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Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements

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Abstract

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.

Graphical abstract: Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements

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Publication details

The article was received on 26 Dec 2016, accepted on 25 May 2017 and first published on 12 Jun 2017


Article type: Paper
DOI: 10.1039/C6AN02724E
Citation: Analyst, 2017, Advance Article
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    Analysis of ribonuclease activity in sub-nanoliter droplets by label-free fluorescence measurements

    J. Choi, B. M. K. Vasamsetti, K. Kim, S. H. Seo, D. Lee, S. Chang, J. Choo and H. Y. Kim, Analyst, 2017, Advance Article , DOI: 10.1039/C6AN02724E

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