2014 Annual Meeting Abstract - Comparing Various Methods of Quantifying Reducing sugars and improving the measurement.

Poster Presentation
Analytical methods

11-P

Comparing Various Methods of Quantifying Reducing sugars and improving the measurement.
Y. Shao (1), (. M. LIN (1)
(1) Purdue University, West Lafayette, IN, U.S.A.

In starch research, measuring reducing sugars is a common analysis; however, it is not always clear which method is proper to use. Also, most of methods are time consuming. In our research group, we need to quantify large numbers of αâ€amylase hydrolysates of starch, which consist of various reducing sugars with a broad concentration range. Our goal is to select proper methods to quantify α-amylase hydrolysates and to improve the efficiency of the measurement. We compared dinitrosalicylic acid (DNS), Somogyi-Nelson (SN), Park-Johnson (PJ), and 2,2'-Bicinchoninate (BCA) assays. DNS is not a proper method as several reports have shown that it has different responses to various reducing sugars. The other methods do not have this disadvantage, thus the rest of this project focused on those assays. We tested 17 different concentrations of glucose solutions (3 μ g/mL˜ 90 μg/mL). BCA can be operated easily in a 96-well plate, however, operating SN and PJ is time consuming. Thus, we improved SN by using a heat-tolerant 96-well plate. We added 35 μL of substrate and same amount of reagent to the plate. We wrapped it with foil before heating in a boiling water bath for 20 min. After cooling, we added the test reagent (35 μL) and water (105 μL). The plate was read at 600 nm. With the improvement, we can measure 32 samples in triplicate in about an hour. We checked R-squared to decide if there is a good linear relationship of concentration and absorbance. The results showed that SN has a good linear relationship between 20 ˜ 90 μg/mL. PJ is known to measure sugar between 0 ˜ 10 μg/mL, and we found BCA can precisely measure between 0 ˜ 20 μg/mL. In conclusion, the modified-SN and BCA can precisely and efficiently measure reducing sugars and only requires a small amount of sample and reagents. Now we do both SN and BCA assays to detect reducing sugars in a broad range of concentration.