[Analyst] Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence mea
We report the results of a label-free analysis of ribonuclease activity using droplet-based microfluidics. The ribonucleolytic activity of ribonucleases (RNases) plays a critical role in cellular functions such as development, survival, growth and differentiation. Altered ribonucleolytic activity and/or the expression level of the RNase A family are known to be associated with pancreatic, bladder, ovarian and thyroid cancers among others. For this reason, the RNase A family is a meaningful protein biomarker that can be used in the diagnosis of cancer and as a target for new drug screening. There are some successful traditional methods for analysing the RNase activity, such as radioactive label-based assay, methylene blue-based assay, gel zymography, as well as other more recently developed methods such as electrochemical assay and fluorescence resonance energy transfer (FRET). However, these methods require analytical samples with a volume ranging from microliters to milliliters, and are not suitable for high-throughput analysis. Therefore, we integrated ethidium bromide (EtBr), which intercalates the chemical itself to nucleic acid, to droplet-based microfluidics for a cost-effective, high-throughput analysis. Put simply, this method is dependent on the amount of intercalated EtBr molecules on RNA. Our assay also uses visible light that is harmless to humans, unlike previous methods that used harmful UV rays, to excite the EtBr molecules. Specifically, we monitored the ribonucleolytic activity of less than 10 nM RNase A in droplets of about 330 picoliters. Also, half the maximal inhibitory concentration (IC50) of the RNase inhibitor was successfully measured in the same volume of droplets at a frequency of 40 hertz.
Jae-Won Choi,a Bala Murali Krishna Vasamsetti,a Kyu-Wan Kim,b Seung Hwan Seo,a Dong-Hun Lee,cd Soo-Ik Chang,a Jaebum Choo*e and Hak Yong Kim*ad Author affiliations * Corresponding authors a Department of Biochemistry, Chungbuk National University, Cheongju 28644, Republic of Korea E-mail: firstname.lastname@example.org Tel: +82-43-261-2307 b Department of Nuclear Medicine, College of Medicine, Seoul National University, Seoul 03087, Republic of Korea c Department of Microbiology, Chungbuk National University, Cheongju 28644, Republic of Korea d School of Biological Sciences, Chungbuk National University, Republic of Korea e Department of Bionano Technology, Hanyang University, Ansan 15588, Republic of Korea E-mail: email@example.com