Improved Single-Molecule Detection of Native Proteins Using Hydrogel-Backed Nanopores

Abstract Accurate identification and quantification of proteins in a solution using nanopores is technologically challenging in part because of the large fraction of missed translocation events due to short event times and limitations of conventional current amplifiers. Previously, we have shown that a nanopore interfaced with PEG(1000)-DMA hydrogel with an average mesh size of 3.1 nm significantly enhances protein residence time inside the nanopore, reducing the number of missed events. Following up on our previous work, here, we explored measurement limits, sensitivity, and further characterization capabilities of our proposed hydrogel-backed nanopore system. We demonstrated the ability of the hydrogel-backed nanopores to sense unlabeled proteins as small as 5.5 kD in size and 10 fM in concentration, without a major restriction on the nanopore size or the experimental setup. Also, we showed that the frequency of protein translocation events scales linearly with the bulk concentration over a wide range of concentrations, and an unknown protein concentration can be determined from an interpolation of the frequency-concentration calibration curve with less than 10% error. Further, we precisely determined protein volumes from measurement data, and we employed an iterative method to determine a protein’s volume when its diameter is comparable to nanopore diameter. We investigated possible mechanisms for detection enhancement enabled by the presence of the hydrogel; we found that the possible gap between the pore mouth and the hydrogel indicates the sensitivity of the hydrogel-backed nanopores. Moreover, we demonstrated that hydrogel-backed nanopores can serve as an effective, reliable, ultra-sensitive, non-destructive, reproducible, and easy-to-operate substitute for commonly used UV-Vis detectors in fast protein liquid chromatography. The hydrogel-backed nanopores resolved protein fractions at much lower concentrations than the minimum concentration detectable by the standard UV-Vis detector. They also measured protein fractions with a higher selectivity and provided a more informative analysis of proteins’ physical properties than the UV-Vis detector. Additionally, we integrated the nanopore with PDMS microchannels to create a fluidic circuit between a chromatographic column and the nanopore to facilitate the continuous and live measurement of column effluents. Finally, we demonstrated that integrating lipid-bilayer coated nanopores with a hydrogel is a suitable platform for acquiring artifact-free and long protein translocation events to analyze a single protein translocation event accurately. Using hydrogel-backed lipid-bilayer coated nanopores, we determined the volumes of IgG, Ovalbumin, and gold nanoparticles (5 nm diameter) from individual single translocation events in agreement with reference values. Further, we observed that higher applied voltages increased the probability of IgG alignment. We determined the volume and the length-to-diameter ratio of IgG molecules at different applied voltages and noticed an expansion in the conformation of IgG molecules with an increase in the voltage; this is likely due to IgG’s flexibility and an intense electric field’s ability to expand the IgG hinges from one another