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

https://doi.org/10.55230/mabjournal.v51i4.16

Authors

  • NOOR SYAFFINAZ NOOR MOHAMAD ZIN Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia; TuAH Industrial Research Lab with Bio Fluid Sdn Bhd, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor.
  • NUR AIN SABRINA AZMI TuAH Industrial Research Lab with Bio Fluid Sdn Bhd, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor.
  • NURSARAH SYAMIMI ANUAR Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia; TuAH Industrial Research Lab with Bio Fluid Sdn Bhd, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor.
  • SYAHIRAH AIN SHAFIE Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia; TuAH Industrial Research Lab with Bio Fluid Sdn Bhd, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor.
  • MUHAMAMMAD AIMAN FARIS MAZNAN Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia; TuAH Industrial Research Lab with Bio Fluid Sdn Bhd, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor.
  • YONG MENG GOH TuAH Industrial Research Lab with Bio Fluid Sdn Bhd, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor; Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
  • NURDIANA SAMSULRIZAL Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia

Keywords:

3,5-Dinitrosalicylic Acid (DNSA), 96-well plate, alpha-amylase enzyme assay, alpha-amylase inhibition

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 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.

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Published

31-10-2022

How to Cite

NOOR MOHAMAD ZIN, N. S. ., AZMI, N. A. S. ., ANUAR, N. S. ., SHAFIE, S. A. ., MAZNAN, M. A. F. ., GOH, Y. M. ., & SAMSULRIZAL, N. . (2022). A 96-WELL-PLATE–BASED METHOD FOR THE ESTIMATION OF ALPHA-AMYLASE ACTIVITY USING MINIATURISES 3,5-DINITROSALICYLIC ACID (DNSA) COLORIMETRIC METHOD. Malaysian Applied Biology, 51(4), 95–102. https://doi.org/10.55230/mabjournal.v51i4.16

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