Enzyme biosensors adsorption

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)... 

In order to make a useful biosensor, enzyme has to be properly attached to the transducer with maintained enzyme activity. This process is known as enzyme immobilization. The choice of immobilization method depends on many factors such as the nature of the enzyme, the type of transducer used, the physiochemical properties of analyte, and the operating conditions [73]. The major requirement out of all these is its maximum activity in immobilized microenvironment. Enzyme-based electrodes provide a tool to combine selectivity of enzyme toward particular analyte and the analytical power of electrochemical devices. The amperometric transducers are highly compatible when enzymes such as urease, generating electro-oxidizable ions, are used [74]. The effective fabrication of enzyme biosensor based on how well the enzyme bounds to the transducer surface and remains there during use. The enzyme molecules dispersed in solutions will have a freedom of their movement randomly. Enzyme immobilization is a technique that prohibits this freedom of movement of enzyme molecules. There are four basic methods of immobilizing enzymes on support materials [75] and they are physical adsorption, entrapment, covalent bonding, and cross-linking, as shown in the Fig. 36. 

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs), AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide (OP) biosensor [40, 41], Another OP biosensor has also been constructed by adsorption of AChE on MWNTs modified thick film [8], More recently AChE has been covalently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite film [11], in which biopolymer chitosan provides biocompatible nature to the enzyme and MWNTs improve the conductive nature of chitosan. Even though these enzyme immobilization techniques have been reported in the last three decades, no method can be commonly used for all the enzymes by retaining their complete activity. 

Besides catalyzing styrene and benzaldehyde, CYP enzymes play an important role in the metabolism of endogenous compounds as well as in pharmacokinetics and toxicokinetics. Joseph [228] developed a biosensor with human CYP3A4 as a novel drugscreening tool. It was constructed by assembling enzyme films on Au electrodes by alternate adsorption of a layer of CYP3A4 on top of a layer of PDDA. The biosensor was applied to detect verapamil, midazolam, quinidine, and progesterone. 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

In recent years the electrochemistry of the enzyme membrane has been a subject of great interest due to its significance in both theories and practical applications to biosensors (i-5). Since the enzyme electrode was first proposed and prepared by Clark et al. (6) and Updike et al. (7), enzyme-based biosensors have become a widely interested research field. Research efforts have been directed toward improved designs of the electrode and the necessary membrane materials required for the proper operation of sensors. Different methods have been developed for immobilizing the enzyme on the electrode surface, such as covalent and adsorptive couplings (8-12) of the enzymes to the electrode surface, entrapment of the enzymes in the carbon paste mixture (13 etc. The entrapment of the enzyme into a conducting polymer has become an attractive method (14-22) because of the conducting nature of the polymer matrix and of the easy preparation procedure of the enzyme electrode. The entrapment of enzymes in the polypyrrole film provides a simple way of enzyme immobilization for the construction of a biosensor. It is known that the PPy-... 

Electron-Mediated Biosensor. Electropolymerization of 1,3-diaminobenzene (1,3-DAB), followed by adsorption of l,r-dimethylferrocene (1,1 -DMF), and immobilization of glucose oxidase, results in an easily and quickly (<2 h) constructed glucose biosensor with excellent linearity and stability (>3 months). Figure 5 shows a proposed schematic of the sensing layer consisting of film/mediator/enzyme. The ferrocene is depicted as circles... 

The hydrophobias are a case where protein nanofibers can play a dual role in creating a biosensor. They can aid in the immobilization of bioactive components within a biosensor and also add further functionality to the transducing element of a biosensor device. Hydrophobins are self-assembling [3-sheet structures observed on the hyphae of filamentous fungi. They are surface active and aid the adhesion of hyphae to hydrophobic surfaces (Corvis et al., 2005). These properties can be used to create hydrophobia layers on glass electrodes. These layers can then facilitate the adsorption of two model enzymes glucose oxidase (GOX) and hydrogen peroxidase (HRP) to the electrode surface. The hydrophobin layer also enhances the electrochemical properties of the electrodes. 

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