Enzyme amplification

An ingenious method of decreasing detection limits in biosensors that is frequently employed is enzyme amplification. An example is enzyme multiplied immunoassay technology (emit). 

Figure 36.3. Typical behaviour of the concentration of the antigen by multiloading and the enzyme amplification that enables to obtain a signal detectable by electrochemistry even for a starting antigen concentration of 1 pM.

Self, C. H. (1985) Enzyme amplification—a general method applied to provide an immunoassisted assay for placental alkaline phosphatase. J. Immunol. Methods 76, 389-393. 

Endo, T., A. Okuyama, Y. Matsubara, et al. 2005. Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls. Anal. Chim. Acta 531 7-13. 

Bates, D. L. (1987) Enzyme amplification in diagnostics. Trends BkotedawL 5, 204-209 Johannsson, A. and Bates, D L (1988) Ampliflcation by second enzymes, in ELISA and Other Sobd Phase Immunoassays (Kemeny,D. M. andChallacombe,S.J.,eds ),John Wiley, NY, pp 85-106. 

It should be noted that the relationship between the final signal output and concentration of the analyte (dose-response) may be one of direct or inverse proportionality, and is dependent on the specific assay format. In addition, a number of different reporter enzymes may be used (e.g., horseradish peroxidase, alkaline phosphatase, p-galactosidase), along with a number of different signaling systems (e.g., substrates that yield chromogenic or fluorescent or chemiluminescent products, activation of signaling enzymes, amplification by biotin-avidin system or polymerase chain reaction). 

The probes are used in immunology and cell biology as biotinylated fluorescent markers for enzyme amplification, membrane probes, DNA probes, and as specific enzyme substrates for determination of proteases, lipases, alkaline phos-... 

Based on the principle of enzyme amplification for enzyme immunoassays adopted in the 1980s, Bobrow and associates developed a catalyzed reporter deposition technique (CARD) to achieve amplified signal for solid-phase immunoassay system and membrane immunoassays. Subsequently, this CARD technique was introduced to IHC in 1992. ... 

Fig. 2 Enzyme amplification for alkaline phosphatase. The primary substrate is NADP+. The product NAD+ is recycled through a coupled-enzyme redox system of alcohol dehydrogenase and diaphorase for continuous generation of the color product Formazan.

Fig. 6 Assay design using a general secondary antibody label. It is more cost-effective to label the secondary antibody against the animal host species than to label the primary antibody. Assay protocols of Figs. 3 (Fig. 6a) and 5 (Fig. 6b) can be modified to use this strategy. Variations of enzyme amplification or biotinylation can also be introduced.

A Johannsson, DH Ellis, DL Bates. Enzyme amplification for LAs detection limit of 1/100 of an attomole. J Immunol Meth 87 7, 1986. 

Of the optical techniques that have become part of the analyst s armory, chemiluminescence is, perhaps, the latest to have come of age. Historically, absorbance spectrophotometry with chromogenic labels has offered the clinical analyst convenience and universality, since UV/visible spectrophotometers are both ubiquitous and amenable to automation. However, direct detection of chromophores without enzyme amplification is limited in sensitivity to 0.1-1.0 jiM, since few molar extinction coefficients are greater than 10. Radiolabel techniques do offer far superior sensitivity, but as the demands for highly sensitive analyses become more voluminous and more widespread in terms of their setting (i.e., both health-... 

Although it is not the purpose of this article to champion one technology over another, it is fair to say that chemiluminescence offers the sensitivity of fluorescence detection without some of the attendant problems of luminescent emission from analytical samples. Thus, chemiluminescence detection can be comparable to and, with the aid of enzyme amplification, even superior to that of I. This is not to say that there are no problems associated with chemiluminescence-based analytical techniques. As we shall see later (Section 2.5), the actual signal from chemiluminescent molecules is, in most cases, a transient flash, lasting... 

Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification Anal. Chem. Acta. 304 25-31... 

Kuhlmann WD, Peschke P (1986) Glucose oxidase as label in histological immunoassays with enzyme-amplification in a two-step technique coimmobilized horseradish peroxidase as secondary system enzyme for chromogen oxidation. Histochemistry 85 13-17. 

This addition caused the anodic current to decrease by >95% within 6 s, indicating that only 62 generated by the XOD/xanthine system is concerned in the anodic current response measured at SOD/Cys/Au. To examine the direct oxidation of 02 at SOD/Cys/Au without the above-mentioned redox mediation via SOD, the response of apo-SOD/Cys/Au toward O2" was also measured. A much smaller response was obtained at apo-SOD/Cys/Au, indicating that the oxidation of O2 at SOD/Cys/Au was based on the SOD enzyme amplification. The minor current... 

Further enhancement in detection sensitivity of reporter enzymes is achievable by enzyme amplification or cascade reactions often termed enzyme cycling assays. One approach is to use alkaline phosphatase as the reporter enzyme. Phosphatase cleavage of NADP forms NAD, which enters cyclic reactions catalyzed by alcohol dehydrogenase and diaphorase. Each turnover of phosphatase substrate initiates a cascade resulting in numerous detectable product molecules. Such approaches, perhaps incorporating chemiluminophores as the terminal product, hold promise for further extension of the sensitivity of immunoenzymatic methods. 

Another system using enzyme amplification has been patented by IQ Bio Ltd, Cambridge, UK. Alkaline phosphatase was used as a DNA label. The electron transfer connected with the reoxidation of NADH back to NAD" may be followed either photometrically or electrochem-ically [167]. 

Work with algae described above shows that the roost sirople to the roost coroplex (antibody/antigen with enzymic amplification) biological systems provide ready-made microstructures and molecular systems for coupling to electrodes for highly sensitive and selective electroanalytical methods. 

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