Amperometric enzyme assays

Amperometry. Amperometric methods measure the current produced at a working electrode in response to an applied potential. Amperometric enzyme assays rely on the production of an oxidizable or reducible species from an enzyme-catalyzed reaction. The applied potential is extreme enough to completely oxidize (at positive potentials) or reduce (at negative potentials) any analyte that contacts the working electrode. In stirred or unstirred solutions, the current produced under such mass-transport-controlled conditions is directly proportional to analyte concentration. 

McNeil, C. J., Spoors, J. A., Cocco, D., Cooper, J. M., Bannister, J. V., Thermostable Reduced Nicotinamide Adenine Dinucleotide Oxidase Application to Amperometric Enzyme Assay , Anal. Chem. 61 (1989) 25-29. 

M. Castano-Alvarez, M.T. Fernandez-Abedul and A. Costa-Garcia, Amperometric PMMA-microchip with integrated gold working electrode for enzyme assays, Anal. Bioanal. Chem., 382 (2005) 303-310. 

Thin-layer Studies. The thin-layer electrochemical system was developed to address the lack of sensitivity of a preliminary bulk amperometric activity assay (77). The first set of thin-layer studies was taken to characterize the thin-layer cells in soluble enzyme solutions and to determine if there were any interferences to the detection of hydrogen peroxide. Preliminary thin-layer studies (23) indicated that the oxidation of hydrogen peroxide could be detected at approximately 1080 mV with only minimal interference from the oxidation of glucose by gold. The addition of chloride ion to the solution further suppressed the glucose electrooxidation interference. 

A sandwich assay for HCG using GOD as label is also described (309). The procedure is based on an amperometric enzyme immunoassay with an electrode-immobilized antibody. The antibody electrode (activated glassy carbon) is used both to separate the assay and to monitor the activity of the bound enzyme label. Two monoclonal antibodies directed against different antigenic sites are used ... 

Enzyme biosensors containing pol3mieric electron transfer systems have been studied for more than a decade. One of the earlier systems was first reported by Degani and Heller [1,2] using electron transfer relays to improve electrochemical assay of substrates. Soon after Okamoto, Skotheim, Hale and co-workers reported various flexible polymeric electron transfer systems appUed to amperometric enz5une biosensors [3-16], Heller and co-workers further developed a concept of wired amperometric enzyme electrodes [17—27] to increase sensor accuracy and stability. 

The signal-concentration dependence for electrochemical biosensors is linear between one and three concentration decades. The lower limit of detection is at 0.2 mmol I" with potentiometric and 1 jUmoll with amperometric enzyme electrodes. The use of amplification reactions allows us to decrease the detection limit to the nanomolar to picomolar range. Whereas the response time of potentiometric enzyme electrodes averages 2-10 min, with amperometric ones an assay can be conducted within a few seconds up to 1 min. This permits up to several hundred determinations per hour to be performed. Increasing complexity of the biochemical reaction system, e.g. by coupled enzyme reactions, may bring about an increase in the overall measuring time. 

Clark et al. [32] have published in 1981 a method employing both enzymes in solution and potentiometrically measuring the concentration of the hydrogen peroxide formed. For an amperometric cholesterol assay Yao et al. [33, 34] used immobilized cholesterol esterase and cholesterol oxidase in a reactor assaying the hydrogen peroxide formed at a peroxidase electrode with the redox pair hexacyanoferrate(II)/hexacyanoferrate(III) as a mediator. 

FIGURE 3-21 Typical amperometric readout during automated flow injection assays of ethanol at an enzyme carbon-paste electrode. Peaks a through h 2 x 10 5 M to 1.6 x 10 4 M ethanol. 

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

Tabata, S. and Dohi, Y., An assay for oligo-(l—>4) —5(1—>4)-glucantransferase activity in the glycogen debranching enzyme system by using HPLC with a pulsed amperometric detector, Carb. Res., 230,179, 1992. 

Sensors for home use glucose sensor operating with a mediator. In the second type of sensor, the amount of glucose is monitored amperometrically via the current. At the heart of the assay is enzymatic oxidation of glucose by glucose oxidase (GOD). The enzyme itself is not electroactive, so a mediator will oxidize it. The mediator of choice is ferrocene (Felcplj), which can readily oxidize to form a stable radical cation (Fe(cp)J ) (see Figure 6.31). 

Figure 3.12 — Interfacing of a fermenter to an FI system. The fermenter medium is continuously recycled by a pump to the filter unit, from which the filtrate is guided to a small reservoir (500 /xL). The sample solution is aspirated through a dialyser, the acceptor stream of which is fed to the injector of the FIA system. The analyte content is assayed amperometrically by using the glucose sensor incorporating the enzyme-containing chemically modified electrode. (Reproduced from [86] with permission of Elsevier Science Publishers).

P.W. Alexander and G.A. Rechnitz, Enzyme inhibition assays with an amperometric glucose biosensor based on thiolate self-assembled mono-layer, Electroanalysis, 12 (2000) 343-350. 

H. Mohammadi, A. Amine, S. Cosnier and C. Mousty, Mercury-enzyme inhibition assays with an amperometric sucrose biosensor based on a trienzymatic-clay matrix, Anal. Chim. Acta, 543 (2005) 143-149. 

Results obtained with the electrochemical biosensor were compared to those obtained from the colorimetric PPI assay with the enzyme in solution and by HPLC (see Table 21.1 of Procedure 21 in CD accompanying this book). All real samples contained microcystin at levels detectable by the amperometric biosensor and the colorimetric PPI... 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

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 . 

Scheme 2. Reaction pathways utilising a redox enzyme linked amperometric measurement via a redox mediator, (b) assay for glucose (a)+(b) assay for creatine, creatine kinase or hexokinase (c) competitive immunoassay using Fc-labelled antigen.

Potentiometric. Not all analytes can be readily assayed via a redox enzyme and in these instances the assay schemes suggest parameters other than electron transfer which may be probed. Indeed, even where a redox enzyme is available for the analyte in question, it is not always desirable to deploy an amperometric technique. 

To aleviate the dissolution of the enzymatic film during the glucose assay procedure, a covering membrane made by addition of 10 pi of Nafion 0.5% was used. The amperometric current increases until a constant value of 20 pA is reached on the fourth glucose assay (Fig. IB). The addition of Nafion film atop of the AQ-enzyme layer doesn t seem to denaturate the enzyme entrapped in the AQ film because the current reached a similar value as the current observed in absence of Nafion. On the other hand, the use of Nafion as covering membrane gives a thicker film that leads to mass transport limitation of the substrates in the AQ film (20). It results in a longer time to obtain the steady-state current. 

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