Plastic Nanofluidic Sensor with a Solid-Phase Bioreactor and Dual Nanopore Reader: Studying Biological Reactions at the Single-Molecule Level

We report a thermoplastic nanofluidic sensor (exonuclease time-of-flight; X-ToF) fabricated via nanoinjection molding that integrates an immobilized nanoscale enzymatic reactor (INER) directly with a dual in-plane nanopore ToF (DNP-ToF) reader, which not only uses the parameters typically used for resistive pulse sensing (RPS)─normalized event amplitude and time width─but the time taken for a single-molecule to travel between two pores in series. This platform enables the label-free monitoring of complex biological reactions at the single-molecule level. A critical hurdle in integrating such bioenzymatic reactions with RPS is reconciling disparate process step requirements, for example salt effects on an enzymatic reaction and high salt needs for RPS. We addressed this through strategic UV/O 3 surface engineering; an optimized 3.5 min dose created effectively charge-neutral nanopores that maximized capture rates while sustaining a robust ensemble electroosmotic flow (5.33 ± 0.33 × 10 –5 cm 2 V –1 s –1 ) and simultaneously preserving enzyme activity. To demonstrate the sensor’s utility, exoribonuclease 1 (Xrn1) was used as a model. X-ToF successfully deduced the dissociation constant of an input Cas9 RNA to Xrn1 (2.4 ± 0.02 μM –1 ) and monitored in real-time ribonucleotide generation from Cas9 with a cleavage rate of 23 nt/s. Ultimately, this platform serves as a highly versatile tool that can be repurposed for DNA, RNA, or protein sequencing simply by changing the identity of the immobilized enzyme.