Electrochemical enzyme immunoassay

Example 6-2 The following standard addition plot was obtained for a competitive electrochemical enzyme immunoassay of the pesticide 2,4-D. A ground water sample (diluted 1 20 was subsequently assayed by the same protocol to yield a current signal of 65 nA. Calculate the concentration of 2,4-D in the original sample. 

Because of this lack of resolving power, much electroanalytical research is aimed at providing increased selectivity. This can be accomplished in two ways. First, electrochemistry can be combined with another technique which provides the selectivity. Examples of this approach are liquid chromatography with electrochemical detection (LCEC) and electrochemical enzyme immunoassay (EEIA). The other approach is to modify the electrochemical reaction at the electrode to enhance selectivity. This... 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique... 

A competitive electrochemical enzyme immunoassay has been demonstrated for digoxin Alkaline phosphatase, which catalyzes the hydrolysis of phenyl phosphate... 

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

Homogeneous electrochemical enzyme immunoassays for both phenytoin and digoxin have been developed. In both cases the label was glucose-6-phosphate dehydrogenase, which catalyzes the reduction of NAD to NADH. The NADH produced was detected by LCEC at a carbon paste electrode. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

H. Dong, C.M. Li, Q. Zhou, J.B. Sun, and J.M. Miao, Sensitive electrochemical enzyme immunoassay microdevice based on architecture of dual ring electrodes with a sensing cavity chamber. Biosen. Bioelectron. 22, 621-626 (2006). 

T.T. Hua, C.E. Lunte, H.B. Halsall and W.H. Heineman, p-Aminophenyl phosphate An improved substrate for electrochemical enzyme immunoassay, Anal. Chim. Acta, 214 (1988) 187-195. 

Q. Gao, Y. Ma, Z.L. Cheng, W.D. Wang and M.R. Yang, Flow injection electrochemical enzyme immunoassay based on the use of an immuno-electrode strip integrate immunosorbent layer and a screen-printed carbon electrode, Anal. Chim. Acta, 488 (2003) 61-70. 

J. Wang, A. Ibanez, M.P. Chatrathi and A. Escarpa, Electrochemical enzyme immunoassays on microchip platforms, Anal. Chem., 73 (2001) 5323-5327. 

Li, X.-M., X.-Y. Yang, and S.-S. Zhang. 2008. Electrochemical enzyme immunoassay using model labels. 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Figure 4-16 Principle of nonseparation electrochemical enzyme immunoassay (NEEIA) concept, configured in sandwich immunoassay mode to detect a given protein analyte. Enzyme label on reporter antibody generates an electroactive product that is detected at surface of porous gold electrode when substrate for enzyme is added to the back side of membrane.

Meyerhoff ME, Duan C, Meuse M, Novel nonseparation sandwich-type electrochemical enzyme immunoassay system for detecting marker proteins in undilute blood, Clin Chem 1995 41 1378-84. 

Diaz-gonzalez, M., GonzaJez-garda, M.B., and Costa-garda, A. (2005) Recent advances in electrochemical enzyme immunoassays. Electroanalysis, 17 (21), 1901-1918. 

Tang, H.T., Lunte, C.E., Halsall, H.B., and Heineman, W.R. (1988) p-Aminophenyl phosphate an improved substrate for electrochemical enzyme immunoassay. Analytica Chimica Acta, 214,187-195. 

DA Palmer, et al. Flow injection electrochemical enzyme immunoassay for theophylline using a protein-A immunoreactor and /t-aminophenyl phosphate// -aminophenol as the detection system. Analyst 117 1679, 1992. 

Samples from patients receiving digoxin therapy were analyzed by the heterogeneous immunoassay LCEC method. Samples were diluted 5/1 with pooled human plasma in order to eliminate an observed antibody matrix effect. Digoxin standards in pooled human plasma were treated similarly and a standard curve constructed. Digoxin levels in patient samples were determined by reference to the standard curve. The values obtained for the samples by the electrochemical enzyme immunoassay method were compared to those obtained by radioimmunoassay. The results for the 54 samples analyzed are presented in Fig. 8. A good correlation was obtained between the two methods (r = 0.93). 

The amplification of the amperometric response of redox species has been studied for p-aminophenol The sensitive detection of this compound is of particular interest since it is the basis for the measurement of enzymes such as alkaline phosphatase and (J-galactosidase, which are frequently used in enzyme immunoassays The electrode active species (p-aminophenol) is liberated in the enzyme catalysed hydrolysis of its aminophenylated substrate which itself exhibit an irreversible electrochemical behavior The utility of p-aminophenyl phosphate in enzyme immunoassays in combination with IDA electrodes has been demonstrated in [6] The unfavorable pH-optimum of alkaline phosphatase is the drawback of this system Because of its neutral pH-optimum p-galactosidase appeared to be more advantageous An illustration of the proposed electrochemical enzyme immunoassay is given in Fig 2... 

O Niwa, Y Xu, H B Halsall, W R Heinemann. Small-volume voltammetnc detection of 4-aminophenol with mterdigitated array electrodes and its application to electrochemical enzyme immunoassay Anal Chem 65 (1993), 1559-1563... 

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