Enzymatic Biosensors with Enhanced Activity Retention for Detection of Organic Compounds

 This technology describes biosensors and methods of producing distal tips for biosensor transducers fur use in detecting one or more analytes selected from organic compounds susceptible to oxygenation and organophosphate compounds susceptible to hydrolysis.

It also sets forth methods of immobilizing the biocomponent to the distal tip, and methods of stabilizing the biocomponent in the tip.

 This technology enables real-time, in situ, reagentless techniques for:

Environmental monitoring of the concentration and toxicity of contaminants (e.g., analytes, such as heavy metals, pesticides, etc.) in surface and groundwater, waste streams, and soil

Medical monitoring of routine blood, saliva and urine, using disposable one-way sensors

In vivo sensors for monitoring crucial parameters during surgery and other procedures

Food and drink contaminant detection, product content, monitoring of raw material conversion, and evaluation of product freshness

Process control, including the monitoring of pH, temperature, and substrate and dissolved gas concentrations in various processes (e.g., fermentation and microbial cell growth)

In the area of defense and security, measuring the presence of chemical warfare agents such as nerve gases, and detecting trace vapors, explosives, and drugs

 Advantages of these distal tips and biosensors include:

• A response time of one hour or less
• A high level of precision (e.g., the ability to detect application-relevant levels at 0-100 µg/L)
• 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, simultaneously or sequentially, with a high level of precision

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

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.

Features of the distal tip include:

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

Immobilization of the biocomponent to a surface of the tip by one or more of the following:

• Entrapping the enzyme within a hydrogel secured to the tip surface
• Entrapping the enzyme within a polymeric network secured to the tip surface
• Encapsulating the enzyme
• Covalently bonding a second component of the biocomponent to the tip surface
• Crosslinking the enzyme to a support material secured to the tip surface
• Absorbing the enzyme into the tip surface

Treatment of the biocomponent for maintaining a period of enzymatic efficacy

Stabilization of the biocomponent

• Molecular modification
• Crosslinking, covalent bonding, entrapment, encapsulation
• Freeze Drying
• Heat Shock
• Addition of Carbohydrates and Polymers
• Freezing

Peroxide Shunt

Development of enzymatic biosensors has been limited where NADH is required as a cofactor, because their long-term use and performance are limited due to problems encountered in supplying NADH. The peroxide shunt described in this patent may remove the need for NADH regeneration, which may increase biosensor lifetime since living cells are not required, where electrons and oxygen are supplied in the form of peroxide, and energy metabolism is no longer necessary.

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