Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers

The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (<= 10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers.

Automated summary

The multiplexed detection of disease biomarkers is an important aspect of improving diagnostic testing quality, reducing analysis costs, and speeding up treatment processes. Although there have been efforts to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their intricate designs and semi-quantitative capabilities limit their effectiveness. On the other hand, fluorescence resonance energy transfer (FRET)-based single-molecule counting shows promise for sensitive and quantitative detection of biomarkers. However, current FRET-based multiplexed sensing methods require multiple excitation sources and/or FRET pairs, which complicates labeling and data analysis. This study presents a nanotweezer (NT)-based sensing strategy that uses a single FRET pair to detect multiple targets, offering a simple and promising approach for sensitive detection of nucleic acid biomarkers.