The voltammetric measurement has been performed by transferring the oxidized TMB solution to the 96X carbon electrode. Thus, linear sweep voltammetry were measured by sweeping the potential from +6.00 to -2.00 mV. The calibration curve couldn 't be plotted with the obtained voltammograms, therefore, the cocaine-benzoylecgonine antibody concentration has been raised to 0.05 µg/ml (10 fold more) and the tracer dilution ratio to 1:10000. That leads to increase the absorbance (OD) detected by ELISA to 4. The obtained voltammograms were investigated and the calibration curve was plotted. It showed non linear four parameter logistic relationship fit between the concentration of cocaine and the potential applied. As shown in the figures, with increasing the OD, the potential peak position shift with different cocaine concentration. …show more content…
Consequently, the concentration of cocaine benzoylecgonine antibody and BEC-HRP conjugate concentration was optimized to enhance the sensitivity of the assay. Therefore, cocaine-benzoylecgonine antibody concentrations (0.05, 0.025, 0.0125, 0.005µg/ mL) were combined with different dilution of BEC-HRP conjugate (1:1000, 1:5000, 1:10000, 1:15000). Non-specific binding was produced with BEC-HRP conjugate dilution of 1:1000. 0.0125 µg/ml cocaine-benzoylecgonine antibody and tracer dilution of 1:10000 were the optimized concentration to produce the lower OD of 2.3. As a result, the C value dropped from 1.2 to 0.3 using carbon working electrode plate. Since, the absorbance and the c value had been raised, the sensitivity of the assay compared to ELISA was expected to decrease. However, by measuring the precision profile of 0.0125 µg/ mL Antibody concentration and the conjugate of dilution (1:15,000), the LOD of 14 ng/L using electrochemical detection was obtained compared to 10 ng/L using