High-Throughput Optical Sensing from Immobilized Biological Nanopores in a Micro-Bilayer Array

Abstract

We have established a solid-state (SS-) nanopore assay with high specificity for nucleic acid targets, enabling applications like epigenetic screening, micro-RNA analysis, and pathogen detection . Despite its proven specificity and sensitivity, translational biomarker detection at clinically relevant concentrations is hindered by very low event rates and long acquisition times. It has been noted that most small-molecule translocations are not detectable due to low signal to noise ratio or fast timescales. Here, we describe the probability that a given translocation will produce a detectable event by considering tunable system parameters, and use it to show that these key experimental variables can influence the measured event rate in a predictable fashion. This model elucidates the dependencies of nanopore/analyte interactions and suggests methods by which measurement sensitivity can be optimized. Event probability can be determined experimentally by comparing expected (R exp ) to observed (R obs ) event rates for dsDNA constructs of various lengths bound to monovalent streptavidin. R exp can be derived analytically from the Nernst-Planck equation and Knudsen diffusion coefficient, and R obs measured as a function of fundamental experimental parameters, including the driving potential, target concentration, nanopore radius, and data acquisition rate. Using these data to generate probability distributions, we show that R obs can be predicted for arbitrary experimental conditions from first principles. With this approach, we are able to better understand and even influence the sensitivity of our SS-nanopore assay. The explicit use of the probability distribution provides insight into the mechanisms that govern event generation, and our approach may be expanded to take into account any fundamental system parameter. Tuning system parameters according to the underlying probability distribution will enable optimized detection of low-abundance or challenging nucleic acid biomarker targets. Ultrasensitive, rapid, and cost-effective sequence-specific detection of nucleic acids (DNAs and RNAs) is of great significance for various applications. There is an increasing interest in developing label-free nucleic acid detection methods due to their potential for minimization and integration. Significant efforts have been made towards nanopore-based label-free sensor that allows nucleic acids to be analyzed electronically. The detection specificity is usually achieved by functionalizing the nanopore area or probe molecules. However, the sensitivity of nanopore to detect nucleic acids is usually limited due to the diffusion limited transport process. In this work, we explored the feasibility of using the nanopore as a real-time single molecule counter during the amplification process. This novel approach combines the label-free nanopore sensor with the exquisite sensitivity offered by amplification for sequence-specific nucleic acid detection. A glass nanopore provides an electronic eye to sample the number of amplicons in real-ti...