Biosensors bi-enzyme

FIGURE 57.14. Schematic representation of the pathway that leads to the generation of current in the bi-enzyme biosensor containing tyrosinase and neuropathy target esterase catalytic domain (NEST). Reproduced with permission from Kohli et al. 

Table I provides a comparison of the performance of the dendrimer-based electrochemical glucose biosensors. It is found that many of the dendrimer electrode assemblies show reasonable linear range and detection limit. Their stability assessment and high sensitivity values indicate good scope for commercialization. The bi-enzyme model is better than the conventional mono-enzyme model in terms of lower detection potential which can help to circumvent the interferences due to other biomolecules during the measurements.

Rahman MM, X-b L, Kim J, Lim BO, Ahammad A, Lee J-J (2014) A cholesterol biosensor based on a bi-enzyme immobilized on conducting poly (thionine) film. Sensor Actuat B Chem 202 536... 

Meng, X., Wei, J., Ren, X., et al., 2013. Simple and sensitive fluorescence biosensor for detection of organophosphorus pesticides using H202-sensitive quantum dots/bi-enzyme. Biosens. Bioelectron. 47, 402-407. 

Peroxidase (POD) may be added to the bi-enzyme system to build a tri-enzyme device. Multienzyme systems pose greater challenges than mono-enzyme ones. However, they produce biosensors that are more specific and less prone to... 

Dithiocarbamate fungicides inhibit aldehyde dehydrogenase. In order to produce an amperometric biosensor with this enzyme also a bi-enzymatic system was designed with the enzyme diaphorase. Reaction of propiraialdehyde and NAD" in the presence of ADH produced NADH which could be detected via its reaction with hexacyanoferrate(in) by diaphorase. The changes of hexacyanoferrate (11) concentrations were monitored amperometrically with a Pt electrode or bi-amperometrically with two platinum electrodes. A bi-amperometric biosensor was also developed in screen-printed configuration with Pt-sputtered carbon paste In aU these biosensors both enzymes were immobilized in a poly(vinyl alcohol)-styrylpyridinium (PVA-SbQ) layer. 

SCHEME 2 Schematic illustration of analytical mechanism of (a) first-, (b) second-, and (c) third-generation 02 biosensors. Note that the reactions shown in (b) and (c) are bi-directional since SODs are enzymes specifically catalyzing the 02 dismutation, i.e. oxidation into 02 and reduction into H202. 

Figure 6.7 illustrates the voltammetric response of the third-generation SOD-based 02 biosensors with Cu, Zn-SOD confined onto cystein-modified Au electrode as an example. The presence of 02" in solution essentially increases both the cathodic and anodic peak currents of the SOD compared with its absence [150], Such a redox response was not observed at the bare Au or cysteine-modified Au electrodes in the presence of 02". The observed increase in the anodic and cathodic current response of the Cu, Zn-SOD/cysteine-modified Au electrode in the presence of 02 can be considered to result from the oxidation and reduction of 02, respectively, which are effectively mediated by the SOD confined on the electrode as shown in Scheme 3. Such a bi-directional electromediation (electrocatalysis) by the SOD/cysteine-modified Au electrode is essentially based on the inherent specificity of SOD for the dismutation of 02", i.e. SOD catalyzes both the reduction of 02 to H202 and the oxidation to 02 via a redox cycle of its Cu (II/I) complex moiety as well as the direct electron transfer of SOD realized at the cysteine-modified Au electrode. Thus, this coupling between the electrode and enzyme reactions of SOD could facilitate the development of the third-generation biosensor for 02". ...

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. 

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... 

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