Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo-type human immunodeficiency virus type 1 at sub-micron scale. Non-infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.

A nanopore device is capable of providing single‐molecule level information of an analyte as they translocate through the sensing aperture—a nanometer‐sized through‐hole—under the influence of an applied electric field. In this study, a silicon nitride (SixNy)‐based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid‐state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.

The gradual tapered geometry of nanopipettes offers a unique perspective on protein transport through nanopores since both a gradual and fast confinement is possible depending on translocation direction. Protein capture rate, unfolding, speed of translocation, and clogging probability are studied by toggling the lithium chloride concentration between 4 M and 2 M. Interestingly, the proteins in this study could be transported with or against the electrophoresis and offer vastly different attributes of sensing and affect whether a protein unfolds during pore transit. A ruleset for studying proteins is developed that prevents irreversible pore clogging and yielded upwards of >100,000 events/nanopore. Minimizing clogging also permitted higher quality data via the use of smaller pores (i.e., <2x the size of the protein) including higher SNR recordings and data acquisition at the highest available bandwidth (100 kHz). The extended duration of experiments further revealed that the capture rate takes ~2 hours to reach a steady state with a value ~3x greater than the initial reading, emphasizing the importance of reaching equilibrated transport for studying the energetics of protein transport (i.e., diffusion vs barrier-limited). Even in the equilibrated transport state, improper lowpass filtering was shown to distort the classification of diffusion-limited vs barrier-limited transport. Finally electric-field induced protein unfolding was found to be most prominent in EO dominant transport whereas EP dominant events show no evidence of unfolding. Thus, our findings showcase the optimal conditions for protein translocations and the impact on studying protein unfolding, transport energetics, and acquiring high bandwidth data.

DOI: https://doi.org/10.1002/elps.201800311 This cover picture shows how we measured the deformation of virus particle using nanopore sensor. A green virus particle is passing through a nanopore on the gray membrane by the electrophoresis. The virus particle deforms inside the nanopore by the electric field gradient which is represented by the transparent sphere around the nanopore. Each blue and red lines are the forward and the backward electric field, which drives the charged virus forward and backward through the nanopore. The yellow and orange glowing dots are representing the potassium and chloride ions, which forms the ionic current across the chambers.

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