Competition Sensors

Two fundamentally different types of competing reactions can be utilized in biosensors enzyme competition, i.e., the competition of different enzymes for one and the same substrate, and substrate competition, 

the competition of different substrates for a single enzyme. The latter may be regarded as an analogy to the determination of competitive inhibitors (see Section 4.2). Similar to enzyme sequences, these competition systems serve to gain access to analytes, not involved in enzyme reactions, which can be electrochemically monitored. Table 12 gives an overview of enzyme and substrate competition sensors. 

The cofactor-dependent competition of two enzymes for glucose is the basis of sensors for the determination of ATP and NAD+ (Pfeiffer et al., 1980). GOD has been coimmobilized with hexokinase and glucose dehydrogenase (GDH), respectively, and attached to the tip of an oxygen 

Substrate fructose hexokinase + glucose-6- Schubert et al. (1986a) 

Scheller et al. (1987c) described a substrate competition electrode for the determination of aniline and phenol (Fig. 94). The analyte competes with hydroquinone for the pseudo-peroxidatic activity of hemoglobin. The decrease of the electrochemical reduction current of benzoquinone in the presence of the alternative substrate served as the measuring signal. 

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Fig. 94. Schematic and measuring curve of a substrate competition sensor for phenols and aniline using immobilized hemoglobin (Hb). (Redrawn from Scheller et al., 1987c).

B. Baruwati, D. K. Kumar, S. V. Manorama, Hydrothermal Synthesis of Highly Crystalline ZnO Nanoparticles A Competitive Sensor for LPG and EtOH. Sensors and Actuators B Chemical 2006,119,676-682. 

Figure 8 Flow injection analysis (FIA) based fluorescent competitive sensor based on molecularly imprinting acrylic polymer. (Reprinted with permission from Suarez-Rodrfguez JL and Dfaz-Garcfa ME (2001) Fluorescent competitive flow-through assay for chloramphenicol using molecularly imprinted polymers. Biosensors and Bioelectronics 16(9-12) 955-961 Elsevier.)...

On another front, to accomplish electrical measurments of individual NWs, free of parasitic effects, and to develop competitive sensors, various fabrication and characterization strategies have been evaluated. For instance, low-current measur ent protocols have been found to aUow the devices to operate long-term withont degradation of their performance (Hemandez-Ramirez et al. 2007a, b). Thus the present state of development of NW-based technologies has led to complete and weh-controUed characterization of proof-of-concept devices, which were previously imattainable (Comini et al. 2009). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

Fig. I Dependence of response of anisotropy sensor on analyte concentration in direct and competition assays (a) and this dependence for direct assay at different correlations between cp and xF (b)...

Sensing schemes have become reliable enough to be of practical utility, instrumentation has become available at costs that make this sensor technology competitive to established sensors, and numerous other sensors schemes have been presented that are of interest, at least from an academic point of view. 

Nonetheless, near-IR is the most widely used IR technique. Less intense water absorptions permit to increase the sampling volume to compensate, to some extent, for the lower near-IR absorption coefficients and the inferior specificity of the absorption bands can for many applications be overcome by application of advanced chemometric methods. Miniaturised light sources, various sensor probes, in particular based on transmission or transflectance layouts, and detectors for this spectral range are available at competitive prices, as are (telecommunications) glass or quartz fibres. 

Similar to IR sensors, process analysis is the prevalent application area. Due to the applicability of standard VIS instrumentation, Raman probes have been used for more than two decades65, 66. Typically, Raman probes are applied where near-IR probes fail and hence are in direct competition to mid-IR probes. 

Enzymes can be used not only for the determination of substrates but also for the analysis of enzyme inhibitors. In this type of sensors the response of the detectable species will decrease in the presence of the analyte. The inhibitor may affect the vmax or KM values. Competitive inhibitors, which bind to the same active site than the substrate, will increase the KM value, reflected by a change on the slope of the Lineweaver-Burke plot but will not change vmax. Non-competitive inhibitors, i.e. those that bind to another site of the protein, do not affect KM but produce a decrease in vmax. For instance, the acetylcholinesterase enzyme is inhibited by carbamate and organophosphate pesticides and has been widely used for the development of optical fiber sensors for these compounds based on different chemical transduction schemes (hydrolysis of a colored substrate, pH changes). 

Enzyme-based optical sensor applications will be further described in this book. They are still the most widespread optical biosensors but work is needed to overcome limitations such as shelf life, long term stability, in situ measurements, miniaturization, and the marketing of competitive devices. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

With the introduction of more sophisticated, consumer-friendly appliances, this trend could be overcome, and European competitiveness could be strengthened. The use of better control systems and the introduction of more and better sensors are a prerequisite for such a development, as we are going to point out in this book. 

When a sensor is used in an appliance, additional components like a low-voltage power supply and control electronics are required. As these components have a big influence on the actual cost price of the product, it is very important that the cost price of the sensor is reduced to the bare minimum, since a lower cost price makes the product more competitive. 

To make a breakthrough in household appliances and other consumer product markets UV sensors have to become significantly cheaper while spectral selectivity as a major key feature must be guaranteed. Most of today s UV photodiodes are made from crystalline semiconductor materials. The cheaper materials (Si) lack spectral selectivity, and the wide band gap materials are very expensive. What they all have in common their top performance regarding sensitivity and speed. Crystalline photodiodes have risetimes of often below 1 s. However, the described processes to be sensed here are not faster than some milliseconds or even much slower. In order to obtain a reasonably-priced SiC or GaN photodiode, the photoactive area is often reduced to below 1 mm2 and barely fills the sensor housing. So far, the top sensitivity offered by the semiconductor has been sacrificed for a competitive... 

Regarding sensors, Draisci et al. [100] reported the development of an electrochemical competitive ELISA for the detection of erythromycin and tylosin in bovine muscle. They used MAbs against these two macrolides and the activity... 

Fig. 37 (a) QD-based sensing of cocaine by the formation of a cocaine-aptamer supramolecular structure that triggers FRET and (b) time-dependent luminescence spectra of the system in the presence of cocaine. The inset shows a calibration curve for variable concentrations of cocaine and a fixed so observation time of 15 min. (c) Schematic of the FRET-based TNT sensor and (d) increase of the QD luminescence upon addition of TNT in the competitive assay format. (Reprinted with permission from [220, 221], Copyright 2009 Royal Society of Chemistry and 2005 American Chemical Society)... 

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