Enzyme-based sensing

Matsue et al. [27] were the first to explore an enzyme-based OECT biosensor. They used Diaphorase as the entrapped enzyme in a polypyrrole transducing layer for the detection of NADH via a redox mediator (the sodium salt of anthraquinone-2-sulfonic acid). The net result was the conversion of polypyrrole from its conducting state to its insulating state in the presence of NADH. The device showed a response time of 15--20 min in the presence of NADH. Later Nishizawa et al. [26] exploited the pH sensitivity of the polypyrrole film for the design and fabrication of OECT sensors for pH and for pencillin. The Penicillinase enzyme was entrapped in a membrane which was coated with a polypyrrole film, in which a decrease in pH was observed in the presence of penicillin due to the hydrolysis of penicillin by Penicillinase. 

Contractor and coworkers were the first to explore the pH sensitivity of polyaniline for the fabrication of an OECT biosensor for the detection 

In all of the above-mentioned work of Contractor and coworkers, enzyme entrapment was carried out at higher pH after the electropolymerization of aniline at much lower pH. Enzymes could not be added along with the elec-tropolymerizing solution because the enzymes become denaturated at such low pHs. This restricted the amount of enzyme that could be entrapped and hence limited the sensitivity of the OECT devices. In 1999, Tripathy and coworkers [29-31] demonstrated that aniline could be polymerized at pHs as high as 5.5, using a strongly acidic polyelectrolyte like poly(styrene sulfonate). Based on these reports, Contractors and coworkers fabricated OECT 

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The majority of research involving optical sensing devices can be separated into four well-studied areas, involving proton, O2, metal, and enzyme-based sensing. Many of these studies involve the use of organic molecules rather than... 

The examples in the preceding text demonstrate how a successful enzyme immobilization process can help to transition enzyme-based sensing applications to real-world products. This is accomplished by providing extended shelf life and operational stability, which are often seen as the biggest drawbacks of using enzymatic techniques outside the laboratory. 

Particularly attractive for numerous bioanalytical applications are colloidal metal (e.g., gold) and semiconductor quantum dot nanoparticles. The conductivity and catalytic properties of such systems have been employed for developing electrochemical gas sensors, electrochemical sensors based on molecular- or polymer-functionalized nanoparticle sensing interfaces, and for the construction of different biosensors including enzyme-based electrodes, immunosensors, and DNA sensors. Advances in the application of molecular and biomolecular functionalized metal, semiconductor, and magnetic particles for electroanalytical and bio-electroanalytical applications have been reviewed by Katz et al. [142]. 

Another approach, developed in our laboratory, consists of the compartmentalization of the sensing layer25"27. This concept, only applicable for multi-enzyme based sensors, consist in immobilizing the luminescence enzymes and the auxiliary enzymes on different membranes and then in stacking these membranes at the sensing tip of the optical fibre sensor. This configuration results in an enhancement of the sensor response, compared with the case where all the enzymes are co-immobilized on the same membrane. This was due to an hyperconcentration of the common intermediate, i.e. the final product of the auxiliary enzymatic system, which is also the substrate of the luminescence reaction, in the microcompartment existing between the two stacked membranes. 

SWNT-based glucose sensors an enzyme-based sensor and an affinity sensor. These results are a promising beginning however, there is much work left to do before the promise of SWNT in vivo sensors is realized. Investigation of new sensing modalities and optimization of current ones is necessary before the best nanotube-based sensing strategy is discovered. 

Table 11.19 Liquid Membrane Electrodes (LME), Gas-Sensing Electrodes (GSME), and Enzyme-Based Biosensors (EBB) [56]...

Peteu, S.F., Emerson, D., Worden, R.M. (1996). A Clark-type oxidase enzyme-based amperometric microbiosensor for sensing glucose, galactose, or choline. Biosens. Bioelectr. 11 1059-71. 

The above approach for measurement of urea using an enzyme-based potentiometric biosensor assumes that the turnover of urea to ammonium at steady state provides a constant ratio of ammonium ions to urea, independent of concentration. This is rarely the case, especially at higher substrate concentrations, resuitmg in a nonlinear sensor response. The hnearity of the sensor is also limited by the fact tiiat hydrolysis of urea produces a local alkaline pH in the vicinity of the ammonium-sensing membrane, partially converting NH to NH3 (pKa = 9.3). Ammonia (NH3) is not sensed by the ISE. The degree of nonlinearity may be reduced by placement of a semipermeable membrane between enzyme and sample to restrict diffusion of urea to the immobilized enzyme layer. 

Electropolymerization of polymers directly onto the surface of an electrode has been used for a number of enzyme-based biosensors. By polymerizing from a solution containing the monomer, as well as the other components of the sensor, enzymes for example, a multifunctional polymer film can be fabricated. As the polymer film grows on the electrode, the enzyme and other components are entrapped in the film [9]. GOD and other enzymes have been incorporated into sensors using electropolymerization. Advantages of electropolymerization are that the film thickness can be easily controlled by the amount of polymerization charge passed, and that the polymer film is deposited only on the sensing electrode. 

On the other hand, a half-sandwich complex of manganese with j -CyD [( /-MeC5H4)Mn(NO)(S2CNMe2)] (Fig. 16.4.3) was found to be an efficient mediator in enzyme-based sensors including those useful for blood glucose sensing such as those involving GOx and horseradish oxidase [44]. 

When the immobilized sensing reagent also contains a bioreceptor, such as an enzyme or an antibody, the device is regarded as a biosensor (23). Such sensors hold great promise as they exploit the inherent ability of the bio molecule to selectively and sensitively recognize a particular chemical spedesln a complex matrix. Enzyme-based sensors produce a signal due to a selective enzyme-catalyzed chemical reaction of an analyte and form a product that is detected by a transduction element in the sensor. The... 

Enzyme based micron sized sensing system with optical readout was fabricated by co-encapsulation of urease and dextran couple with pH sensitive dye SNARE-1 into polyelectrolyte multilayer capsules. The co-precipitation of calcium caibonate, urease, and dextran followed up by multilayer film coating and Ca- extracting by EDTA resulted in formation of 3.5-4 micron capsules, what enable the calibrated fluorescence response to urea in concentration range from 10 to 10 M. Sensitivity to urea in concentration range of 10 to 10 M was monitored on capsule assemblies (suspension) and on single capsule measurements. Urea presence can be monitored on single capsule level as illustrated by confocal fluorescent microscopy. 

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