Construction of Biosensors

Biosensor construction involves combining two elements with different characteristics. This entails three steps. First a bioreceptor is chosen, then a transducer, and finally the biological component is fixed to the transducer. 

The bioreceptor plays the role of a molecular recognition device. In the presence of the substance under investigation, it must produce a physicochemical effect that is detectable by the transducer. This may involve one of a number of processes, such as biocatalysis, immunological coupling, or chemoreception. 

The biocatalyst employed is usually an enzyme, of which a large number are commercially available, for example, glucose oxidase and urease. Enzymes may also be extracted from one or more biological sources for extremely specific needs, and can be used alone or together with their cofactcns, such as NAD and NADP. 

The use of commercialized enzymes has a number of advantages, such as batch reproducibility, known characteristics and lifetimes, and rapid availability. Commercialized enzymes thus play an important role in the biosensors presendy on the market. The disadvantage of purified enzymes lies in the fact that they are not always very stable, and often require the presence of their cofactors for proper operation. In some systems, a single enzyme is inadequate for the entire transformation desired and several are required for the whole enzymatic sequence. 

In analytical devices, the biologically active molecules, such as enzymes, cells and antibodies, are used repeatedly. These are, therefore, fixed to the carrier materials. There are several advantages for immobilising the enzymes for application in analytical chemistry. These include stabilisation of enzymes and retention of enzyme activity for long periods of time. 

Biosensors provide a powerful and inexpensive alternative to conventional analytical strategies for assaying chemical species in complex matrices. A biosensing device incorporates a biological molecular recognition component connected to a transducer. The main aim of a transducer is to produce a continuous or discrete electronic signal, which is directly proportional to the concentration of an analyte. In biosensors, the following sequence of events take place  

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For application of protein-immobilized porous materials to sensor fields, use of an electroactive substance as the framework material is important. DeLouise and Miller demonstrated the immobilization of glutathione-S-transferase in electrochemically etched porous silicon films [134], which are attractive materials for the construction of biosensors and may also have utility for the production of immobilized enzyme bioreactors. Not limited to this case, practical applications of nanohybrids from biomolecules and mesoporous materials have been paid much attention. Examples of the application of such hybrids are summarized in a later section of this chapter. 

Some non-silica sol-gel materials have also been developed to immobilize bioactive molecules for the construction of biosensors and to synthesize new catalysts for the functional devices. Liu et al. [33] proved that alumina sol-gel was a suitable matrix to improve the immobilization of tyrosinase for detection of trace phenols. Titania is another kind of non-silica material easily obtained from the sol-gel process [34, 35], Luckarift et al. [36] introduced a new method for enzyme immobilization in a bio-mimetic silica support. In this biosilicification process precipitation was catalyzed by the R5 peptide, the repeat unit of the silaffin, which was identified from the diatom Cylindrotheca fusiformis. During the enzyme immobilization in biosilicification the reaction mixture consisted of silicic acid (hydrolyzed tetramethyl orthosilicate) and R5 peptide and enzyme. In the process of precipitation the reaction enzyme was entrapped and nm-sized biosilica-immobilized spheres were formed. Carturan et al. [11] developed a biosil method for the encapsulation of plant and animal cells. 

Semiconductor fabrication techniques have also been successfully applied to the construction of conventional transducers sensitive to hydrogen peroxide, oxygen, and carbon dioxide, A hydrogen peroxide-sensitive silicon chip was made by using metal deposition techniques (28,29). The combination of the hydrogen peroxide-sensitive transducer and enzyme-immobilized membranes gave a miniaturized and multifunctional biosensor. Similarly, an oxygen- and a carbon dioxide-sensitive device was made cmd applied to the construction of biosensors (25, 30, 31). 

The immobilization of biomolecules on solid surfaces offers considerable advantages to enzymes in solution. Immobilization via physical attraction is often not reliable because of problems concerning leaching and loss of the biomolecule [1]. Therefore, covalent attachment of the enzymes to solid surfaces is advantageous, especially in biotechnology, where the biomolecule should be separated fix)m the reaction mixture, or for the construction of biosensors, where a biocatalyst would be used in a detector system. 

Plant and animal tissues have long been successfully employed as biocatalsrtic components in the construction of biosensors, but it is quite unusual to find such a recognition system coupled to the microdialysis sampling techniques. 

Although different nanomaterials such as nanoparticles, nanowires and nanotubes are used for the construction of biosensor, this chapter is mainly devoted to the use of AuNPs for the construction of electrochemical biosensor and their analytical performances. Further, in this chapter we restrict ourselves in the electrochemical sensing of glucose, ascorbic acid, uric acid and dopamine derivatives using the AuNPs modified electrodes. 

There is an enormous variety of nanomaterials that can potentially be employed in biosensor architectures. The most prominent among them are metal nanoparticles [304], quantum dots [308], and carbon nanotubes [309-311]. AH of them have been employed in biosensors though not necessarily exclusively electrochemical biosensors. Quantum dots (QDs) offer unique absorption properties making them highly suitable for the construction of biosensors with optical readout. The most diverse electrochemical nanobiosensors are, however, obtained from carbon nanotubes (CNTs) which offer a wide range of different apphcations. 

Different biospecific interaction processes may be considered for the construction of biosensors. Enzymes, antibodies, lectins, hormones, microorganisms, organelles, or tissue sections can be used to act as molecular... 

As shown in Section 2.3, various biospecific recognition systems and interactions taking place without analyte conversion can be utilized for the construction of biosensors. When the binding of the analyte to an immobilized biomolecule or receptor system is reversible the sensor becomes reusable. Since the physicochemical changes caused by the binding are mostly very slight, in many cases auxiliary reactions have to be coupled. 

Ion-selective electrodes with a liquid membrane are more reliable than ion-selective electrodes with a solid membrane because of the uniformity of the active material partition in the membrane. For the construction of biosensors the maximum reliability is obtained by using graphite paste as the support. As of the present, for in vivo tests only sensors based on plastic membranes have been used. The main problem associated with using them for in vivo tests is the biocompatibility of the materials.147 149 The membrane biocompatibility, the matrix biocompatibility, and the electroactive material biocompatibility are important factors. The matrix biocompatibility is assured by the biocompatibility of the polymer and by the biocompatibility of the plasticizer. The ratio between the quantity of polymer and quantity of plasticizer affects the response of electrochemical sensors because the matrix of the solid membrane electrodes plays the same role as does the solvent in liquid membrane electrodes. 

The question of whether natural chemoreception can provide biosensors, or whether the construction of biosensors can be deduced from the lessons taught by natural processes, is conditional in both circumstances. In many cases the immediate use or a direct transfer of chemoreceptive tissues, cells, or molecular receptors for sensor construction is very limited in terms of longevity and reproducibility, and often would only be possible for the preparation of one-shot devices. 

S.X. Zhang, N. Wang, Y.M. Niu, and C.Q. Sun, Immobilization of glucose oxidase on gold nant tarti-cles modified Au electrode for the construction of biosensor. Sens. Actuators, B Chem. 109, 367-374 (2005). 

The most often used recognition elements for the construction of biosensors are enzymes. Their catalytic activity usually derives from prosthetic groups (nonproteins such as heme, FAD, or pyridox-alphosphate) or metal ions. The prosthetic groups are usually covalently bound to the enzyme, while coenzymes or cosubstrates are only associated with it, binding in close proximity to the substrate binding site during the catalytic action. For their application in biosensors, enzymes have to be isolated from the biological... 

Moreover, in recent years, enzymatic sensors reduce the time needed for analysis and may offer a rapid and reliable screening method for industrial food quality testing. Enzyme sensors able to detect the presence of BAs in dry fermented sausages have been developed, and they can constitute a useful tool for quality control in the meat industry (Hemdndez-Cdzares, Aristoy, Toldra, 2012). An overview of the developments and issues in the construction of biosensors for the detection of most common BAs found in food has been recently reported (Kivirand Rinken, 2011). 

The use of plant and animal tissues, instead of isolated enzymes, for the construction of biosensors has attracted much interest in the last three decades. 

Several biological materials, such as animal tissues, bacterial cells, and plant tissues, have been used for this purpose. In earlier studies, animal materials, such as heart, liver, and kidney, were used exclusively for the construction of biosensors. However, since the introduction of the first plant-tissue-based electrochemical biosensor by Kuriyama and Rechnitz in 1981, the use of plant tissues has attracted the most interest for the development of tissue-based biosensors. The popularity of the use of plant tissues as natural enzyme sources for the construction of biosensors was further enhanced by the introduction of a banana tissue biosensor, known as the bananat-rode , in 1985. Since then several other plant tissues, such as fruits, leaves, roots, seeds, and vegetables, as listed in Table 1, have been used for the development of a wide range of tissue-based biosensors. 

Shumyantseva V, Buiko T, Archakov A (2005) Electrochemical reduction of cytochrome P450 as an approach to the construction of biosensors and bioreactors. J Inorg Biochem 99 1051-1063... 

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