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BACKGROUND OF THE INVENTION
Determination of glucose concentration has applications in clinical
settings, such as for the day-to-day monitoring of glucose levels in
individuals in whom glucose homeostasis is not maintained (e.g., in
diabetes or hypoglycemia) and in biomedical research.
Current methods for determining glucose concentrations include various
colorimetric reactions, measuring a spectrophotometric change in the
property of any number of products in a glycolytic cascade or measuring
the oxidation of glucose using a polarographic glucose sensor. For
example, U.S. Pat. No. 4,401,122 discloses an in vivo method for measuring
glucose, which involves placing an enzyme (e.g., glucose oxidase) either
in or under the skin and detecting the enzymatic reaction product (e.g.,
oxygen) directly through the skin using polarographic or enzyme
electrodes. The amount of enzymatic reaction product detected is a measure
of the amount of substrate.
Although conventional assays have proven reliable, the reagents on which
they rely become exhausted in the presence of glucose. Therefore, these
assays require the use of disposable sticks or replaceable cartridges,
which can be expensive and inconvenient for the active user.
Meadows and Schultz describe another method by which blood glucose levels
can be determined using optical means. They describe a fiber optic glucose
sensor based on the competitive binding of glucose and
fluorescein-labelled dextran (FITC-dextran) to rhodamine-labelled
concanavalin A (Rh-Con A), Meadows, D. and J. S. Schultz, Talanta,
35:145-150 (1988).
The Meadows and Schultz optical sensor is attended by many problems, which
means it is of limited use in a clinical setting or in monitoring blood
glucose levels in individuals on a day to day basis. First, as mentioned
in the article, the sensor can only detect glucose concentrations up to
2.00 mgs/ml. Although the normal physiologic blood glucose concentration
in man is approximately 1.00 mg/ml., the concentration of glucose in
diabetic blood can often exceed 3.00-4.00 mg/ml., which is well beyond the
upper limit of the sensor described.
Second, Meadow's and Schultz's sensor has a short life because, as
mentioned in the article, the dextran aggregates and becomes precipitated.
Third, only 45% of the fluorescence is quenched using the Meadows and
Schultz optical sensor. This effect may not be dramatic enough to be
detected.
Finally, the in vivo use of a fiber optic is clinically impractical because
in order to work, it must pierce the skin. Therefore, it requires an
invasive technique and puts the patient at significant risk for developing
serious infection. This is particularly true in diabetic patients who are
known to have reduced resistance to infection.
An ideal glucose sensor should be capable of detecting a wide range of
glucose concentrations (e.g., concentrations ranging from 0.5 to 5.00
mg/ml.). It should also be reliable, reusable and easy to use. In
addition, an in vivo sensor should be non-invasive. Such a sensor would be
of great value in markedly improving therapy in diabetic patients. It
would also have a number of other research and clinical applications.
SUMMARY OF THE INVENTION
The present invention relates to a method of detecting and quantifying
glucose in a body fluid using non-radiative fluorescence resonance energy
transfer (FRET) and devices useful in carrying out the present method of
glucose detection and measurement.
In the method of the present invention, the body fluid is contacted with a
specific binding pair, the two members of which are a labelled ligand and
a labelled carbohydrate-containing receptor (referred to as a labelled
glycoconjugate) which binds specifically to the ligand in competition with
glucose. Each member of the binding pair is labelled with a
light-absorbing material. In general, two light-absorbing materials with
overlapping excited state energy levels are used, one affixed to the
ligand and one to the glycoconjugate.
In particular, in the method of the present invention, two
fluorescently-labelled substances, a ligand (e.g., lectin, monoclonal
antibody) and a carbohydrate-containing receptor or glycoconjugate which
binds specifically to the ligand in competition with glucose are brought
into contact with the body fluid, either within the body or as a sample
obtained using known methods. Each member of the specific binding pair is
labelled with a fluorophore; generally, they are labelled with two
different fluorophores. For the purposes of FRET, the ligand can be
labelled with either a donor or an acceptor molecule. If the ligand is
labelled with a donor, the glycoconjugate is generally labelled with an
acceptor. If the ligand is labelled with an acceptor, the glycoconjugate
is generally labelled with a donor.
FRET occurs only when the donor and acceptor are in sufficiently close
proximity to one another (generally, within 100 angstroms). In the method
of the present invention, FRET results when the ligand and glycoconjugate
in the sample bind. However, in the presence of glucose, these molecules
are competed off (i.e., glucose competes with the glycoconjugate in
binding with the ligand). Thus, the presence and concentration of glucose
in a sample are indicated as a decrease in the efficiency of energy
transfer. Competition with glucose occurs in a dose-dependent manner and
is reversible. In addition, because the glycoconjugates are stable, the
devices of the invention can be reused for extended periods of time.
The method of the present invention makes use of the fact that when two
light-absorbing materials with overlapping excited state energy levels are
in sufficiently close proximity, a resonance dipole-induced dipole
interaction occurs and, as a result, the excited state energy of the donor
molecule is transferred to the acceptor molecule, resulting in quenching
of the donor fluorescence and sensitized emission of the acceptor.
Such materials, which are attached to or incorporated in members of the
specific binding pairs (i.e., the ligands and the glycoconjugates) used in
the method of the present invention, can be, for example, fluorescent
materials, such as fluorescein and rhodamine. Alternatively, one (the
donor) can be fluorescent in nature and the other (the acceptor)
nonfluorescent. The reverse is also possible (i.e., nonfluorescent donor,
fluorescent acceptor).
A number of devices can be constructed and used to detect glucose
concentration in blood or other samples (e.g., urine or extracellular
fluid), either in vivo or in vitro, using the method of the present
invention. In its broader aspect, the in vivo embodiment of this invention
is directed to measurement of glucose by placing reactants (e.g.,
fluorescently-labelled ligand and glycoconjugate labelled with a second
fluorophore) in communication with (e.g., contacting) glucose present in a
body fluid. For example, the reactants can be placed in, on, or under the
skin, and cutaneous measurement of glucose carried out. Alternatively, the
two reactants can be introduced into an organ or vessel in which
communication of glucose with the reactants is possible. In the embodiment
in which reactants are placed in, on or under the skin, glucose is
detected and quantified by illuminating the skin (e.g., at the excitation
wavelength of the donor). The measure of energy transfer, as detected by a
fluorimeter, is then either the ratio of fluorescence intensities at the
two emission maximum wavelengths or the quenching of the donor
fluorescence at its emission maximum as a function of glucose
concentration.
A variety of modes of placing the reactants in communication with glucose
may be employed in accordance with the principles of the in vivo
embodiment of this invention. In addition, any mode of placing the
reactants in communication with glucose can be modified to provide
feedback for an insulin pump.
In its broader aspect, the in vitro embodiment of this invention is
directed to placing the reactants in communication with a sample of body
fluid containing glucose (e.g., blood, urine, extracellular fluid), such
as by contacting the fluid with a dipstick on which the reactants are
affixed. Glucose is detected and quantified by placing the reactants in
communication with the glucose-containing body fluid in a fluorimeter. As
with the in vivo embodiment, a variety of modes of placing the reactants
in communication with glucose may be employed.
The method and devices of the present invention can be used to detect a
wide range of glucose concentrations (e.g., concentrations ranging from
0.5 to 18 mg/ml.). In addition, the method is reliable, because the
reactants do not interfere with the determination by aggregating and the
size of the effect, as measured by the ratio of fluorescence intensities
at the emission maximum wavelengths of the donor and/or acceptor or the
quenching of the donor at its emission maximum as a function of glucose,
is large enough that it is easily detected.
Also, because the reactants are not consumed, the devices are reusable for
extended periods. Finally, the in vivo embodiments are completely
non-invasive or are non-invasive after one implant.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graphic representation of absorbance and emission spectra of
donor and acceptor molecules.
FIG. 1B is a representation of non-radiative energy transfer.
FIG. 2 is a graphic representation of the use of FRET to measure glucose
concentrations in a sample.
FIG. 3 is a graph representing the effect that increasing concentrations of
glucose have on FRET between the fluorescently labelled ligand,
Rhodamine-ConA (RC) and the fluorescently labelled glycoconjugate,
fluorescein BSA-Glucose (FBG) over time.
FIG. 4 is a graph representing FRET between FBG and RC dialyzed against
various concentrations of glucose in Hanks Buffered Salt Solution (HBSS).
FIG. 5 is a bar graph of Fl.sub.520 /Fl.sub.600 ratio for a sample of FBG
and RC dialyzed sequentially against variour different glucose
concentrations in the following: (reading left to right on the X axis) (O)
Hanks Buffered Salt Solution; (HS) normal horse serum; (+5) horse serum +5
mM glucose; (HS) horse serum; (HO) normal horse serum +10 mM glucose; (HS)
normal horse serum; (O) Hanks Balanced Salt Solution; (+10) normal horse
serum +10 mM glucose; (HS) horse serum; (O) Hanks Balanced Salt Solution.
FIG. 6A is a graph representing FRET between FBG and RC with no glucose
present (control).
FIG. 6B is a graph representing FRET between FBG and RC microdialyzed
against blood containing 3.2 mg/ml. of glucose, similar to what might be
found in diabetic patients.
FIG. 7 is a graph representing FRET in response to 150 uls of a mixture of
RC and FBG microdialyzed against blood containing varying concentrations
of glucose.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of detecting and quantifying
glucose in a body fluid and to devices useful in carrying out the present
method of glucose detection and measurement. The present method relies on
the process of non-radiative fluorescence resonance energy transfer (FRET)
to determine the occurrence and extent of binding between members of a
specific binding pair which is competitively decreased by glucose. Members
of the binding pair are a ligand (e.g., a lectin, monoclonal antibody) and
a carbohydrate-containing receptor (referred to as a glycoconjugate),
which binds specifically to the ligand in competition with glucose. Both
the ligand and the glycoconjugate are fluorescently labelled, but
typically are not labelled with the same fluorophore. They are brought
into contact with a sample (e.g., blood, urine, extracellular fluid) in
which glucose concentration is to be determined.
The present method is particularly useful in the day-to-day monitoring of
glucose concentrations in individuals in whom glucose homeostasis is
compromised (e.g., diabetic or hypoglycemic individuals) and in biomedical
research.
Basic Elements of FRET
FRET generally involves the non-radiative transfer of energy between two
fluorophores, one an energy donor (D) and the other an energy acceptor
(A). Any appropriately selected donor-acceptor pair can be used, provided
that the emission of the donor overlaps with the excitation spectra of the
acceptor and both members can absorb light energy at one wavelength and
emit light energy of a different wavelength.
The method is described below with particular reference to fluorescein and
rhodamine as the donor-acceptor pair. As used herein, the term fluorescein
refers to a class of compounds which includes a variety of related
compounds and their derivatives. Similarly, as used herein, the term
rhodamine refers to a class of compounds which includes a variety of
related compounds and their derivatives. Other examples of donor/acceptor
pairs are NBD N-(7-mitrobenz-2-oxa-1,3-diazol-4-yl) to rhodamine, NBD or
fluorescein to eosin or erythrosis, dansyl to rhodamine, acridine orange
to rhodamine.
Alternatively, one molecule (the donor) can be fluorescent and the other
(the acceptor) can be nonfluorescent. It is also possible to make use of a
donor-acceptor pair in which the acceptor is not normally excited at the
wavelength used to excite the (fluorescent) donor; however, nonradiative
FRET causes acceptor excitation.
Although the donor and the acceptor are referred to herein as a "pair", the
two "members" of the pair can, in fact, be the same substance. Generally,
the two members will be different (e.g., fluorescein and rhodamine). It is
possible for one molecule (e.g., fluorescein, rhodamine) to serve as both
donor and acceptor; in this case, energy transfer is determined by
measuring depolarization of fluorescence.
The concept of FRET is represented in FIG. 1. The absorbance and emission
of donor, designated A(D), and E(D), respectively, and the absorbance and
emission of acceptor, designated A(A) and E(A), respectively, are
represented graphically in FIG. 1A. The area of overlap between the donor
emission and the acceptor absorbance spectra (which is the overlap
integral) is of importance. If excitation occurs at wavelength I, light
will be emitted at wavelength II by the donor, but not at wavelength III
by the acceptor because the acceptor does not absorb light at wavelength
I.
The non-radiative transfer process which occurs is represented in FIG. 1B.
D molecule absorbs the photon whose electric field vector is represented
by E. The excited state of D is shown as a dipole with positive charge on
one side and negative charge on the other. If an acceptor molecule (A) is
sufficiently close to D (e.g., typically less than 100 Angstroms), an
oppositely charged dipole is induced on it (it is raised to an excited
state). This dipole-induced dipole interaction falls off inversely as the
sixth power of donor-acceptor intermolecular distance.
Classically, partial energy transfer can occur. However, this is not what
occurs in FRET, which is an all or nothing quantum mechanical event. That
is, a donor is not able to give part of its energy to an acceptor. All of
the energy must be transferred and energy transfer can occur only if the
energy levels (i.e., the spectra) overlap. When A leaves its excited
state, the emitted light is rotated or depolarized with respect to the
incident light. As a result, FRET manifests itself as a decrease in
fluorescence intensity (i.e., decrease in donor emission) at II, an
appearance of fluorescence intensity at III (i.e., an increase in
sensitized emission) and a depolarization of the fluorescence relative to
the incident light.
A final manifestation of FRET is in the excited state lifetime.
Fluorescence can be seen as an equilibrium process, in which the length of
time a molecule remains in its excited state is a result of competition
between the rate at which it is being driven into this state by the
incident light and the sum of the rates driving it out of this state
(fluorescence and non-radiative processes). If a further nonradiative
process, FRET, is added (leaving all else unchanged), decay is favored,
which means donor lifetime at II is shortened.
When two fluorophores whose excitation and emission spectra overlap are in
sufficiently close proximity, the excited state energy of the donor
molecule is transferred by a resonance dipole-induced dipole interaction
to the neighboring acceptor fluorophore. In FRET, a sample or mixture is
illuminated at a wavelength which excites the donor but not the acceptor
molecule directly. The sample is then monitored at two wavelengths: that
of donor emissions and that of acceptor emissions. If donor and acceptor
are not in sufficiently close proximity, FRET does not occur and emissions
occur only at the donor wavelength. If donor and acceptor are in
sufficiently close proximity, FRET occurs. The results of this interaction
are a decrease in donor lifetime, a quenching of donor fluorescence, an
enhancement of acceptor fluorescence intensity, and depolarization of
fluorescence intensity. The efficiency of energy transfer, E.sub.t, falls
off rapidly as the distance between donor and acceptor molecule, R,
increases. For an isolated donor acceptor pair, the efficiency of energy
transfer is expressed as:
E.sub.t =1/[1+(R/R.sub.o).sup.6 ] (1)
where R is the separation distance between donor and acceptor and R.sub.o
is the distance for half transfer. R.sub.o is a value that depends upon
the overlap integral of the donor emission spectrum and the acceptor
excitation spectrum, the index of refraction, the quantum yield of the
donor, and the orientation of the donor emission and the acceptor
absorbance moments. Forster, T., Z Naturforsch. 4A, 321-327 (1949);
Forster, T., Disc. Faraday Soc. 27, 7-17 (1959).
Because of its 1/R.sup.6 dependence, FRET is extremely dependent on
molecular distances and has been dubbed "the spectroscopic ruler".
(Stryer, L., and Haugland, R. P., Proc. Natl. Acad. Sci. USA, 98:719
(1967). For example, the technique has been useful in determining the
distances between donors and acceptors for both intrinsic and extrinsic
fluorophores in a variety of polymers including proteins and nucleic
acids. Cardullo et al. demonstrated that the hybridization of two
oligodeoxynucleotides could be monitored using FRET (Cardullo, R., et al.,
Proc. Natl. Acad. Sci., 85:8790-8794 (1988)).
Concept of Using FRET to Measure Glucose Concentrations
The concept of using FRET to measure glucose concentrations in solution is
represented in FIG. 2. One macromolecule (designated M) includes a number
of covalently-bound fluorophores and glucose residues and is referred to
as a glycoconjugate. A second macromolecule (designated L) includes a
ligand which has a high degree of specificity for glucose (e.g.,
concanavalin A) and a fluorophore which is generally not the same
fluorophore as that on the first macromolecule.
One of these fluorophores is chosen to be a donor and the other is an
acceptor as described previously. For the purposes of this illustration,
the donor molecule has been placed on the glycoconjugate and the acceptor
has been placed on the ligand. The association can then be diagrammed as:
DMG+AL.fwdarw.DMG--LA,
where DMG stands for Donor-Macromolecule-Glucose, AL stands for
Acceptor-Ligand, and DMG-LA represents the association between the glucose
present in the first complex and the ligand present in the second complex.
Upon association, the two macromolecules are now close enough to allow
energy transfer between the donor and the acceptor to occur.
The presence of free glucose introduces a competitive inhibitor into the
formula because free glucose competes with the conjugated glucose for the
ligand. Thus, increasing concentrations of glucose produces a decrease in
the amount of ligand binding to the glycoconjugate. At relatively low
concentrations of glucose, the transfer efficiency will remain high, since
little of the macromolecular association will be affected. At high
concentrations of glucose, the transfer efficiency will be low, due to the
fact that the glucose has successfully competed the ligand off of the
complementary macromolecule.
As described in the following sections, it has been shown that it is
possible to obtain a reliable, repeatable measure of glucose in a sample
containing glucose concentrations within the range typically found in
normal individuals and in those in whom glucose homeostasis is altered
(e.g., in diabetic and hypoglycemic patients). Further, it has been shown
that the reactants used (i.e., fluorescently-labelled ligand and
glycoconjugate) are stable and can be reused.
Competition experiments in which FRET was measured for various
concentrations of glucose in Hanks Buffered Salt Solution (HBSS) were
conducted. These experiments are described in detail in Example 3. Spectra
were collected by exciting fluorescein at 472 nm and scanning the emission
from 500-650 nm. Typically, fluorescence intensities were monitored at the
emission maxima for fluorescein (about 520 nm) and rhodamine (about 596
nm). The measure of energy transfer in these studies was either the ratio
of fluorescence intensities at 520 nm and 596 nm (i.e., FI 520/FI 596) as
a function of glucose concentration or the quenching of fluorescein at 520
nm.
A number of observations were made during these trials that indicate that
the method of the subject invention provides a reliable means of detecting
glucose over a wide range (i.e., glucose concentrations found in normal,
hypoglycemic and diabetic subjects). Firstly, the compounds were found to
be stable over 2-4 weeks at room temperature. FIG. 3 shows that the
compounds exhibited the same fluorescence properties over a two week time
period in response to glucose concentration. Data in the graph reflect the
change in fluorescence intensity at 520 nm from 0 mM glucose as a function
of glucose concentration.
Secondly, the glucose concentration determined by FRET is in accordance
with measurements using other meters. In a double blind experiment, FRET
was able to predict glucose concentrations within .+-.10% of those
measured using a Direct 30/30 Glucometer in a range of 60-300 mg/dL.
In addition, FIG. 4 shows that the FRET method was able to predict glucose
concentrations accurately at concentrations up to 31 mM glucose
(.about.600 mg/dL), which is well within and in excess of the desired
range. Normal glucose concentrations in blood are usually between 80 and
120 mg/dL and diabetic levels can exceed 500 mg/dL. In the range of 0 to
31 mM glucose, the response by FRET was nearly linear with a coefficient
of determination (r.sup.2) of 0.983. Therefore, the sensor is reliable for
detecting glucose concentrations over the entire physiologic range (i.e.,
for normal and hyperglycemic (diabetic) individuals.
The components of the present invention were also found to be reusable.
When FBG and RC were placed into 8-11 kD cutoff dialysis tubing and
dialyzed against different glucose concentrations in serum, FRET
accurately determined these concentrations in accordance with solution
studies. The initial volume of FBG and RC in HBSS was 2 ml and dialyzed
against different glucose concentrations in 50 ml volumes. In general, it
took approximately 30 minutes to reach a plateau level in the FI.sub.520
/FI.sub.600 ratio. As shown in FIG. 5, the response is reversible as the
dialysis tubing was changed into different glucose concentrations.
Experiments in horse serum showed that the response was not affected by
dialyzed components, but did shift the baseline due to light scattering.
The method of the subject invention can be used to detect and quantify
glucose in samples of a size appropriate for obtaining from an individual
(e.g., 10-100 .mu.l). In assessments carried out using these microsamples
of normal human blood, the glucose level was found to be essentially the
same as a measurement made simultaneously on a conventional glucose meter.
The ability of the present method to determine the glucose concentration in
hyperglycemic blood (i.e., a sample of normal blood augmented with glucose
to produce glucose levels observed in diabetic individuals) was also
assessed. FIG. 6 shows the raw data scans from this experiment. The first
(FIG. 6A) is an emission scan from 500 nm of a sample excited at 472 nm
which does not contain glucose. It shows considerable energy transfer, as
evidenced by the ratio of the two emission peaks. The rhodamine (second)
peak is actually higher than the fluorescein (first) peak.
The second scan depicted in FIG. 6B shows the effect of dialyzing against a
blood sample with hyperglycemic levels of glucose, here 317 mg/dL. The
energy transfer decreases as seen both in the increase of the fluorescein
emission peak and a concurrent decrease in the sensitized rhodamine
emission.
The present method was shown to be effective in assessing glucose levels in
blood and is sensitive in the hyperglycemic range in the following way:
150 .mu.l of a mixture of Rh-Con A and FBG was microdialyzed for 15
minutes against 1500 .mu.l samples of hyperglycemic blood. FIG. 7 shows
that the initial fluorescein/rhodamine fluorescence ratio was just above
1.6 and that the fluorescein/rhodamine fluorescence ratio increased as a
function of glucose concentration, saturating at about 40 mM glucose.
Based on the method of the subject invention a number of devices can be
constructed to detect glucose concentration in blood either in vivo or in
vitro. These devices can remain active for extended periods of time (e.g.,
one month or more) before having to be replaced.
In vivo embodiments of this invention are directed to cutaneous measurement
of glucose by placing the reactants (i.e., fluorescently labelled ligands
and glycoconjugates labelled with a second fluorophore) in communication
with (e.g., contacting) glucose. The reactants can be placed in, on, or
under the skin. Alternatively, the reactants can be placed within an organ
or a vessel (e.g., a vein or artery) in which they are in communication
with glucose, which can then be measured by the present method. In the
embodiment in which the reactants are positioned in, on or under the skin,
glucose is detected by illuminating the skin at the donor excitation
wavelength and monitoring the wavelength for the two fluorescent
materials. For example, if the fluorescent materials are fluorescein and
rhodamine, fluorescence intensities are monitored at 520 nM and 596 nM
(i.e., the respective emission maximum wavelengths).
The measure of energy transfer, as detected by a fluorimeter, is then
either the ratio of fluorescence intensities at the two emission maximum
wavelengths (e.g., FI 520/FI 596) or the quenching of the donor (e.g.,
fluorescein) fluorescence at its emission maximum as a function of glucose
concentration.
A variety of modes of placing the reactants in communication with glucose
may be employed in accordance with the principles of the in vivo
embodiment of this invention. The reactants can be introduced into the
body in any type of supporting or surrounding material which retains the
reactants at the desired location and also allows contact or communication
with glucose such that it can be measured (i.e., its concentration can be
determined by the present method). For example, the reactants may be
encapsulated in a microdialysis vessel or in spheres having an inner
diameter of about 1 mm and 50-100 .mu.m wall thickness and sealed ends.
The encapsulated glucose sensor can be implanted intracutaneously anywhere
in the body. In another procedure, reactants may be mixed with silicone or
fluorocarbon oils and injected subcutaneously. The reactants may also be
tattooed onto the skin or contained in a transcutaneous patch.
Alternatively, the reactants may be modified in such a way that when
injected subcutaneously, they become bound to cell structure and therefore
remain fixed in situ under the skin. For example, RC is known to bind to
cells. In addition, the albumin of the FBG complex can be engineered to
include a reactive group that binds cells.
In order to determine whether such an in vivo glucometer is feasible,
experiments were conducted in which solutions of RC (150 .mu.g/ml) and FBG
(2 .mu.g/ml) were injected into mouse skin. Illumination with a laser at
the appropriate wavelengths produced strong signals from both the
fluorescein and the rhodamine; energy transfer occurred and was detected.
Any in vivo mode of placing the reactants in communication with glucose can
be modified to include an insulin pump. The pump could therefore inject
insulin into a patient upon detection of inappropriately high glucose
levels.
In its broader aspect, the in vitro embodiment of this invention is
directed to placing the reactants in communication with a sample of blood
or other bodily fluids containing glucose (e.g., urine, extracellular
fluid) that has been removed from the body. Glucose is detected and
quantified by placing the reactants in communication with the
glucose-containing bodily fluid in a fluorimeter.
A variety of modes of placing the reactants in communication with glucose
may be employed in accordance with the in vitro embodiment of this
invention. For example, the reactants may be adhered to a solid substrate
(e.g., a stick) or may be contained in a chamber (e.g., a microdialysis
vessel). The reactants may also be contained in a pen cartridge that
dispenses an appropriate volume of the reactants into the blood or other
bodily substance containing glucose.
The subject invention will now be illustrated by the following examples,
which are not to be seen as limiting in any way.
EXAMPLE 1
Preparation of FBG and RC
Fluorescein-BSA-Glucose (FBG) purchased from Sigma and Rhodamine-ConA (RC)
purchased from Molecular Probes were dissolved in Hanks Buffered Salt
Solution (HBSS) from Gibco to a final concentration of 2 mg/ml. The
solutions were then centrifuged at 10,000 g for 30 min. to remove large
particulates. The supernatant was then collected and placed on a 10,000 MW
Amicon ultrafiltration device and centrifuged at 2,000 g until the
ultra-filtrate had passed through the membrane. The retained material was
then resuspended in 2 mls. of HBSS and this procedure was repeated until
no free fluorescein or rhodamine was detected in the ultra-filtrate.
The final retained material was collected and resuspended in 2 ml. of HBSS
and spun one more time at 10,000 g for 30 min. The supernatant was
collected and stored at 4.degree. C. until used for energy transfer
experiments.
EXAMPLE 2
Determination of the Optimal Concentrations of Fluorescein-BSA-Glucose and
Rhodamine-Con A
Before competition experiments could be performed with mannose and glucose,
optimal concentrations of Fluorescein-BSA-Glucose (FBG) and Rhodamine-Con
A (RC) had to be determined. In any solution study using energy transfer,
one must avoid the trivial possibility that FRET is occurring simply
because the molecules are close enough to one another in solution. Thus,
FRET solution studies should ideally be done at concentrations less than 1
mM because at that [] the mol. are separated by 120A units (far enough
apart so specifically interact).
Conversely, in these experiments, the concentrations of fluorophore must be
high enough to be detected and the concentration of the two species (FBG
and RC) must be above their binding constants (K.sub.b s).
Initial concentrations of FBG and RC were approximately 2 mg/ml. Maximum
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