Optical enzyme-based sensors analytes

Optical Enzyme-Based Sensors for Reagentless Detection of Chemical Analytes, Chapter 4... 

Enzyme sensors can measure analytes that are the substrates of enzymatic reactions. Thermometric sensors can measure the heat produced by the enzyme reaction [31], while optical or electrochemical transducers measure a product produced or cofactor consumed in the reaction. For example, several urea sensors are based on the hydrolysis of urea by urease producing ammonia, which can be detected by an ammonium ion-selective ISE or ISFET [48] or a conductometric device [49]. Amperometric enzyme sensors are based on the measurement of an electroactive product or cofactor [50] an example is the glucose oxidase-based sensor for glucose, the most commercially successful biosensor. Enzymes are incorporated in amperometric sensors in functionalised monolayers [51], entrapped in polymers [52], carbon pastes [53] or zeolites [54]. Other catalytic biological systems such as micro-organisms, abzymes, organelles and tissue slices have also been combined with electrochemical transducers. 

Hale Z M and Payne F P 1994 Fluorescent sensors based on tapered single-mode optical fibres Sensors Actuators 17 233 0 Luo S and Walt D R 1989 Avidin-biotin coupling as a general method for preparing enzyme-based fiber-optic sensors Anal. Chem. 61 1069-72 Li L and Walt D R 1995 Dual-analyte fiber-optic sensor for the simultaneous and continuous measurement of glucose and oxygen Anal. Chem. 67 3746-52 Tan W, Shi Z-Y, Smith S, Bimbaum D and Kopelman R 1992 Submicrometer intracellular chemical optical fiber sensors Science 258 778-81 Tan W, Shi Z-Y and Kopelman R 1992 Development of submicron chemical fiber optic sensors Anal. Chem. 64 2985-90... 

Table 18-2. Selection of biochemical analytes for which enzyme-based optical sensors have been described, and respective transducers. ...

Electrochemical sensor fabrication has dominated the analytical application of polymers. In some sensors the polymer film acts as a membrane for the preconcentration of ions or elements before electrochemical detection. Polymers also serve as materials for electrode modification that lower the potential for detecting analytes. In addition, some polymer films function as electrocatalytic surfaces. Using a polymer in biosensors is a very rapidly developing area of electroanalytical chemistry. Polymeric matrix modifiers have been applied as diffusional barriers in constructing not only sensitive amperometric biosensors, but also electrochemical sensors that apply potentiometric, conductimetric, optical, and gas-sensing transducer systems. The principles, operations, and application of potentiometric, conductimetric, optical and gas sensors are described in Refs. 13, 39-41. In this chapter, we focus mainly on amperometric biosensors based on redox enzymes. 

Another principal that has been evaluated for the construction of optically based sensors is the use of chemiluminescence. In these cases an enzyme system specific for the analyte are coupled to reactions that produce light through chemiluminescence. In principle, systems of this type could be very sensitive, first of all due to the amplification factor of enzyme reactions, and secondarily because fluorescence measurements are among the most sensitive of optical techniques. 

Finally, in optical-based sensors and assays, there is the development of label-free bioanalytic detection on prepared membrane surfaces, ultra sensitive detection of microbial agents, the development of fiber-optic based enzyme sensors, multi-analyte breast cancer immunoassays, and photodynamic therapy of osteosarcoma in veterinary patients. 

A review of chemical sensors and arrays [6] focuses on conducting polymers as inherent receptors, the modification of conducting polymers with receptors, the use of conducting polymers as transducers as well as some applications in combinatorial and high-throughput assays. Fundamental aspects of redox-related conductivity and pH-sensitive conductivity are included, and applications to chemical- and enzyme-based biosensors are summarized based on analyte. A variety of detection methods (electrical, electrochemical, and optical) are surveyed. 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

The sensing microzone of the flow-through sensor depicted in Fig. 5.9.B1 integrates gas-diffusion and detection with two analytical reactions [28], viz. (a) the urease-catalysed formation of ammonium ion by hydrolysis of urea (the analyte), which takes places on a hydrophilic enzyme membrane in contact with the sample-donor stream, which contains a gel where the enzyme is covalently bound and (b) an acid-b reaction that takes place at the microzone on the other side of the diffusion membrane and involves Bromothymol Blue as indicator. This is a sandwich-type sensor including a hydrophilic and a hydrophobic membrane across which the sample stream is circulated —whence it is formally similar to some enzyme electrodes. Since the enzymatic conversion of the analyte must be as efficient as possible, deteetion (based on fibre optics) is performed after the donor and acceptor streams have passed through the sensor. Unlike the previous sensor (Fig. 5.9.A), this does not rely on the wall-jet approach in addition, each stream has its own outlet and the system includes two sensing microzones... 

In amperometry, we measure the electric current between a pair of electrodes that are driving an electrolysis reaction. One reactant is the intended analyte and the measured current is proportional to the concentration of analyte. The measurement of dissolved 02 with the Clark electrode in Box 17-1 is based on amperometry. Numerous biosensors also employ amperometry. Biosensors8-11 use biological components such as enzymes, antibodies, or DNA for highly selective response to one analyte. Biosensors can be based on any kind of analytical signal, but electrical and optical signals are most common. A different kind of sensor based on conductivity—the electronic nose —is described in Box 17-2 (page 360). 

Similar optical biosensors have been prepared for many other analytes. For example, a cholesterol optical biosensor has been devised based on fluorescence quenching of an oxygen-sensitive dye that is coupled to consumption of oxygen resulting from the enzyme-catalyzed oxidation of cholesterol by the enzyme cholesterol oxidase. Serum bilirubin has been detected using bilirubin oxidase, coimmobilized with a ruthenium dye, on an optical fiber.The bilirubin sensor was reported to exhibit a lower detection limit of iO Xmol/L, a linear range up to 30mmol/L, and a typical reproducibihty of 3% (CV), certainly adequate for clinical application. 

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