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Chili hotness is very much dependent on the concentration of capsaicin present in the chili fruit. A new biosensor based on a horseradish peroxidase enzyme-capsaicin reaction mediated by ferrocene has been successfully developed for the amperometric determination of chili hotness. The amperometric biosensor is fabricated based on a single-step immobilization of both ferrocene and horseradish peroxidase in a photocurable hydrogel membrane, poly(2-hydroxyethyl methacrylate). With mediation by ferrocene, the biosensor could measure capsaicin concentrations at a potential 0.22 V (vs. Ag/AgCl), which prevented potential interference from other electroactive species in the sample. Thus a good selectivity towards capsaicin was demonstrated. The linear response range of the biosensor towards capsaicin was from 2.5-99.0 µM with detection limit of 1.94 µM. A good relative standard deviation (RSD) for reproducibility of 6.4%-9.9% was obtained. The capsaicin biosensor demonstrated long-term stability for up to seven months. The performance of the biosensor has been validated using a standard method for the analysis of capsaicin based on HPLC.

The evaluation of chilli hotness using amperometric capsaicin biosensor-based enzyme that was immobilized covalently to the surface of modified acrylic microspheres is the first of its kind presented in this work. The immobilization of enzyme covalently to the surface of microspheres via succinimide groups prevented the leaching of the enzyme. The enzymatic reaction between horseradish peroxidase-capsaicin in the presence of hydrogen peroxide, which was mediated by vinyl ferrocene, enabled the current measurement at a low potential (0.22 V). Besides, the pungency level of the chilli that was proportional to the capsaicin concentration was measured using this method. This biosensor gave a linear response range towards capsaicin from 0.75–24.94 μM (R 2 = 0.992) with a detection limit at 0.39 μM. Moreover, the relative standard deviation (RSD) for reproducibility study was 8.2% (n = 7). Therefore, this biosensor was successfully applied for evaluation of chilli hotness in chilli sample, as well as in comparison with a standard method that employed the HPLC method. [ABSTRACT FROM AUTHOR]

Abstract: The fabrication of an optical biosensor based on stacked immobilisation of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in sol–gel/butyl acrylate hybrid film and horseradish peroxidase (HRP) in chitosan film for capsaicin detection was studied. The optimum volume ratio of sol–gel to butyl acrylate was 1:0.5 (v/v). Peroxidase was immobilised in chitosan film deposited on top of the hybrid sol–gel/butyl acrylate film containing MBTH. The reaction between capsaicin and MBTH in the presence of HRP and hydrogen peroxide as oxidative agent was studied. The finding indicated that HRP catalysed the oxidative coupling reaction between MBTH and capsaicin thus producing red-coloured azo-dye compounds that could be observed at the wavelength of 505nm. The colour intensity of the product was found to increase in proportion to the capsaicin concentration after 20–30s of exposure. The linearity of the biosensor towards capsaicin was in the concentration range of 0.2–4.0mM with detection limit of 0.17mM. Relative standard deviation (RSD) values of reproducibility were 5.57–6.33%. The developed biosensor correlated well with the standard method, HPLC for the determination of capsaicin concentration in real samples. [Copyright &y& Elsevier]