Enzymatic Biosensors with Enhanced Activity Retention for Detection of Organic Coupounds

This invention advances the art of enzymatic biosensor technology. It describes the biosensor, and sets forth methods of producing distal tips for biosensor transducers. These distal tips are used in detecting one or more analytes selected from organic compounds that are susceptible to dehalogenation, oxygenation or deamination, and from organosulfate compounds and organophosphate compounds susceptible to hydrolysis. Also disclosed are biosensor arrays, methods of detecting and quantifying analytes within a mixture, and devices and methods for delivering reagents to enzymes disposed within the distal tip of a biosensor.

This technology enables real-time, in situ, reagentless techniques for monitoring organic chemicals (pollutants, pesticides, contaminants, etc.) in soil, groundwater, drinking water, waste water, and other aqueous environments.

Medical uses include disposable one-way sensors (assays) for routine blood, saliva and urine testing, and in vivo sensors for monitoring crucial parameters during surgery and other procedures.

The biosensor may be made small enough to be placed inside a catheter for measurements within blood vessels.

Food and drink industry applications include contaminant detection, verification of product content, monitoring of raw material conversion and evaluation of product freshness.

Process control applications include monitoring reactant, product, and dissolved gas concentrations in various processes, such as fermentation and microbial cell growth.

Defense and homeland security industry applications include measuring the presence of chemical warfare agents such as nerve gases, detection of trace vapors, explosives, and drugs.

Advantages of these distal tips and biosensors include:

• Disposable distal tips
• Continuous real-time monitoring over an extended period of time
• Simplicity and versatility, by allowing the collection of information about the physical properties of a wide range of analytes without requiring sophisticated equipment and complicated procedures
• The simplicity of design may reduce fabrication costs, and allows for use at the site of contamination, making information readily available
• The structural design includes a combination of immobilization and stabilization techniques for preserving enzymatic efficacy, thereby providing a robust distal tip that may be used or stored for prolonged periods
• A bundle of biosensors may be provided so that multiple analytes - including closely related analytes - can be qualitatively and quantitatively detected with a high level of precision
• Particular analytes of interest include, but are not limited to, S-triazine pesticides, chlorinated ethenes, organophosphates (i.e. methyl parathion, dichlorpyrofos, diazinon, sarin), organosulfates (i.e. heparin), and amine-substituted chemicals (i.e. melamine, ammeline, biotin, and aniline).
• A response time of one hour or less
• High level of precision; e.g. ability to detect chlorinated ethenes at 0-100 µg/L (application-relevant levels).

Enzymes are the preferred biocomponents of biosensors because they are catalytic, specific to a particular analyte, and fast-acting. The technology of this biosensor allows for the use of whole cells or isolated enzymes, and its methods of producing distal tips for the transducers enable detection of one or more analytes, simultaneously or sequentially.

This biosensor is portable, has a fast response time, and a low manufacturing cost, allowing for an excellent Return on Investment (ROI).

Features of the distal tip include:

A biocomponent comprising at least one enzyme for carrying out a dehalogenation, oxidation, deamination, or hydrolysis of an analyte;

The biocomponent is immobilized to a surface of the tip;

Treatment of the biocomponent for maintaining a period of enzymatic efficacy; and

The biocomponent stabilized by one or more of:

• crosslinking of a surface of the immobilized biocomponent,
• crosslinking a polymer layer to the biocomponent,
• adding a gel-hardening agent to the biocomponent,
• adding a stabilizing agent to the biocomponent, and/or
• modifying a component used to immobilize the biocomponent to the surface of the tip.

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