Electrochemical immunoassay amperometric detection

Based on many of the advances described above in electrochemical approaches to immunoassay, it is tempting to conclude that commercialization of some of the approaches is imminent. This may be true, but the historical use of optical methods for many clinical chemistry tests coupled with their rapidly growing use in immunoassay is a difficult barrier for any radically different method to overcome, though electrochemical sensors have become more important in the clinical chemistry laboratory over the last decade. In any event, to be successful ECIA methods will have to demonstrate clear superiority over existing and emerging technologies in both cost and performance. Some of the more recently described approaches such as those using enzyme amplified amperometric detection and ecLIA appear... 

As an example of microchip-based electrochemical immunoassays, we describe here the protocol established for the analysis of interleukin IB by enzyme linked immunosorbent assay (ELISA) with amperometric detection at the sub-pM level in DiagnoSwiss microfluidic chip called Immuchip . 

Ho, W. O., Athey, D., and McNeil, C. J. Amperometric detection of alkaline phosphatase activity at a horseradish peroxidase enzyme electrode based on activated carbon - Potential application to electrochemical immunoassay. Biosens. Bioelectron. 1995,10, 683-691. 

Potentiometric, capacitive, and amperometric transducers have been used for direct and indirect electrochemical immunoassays. However, due to their fast detection, broad linear range, and low detection limit, amperometric transducers are preferred. 

Five types of amperometric detection have been applied for enzyme-based ECIA. They are flow-injection analysis with electrochemical detection (FIAEC), liquid chromatography with electrochemical detection (LCEC), amperometric detection with interdigitated array electrodes (IDA), rotating disk electrode (RDE) amper-ometry, and scanning electrochemical microscopy (SECM). Of these, the two conventional types, FIAEC and LCEC, shall be discussed in this section, leaving the discussion of the other types to Section V on miniaturized immunoassays. 

The ability to combine small volumes with excellent sensitivity has made amperometric detection with RDE ideal for bead-based immunoassays. Direct comparison of the results obtained in the above Bugbead assay with those obtained with FIAEC detection showed that the detection limit of RDE was 2 orders of magnitude better than for EIAEC [53]. The other important feature of RDE is the extremely short detection time that could be reached by combining enzyme turnover with electrochemical detection. The RDE detection time of 2 min in the above Bugbead assay is over 10 times faster than the typical detection time for HAEC that requires a 20 min enzyme substrate incubation. It is also clear... 

The microplate model has often been used as a starting platform for electrochemical array development. In a report from Tang et al. [14], a multichannel array consisting of eight channels of Pt electrodes, fitted to the dimensions of standard microplate wells, has been developed, with the required multichannel potentiostat for amperometric detection of the product of an enzymatic immunoassay label. Alkaline phosphatase was used as the model immunoassay label in a model rabbit/anti-rabbit IgG study, where the second IgG is labeled with enzyme. The enzyme product provided the amperometric signal with a detection limit in the low pM range. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

Frew, J.E., Foulds, N.C., Wilshere, ).M., Forrow, N.J., and Green, M.J. (1989) Measurement of alkaline phosphatase activity by electrochemical detection of phosphate esters. Application to amperometric enzyme immunoassay. Journal of Electroanalytical Chemistry,... 

---