Colourimetric assays

Figure 8.14 Colourimetric assay. Acetyl Choline Esterase (AChE) assay system involving thio-acetylcholine that is hydrolyzed to thio-choline. This in turn combines with colourless reagent 5,5 -dithio-bis(nitrobenzoic acid) (DTNB) to form yellow coloured 5-thio-2-nitro-benzoic acid (TNB).

Figure 8.15 Colourimetric assay. Carbonic Anhydrase (CA) assay system. Changes in pH with enzyme catalysis are followed with great precision and accuracy by pH-sensitive dye that changes absorbance profile with pH-change.

Figure 8.18 Colourimetric assay. Malate dehydrogenase (MDH) assay system. Oxalate is onward converted to L-malate by means of enzyme MDH that uses the reverse-colourimetric reductant NADH to effect catalytic reduction.

The phenol-sulphuric acid colourimetric assay can be used for the quantitative determination ( 3-4%) of glycosides, oligosaccharides, glyc[Pg.341]

The other major class of DNAzyme reported mimics the action of enzymes like horseradish peroxidase, utilising in the process hydrogen peroxide as oxidant. Various reports describe mechanistic details of this class of DNAzyme, and a number of modifications have also been described. These include the modification by addition of PEG to the DNAzyme to make the DNAzyme active in organic solvents, the effect of backbone modifications, and a variant of the DNAzyme that requires Pb(II) ions for activity. A variant of the DNAzyme has been reported that mimics a NADH oxidase, and variants are reported that act as logic-controlled biofuel cells, " " and for the generation of DNA nanostructures. Publications have been described that use the peroxidase DNAzyme as a colourimetric assay for the detection of PCR products, for a fluorimetric assay of genetically-modified... 

S144 Harding, R.S. and Virapen, K.V. (1986). Evaluation of the ARIS colourimetric phenobarbital assay. Clin. Chem. 32, 1085, Abstr. 177. 

Figure 8.17 Enzyme coupled assay. Triose Phosphate Isomerase (TIM) assay system with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) colourimetric coupled assay for detection purposes. In this version of the TIM assay, dihydroxyacetone phosphate (DHAP) is converted enzymically to glyceraldehyde 3-phosphate (GAP) that is onward converted to glycerol phosphate (GP) by means of the coupled enzyme GAPDH enzyme that uses the reverse-colourimetric reductant NADH.

Reversible dye binding systems can be developed into colourimetric and fluorescent assays for analytical detection. As shown in Figure 11.3, two different binding schemes can be envisioned, (a) competitive dye/analyte inclusion, and (b) cooperative dye/analyte inclusion. The competitive inclusion process is the basis for the dye displacement assay where the analyte displaces the dye from the container molecule. For this process to be visualized, the properties of complexed and uncomplexed dyes must be markedly dif-... 

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