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Sensors and Actuators B: Chemical
Volume 332, 2021, 129452

Utilization of chromogenic enzyme substrates for signal amplification in multiplexed detection of biomolecules using surface mass spectrometry

Hee-Kyung Naa, Hyun Kyong Shona, Hye Young Sonb, Eunji Jangb, Sunho Joha,c, Yong-Min Huhb, David G.Castnerd, Tae Geol Leea,c

Center for Nano-Bio Measurement, Korea Research Institute of Standards and Science (KRISS), Daejeon, 34113, Republic of Korea.


MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

Keywords: Enzyme-based signal amplification in mass spectrometry, miRNA sensing, Molecular signal enhancer, Single base mismatch discrimination, ToF-SIMS.

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