Engineered proteins for analyte sensing

Details Engineered proteins for analyte sensing Engineered proteins for analyte sensing

1999-07-15

2001-03-06

Leary, Louise N. (Department: 1623)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving glucose or galactose

C435S023000, C435S025000, C435S968000, C536S001110, C536S123130, C536S123100

Reexamination Certificate

active

06197534

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the determination of the presence or concentration of an analyte, such as a sugar, in a sample, using a labeled protein sensor.
2. Description of the Related Art
A bibliography follows at the end of the Detailed Description of the Invention. The listed references are all incorporated herein by reference.
Diabetes results in long-term health consequences including cardiovascular disease and blindness. These adverse long-term health consequences result from erratic levels of blood glucose in diabetics. To control the long-term complications associated with diabetes, blood glucose levels must be tightly regulated. This requires careful monitoring of blood glucose involving the unpleasant procedure of drawing blood.
The need for real-time measurement of blood glucose has resulted in efforts to develop non-invasive and minimally invasive methods to monitor blood glucose. A wide variety of methods have been proposed, including near infrared spectroscopy [
1
-
3
], optical rotation [
4
,
5
], amperometric [
6
,
7
], calorimetric [
8
,
9
] and fluorescence detection [
10
-
15
]. In spite of intensive efforts, no method is presently available for non-invasive measurement of blood glucose.
Most glucose sensors that use biological elements for signal transduction use electrochemical or calorimetric detection of glucose oxidase activity. This method is associated with difficulties including the influence of oxygen levels, inhibitors in the blood, and problems with electrodes. In addition, detection results in consumption of the analyte which can cause difficulties when measuring low glucose concentrations. Electrochemical measurements are known to require frequent calibration, which is not acceptable for a continuous glucose monitor.
Using fluorescence, glucose can be measured using fluorophores which respond either to glucose or to proteins such as concanavalin A (ConA). Glucose assays based on proteins are typically competitive assays in which glucose disrupts the binding of ConA to a glucose containing polymer such as dextran. ConA binding to the glucose polymer is typically detected by resonance energy transfer (RET).
While a practical glucose sensor such as ConA-dextran may be used to successfully detect glucose by competitive RET assay, it is not completely reversible [
13
-
15
]. ConA and dextran form aggregates which with time become increasingly resistant to disruption by glucose. A sensor is not useful for glucose monitoring unless binding is reversible.
Another approach to developing a biosensor is to genetically engineer a protein for site-specific positioning of allosteric signal transducing molecules. Structural principles are used to take advantage of cooperative interactions between the signaling molecule and ligand binding. This technique has been applied to Maltose binding protein and Glucose/Galactose binding protein of
Escherichia coli
(GGBP) [
16
,
17
]. Structural studies of GGBP reveal two domains, the relative positions of which change upon the binding of glucose [
18
]. Such conformational changes can be expected to result in spectral changes of environmentally sensitive probes, or changes in the transfer efficiency between donor and acceptor pairs covalently bound to the protein. Spectral changes of environmentally sensitive probes have been reported for GGBP [
17
].
However, there remains a need in the art for improved methods for determining the presence or concentration of glucose using fluorescent sensing molecules.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to a method for determining the presence or concentration of an analyte in a sample, comprising the steps of:
a) providing a protein sensing molecule that is capable of binding said analyte in said sample, said molecule having a detectable quality that changes in a concentration-dependent manner when said molecule is bound to said analyte;
b) exposing said sensing molecule to said sample; and
c) measuring any change in said detectable quality to thereby determine the presence or concentration of said analyte in said sample.
In another aspect, the present invention provides a sensor for determining the presence or concentration of an analyte in a sample, which comprises:
a) a protein sensing molecule that is capable of binding to the analyte in said sample, said molecule having a detectable quality that changes in a concentration-dependent manner when said molecule is bound to the analyte;
b) a radiation source which is capable of causing said sensing molecule to emit said detectable quality; and
c) means for detecting changes in said detectable quality in response to said analyte binding.


REFERENCES:
Lakowicz et al, J. Biomed. Opt., vol. 4(4), p443-449, 1999.
Jacques et al., Controlled removal of human stratum corneum by pulsed laser.The Journal of Investigative Dermatology, 88(1) (1987) 88-93.
Rabinovitch et al., Noninvasive glucose monitoring of the aqueous humor of the eye: Part 1. Measurement of very small optical rotations.Diabetes Care5(3) (1982) 254-258.
Castellano et al., A water-soluble luminescence oxygen sensor.Photochemistry and Photobiology, 67(2) (1998), 179-183.
Feddersen et al., Digital parallel acquisition in frequency domain fluorimetry.Rev. Sci. Instrum., 60 (9) (1989), 2929-2936.
Lakowicz et al., Optical sensing of glucose using phase-modulation fluorimetry.Analytica Chimica Acta, 271 (1993) 155-164.
Sipior et al., Single quantum well light emitting diodes demonstrated as excitation sources for nanosecond phase-modulation fluorescence lifetime measurements.Rev. Sci. Instrum.67(11), (1996), 3795-3798.
Berndt et al., Phase-modulation fluorometry using a frequency-doubled pulsed laser diode light source.Rev. Sci. Instrum., 61(7) (1990), 1816-1820.
Berndt et al., Electroluminescent lamp-based phase fluorometer and oxygen sensor.Analytical Biochemistry, 201 (1992), 319-325.
Sipior et al., Blue light-emitting diode demonstrated as an ultraviolet excitation sourcefor nanosecond phase-modulation fluorescence lifetime measurements.Rev. Sci. Instrum., 68(7) (1997), 2666-2670.
Tolosa et al., Optical assay for glucose based on the luminescence decay time of the long wavelength dye Cy5™ .Sensors and Actuators, B45 (1997) 93-99.
Tamada et al., Measurement of glucose in diabetic subjects using noninvasive transdermal extraction.Nature Medicine1(11) (1995), 1198-1201.
Scholle et al., Sequence of themg1Bgene fromEscherichia coliK12: Comparison of wild-type and mutant galactose chemoreceptors.Mol. Gen. Genet208 (1987) 247-253.
Lakowicz et al., Low-frequency modulation senors using nanosecond fluorophores.Anal. Chem. 70 (1998) 5115-5121.
Tolosa et al., Lifetime-based sensing of glucose using energy transfer with a long lifetime donor.Analytical Biochemistry250 (1997), 102-108.
Robinson et al, Noninvasive glucose monitoring in diabetic patients: A preliminary evaluation.Clin. Chem., 38/9 (1992) 1618-1622.
Heise et al., Noninvasive blood glucose sensors based on near-infrared spectroscopy.Artif. Organs, 18(6) (1994), 439-447.
Burmeister et al., Phantoms for noninvasive blood glucose sensing with near infrared transmission spectroscopy.Photochemistry and Photobiology, 67(1) (1998), 50-55.
March et al., Ocular glucose sensor.Trans. Am. Soc. Artif Intern. Organs, 28 (1982), 232-235.
Meadows et al., Fiber-optic biosensors based on fluorescence energy transfer.Talanta, 35(2) (1998) 145-150.
Schultz et al., Affinity sensor: A new technique for developing implantable sensors for glucose and other metabolites.Diabetes Care5(3) (1982) 245-253.
Schultz et al., Affinity sensors for individual metabolites.Biotechnology and Bioengineering Symp. 9 (1979) 65-71.
Claremont, Biosensors: clinical requirements and scientific promise.Journal of Medical Engineering and Technology, 11(2) (1987) 51-56.
Ito et al., Development of a transcutaneous blood-constituent monitoring method using a suction effusion fluid collecti

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