Positive response biosensors and other sensors

Details Positive response biosensors and other sensors Positive response biosensors and other sensors

2001-05-07

2004-06-15

Leary, Louise N. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S004000, C435S019000, C435S183000, C435S283100, C435S287100, C435S963000, C435S007100

Reexamination Certificate

active

06750033

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to positive response sensors and, particularly, to enzymatic biosensors in which two reaction schemes provide a positive response.
There are many types of sensors designed to detect the presence of chemical species, for example, on surfaces or within solutions. Such sensors exhibit signals based on a wide variety of chemical, electrical, or physical responses. Many such sensors are based upon “negative responses”. In negative response sensors, the chemical analyte of interest inhibits or retards a chemical or physical process that would otherwise take place within the sensor in the analyte's absence. The term “negative response sensor” thus generally refers sensors in which the presence of a target analyte results in the absence of or the reduction of a signal change or a signal change.
Enzymatic proteins are remarkable natural catalysts in that they selectively catalyze many reactions under relatively mild reaction conditions. Enzymes also offer the potential to perform sterio- and regio-selective reactions not readily accomplished with conventional chemistry. As used herein, the term “enzyme” refers generally to proteins that catalyze biochemical reactions. These “biopolymers” include amide-linked amino acids and typically have molecular weights of 5,000 or greater. A compound for which a particular enzyme catalyzes a reaction is typically referred to as a “substrate” of the enzyme.
In general, six classes or types of enzymes (as classified by the type of reaction that is catalyzed) are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases. Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
Enzymes have been known since the early 1960's to be useful tools for detecting the presence of chemical species. Rogers, K. R., Biosensors Bioelectronics, 10, 533 (1995). A number of enzymatic biosensors have been designed to detect a variety of different compounds including, for example, glucose, creatinine, urea, and cholinesterase inhibitors. Parente, A. H., Marques, E. T. Jr.,
Appl. Biochem. Biotechnol.
37, 3, 267 (1992); Yang, S., Atanasov, P., Wilkins, E.,
Ann. Biomed. Eng.,
23, 6, 833 (1995). U.S. Pat. No. 5,858,186 describes a urea-based biosensor in which substrate hydrolysis is monitored with a pH electrode. U.S. Pat. Nos. 5,945,343 and 5,958,786 describe enzyme-based polymer sensors which fluoresce in the presence of ammonia, which is enzymatically produced from urea and creatinine respectively. In addition U.S. Pat. No. 4,324,858 describes the utilization of cholinesterase for the colorimetric detection of organophosphorus pesticides and nerve agents. A related patent, U.S. Pat. No. 4,525,704 describes the use of cholinesterases and electrical currents in detecting toxic gases.
Generally, enzymatic biosensors function by one of two methods: (1) the sensing enzyme converts an otherwise undetectable compound into another or series of compounds which can be detected by visual, chemical, or electrical techniques; or (2) the enzyme is inhibited by the presence of the compound of interest and enzyme inhibition is linked to a measurable quantity.
Independent of the method of use, the signals of enzyme-based biosensors are often limited in practical application by the nature of enzyme activity. Only in the case of enzyme substrate detection does the sensor provide a positive response in the presence of target analyte. In other words a noticeable change in the sensor indicates the presence of a target analyte. If the detection of enzyme inhibitors or the detection of substrate deficiency is desired, existing approaches rely on negative response signals, or the absence or reduction of an enzymatic reaction, to indicate the presence of inhibitors or the absence of target compounds.
For example, many commercially available nerve agent sensors are based on the inhibition of cholinesterases. The presence of nerve agents blocks the catalytic side on cholinesterase, disabling its ability to catalyze reactions. Such a sensor is employed by exposing the sensing enzyme (cholinesterase) to a questionable environment. Cholinesterase substrate is later applied. Depending upon the substrate or assay system employed, cholinesterase activity may result in a pH change, color change or fluorescent signal. In each of these negative response systems, a signal change occurs only in the absence of analyte (nerve agents). The initial signal of the sensor is unchanged in the presence of analyte. Kumaran, S., and Morita, M.
Talanta,
42, 649 (1995). Campanella, L., Colapicchioni, C., Favero, G., Sammartino, M. P. and Tomassetti, M.
Sensors and Actuators B,
33, 25 (1996). Hart, A. L., Collier, W. A., and Janssen, D.
Biosensors & Bioelectronics,
12, 545-654 (1997). Cho, Y. A., Lee, H. S., Cha, G. S., and Lee, Y. T.
Biosensors & Bioelectronics,
14, 387-390 (1999). Bachmann, T. T., and Schmidt, R. D. Analytica Chimica Acta, 401, 95 (1999). Diaz, A., and Ramos Peinado, M. C.
Sensors and Actuators B,
38-39, 426 (1997).
It is very desirable to develop sensors and sensing method through which the non-intuitive nature of negative response sensors can be changed to a more intuitive positive response system.
SUMMARY OF THE INVENTION
In general, the present invention provides sensors and methods in which the non-intuitive nature of a previously negative response sensor is changed to a more intuitive, positive response system. The present invention is well suited for application in enzymatic biosensors and enzymatic biosensing methods.
In one aspect, the present invention provides a sensor for detecting an analyte in an environment including a first reaction system including at least a first enzyme and at least one substrate for the first enzyme. The analyte inhibits the reaction of the substrate catalyzed by the first enzyme (in other words, the analyte inhibits the first enzyme). The sensor further includes at least a second reaction system that reacts to produce a first detectable state when the first enzyme is inhibited. In some embodiments, the reaction of the first reaction system can produce a second detectible state, different from the first detectible state.
In one embodiment, the reaction of the first reaction system (that is, the reaction of the substrate catalyzed by the first enzyme) causes pH to change in a first direction, and the reaction of the second reaction system causes pH to change in a second direction, opposite of the first direction. The first enzyme can, for example, be a hydrolase, which catalyze hydrolysis reactions, typically resulting in a pH change.
The second reaction system can, for example, include a second enzyme and a substrate for the second enzyme. The second reaction system can also involve a non-enzymatic, chemical reaction. In the case that the second reaction system includes a second enzyme, the first enzyme can, for example, be a hydrolase and the second enzyme can, for example, be a different hydrolase.
The first enzyme and/or the second enzyme can, for example, be immobilized in a polymer medium (for example, in a sponge-like polyurethane) or be in solution. Substrates can, for example, be added to the polymer medium in solution or as a powder.
The first detectible state can, for example, be a colorimetric change. As used herein, the phrase “colorimetric change” refers generally to a detectible change in color. The colorimetric change can be detectible with the human eye or with instrumentation as known in the art.
As set forth above, the reaction of the first reaction

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