Phurpa Dema Thungon
Indian Institute of Technology - Guwahati, India
Phurpa Dema Thungon is a PhD scholar under Prof. Pranab Goswami in the Department of Biosciences and Bioengineering, IIT Guwahati, India. Her thesis is on the development of Lab-on-Chip alcohol biosensors. She holds expertise in paper microfluidics, optical biosensors, and nanotechnology. She also is trained in developing silk films and enzyme studies.
Motivation: The quantitative measurement of alcohol have great importance in chemical and alcohol industries, to curtail drunk driving and upcoming rise of bioethanol fuel industry. However, the analytical methods for the determination of alcohols like high-performance liquid chromatography (HPLC) and gas chromatography (GC), although precise are complex, time-consuming, require trained operators. Therefore, we aim in developing sensitive alcohol biosensor for on-site application. Enzymes are an excellent tool for developing biosensors due to their high selectivity, specificity and activity under mild experimental conditions. However, maintaining the stability of the enzyme is of particular importance.
Methods: Alcohol Oxidase (AOx) is an important enzyme which catalyses the oxidation of alcohols into their corresponding aldehydes (equation 1), along with the production of hydrogen peroxide (H2O2). This reaction was paired with peroxidase and dyes ABTS (2,2_-azino-di[3-ethylbenzthiozoline-6-sulfonic acid]) to develop a colorimetric sensors (equation 2).
SF is a unique protein biopolymer, that possesses inherent high stability to changes in temperature and moisture, due to the extensive network of physical cross-links (β-sheets) formed during the assembly process. AOx was entrapped in Silk fibroin (SF) films to improve it’s stability. A 2D microfluidic paper-based analytical device (μPAD) was prepared using AKD inject printing to produce two pattern hydrophilic zones and simple folding. One zone was used for sample and the other as detection zone for the colorimetric detection of alcohol. AOx entrapped silk film was placed on the detection zone. A small volume of samples was added to the μPADs, which was photographed after 15 min using a phone camera and the pixel intensity was obtained using Image J software.
Conclusion & Significance: AOx was successfully entrapped and stabilized in SF films with the highest recorded residual activity. AOx and HRP were co-entrapped in SF films for better interaction between the enzymes. The hybrid µPAD was used for optical detection of methanol with dynamic range 0.001 % to 0.1% and L.O.D 0.03 ± 0.001%.