Stiffness measurement of nanosized liposomes using solid-state nanopore sensor with automated recapturing platform.

This paper describes a method to gauge the stiffness of nanosized liposomes - a nanoscale vesicle - using a custom-made recapture platform coupled to a solid-state nanopore sensor. The recapture platform electrically profiles a given liposome vesicle multiple times through automated reversal of the voltage polarity immediately following a translocation instance to re-translocate the same analyte through the nanopore - provides better statistical insight at the molecular level by analyzing the same particle multiple times compared to conventional nanopore platforms. The capture frequency depends on the applied voltage with lower voltages (i.e., 100 mV) permitting higher recapture instances than at higher voltages (>200 mV) since the probability of particles exiting the nanopore capture radius increases with voltage. The shape deformation was inferred by comparing the normalized relative current blockade ( ΔI/I0̂) at the two voltage polarities to that of a rigid particle, i.e., polystyrene beads. We found that liposomes deform to adopt a prolate shape at higher voltages. This platform can be further applied to investigate the stiffness of other types of soft matters, e.g., virus, exosomes, endosomes, and accelerate the potential studies in pharmaceutics for increasing the drug packing and unpacking mechanism by controlling the stiffness of the drug vesicles.

Mechanical characterization of HIV-1 with a solid-state nanopore sensor.

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.

Hydrophilic and size-controlled graphene nanopores for protein detection.

This paper describes a general approach for transferring clean single-layer graphene onto silicon nitride nanopore devices and the use of the electron beam of a transmission electron microscope (TEM) to drill size-controlled nanopores in freely suspended graphene. Besides nanopore drilling, we also used the TEM to heal and completely close the unwanted secondary holes formed by electron beam damage during the drilling process. We demonstrate electron beam assisted shrinking of irregularly shaped 40-60 nm pores down to 2 nm, exhibiting an exquisite control of graphene nanopore diameter. Our fabrication workflow also rendered graphene nanopores hydrophilic, allowing easy wetting and use of the pores for studying protein translocation and protein-protein interaction with a high signal to noise ratio.

Nanoparticle mechanics: deformation detection via nanopore resistive pulse sensing.

Solid-state nanopores have been widely used in the past for single-particle analysis of nanoparticles, liposomes, exosomes and viruses. The shape of soft particles, particularly liposomes with a bilayer membrane, can greatly differ inside the nanopore compared to bulk solution as the electric field inside the nanopores can cause liposome electrodeformation. Such deformations can compromise size measurement and characterization of particles, but are often neglected in nanopore resistive pulse sensing. In this paper, we investigated the deformation of various liposomes inside nanopores. We observed a significant difference in resistive pulse characteristics between soft liposomes and rigid polystyrene nanoparticles especially at higher applied voltages. We used theoretical simulations to demonstrate that the difference can be explained by shape deformation of liposomes as they translocate through the nanopores. Comparing our results with the findings from electrodeformation experiments, we demonstrated that the rigidity of liposomes can be qualitatively compared using resistive pulse characteristics. This application of nanopores can provide new opportunities to study the mechanics at the nanoscale, to investigate properties of great value in fundamental biophysics and cellular mechanobiology, such as virus deformability and fusogenicity, and in applied sciences for designing novel drug/gene delivery systems.

Use of solid-state nanopores for sensing co-translocational deformation of nano-liposomes.

Membrane deformation of nano-vesicles is crucial in many cellular processes such as virus entry into the host cell, membrane fusion, and endo- and exocytosis; however, studying the deformation of sub-100 nm soft vesicles is very challenging using the conventional techniques. In this paper, we report detecting co-translocational deformation of individual 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) nano-liposomes using solid-state nanopores. Electrokinetic translocation through the nanopore caused the soft DOPC liposomes (85 nm diameter) to change their shape, which we attribute to the strong electric field strength and physical confinement inside the pore. The experiments were performed at varying transmembrane voltages and the deformation was observed to mount up with increasing applied voltage and followed an exponential trend. Numerical simulations were performed to simulate the concentrated electric field strength inside the nanopore and a field strength of 14 kV cm(-1) (at 600 mV applied voltage) was achieved at the pore center. The electric field strength inside the nanopore is much higher than the field strength known to cause deformation of 15-30 µm giant membrane vesicles. As a control, we also performed experiments with rigid polystyrene beads that did not show any deformation during translocation events, which further established our hypothesis of co-translocational deformation of liposomes. Our technique presents an innovative and high throughput means for investigating deformation behavior of soft nano-vesicles.

Low aspect ratio micropores for single-particle and single-cell analysis.

This paper describes microparticle and bacterial translocation studies using low aspect ratio solid-state micropores. Micropores, 5 µm in diameter, were fabricated in 200 nm thick free-standing silicon nitride membranes, resulting in pores with an extremely low aspect ratio, nominally 0.04. For microparticle translocation experiments, sulfonated polystyrene microparticles and magnetic microbeads in size range of 1-4 µm were used. Using the microparticle translocation characteristics, we find that particle translocations result in a change only in the pore's geometrical resistance while the access resistance remains constant. Furthermore, we demonstrate the ability of our micropore to probe high-resolution shape information of translocating analytes using concatenated magnetic microspheres. Distinct current drop peaks were observed for each microsphere of the multibead architecture. For bacterial translocation experiments, nonflagellated Escherichia coli (strain HCB 5) and wild type flagellated Salmonella typhimurium (strain SJW1103) were used. Distinct current signatures for the two bacteria were obtained and this difference in translocation behavior was attributed to different surface protein distributions on the bacteria. Our findings may help in developing low aspect ratio pores for high-resolution microparticle characterization and single-cell analysis.