Biosensors Using Coupled Enzyme Reactions

Of all types of biosensors, metabolism sensors based on the molecular analyte recognition and conversion have been most intensively studied. According to the degree of integration of the biocomponents they can be classified into monoenzyme sensors, biosensors using coupled enzyme reactions, organelle, microbial, and tissue-based sensors. The sequence of the following sections corresponds to this classification. 

Fig. 140. Internal signal processing in biosensors using coupled enzyme reactions. (Redrawn from Scheller et al., 1985b).

In biosensors, interactions between the immobilized biomolecule and the sample, which is often a highly complex matrix, may cause undesired binding events or measuring effects. Particularly in biosensors that use coupled enzyme reactions the substrates of each reaction will interfere therefore, increasing complexity of biosensors results in a decreased selectivity. Interferences can also occur on the level of the transducer reaction. 

The signal-concentration dependence for electrochemical biosensors is linear between one and three concentration decades. The lower limit of detection is at 0.2 mmol I" with potentiometric and 1 jUmoll with amperometric enzyme electrodes. The use of amplification reactions allows us to decrease the detection limit to the nanomolar to picomolar range. Whereas the response time of potentiometric enzyme electrodes averages 2-10 min, with amperometric ones an assay can be conducted within a few seconds up to 1 min. This permits up to several hundred determinations per hour to be performed. Increasing complexity of the biochemical reaction system, e.g. by coupled enzyme reactions, may bring about an increase in the overall measuring time. 

Types of Coupled Enzyme Reactions Used in Biosensors... 

Coupled enzyme reactions mimic certain metabolic situations in organelles and cells that have themselves already been exploited in biosensors [367], [368], The use of enzyme sequences facilitates ... 

Table 13.2 summarises the different approaches used to construct enzyme electrochemical biosensors for application to food analysis based on the different types of enzymes available. Generally, the main problems of many of the proposed amperometric devices have been poor selectivity due to high potential values required to monitor the enzyme reaction, and poor sensitivity. Typical interferences in food samples are reducing compounds, such as ascorbic acid, uric acid, bilirubin and acetaminophen. Electrocatalysts, redox mediators or a second enzyme coupled reaction have been used to overcome these problems (see Table 13.2), in order to achieve the required specifications in terms of selectivity and sensitivity. 

This review is a survey of the research on the direct electron transfer (DET) between biomolecules and electrodes for the development of reagentless biosensors. Both the catalytic reaction of a protein or an enzyme and the coupling with further reaction have been used analytically. For better understanding and a better overview, this chapter begins with a description of electron transfer processes of redox proteins at electrodes. Then the behaviour of the relevant proteins and enzymes at electrodes is briefly characterized and the respective biosensors are described. In the last section sensors for superoxide, nitric oxide and peroxide are presented. These have been developed with several proteins and enzymes. The review is far from complete, for example, the large class of iron-sulfur proteins has hardly been touched. Here the interested reader may consult recent reviews and work cited therein [1,19]. 

Reports on the use of monomeric MPc complexes for the electrocatalysis of phenols are rare, hence the use of some polymeric MPc complexes will be included in this section. Biosensors based on enzymes have been developed for many electrochemical analyses. The use of MPc complexes as part of the enzyme improves the sensitivities of the biosensors. Ozsoz et al. used CoPc monomer dispersed in mushroom tissue electrode as a catalyst for the analysis of phenolic compounds The enzymatic reaction between mushroom and the phenolic compounds was coupled with the catalytic activity of CoPc. The CoPc dispersed electrodes gave shorter response times and lower potentials compared to conventional tissue biosensors . 

Polyaniline is the conducting polymer most commonly used as an electrocatalyst and immobilizer for biomolecules [258-260]. However, for biosensor applications, a nearly neutral pH environment is required, since most biocatalysts (enzymes) operate only in neutral or slightly acidic or alkaline solutions. Therefore, it has been difficult or impossible to couple enzyme catalyzed electron transfer processes involving solution species with electron transport or electrochemical redox reactions of mostly polyaniline and its derivatives. Polyaniline is conducting and electroactive only in its protonated (proton doped) form i.e., at low pH valnes. At pH values above 3 or 4, polyaniline is insulating and electrochemically inactive. Self-doped polyaniline exhibits redox activity and electronic conductivity over an extended pH range, which greatly expands its applicability toward biosensors [209, 210, 261]. Therefore, the use of self-doped polyaniline and its derivatives could, in principle. 

---