Amperometric immunosensors

The amperometric immunosensors reported so far rely on various methodological principles including use of a Clark electrode for detecting oxygen formation or depletion, an electrochemically active product yielded in an enzyme reaction or an antigen labelled with an electroactive species. 

Several amperometric immunosensors have been developed for monoclonal antibodies (IgG), a-interferon and the pesticide 2,4-dichlorophenoxy-acetic acid (2,4-D) by using a flow-cell with the catching antibody covalently bound to a cellulose acetate or activated nylon membrane as shown in Fig. 3.31. B. With multiepitope antigens (e.g. a protein), after the antigen is bound and washed, a second enzyme-labelled antibody is used to form a sandwich 

Some electrochemically active substances that can generate photons on an electrode surface are suitable labels for homogeneous immunoassays. A labelled antigen exhibits an electrochemical reactivity and produces luminescence, but when it is immunochemically complexed, the labelled antigen loses its electrochemiluminescent properties. One optical immunosensor for homogeneous immunoassays was assembled by spattering platinum on the end surface of an optical fibre. Spattered platinum maintains optical transparency and functions as an electrode. An optical electrode efficiently 

In amperometry, the current produced by the oxidation or reduction of an electroactive analyte species at an electrode surface is monitored under controlled potential conditions. The magnitude of the current is then related to the quantity of analyte present. However, as both antibody and antigen are not intrinsically electroactive, a suitable label must be introduced to the immunocomplex to promote an electrochemical reaction at the immunosensors. In this respect, enzyme labels including the 

Electrochonical Sensors, Biosensors and Their Biomedical Applications 

They found that the hydrolysis products of 4-AP and 1-naphthol produced well-defined anodic responses at low potentials at a bare SPCE. However, the presence of antibody immobilized on the electrode surface slowed the diffusion of 4-AP towards the electrode surface. In addition, 4-AP may interact with polyphenols on the electrode surface, thus reducing the electroactive working area of the electrode by fouhng. In contrast, diffusion of 1-naphthol to the electrode surface was not hindered by immobilized antibody. This feature, along with its low cost, ease of availability, and high solubility, resulted in 1-NP being the preferred AP substrate in their work. 

A similar study has also been conducted to determine the suitabihty of ascorbic acid 2-phosphate (AAP) as an alternative substrate to 4-AP for AP under identical conditions [48], Although 4-APP and AAP were suitable substrates for amperometric immunosensors, 4-APP was superior owing to its sixfold faster enzymatic reaction and lower detection potential (approximately 200-400mV). Notably, the lower detection potential for the hydrolysis product of 4-APP minimizes interferences from other species and hence improves the sensitivity of the immunosensor. 

A comparison of the products of AP hydrolysis of HQDP (HQ), PP, and 1-NP using cyclic voltammetry revealed that HQ produced well-defined peaks, and that the oxidation of HQ is reversible. More importantly, no apparent passivation of the electrode surface was observed even at high millimolar concentrations after 50 scans. Following a series of investigations, this non-fouling nature of HQ was attributed to the non-accumulation of its oxidation products on the electrode surface and the good diffusional properties of HQ at the electrode-solution interface. Another positive feature of HQDP as a substrate for AP is a tenfold greater oxidation current response of HQ compared to those obtained in the presence of PP or 1-NP. Qverall, HQDP provides a suitable and attractive alternative substrate system for AP in the development of amperometric immunosensors. 

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J. Zeravik, T. Ruzgas, and M. Franek, A highly sensitive flow-through amperometric immunosensor based on die peroxidase chip and enzyme-channeling principle. Biosens. Bioelectron. 18,1321—1327 (2003). 

K. Grennan, G. Strachan, A.J. Porter, A.J. Killard, and M.R. Smyth, Atrazine analysis using an amperometric immunosensor based on single-chain antibody fragments and regeneration-free multi-calibrant measurement. Anal Chim. Acta 500, 287-298 (2003). 

T. Kalab and P. Skladal, A disposable amperometric immunosensor for 2,4-dichlorophenoxyacetic acid. 

Due to the broad substrate specificity of AP, and the drive for higher efficiency, several studies have recently investigated the suitability of alternative substrates to the common 4-APP [47, 48], For example, Pemberton et al. compared 4-APP and 1-naphthyl phosphate (1-NP) as AP substrates in an amperometric immunosensor for progesterone [47], The signal generation scheme when 1-NP is used as a substrate is illustrated in Scheme 2. 

In some instances, the design of an amperometric immunosensor may be such that the enzyme is located some distance away from the electrode surface, or the presence of interfering substances in biological samples may require using an alternative electron transfer pathway. This usually involves a redox-active species with a small molecular... 

Y.-M. Zhou, Z.-Y. Wu, G.-L. Shen, and R.-Q. Yu, An amperometric immunosensor based on Nafion-modified electrode for the determination of Schistosoma japonicum antibody. Sensors and Actuators, B Chemical 89, 292-298 (2003). 

T.-S. Zhong and G. Liu, Silica sol-gel amperometric immunosensor for Schistosoma japonicum antibody assay. Anal. Sci. 20, 537-541 (2004). 

C. Singh, G.S. Agarwal, G.P. Rai, L. Singh, and V.K. Rao, Specific detection of Salmonella typhi using renewable amperometric immunosensor. Electroanalysis 17, 2062-2067 (2005). 

R.E. Ionescu, C. Gondran, S. Cosnier, L.A. Gheber, and R.S. Marks, Comparison between the performances of amperometric immunosensors for cholera antitoxin based on three enzyme markers. Talanta... 

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R.M. Pemberton, J.P Hart, P Stoddard, and J.A. Foulkes, A comparison of 1-naphthyl phosphate and 4 aminophenyl phosphate as enzyme substrates for use with a screen-printed amperometric immunosensor for progesterone in cows milk. Biosens. Bioelectron. 14, 495-503 (1999). 

E.J. Moore, M. Pravda, M.P Kreuzer, and G.G. Guilbault, Comparative study of 4-aminophenyl phosphate and ascorbic acid 2-phosphate, as substrates for alkaline phosphatase based amperometric immunosensor. Anal. Lett. 36, 303-315 (2003). 

J. Wang, P. Pamidi, and K.R. Rogers, Sol-gel-derived thick-film amperometric immunosensors. Anal. Chem. 70, 1171-1175 (1998). 

M. Santandreu, F. Cdspedes, S. Alegret, and E. Martinez-Fiibregas, Amperometric immunosensors based on rigid conducting immunocomposites. Anal. Chem. 69, 2080-2085 (1997). 

R. Blonder, E. Katz, Y. Cohen, N. Itzhak, A. Riklin, and I. Willner, Application of redox enzymes for probing the antigen—antibody association at monolayer interfaces development of amperometric immunosensor electrodes. Anal. Chem. 68, 3151—3157 (1996). 

J. Li, L.T. Xiao, G.M. Zeng, G.H. Huang, G.L. Shen, and R.Q. Yu, Amperometric immunosensor based on polypyrrole/poly(m-phenylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(D2-isopentenyl) adenosine assay. Anal. Biochem. 321, 89—95 (2003). 

Z. Dai, E Yan, J. Chen, and H.X. Ju, Reagentless amperometric immunosensors based on direct electrochemistry of horseradish peroxidase for determination of carcinoma antigen-125. Anal. Chem. 75, 5429-5434 (2003). 

C.H. Liu, K.T. Liao, and H.J. Huang, Amperometric immunosensors based on protein A coupled poly-aniline-perfluorosulfonated ionomer composite electrodes. Anal. Chem. 72, 2925-2929 (2000). 

I. Willner, R. Blonder, and A. Dagan, Application of photoisomerizable antigenic monolayer electrodes as reversible amperometric immunosensors. J. Am. Chem. Soc. 116, 9365-9366 (1994). 

H.S. Jung, J.M. Kim, J.W. Park, H.Y. Lee, and T. Kawai, Amperometric immunosensor for direct detection based upon functional lipid vesicles immobilized on nanowell array electrode. Langmuir 21, 6025-6029 (2005). 

C.X. Lei, Y. Yang, H. Wang, G.L. Shen, and R.Q. Yu, Amperometric immunosensor for probing complement III (C3) based on immobilizing C3 antibody to a nano-Au monolayer supported by sol-gel-derived carbon ceramic electrode. Anal. Chim. Acta 513, 379-384 (2004). 

FIGURE 16.9 Principle of reagentless amperometric immunosensor based on immobilized antigen, competitive immunological reaction, and direct electrochemistry of HRP label (adapted from [138]). 

Electrochemical immunosensors are a powerful tool for the analysis of antibacterials in food and different configurations have been presented during recent years. For example, an amperometric immunosensor was reported by Wu et al. [182], for penicillin quantification in milk, with a linear range from 0.25 to 3 ng/ml and a limit of detection of 0.3 pg/L [182]. Other types of transduction have been also explored, like a label-free impedimetric flow injection immunosensor for the detection of penicillin G. 

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