A 96-WELL-PLATE–BASED METHOD FOR THE ESTIMATION OF ALPHA-AMYLASE ACTIVITY USING MINIATURISES 3,5-DINITROSALICYLIC ACID (DNSA) COLORIMETRIC METHOD

• Published in: Malaysian Applied Biology
• Main Author: Zin N.S.N.M.; Azmi N.A.S.; Anuar N.S.; Shafie S.A.; Maznan M.A.F.; Goh Y.M.; Samsulrizal N.
• Format: Article
• Language: English
• Published: Malaysian Society of Applied Biology 2022
• Online Access: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85140997980&doi=10.55230%2fmabjournal.v51i4.16&partnerID=40&md5=19269d5d68a592e9a0623621831a9490

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 α-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 α-amylase inhibitory activity and would provide reproducible results. © 2022, Malaysian Society of Applied Biology. All rights reserved.