1. A 96-WELL-PLATE-BASED METHOD FOR THE ESTIMATION OF ALPHA-AMYLASE ACTIVITY USING MINIATURISES 3,5-DINITROSALICYLIC ACID (DNSA) COLORIMETRIC METHOD.
- Author
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MOHAMAD ZIN, NOOR SYAFFINAZ NOOR, AZMI, NUR AIN SABRINA, ANUAR, NURSARAH SYAMIMI, AIN SHAFIE, SYAHIRAH, MAZNAN, MUHAMAMMAD AIMAN FARIS, YONG MENG GOH, and SAMSULRIZAL, NURDIANA
- Subjects
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ALPHA-amylase , *AMYLASES , *DETECTION limit - Abstract
The DNSA assay has been widely employed for the in vitro detection and quantification of alpha-amylase inhibitory activity. However, the conventional method is associated with inconsistencies between protocols and requires a large volume of samples and other assay reagents that can compromise accurate quantitation. Therefore, the study aimed to develop a reliable, simple, and rapid analytical method for determining a-amylase activity. The developed method was carried out in 96-well microplates with a total volume of 250 µL and a total assay time of 1 hr, including pre-incubation. The method was validated for linearity, the limit of detection (LOD), the limit of quantitation (LOQ), and precision. A higher coefficient of determination (R²) value was observed for the developed method as compared to the conventional method (0.9983 ± 0.0003 vs 0.9667 ± 0.0383). The coefficient of variation (CV%) of each data point was less than 15%, indicating excellent data precision. The optimum assay conditions were identified at 2 U/mL of enzyme solution and 5% (w/v) starch solution at 50 °C incubation temperature with an IC50 value of 0.026 ± 0.005 mg/mL. It is concluded that the developed method is practical, precise, and accurate for estimating a-amylase inhibitory activity and would provide reproducible results. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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