Electrochemical immunoassay detection

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... 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

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. 

K.R. Wenmeyer, H.B. Halsall, W.R. Heineman, C.P. Voile, and I.W. Chen, Competitive heterogeneous enzyme immunoassay for digoxin with electrochemical detection. Anal. Chem. 58, 135-139 (1986). 

F.J. Hayes, H.B. Halsall, and W.R. Heineman, Simultaneous immunoassay using electrochemical detection of metal ion labels. Anal. Chem. 66, 1860-1865 (1994). 

This approach separates the steps relative to the immunoreaction from the step of electrochemical detection and for this reason the working electrode surface is easily accessible by enzymatic product, which diffuse onto bare electrode surface [28,33] (Fig. 25.3). Using this strategy, finding the optimum conditions for the immunoassay on the magnetic beads and for electrochemical detection on the transducer (carbon screen-printed electrodes) is much easier than in the usual one (electrode) surface systems, because optimum conditions for immunoassay do not conform with those for electrochemical detection and vice versa. 

This configuration based on the use of two surfaces, magnetic beads for immunoassay and screen-printed electrodes for electrochemical detection, allows to obtain a faster and a more sensitive detection of the immunoreaction than using a unique surface (screen-printed electrode) in this case it is possible to perform the electrochemical measurement in faster times (less then 30 min) and improve the sensitivity (around two magnitude orders). For this reason, this approach is advised in the development of an electrochemical immunosensor specific to any analyte. 

Using this strategy, finding the optimum conditions for the immunoassay on the magnetic beads and for electrochemical detection on the transducer (carbon screen-printed electrodes) is much easier than in the traditional format. 

The specific electrochemical behaviour of IDAs is result of its design [97], i.e. two arrays intercalated and individually addressed in a bipotentiostatic system where reversible redox species can be cycled between one array (generator) and the other array (collector) (Fig. 32.3). The feedback obtained, greatly enhances the current and high sensitive detection can be achieved. An important application of IDAs is the electrochemical detection of p-aminophenol when it is generated from p-aminophenyl phosphate, by enzymatic reaction with alkaline phosphatase (like enzymatic label), in geno- [98-100] and immunoassays [101-103]. Another interesting feature of IDAs is the possibility of... 

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]). 

The use of ELISA is broad and it finds applications in many biological laboratories over the last 30 years many tests have been developed and vahdated in different domains such as clinical diagnostics, pharmaceutical research, industrial control or food and feed analytics for instance. Our work has been to redesign the standard ELISA test to fit in a microfluidic system with disposable electrochemical chips. Many applications are foreseen since the biochemical reagents are directly amenable from a conventional microtitre plate to our microfluidic system. For instance, in the last 5 years, we have reported previous works with this concept of microchannel ELISA for the detection of thromboembolic event marker (D-Dimer) [4], hormones (TSH) [18], or vitamin (folic acid) [24], It is expected that similar technical developments in the future may broaden the use of electroanalytical chemistry in the field of clinical tests as has been the case for glucose monitoring. This work also contributes to the novel analytical trend to reduce the volume and time consumption in analytical labs using lab-on-a-chip devices. Not only can an electrophoretic-driven system benefit from the miniaturisation but also affinity assays and in particularly immunoassays with electrochemical detection. 

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). 

In enzyme electrochemical immunoassays the antigen or a second antibody is labeled with an enzyme that catalyzes the production of an electrochemically detectable product and the rate of the product formation is taken to quantify the antigen. 

Staub, C., Robyr, C., Analysis of Morphine in Hair by Radio-Immunoassay and HPLC with Electrochemical Detection, in Forensic Toxicology Proceedings of the 25th International Meeting, June 27-30, 1988, Groningen, The Netherlands, Uges, D. R. A., de Zeeuw, R. A., Eds., University Press, Groningen, 1988, 230. 

Electrochemical Detection, Immunoassay with (Heineman, Halsall, Wehmeyer,... 

Immunoassay with Electrochemical Detection, see Electrochemical Detection,... 

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. 

Electrochemical immunoassays include a wide variety of devices based on the coupling of immunological reactions with electrochemical transduction. All of them involve the immobilization of an immunoreagent component on the surface of the electrode transducer. Electrochemical detection is based on the direct intrinsic redox behavior either of an analyte species or of some reporter molecule. For the detection no expensive equipment is needed, with the measurement of either a simple current or a voltage charge. Different electrochemical detection strategies are used, but ampero-metric detection is most widely used. Potentiometric and conductometric detection are applied in different assays as well. 

In order to be used as an immunoassay label, an elec-trochemically active compound has to possess suitable electrochemical properties. It has to be soluble in aqueous media and should be stable in solution over a wide pH range. To be detectable, it must allow highly selective electrochemical detection or possess chemical properties to allow selective membranes to be used in the measurement electrode. 

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