Enzyme-based biosensor detection

Electrochemical biosensors are analytical devices in which an electrochemical device serves as a transduction element. They are of particular interest because of practical advantages, such as operation simplicity, low expense of fabrication, and suitability for real-time detection. Since the first proposal of the concept of an enzyme-based biosensor by Clark, Jr [1], significant progress in this field has been achieved with the inherited sensitivity and selectivity of enzymes for analytical purposes. 

Enzyme-based biosensors are very suitable for the antioxidant status evaluation, since they show excellent selectivity for biological substances and can directly determine and/or monitor antioxidant compounds in a complex media such as biological or vegetable samples without needing a prior separation step. During the course of the catalytic reaction on the electroactive substrates, the current produced at an applied potential is related to the concentration of a specific biomarker, for which the biosensor is selective. HRP-based biosensors for antioxidant status evaluation have been applied in the detection of superoxide radical [119], nitric oxide [120], glutathione [119, 121], uric acid [122, 123], and phenolic compounds [124—126],... 

Enzyme-Based Biosensors with Amperometric Detection... 

Electrochemical biosensors have been divided into two basic types enzyme-based sensor and electrochemical probe-based sensor. Alkaline phosphatase (ALP) and horse radish peroxidase (HRP) have been often employed for enzyme-based biosensors using p-nitrophenyl phosphate (PNP), a-naphtyl phosphate, 3-3, 5,5 -tetramethylbenzidine (TMB) and 2,2 -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrates of electrochemically active species, and ferrocene (Fc) and methylene blue as the electrochemical mediators. In general, enzymatic amplification of electrochemical signals enables highly sensitive detection of analytes. On the other hand, a direct detection of analytes by using electrochemical probes allows a more rapid time-response onto the detector surface and needs no enzymatic reaction. Based on the reason, a direct detection of analytes by using electrochemical probes has been... 

For enzyme-based biosensors the mode of detection is based on the catalytic activity and/or binding capacity. Because of the protein nature of almost all enzymes, the catalytic activity depends on the conformation. Exceptions are catalytic ribonucleic acids called DNA biosensors or genosensors. DNA fragments are used as probes for detecting low concentrations of DNA in large samples (see also Part I, Chapters 2 and 3). Because of the highly diluted DNA concentration, microelectromechanical systems which are able of performing PCRs are employed. 

Enzymes-based biosensors are well reported in the literature for chemical toxicity screening. The sensor devices produced using enzymes are usually simple and easy to fabricate, inexpensive, and sensitive to low levels of toxicants. Immobilization of enzymes on the electrode surface can include adsorption, covalent attachment, or film deposition using a range of procedures [68-70]. The sensor system relies primarily on two enzyme mechanisms catalytic transformation of a pollutant and detection of pollutants that inhibit or mediate the enzyme s activity. In catalytic enzyme biosensor, the enzyme specific for the substrate of interest (toxin in this case)... 

Enzyme inhibition sensors are the most commonly reported enzyme-based biosensors for the detection of toxic compounds and heavy metal ions. The sensors are based on the selective inhibition of specific enzymes by classes of compounds or by the more general inhibition of enzyme activity. Most of the research carried out has been directed toward the detection of organophosphorus and carbamate insecticides and the triazine herbicides and metal ions analysis [72,73]. Several enzymes have been used in inhibition sensors for pesticides and heavy metal analysis using water, soil, and food samples including choline esterase, horseradish peroxidase, polyphenol oxidase, urease, and aldehyde dehydrogenase. 

Me Ardle, F.A., and K.G. Persaud. 1993. Development of an enzyme-based biosensor for atrazine detection. Analyst 118 419. 

The development of antibody-based biosensors presents more difficulties than enzyme-based biosensors as the antigen-antibody interactions are not readily reversible because of the high values of the affinity constants. Another limitation is that the physicochemical changes resulting from the immunochemical reaction are often insufficient to provide detection limits comparable with those of conventional analysis. As a consequence, indirect systems have been developed that rely on the use of enzyme-or fluorescent-tagged reagents. Both competitive and sandwich formats are used. Evanescent wave-induced fluorescence is frequently chosen to avoid possible interferences from the bulk media. For... 

HPLC with UV-based diode array detection (DAD-UV) or electrochemical detection is normally used to determine ascorbic acid. Many types of electrochemical determinations of ascorbic acid have been proposed. Although the electrochemical determinations using enzyme-based biosensors exhibited high specificity and sensitivity, these methods suffer in the fabrication of the electrodes and in automatic analysis. Recently, chemically modified screen-printed electrodes have been constructed for the determination of ascorbic acid. This is one of the most promising routes for mass production of inexpensive, reproducible, and reliable electrochemical sensors. 

CAD-Kit. The authors have developed and fielded an enzyme-based biosensor kit that detects nerve, blood and blister agents, and TICs (toxic industrial chemicals)/TIMs (toxic industrial materials) on surfaces (22,23). The penlike sensors of the Chemical Agent Detection Kit (CAD-Kit) consist of a spongelike polyurethane tip that contains immobilized enzymes. These enzymes are used to detect several different chemical compovmds. 

Amperometric or voltammetric biosensors typically rely on an enzyme system that catalyt-ically converts electrochemically non-active analytes into products that can be oxidized or reduced at a working electrode. Although these devices are the most commonly reported class of biosensors, they tend to have a small dynamic range due to saturation kinetics of the enzyme, and a large overpotential is required for oxidation of the analyte this may lead to oxidation of interfering compounds as well (e.g., ascorbate in the detection of hydrogen peroxide). In addition to the use in enzyme-based biosensors, amperometric transducers have also been used to measure enzyme-labelled tracers for affinity-based biosensor (mainly immunosensors and genosensors). Enzymes which are commonly used for this purpose include horseradish peroxidase (HRP) [17] and alkaline phosphatase (AP) [18,19,21]. 

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