Enzyme assays, electrochemical detection

The anti-DNA antibody has been used as a marker molecule of Systemic Lupus Erthematosus (SLE) which is a severe autoimmune disease. Enzyme immunoassay is the most reliable, widely used method of assay however, the electrochemical detection method reported here should be interesting for the purpose of a rapid and convenient diagnostic tool of SLE. 

Z.P. Aguilar and I. Fritsch, Immobilized enzyme-linked DNA-hybridization assay with electrochemical detection for Cryptosporidium parvum hsp70 mRNA. Anal. Chem. 75, 3890-3897 (2003). 

J. Rossier and H. Girault, Enzyme linked immunoabsorbent assay on a microchip with electrochemical detection, Lab on a Chip, 1 (2001) 153-157. 

R.M. Pemberton, J.P. Hart and T.T. Mottram, An assay for the enzyme lV-acetyl-/ -n-glucosaminidase (NAGase) based on electrochemical detection using screen-printed carbon electrodes (SPCEs), Analyst, 126 (2001) 1866-1871. 

J. Wang, M.P. Chatrathi, A. Ibanez and A. Escarpa, Micromachined separation chips with post-column enzymatic reactions of class enzymes and end-column electrochemical detection assays of amino acids, Electroanalysis, 14 (2002) 400-404. 

This chapter presents an approach to perform enzyme linked immunosorbent assays (ELISA) in a microfluidic format with electrochemical detection. This field of analytical chemistry has shown a strong activity in recent years, and many reports have presented the use of capillary-sized reactors for running immunoassays either in homogeneous format (where the antigen-antibody complex and the labelled revelation reagents are separated prior to detection, as for instance by capillary electrophoresis [1-3]) or in heterogeneous format (where the antibody is immobilised on the inner surface of the microsensor device [4] or on microbeads [5,6]). 

Acetylcholineesterase and choline oxidase Enzymes were co-immobilized on chemically preactivated immun-dyne polyamide membrane (thickness 120 pm, size cut-off 3 pm). Applying 20 pL of 1% ChO solution in 0.1 M-phosphate buffer (pH 6). Assay was based on electrochemical detection of the generated h2o2. The response time was 2min. Detection limit was 50 nM and the response was rectilinear up to 20 pM. [71]... 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

The enzyme assay components including internal standards are resolved within 3 minutes on a 4.6 mm x 3.3 cm, 3 gm reversed-phase ODS column (Perkin-Elmer). The mobile phase contained 0.100 M citric acid, 0.50 mW tetrapentylammonium chloride (an ion-pairing agent), 50 fiM EDTA, and 12.5% acetonitrile (v/v) titrated to pH 4.70 with NaOH. Eluted compounds were detected with an electrochemical detector. The separation obtained is shown in Figure 9.16. 

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. 

Doyle, M.J. Halsall, H.B. Heineman, W.R. Enzyme-linked immunoasorbent assay with electrochemical detection for alpha-1-acid glycoprotein. Anal. Chem. 1982, 56, 2318-2322. 

The electrochemical detection utilized the re-oxidation of hexacyano-ferrate(II) on a platinum electrode. For pyruvate determination this assay was extended to a 3-enzyme system by the addition of glutamate p5u-uvate transaminase, which produces alanine from pyruvate. All enz5unes were used in solution in a reaction chamber of approximately 2 pi directly in front of the electrode. The cofactor NAD" " was coupled to dextran with a molecular weight of 40,000 to avoid its replacement for each assay. As the sensor responded to L-alanine and pyruvate again a differential measurement was required when a sample contained both compounds. It was applied to off-line monitoring of a cultivation of S. cerevisiae and data showed good correlation to the photometric assays. 

Enzyme DNA hybridization assays with electrochemical detection can offer enhanced sensitivity and reduced instrumentation costs in comparison with their optical counterparts. Efforts to prevent non-specific binding of the codissolved enzyme and to avoid fouling problems by selecting conditions suitable to amplify the electrode response have been reported by Heller and co-workers [107]. A disposable electrochemical sensor based on an ion-exchange film-coated screen-printed electrode was described by Limoges and co-workers for an enzyme nucleic acid hybridization assay using alkaline phosphatase [108] or horseradish peroxidase [109]. In another methodology to improve sensitivity, a carbon paste electrode with an immobilized nucleotide on the electrode surface and methylene blue as hybridization indicator was coupled, by Mascini and co-workers [110], with PGR amplification of DNA extracted from human blood for the electrochemical detection of virus. 

Modern tHcy methods include enzyme immunoassays and chromatographic-based methods. In practice, immunoassays " are most often used for routine purposes (e.g., fluorescence polarization immunoassay IFPIA] as run on Abbott s IMx and AxSYM platforms) Chromatographic assays include amino acid analysis high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection ° " capillary electrophoresis with fluorescence detection gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem MS (MS-MS). 

Under both optimum and shortened assay conditions the labeled antibody bound by the lowest detectable rabbit IgG standard sufficed to produce a phenol concentration in the 10 to 100 nmol/liter range. Phenol concentrations in these ranges did not strain the detection capabilities of the electrochemical technique. For both the optimum and shortened assays the detection limit was not set by the ability to quantitate the phenol produced by the enzyme, but rather by the amount of nonspecific adsorption of the antibody-enzyme conjugate demonstrated by the 0 ng/liter standard. This nonspecific binding corresponded to a phenol concentration in the 10 nmol/liter region. 

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