Anmiv S. Prabhu

Chemical, thermal, and electric field induced unfolding of single protein molecules studied using nanopores.

Single-molecule experimental techniques have recently shown to be of significant interest for use in numerous applications in both the research laboratory and industrial settings. Although many single-molecule techniques exist, the nanopore platform is perhaps one of the more popular techniques due to its ability to act as a molecular sensor of biological macromolecules. For example, nanopores offer a unique, new method for probing various properties of proteins and can contribute to elucidating key biophysical information in conjunction with existing techniques. In the present study, various forms of bovine serum albumin (BSA) are detected including thermally refolded BSA, urea-denatured BSA, and multiple forms of BSA detected at elevated electric field strengths (with and without urea). We also provide excluded volume measurements for each of these states that normally are difficult to obtain due to unknown and unstable protein conformations.

Chemically modified solid state nanopores for high throughput nanoparticle separation

The separation of biomolecules and other nanoparticles is a vital step in several analytical and diagnostic techniques. Towards this end we present a solid state nanopore-based set-up as an efficient separation platform. The translocation of charged particles through a nanopore was first modeled mathematically using the multi-ion model and the surface charge density of the nanopore membrane was identified as a critical parameter that determines the selectivity of the membrane and the throughput of the separation process. Drawing from these simulations a single 150 nm pore was fabricated in a 50 nm thick free-standing silicon nitride membrane by focused-ion-beam milling and was chemically modified with (3-aminopropyl)triethoxysilane to change its surface charge density. This chemically modified membrane was then used to separate 22 and 58 nm polystyrene nanoparticles in solution. Once optimized, this approach can readily be scaled up to nanopore arrays which would function as a key component of next-generation nanosieving systems.

Nanopore-Based Devices for Bioanalytical Applications

With over a decade passed since the first reported use of a Staphylococcal α-hemolysin pore to study single molecules of single-stranded DNA, research in the field of nanopores has advanced rapidly. We discuss the technological progression of nanopore-based devices from the initial use of α-hemolysin pores to the advent of solid-state nanopores to the burgeoning of organic-inorganic hybrid pores driven by the desire to achieve fast and inexpensive DNA sequencing. Additional nanopore-based efforts are also discussed that study other classes of molecules, such as proteins. We discuss the use of nanopores for protein folding and binding analysis. In addition to single-molecule analysis, we report on the introduction of nanopore arrays on thin film membranes for ultrafiltration. Owing to their reduced spatial dimensionality, such membranes offer greater control over how the pores interact with analytes thus leading to very efficient separation. With several technical hindrances yet to be overcome, the devices we report are still works in progress. The realization of these devices will enhance laboratory processes by permitting superior spatial and temporal analytical resolution at the single-molecule level resulting in laboratory capacities of great impact.

Modeling and simulation of nanoparticle separation through a solid-state nanopore

Recent experimental studies show that electrokinetic phenomena such as electroosmosis and electrophoresis can be used to separate nanoparticles on the basis of their size and charge using nanopore‐based devices. However, the efficient separation through a nanopore depends on a number of factors such as externally applied voltage, size and charge density of particle, size and charge density of membrane pore, and the concentration of bulk electrolyte. To design an efficient nanopore‐based separation platform, a continuum‐based mathematical model is used for fluid. The model is based on Poisson–Nernst–Planck equations along with Navier–Stokes equations for fluid flow and on the Langevin equation for particle translocation. Our numerical study reveals that membrane pore surface charge density is a vital parameter in the separation through a nanopore. In this study, we have simulated high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) as the sample nanoparticles to demonstrate the capability of such a platform. Numerical results suggest that efficient separation of HDL from LDL in a 0.2 M KCL solution (resembling blood buffer) through a 150 nm pore is possible if the pore surface charge density is ∼ −4.0 mC/m2. Moreover, we observe that pore length and diameter are relatively less important in the nanoparticle separation process considered here.

SEM-induced shrinking of solid-state nanopores for single molecule detection

We have investigated the mechanism by which the diameter of solid-state nanopores is reduced by a scanning electron microscope. The process depends on beam parameters such as the accelerating voltage and electron flux and does not involve simple electron-beam-induced deposition of hydrocarbon contaminants. Instead, it is an energy-dependent process that involves material flow along the surface of the nanopore membrane. We also show that pores fabricated in this manner can detect double stranded DNA.

Single molecule protein biophysics using chemically modified nanopores

The kinetics of protein folding and binding are not only scientifically relevant to understanding the complex molecular machine-like functionality of proteins inside of cells but can also help elucidate disease pathways and lead to better therapeutic agents. Using nanopores to investigate these kinetics holds great potential for such proteomic studies in which the structure and function of proteins can be rapidly screened. In this study, we achieve part of this goal by detecting the folded and unfolded states of BSA. Furthermore, we also show that protein sensing can be performed on more biologically significant protein domains such as PDZ2. To achieve this goal, pore fabrication methods and chemical surface modifications were investigated and optimized for efficient protein sensing.

Case Studies Using Solid-State Pores

Biological nanopores have been widely used for studying DNA and RNA translocations, protein binding and many other interesting applications as discussed in Chapter 5; however, the pores and the lipid bilayer in which they are suspended suffer from limitations like fixed pore diameter and length, mechanical instability and can operate over very limited ranges of pH and voltages. These limitations of biological nanopores have been addressed by the use of solid-state nanopores which are artificially drilled holes in silicon nitride (or silicon oxide, or graphene) membranes. The solid state technology makes it possible to fabricate robust nanopores with variable pore dimensions (discussed in Chapter 6) which can be used over a much wider range of experimental conditions. These solid state pores have perfectly complemented the biological nanopores for single molecule detection and analysis by expanding the experimental repertoire; and by incorporating new electrical and/or optical detection strategies. This chapter will focus on various applications for which solid state nanopores have been used. We will start with DNA and RNA translocations and then move to DNA unzipping, optical detection, DNA sequencing and finally end with protein analysis using the solid state nanopores.

Modeling and Simulation of Translocation Phenomenon in a Solid State Nanopore for Nanoparticle Separation

Solid state nanopore is a potential candidate for separation of nanoparticles or biomolecules such as proteins, DNA, and RNA. However, efficient separation of particles through nanopores is a challenging task as a number of factors such as externally applied voltage, size and charge density of particle, size and charge density of membrane pore, and the concentration of bulk electrolyte influence the translocation behavior of nanoparticles through pores. This paper uses a mathematical model based on Poisson–Nernst–Plank equations along with Navier-Stokes equations to systematically study these factors. Membrane pore surface charge is found to be a vital parameter in this seperation process. Numerical results reveal that efficient separation of high density lipoprotein (HDL) from low density lipoprotein (LDL) in a 0.2 M KCL solution (resembling blood buffer) through a 150 nm pore is possible if the pore surface charge density is around −4.0 mC/m2 .Copyright

High Throughput Nanofluidic Architectures for Nanoparticle Separation

High blood cholesterol levels and associated complications are a major health concern the world over and current techniques to deal with this, especially low density lipoprotein (LDL) apheresis, have significant room for improvement. We had previously reported a proof of concept technique that relies on silicon nitride based solid state nanopores to separate high density lipoprotein (HDL) like particles from LDL like particles. A mathematical model to describe the setup is reported. This model revealed that charge density of the pore surface was critical in determining the efficiency of separation. Accordingly we chemically modified our nanopores with (3-aminopropyl)-triethoxysilane (APTES) to achieve more efficient, high throughput particle separation. Such a technique could make it possible to develop safe and affordable LDL apheresis devices in the future.Copyright

Synthetic Nanoscale Architectures for Lipoprotein Separation

Current low density lipoprotein (LDL) apheresis procedures are expensive and time consuming. We report here a novel technique to detect and separate nanoparticles using solid state nanopores. Our technique relies on the resistive pulse phenomenon used in coulter counters. We used a 150nm diameter nanopore to detect nanoparticles that closely resembled HDL and LDL in terms of their size and surface charge. Statistical analysis of the translocation data revealed that our setup preferentially allowed the particles resembling HDL to pass thorough while restricting the translocation of the particles that resembled LDL.Copyright