereas the second receptor can have only one boronic acid group
(for example, it is a receptor of the formula (III) as defined
herein). A diboronic acid, such as those of formulae (I) or (II)
above, may bind to both glucose and a mannitol interferent, whereas
a monoboronic acid, such as that of formula (III), may bind more
strongly to a mannitol interferent than to glucose.
[0104] The second receptor and second fluorophore can be bound to
one another and may further be bound to a polymeric matrix such as
a hydrogel, or to a dendrimer. Examples of suitable hydrogels and
dendrimers are those described in WO 2011/101624, the content of
which is incorporated herein by reference. Where a polymeric matrix
is used, the particles of polymeric matrix may simultaneously carry
the first receptor and first fluorophore together with the second
receptor and second fluorophore. In some cases, the second receptor
and second fluorophore can be bound to a different polymeric
matrix.
[0105] Alternatively, the second receptor and second fluorophore
may be not directly bonded to one another, for example as described
above with reference to the first receptor and first
fluorophore.
Comparing the Measurements and Thereby Determining Whether any
Interferent is Present in the Sample
[0106] Methods provided herein can include comparing the first
measurement (e.g., the emission of the first fluorophore) and the
second measurement (e.g., the emission of the second fluorophore)
and thereby determining whether any interferent is present in the
sample. Preferably, where it is determined that interferent is
present in the sample the method further includes either (a)
correcting for the presence of any interferent in the sample, or
(b) obtaining a subsidiary measurement of glucose concentration at
a later time point when the interferent is (substantially) no
longer present in the sample (e.g., when it has been metabolised in
vivo). Thus, the step of correcting for the presence of any
interferent in the sample or obtaining a subsidiary measurement of
glucose concentration can be understood to mean "obtaining a
measurement of the amount of glucose in the sample, said
measurement having substantially no or a reduced contribution from
the interferent", i.e., "quantifying the amount of glucose in the
sample".
[0107] If the step of "comparing the first measurement and the
second measurement" leads to the conclusion that there is
(substantially) no interferent in the sample, then one or both of
the measurements can be used to determine the glucose concentration
without any additional correction or subsidiary measurement
step).
[0108] The expression "correcting for the presence of any
interferent in the sample" takes a broad meaning and includes
procedures such as mathematically eliminating the contribution made
by the interferent to the first (or second) measurement.
[0109] In the context of a method provided herein involving two
fluorophores, the skilled person would readily appreciate that
because these two fluorophores have different sensitivities to the
interferent, comparison of their emission behaviour in a single
sample (containing specific glucose and interferent concentrations)
can be used to eliminate (by mathematical analysis) the interferent
contribution from either measurement and thereby to obtain a
correct value for the glucose concentration in the sample. It will
be appreciated that implicit in this ability to eliminate the
interferent contribution is the ability to calculate the
concentrations of both glucose and interferent in the sample.
[0110] Some exemplary but non-limiting mathematical equations
parameterising the association behaviour of a mixed solution of
glucose and interferent in the presence of one or two receptors are
presented in the Examples section below. It should be emphasised
that these equations are generally applicable to the methods
provided herein and are not in any way limited to the specifically
described methods and systems (e.g., the particular hydrogels or
interferent species) that are used in these Examples.
[0111] In certain cases in which the second measurement method
includes electrochemically measuring the amount of glucose in the
sample, the step of "comparing the first measurement and the second
measurement, and thereby determining whether any interferent is
present in the sample" can include initially assessing whether the
first (fluorescence emission--susceptible to interference from
mannitol) and second measurements (electrochemical--not susceptible
to interference from interferent) indicate substantially the same
glucose concentration. If there is no significant difference, then
it will be appreciated that no further steps are required
specifically to account for interference (there being no
interferent in the sample). However, if there is a significant
difference, then the presence of interferent in the sample can be
accounted for by carrying out further measurement(s) at later time
point(s), i.e. after the interferent has been sufficiently
metabolised in vivo, as described elsewhere herein.
[0112] Similarly, in certain cases when the second measurement
method includes electrochemically measuring the amount of
interferent in the sample, the step of "comparing the first
measurement and the second measurement, and thereby determining
whether any interferent is present in the sample" can include
initially assessing whether the second (electrochemical)
measurement is indicative of interferent being present in the
sample. If not, then it will be appreciated that no further steps
are required specifically to account for interference (there being
no interferent in the sample). However, if the second measurement
does indicate the presence of interferent, then this presence of
interferent in the sample can be accounted for by carrying out
further measurement(s) at later time point(s), i.e. after the
interferent has been sufficiently metabolised in vivo, as described
elsewhere herein.
[0113] Methods and systems provided herein can includes a first
indicator system that is pre-calibrated for detection sensitivity
to both glucose and interferent. The second indicator system can
also be pre-calibrated if present.
The Glucose Sensor
[0114] The present document also provides a glucose sensor. The
glucose sensor can be adapted for carrying out the methods provided
herein. In some cases, the glucose sensors provided herein can be
adapted to carry out a method in which the second measurement
method involves detecting fluorescence emission from a second
fluorophore, as described herein.
[0115] The constituent elements of the glucose sensors provided
herein can correspond to those described in the discussion of the
methods provided herein above. For example, the sensing region, the
first indicator system, the first receptor, the first fluorophore,
the second indicator system, the second receptor, the second
fluorophore and the optical waveguide, and preferred aspects
thereof, are all as herein described with reference to the methods
provided herein as discussed above. Since both the first indicator
system and the second indicator system are comprised within the
sensing region, they are configured such that when contacting the
glucose sensor with the sample, both the first indicator system and
the second indicator system come into contact with the sample.
Thus, typically the sensing region contains a cell or chamber in
which both the first and second indicator systems are
contained.
[0116] The glucose sensor may be an equilibrium glucose sensor.
[0117] For the avoidance of doubt, it is emphasised that throughout
the present specification, references to a receptor (e.g., the
first or second receptor) being "for binding to glucose" means that
the receptor is capable of binding to glucose (i.e., has a non-zero
association constant with glucose). Consequently the indicator
system comprising such a receptor, together with a corresponding
fluorophore, is sensitive to the concentration of glucose in the
sample.
The Interferent
[0118] As used herein, an "interferent" is a substance that is
capable of interfering with the binding of glucose to the first
receptor comprised in the sensing region of the glucose sensor.
Typically, the substance is capable of interfering with the glucose
binding by being capable itself of binding to the first receptor
(i.e., and thus of competing with glucose for the receptor sites in
the glucose sensor). However, other means of interfering can be
addressed by the methods and sensors described herein, for example
where the substance is capable of interacting with (e.g., binding
to) the glucose in such a way as to modify the ability of the
glucose to bind to the first receptor.
[0119] Typically an interferent that is "capable of interfering
with the binding of glucose to the first receptor" is an
interferent that has a non-zero association constant with the first
receptor (and therefore can compete with glucose for the receptor).
For the example, the association constant of the interferent with
the first receptor may be at least 1%, or at least 10%, or least
25% of the association constant of glucose with the first
receptor.
[0120] Typically the interferent is a substance other than protons
or hydroxyl ions. Typically the interferent is a substance that is
capable of interfering with the binding of glucose to the first
receptor comprised in the sensing region of the glucose sensor by a
means other than by merely modifying the pH of the sample.
[0121] The sample may comprise one interferent or a plurality of
different (i.e., chemically different) interferents. Where a
plurality of different interferents are present, the methods
provided herein can still enable the amount of glucose in the
sample to be quantified.
[0122] The interferent may be a sugar alcohol or a saccharide other
than glucose. Sugar alcohols and saccharides of particular interest
include those that may be present in significant quantities in the
blood.
[0123] The interferent may comprise a cis-diol group. For example,
the interferent may be a polyol (e.g., a sugar alcohol or a
saccharide) that comprises a cis-diol group. Substances that
comprise a cis-diol group are particularly likely to be capable of
binding to boronic acid receptors such as the first receptor
comprised in the glucose sensors described herein. For the
avoidance of doubt, the term "comprise(s) a cis-diol group" does
not mean that no hydroxyl groups may be present in the interferent
other than those comprised in the cis-diol group. Thus, for
example, the interferent may be a polyol (e.g., a sugar alcohol or
saccharide) containing at least a cis-diol group and may therefore
contain two, three, four, five, six, or even more than six hydroxyl
groups in total.
[0124] The interferent may be a sugar alcohol or saccharide
selected from the group consisting of mannitol, sorbitol,
galactitol, inositol, fructose, galactose and arabinose. The
interferent may be mannitol.
[0125] The interferent may also be an amine, i.e. a compound that
contains an amine functional group. The amine functional group may
be a primary, secondary or tertiary amine group. Amines of
particular interest include those that may be present in
significant quantities in the blood. The amine may, for example, be
selected from glutamine and catechol amines (e.g., epinephrine,
norepinephrine and dopamine).
[0126] The interferent may have a molecular weight of less than
1000 daltons, such as less than 500 daltons, or less than 300
daltons, or less than 200 daltons. The present systems and methods
may be particularly useful in the context of these low molecular
weight interferents, since it may be difficult or impossible to
exclude such interferents from the sensing region of the glucose
sensor through use of a barrier layer of the type described
elsewhere herein.
[0127] Methods and sensors provided herein are described below with
reference to particular Examples. The invention is not intended to
be limited to these particular Examples.
EXAMPLES
[0128] The following hydrogels were used in the working
Examples:
##STR00004##
Example 1
[0129] This Example shows that when a second measurement (for
example an electrochemical measurement) is obtained that determines
the concentration of a mannitol interferent, the first measurement
can be corrected based on that known mannitol concentration to
obtain a corrected value of the glucose concentration.
General Equations for Competitive Binding for Two Analytes Using
One Receptor
[0130] The following equilibria are present when a host, "H", can
bind to the analytes D-glucose, "G", and D-mannitol, "M":
##STR00005##
Three equilibria are present:
H+G-HG (1)
H+M-HM (2)
HG+M-HM+G (3),
leading to three association constants:
K G = [ HG ] [ H ] [ G ] ( 4 ) K M = [ HM ] [ H ] [ M ] ( 5 ) K
Disp = [ HM ] [ G ] [ HG ] [ M ] = K M K G . ( 6 ) ##EQU00001##
From mass balance, it is also known that
[G]=[G].sub.i-[HG] (7)
[M]=[M].sub.i-[HM] (8)
[H]=[H].sub.i-[HG]-[HM] (9),
where [X]=equilibrium concentration and [X].sub.i=initial
concentration. Substituting (7) and (9) into (4) gives
K G = [ HG ] [ [ G ] i - [ HG ] ] [ [ H ] i - [ HG ] - [ HM ] ] (
10 ) ##EQU00002##
Rearrangement of (10) leads to
[ HG ] 2 + [ [ HM ] - [ G ] i - [ H ] i - 1 K G ] [ HG ] + [ G ] i
[ [ H ] i - [ HM ] ] = 0 ( 11 ) ##EQU00003##
Assuming that K.sub.G[H].sub.i<<1, the squared term is
negligible and therefore
[ HG ] = [ G ] i [ [ H ] i - [ HM ] ] [ G ] i + [ H ] i - [ HM ] +
1 K G ( 12 ) ##EQU00004##
It can also be assumed that [HM] and [H].sub.i<<[G].sub.i,
and 1/K.sub.g. Therefore (12) simplifies to
[ HG ] = [ G ] i [ [ H ] i - [ HM ] ] [ G ] i + 1 K G , ( 13 )
##EQU00005##
which can be rewritten as
[ HG ] [ H ] i = [ 1 - [ HM ] [ H ] i ] K G [ G ] i 1 + K G [ G ] i
( 14 ) ##EQU00006##
By substituting (8) and (9) into (5) and carrying out the same
manipulations one obtains
[ HM ] [ H ] i = [ 1 - [ HG ] [ H ] i ] K M [ M ] i 1 + K M [ M ] i
( 15 ) ##EQU00007##
(14) and (15) can be solved for [HG] and [HM] by writing them in
the matrix form
[ 1 + K G [ G ] i K G [ G ] i K M [ M ] i 1 + K M [ M ] i ] [ [ HG
] [ H ] i [ HM ] [ H ] i ] = [ K G ? [ ? ] K M [ ? ] ] ? indicates
text missing or illegible when filed ( 16 ) ##EQU00008##
and inverting the matrix:
[ [ HG ] [ H ] i [ HM ] [ H ] i ] = 1 .DELTA. [ 1 + K M [ M ] i - K
G [ G ] i - K M [ M ] i 1 + K G [ G ] i ] [ K G [ ? ] K M [ ? ] ] ,
? indicates text missing or illegible when filed ( 17 )
##EQU00009##
where the determinant is
.DELTA. = 1 + K G [ G ] i + K M [ M ] I Thus , ( 18 ) [ HG ] [ H ]
i = K G [ G ] i 1 + K G [ G ] i + K M [ ? ] ( 19 ) [ HG ] [ H ] i =
K M [ M ] i 1 + K G [ G ] i + K M [ ? ] ? indicates text missing or
illegible when filed ( 20 ) ##EQU00010##
For intensity measurements
i = I - I 0 ( 21 ) .DELTA. I = I .dwnarw. ( - I .dwnarw. 0 ( 22 ) i
= [ [ HG ] [ H ] i ] .DELTA. I G + [ [ HG ] [ H ] i ] .DELTA. I M (
23 ) ##EQU00011##
Substituting (19) and (20) into (23) gives
i = [ K G [ G ] i 1 + K G [ G ] i + K M [ M ] i ] .DELTA. I G + [ K
M [ M ] i 1 + K G [ G ] i + K M [ M ] i ] .DELTA. I M ( 24 )
##EQU00012##
Substituting (21) and (22) into (24) and rearranging leads to
I = I 0 + I .infin. G K G [ G ] i + I .infin. M K M [ M ] i 1 + K G
[ ? ] + K M [ ? ] ? indicates text missing or illegible when filed
( 25 ) ##EQU00013##
[0131] Assuming that I.sub..infin.G=I.sub..infin.M results in a
further simplification of (25) to (26), which allows calculation of
the intensity of a fluorophore emission when two analytes are
present (in this case glucose and mannitol). Rearrangement of (26)
leads to (27) and (28) which allow one to determine the
concentration of a first analyte if the concentration of the second
analyte and the fluorescent response of a sensor are known:
I = I 0 + I .infin. [ K G [ G ] i + K M [ M ] i ] 1 + K G [ ? ] i +
K M [ ? ] ( 26 ) [ G ] i = I - I 0 + ( I - I .infin. ) K M [ M ] i
K G ( I .infin. - I ) ( 27 ) [ M ] i = I - I 0 + ( I - I .infin. )
K G [ G ] i K M ( I .infin. - I ) ? indicates text missing or
illegible when filed ( 28 ) ##EQU00014##
Calibration of Sensor Containing Hydrogel 3 Against D-Mannitol and
D-Glucose
[0132] Hydrogel 3 was used to demonstrate that a sensor can be
calibrated against both D-mannitol and D-glucose. The results of
these calibrations are shown below in Table 1.
TABLE-US-00001 TABLE 1 Calibration constants for hydrogel 3 vs.
D-glucose and D-mannitol. [Glc]/mM I [Man]/mM I 0.00 0.9776 0.00
0.9601 17.48 1.9571 11.64 1.6330 34.23 2.3871 21.85 2.0025 K.sub.G
= 0.0346 K.sub.M = 0.0274 I.sub.0 = 0.9776 I.sub.0 = 0.9601
I.sub..infin. = 3.5782 I.sub..infin. = 3.7463 Mod.sub.5mM % = 28.2
Mod.sub.5mM % = 25.9
[0133] It can be observed from K.sub.G and K.sub.M that the
di-boronic acid receptor of hydrogel 3 is more selective for
D-glucose than D-mannitol. I.sub.0 for both analytes are the same
and the differences observed in measuring I.sub..infin. are
probably due to error from the calibrations.
Example 2
[0134] This Example shows that a glucose sensor incorporating two
receptors that have different relative association constants for
glucose and mannitol can be used to correct for the presence of
mannitol interferent, and therefore obtain accurate glucose
concentration measurements, in a sample containing both glucose and
mannitol.
General Equations for Competitive Binding for Two Analytes Using
Two Receptors
[0135] The following equilibria are present when two hosts,
"H.sub.1" and "H.sub.2", can bind to the analytes D-glucose, "G",
and D-mannitol, "M".
##STR00006##
The equilibria present are:
H.sub.1+G-H.sub.1G (29)
H.sub.1+M-H.sub.1M (30)
H.sub.1G+M-H.sub.1M+G (31)
H.sub.2+G-H.sub.2G (32)
H.sub.2+M-H.sub.2M (33)
H.sub.2G+M-H.sub.2M+G (34),
leading to six association constants:
K G 1 = [ H 1 G ] [ H 1 ] [ G ] ( 35 ) K M 1 = [ H 1 M ] [ H 1 ] [
M ] ( 36 ) K Disp = [ H 1 M ] [ G ] [ H 1 G ] [ M ] = K M 1 K G 1 (
37 ) K G2 = [ H 2 G ] [ H 2 ] [ G ] ( 38 ) K M 2 = [ H 2 M ] [ H 2
] [ M ] ( 39 ) K Disp = [ H 2 M ] [ G ] [ H 2 G ] [ M ] = K M 2 K G
2 ( 40 ) ##EQU00015##
From mass balance it is also known that:
[G]=[G].sub.i-[H.sub.1G]-[H.sub.2G] (41)
[M]=[M].sub.i-[M.sub.1G]-[M.sub.2G] (42)
[H.sub.1]=[H.sub.1].sub.i-[H.sub.2G]-[H.sub.2M] (43)
[H.sub.2]=[H.sub.2].sub.i-[H.sub.2G]-[H.sub.2M] (44),
where [X]=equilibrium concentration and [X].sub.i=initial
concentration. Substituting (41) and (43) into (35) gives
K G 1 = [ H 1 G ] [ [ G ] i - [ H 1 G ] - [ H 2 G ] ] [ [ H ] i - [
H 1 G ] - [ H 1 M ] ] ( 45 ) ##EQU00016##
Rearrangement of (45) leads to
[ H 1 G ] 2 + [ [ H 1 M ] + [ H 2 G ] - [ G ] i - [ H 1 ] i - 1 K G
1 ] [ H 1 G ] + [ [ H 1 ] i - [ H 1 M ] ] [ [ G ] i - [ H 2 G ] ] =
0 ( 46 ) ##EQU00017##
Assuming that K.sub.G[H].sub.i<<1 means the squared term is
negligible and therefore that
[ H 1 G ] = [ [ H 1 ] i - [ H 1 M ] ] [ [ G ] i - [ H 2 G ] ] [ G ]
i + [ H 1 ] i - [ H 1 M ] - [ H 2 G ] + 1 K G 1 ( 47 )
##EQU00018##
Since host concentrations are assumed to be extremely small one can
also approximate this further by dropping the [H.sub.2G] and
[H.sub.1M] terms compared to [G].sub.i and 1/K.sub.g1
[ H 1 G ] = [ G ] 1 [ [ H 1 ] i - [ H 2 G ] ] [ G ] i + 1 K G 1 , (
48 ) ##EQU00019##
which can be rewritten as
[ H 1 G ] [ H 1 ] i = [ 1 - [ H 1 M ] [ H 1 ] i ] K G 1 [ G ] i 1 +
K G 1 [ G ] i ( 49 ) ##EQU00020##
If one substitutes (42) and (43) into (36) and carries out some
manipulations one obtains
[ H 1 M ] [ H 1 ] i = [ 1 - [ H 1 G ] [ H 1 ] i ] K M 1 [ M ] I 1 +
K M 1 [ M ] I ( 50 ) ##EQU00021##
(49) and (50) can be solved for [H.sub.1G] and [H.sub.1M] by
writing them in the matrix form
[ 1 + K G 1 [ G ] i K G 1 [ G ] i K M 1 [ M ] I 1 + K M 1 [ M ] I ]
[ [ H 1 G ] [ H 1 ] i [ H 1 M ] [ H 1 ] i ] = [ K G 1 [ G ] i K M 1
[ M ] I ] , ( 51 ) ##EQU00022##
and inverting the matrix
[ [ H 1 G ] [ H 1 ] i [ H 1 M ] [ H 1 ] i ] = 1 .DELTA. [ 1 + K M 1
[ G ] i - K G 1 [ G ] i - K M 1 [ M ] I 1 + K G 1 [ G ] i ] [ K G 1
[ G ] i K M 1 [ M ] I ] , ( 52 ) ##EQU00023##
where the determinant is
.DELTA. = 1 + K G 1 [ G ] i + K M 1 [ M ] I Thus , ( 53 ) [ H 1 G ]
[ H 1 ] i = K G 1 [ G ] i 1 + K G 1 [ G ] i + K M 1 [ M ] I ( 54 )
[ H 1 M ] [ H 1 ] i = K M 1 [ M ] I 1 + K G 1 [ G ] i + K M 1 [ M ]
I ( 55 ) ##EQU00024##
For the second host, and using the same method and approximations,
one obtains
[ H 2 G ] [ H 2 ] i = K G 2 [ G ] i 1 + K G 2 [ G ] i + K M 2 [ M ]
I ( 56 ) [ H 2 M ] [ H 2 ] i = K M 2 [ M ] I 1 + K G 2 [ G ] i + K
M 2 [ M ] I ( 57 ) ##EQU00025##
The light intensities for the two fluorophores can be given by
i 1 = [ [ H 1 G ] [ H 1 ] i ] .DELTA. I G 1 + [ [ H 1 M ] [ H 1 ] I
] .DELTA. I M 1 ( 58 ) i 2 = [ [ H 2 G ] [ H 2 ] i ] .DELTA. I G 2
+ [ [ H 2 M ] [ H 2 ] I ] .DELTA. I M 2 ( 59 ) ##EQU00026##
(54) and (55) can then be substituted into (58) to give
i 1 = K G 1 [ G ] i .DELTA. I G 1 + K M 1 [ M ] i .DELTA. I M 1 1 +
K G 1 [ G ] I + K M 1 [ M ] I ( 60 ) ##EQU00027##
Expanding and rearranging gives
i.sub.1=(.DELTA.I.sub.G1-i.sub.1)K.sub.G1[G].sub.i+(.DELTA.I.sub.M1-i.su-
b.1)K.sub.M1[M].sub.i (61)
Similarly, from substituting (56) and (57) into (59)
i.sub.2=(.DELTA.I.sub.G2-i.sub.2)K.sub.G2[G].sub.i+(.DELTA.I.sub.M2-i.su-
b.2)K.sub.M2[M].sub.i (62)
Writing (61) and (62) in a matrix form and inverting gives
[ [ G ] i [ M ] i ] = 1 .DELTA. [ ( .DELTA.I M 2 - i 2 ) K M 2 - (
.DELTA. I M 1 - i 1 ) K M 1 - ( .DELTA. I g 2 - i 2 ) K G 2 (
.DELTA. I G 1 - i 1 ) K G 1 ] [ i 1 i 2 ] , ( 63 ) ##EQU00028##
where the determinant is
.DELTA. = ( .DELTA. I G 1 - i 1 ) K G 1 ( .DELTA. I M 2 - i 2 ) K M
2 - ( .DELTA. I G 2 - i 2 ) K G 2 ( .DELTA. I M 1 - i 1 ) K M 1
Thus , ( 64 ) [ G ] i = ( .DELTA. I M 2 - i 2 ) K M 2 i 1 - (
.DELTA. I M 1 - i 1 ) K M 1 i 2 ( .DELTA. I G 1 - i 1 ) K G 1 (
.DELTA. I M 2 - i 2 ) K M 2 - ( .DELTA. I g 2 - i 2 ) K G 2 (
.DELTA. I M 1 - i 1 ) K M 1 ( 65 ) ##EQU00029##
Substituting (21) and (22) into (65) gives
[ G ] i = K M 2 ( I .infin. M 2 - I 2 ) ( I 1 - I 01 ) - K M 1 ( I
.infin. M 1 - I 1 ) ( I 2 - I 02 ) K G 1 K M 2 ( I .infin. G 1 - I
1 ) ( I .infin. M 2 - I 2 ) - K G 2 K M 1 ( I .infin. g 2 - I 2 ) (
I .infin. M 1 - I 1 ) , ( 66 ) ##EQU00030##
and similarly
[ G ] i = K G 1 ( I .infin. G 1 - I 1 ) ( I 2 - I 02 ) - K G 2 ( I
.infin. G 2 - I 2 ) ( I 1 - I 01 ) K G 1 K M 2 ( I .infin. G 1 - I
1 ) ( I .infin. M 2 - I 2 ) - K G 2 K M 1 ( I .infin. g 2 - I 2 ) (
I .infin. M 1 - I 1 ) . ( 67 ) ##EQU00031##
Results Obtained on a Sensor Containing Hydrogel 4 Against
D-Mannitol and D-Glucose
[0136] Two separate boronic acid sensors were immobilised within
hydrogel 4. One of these was a di-boronic acid that had similar
association constants for both D-glucose and D-mannitol. The second
was a mono-boronic acid that was more selective towards D-mannitol.
FIG. 7 shows the fluorescent spectra of hydrogel 4 when it was
excited at selected wavelengths with increasing D-glucose or
D-mannitol concentrations. It is possible to calculate association
constants for each of these sensors independently and these results
are shown in Table 2 below.
TABLE-US-00002 TABLE 2 Association constants for hydrogel 4 to
D-glucose and D-mannitol when excited at two differing wavelengths.
D-Glucose D-Mannitol .lamda..sub.ex = 350 .lamda..sub.ex = 405
.lamda..sub.ex = 350 .lamda..sub.ex = 405 .lamda..sub.em = 380
.lamda..sub.em = 487 .lamda..sub.em = 380 .lamda..sub.em = 487
I.sub.0 1.000 1.000 1.000 1.000 I.sub..infin. 3.915 2.725 4.077
4.699 K/mM.sup.-1 0.005 0.020 0.063 0.009
[0137] The selectivity of the two boronic acid receptors for
D-glucose and D-mannitol are different from each other. The
algorithms (66) and (67) can therefore be used to determine both
analyte concentrations in a solution containing glucose and
mannitol and thus to correct for the presence of mannitol
interferent when seeking to obtain an accurate measurement of
glucose concentration.
* * * * *