Abstract
The development and optimisation of the AT-cut quartz crystal sensor for hydrogen peroxide is described. The optimised hydrogen peroxide biosensor was subsequently used to determine the total cholesterol concentration in buffer and serum, plus creatinine in buffer, through a series of enzyme reactions that produced hydrogen peroxide from the analyte of interest. The detection of hydrogen peroxide was achieved through the oxidation of the benzidines, 3,3’-diaminobenzidine (DAB), 3,3’-dimethoxybenzidine (DMOB), and 3,3’,5,5’-tetramethylbenzidine (TMB), in the presence of horseradish peroxidase (HRP). The oxidised product is insoluble, but was found to remain in suspension, reducing the crystal’s ability to defect its formation. Through the inclusion of the non-ionic surfactants, Tween 80 and Triton X-100, it was found that greater adsorption of the oxidised benzidine occurred, leading to increased impedance shifts. The presence of Triton X-100 gave an improved response time e. g. with DAB the response was reduced from 21 min to 10 min. The use of polymers with the hydrogen peroxide sensor was found to have no beneficial effect, apart from poly(vinyl chloride), which increased the impedance shift. In the presence of Triton X-100, the response was the same as that for a bare crystal, suggesting the possibility that polymers may be used to reduce crystal fouling by the oxidised benzidine. The presence of the enzymes, glucose oxidase and β-amylase in the reaction mixture increased the impedance response by about 10 and 2-fold respectively, and reduced the response time to 15 min from 68 min, at the concentrations used. All the proteins used had various effects upon the response, and this indicates that the method will suffer from interference (both positive and negative), due to proteins present in the biological sample. The optimised hydrogen peroxide biosensor using Triton X-100 and DAB was used to determine total cholesterol in buffer and serum, using the additional two enzymes, cholesterol esterase (ChE) and cholesterol oxidase (ChOx). The response of the sensor to LDL (low-density lipoprotein) cholesterol gave a response time of less than 25 min. Using the optimised biosensor, a linear response for free and LDL cholesterol was obtained between 50 - 300 μM, and 25 - 400 μM, respectively. HDL (high-density lipoprotein) cholesterol was found to solubilise the oxidised DAB, and hence act as an interferent in the cholesterol determination. A linear response for cholesterol determination (at 300 μM) in serum was obtained. The hydrogen peroxide biosensor with DAB and Triton X-100 was used to measure creatinine in buffer, through the addition of the enzymes, sarcosine oxidase (SO), creatinase (CI), and creatininase (CA). The creatinine determination at the concentration of the enzymes used, reached an endpoint in under 3 hr. The potential of the quartz crystal to determine low molecular solutes through a series of enzyme-based reactions, using the hydrogen peroxide detection strategy has been demonstrated.