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  1. null (Ed.)
    Nanopore probing of molecular level transport of proteins is strongly influenced by electrolyte type, concentration, and solution pH. As a result, electrolyte chemistry and applied voltage are critical for protein transport and impact, for example, capture rate ( C R ), transport mechanism ( i.e. , electrophoresis, electroosmosis or diffusion), and 3D conformation ( e.g. , chaotropic vs. kosmotropic effects). In this study, we explored these using 0.5–4 M LiCl and KCl electrolytes with holo-human serum transferrin (hSTf) protein as the model protein in both low (±50 mV) and high (±400 mV) electric field regimes. Unlike in KCl, where events were purely electrophoretic, the transport in LiCl transitioned from electrophoretic to electroosmotic with decreasing salt concentration while intermediate concentrations ( i.e. , 2 M and 2.5 M) were influenced by diffusion. Segregating diffusion-limited capture rate ( R diff ) into electrophoretic ( R diff,EP ) and electroosmotic ( R diff,EO ) components provided an approach to calculate the zeta-potential of hSTf ( ζ hSTf ) with the aid of C R and zeta potential of the nanopore surface ( ζ pore ) with ( ζ pore – ζ hSTf ) governing the transport mechanism. Scrutinization of the conventional excluded volume model revealed its shortcomings in capturing surface contributions and a new model was then developed to fit the translocation characteristics of proteins. 
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  2. Abstract

    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.

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  3. Abstract

    Stability, long lifetime, resilience against clogging, low noise, and low cost are five critical cornerstones of solid‐state nanopore technology. Here, a fabrication protocol is described wherein >1 million events are obtained from a single solid‐state nanopore with both DNA and protein at the highest available lowpass filter (LPF, 100 kHz) of the Axopatch 200B–the highest event count mentioned in literature. Moreover, a total of ≈8.1 million events are reported in this work encompassing the two analyte classes. With the 100 kHz LPF, the temporally attenuated population is negligible while with the more ubiquitous 10 kHz, ≈91% of the events are attenuated. With DNA experiments, the pores are operational for hours (typically >7 h) while the average pore growth is merely ≈0.16 ± 0.1 nm h−1. The current noise is exceptionally stable with traces typically showing <10 pA h−1increase in noise. Furthermore, a real‐time method to clean and revive pores clogged with analyte with the added benefit of minimal pore growth during cleaning (< 5% of the original diameter) is showcased. The enormity of the data collected herein presents a significant advancement to solid‐state pore performance and will be useful for future ventures such as machine learning where large amounts of pristine data are a prerequisite.

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  4. Abstract

    Electrolyte chemistry plays an important role in the transport properties of analytes through nanopores. Here, we report the translocation properties of the protein human serum transferrin (hSTf) in asymmetric LiCl salt concentrations with either positive (Ctrans/Ccis< 1) or negative chemical gradients (Ctrans/Ccis> 1). Thecisside concentration was fixed at 4 M for positive chemical gradients and at 0.5 M LiCl for negative chemical gradients, while thetransside concentration varied between 0.5 to 4 M which resulted in six different configurations, respectively, for both positive and negative gradient types. For positive chemical gradient conditions, translocations were observed in all six configurations for at least one voltage polarity whereas with negative gradient conditions, dead concentrations where no events at either polarity were observed. The flux of Li+and Clions and their resultant cation or anion enrichment zones, as well as the interplay of electrophoretic and electroosmotic transport directions, would determine whether hSTf can traverse across the pore.

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  5. Abstract

    Recently, we developed a fabrication method—chemically‐tuned controlled dielectric breakdown (CT‐CDB)—that produces nanopores (through thin silicon nitride membranes) surpassing legacy drawbacks associated with solid‐state nanopores (SSNs). However, the noise characteristics of CT‐CDB nanopores are largely unexplored. In this work, we investigated the 1/fnoise of CT‐CDB nanopores of varying solution pH, electrolyte type, electrolyte concentration, applied voltage, and pore diameter. Our findings indicate that the bulk Hooge parameter (αb) is about an order of magnitude greater than SSNs fabricated by transmission electron microscopy (TEM) while the surface Hooge parameter (αs) is ∼3 order magnitude greater. Theαsof CT‐CDB nanopores was ∼5 orders of magnitude greater than theirαb, which suggests that the surface contribution plays a dominant role in 1/fnoise. Experiments with DNA exhibited increasing capture rates with pH up to pH ∼8 followed by a drop at pH ∼9 perhaps due to the onset of electroosmotic force acting against the electrophoretic force. The1/fnoise was also measured for several electrolytes and LiCl was found to outperform NaCl, KCl, RbCl, and CsCl. The 1/fnoise was found to increase with the increasing electrolyte concentration and pore diameter. Taken together, the findings of this work suggest the pH approximate 7–8 range to be optimal for DNA sensing with CT‐CDB nanopores.

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  6. Abstract

    In this work, we present a step‐by‐step workflow for the fabrication of 2D hexagonal boron nitride (h‐BN) nanopores which are then used to sense holo‐human serum transferrin (hSTf) protein at pH ∼8 under applied voltages ranging from +100 mV to +800 mV. 2D nanopores are often used for DNA, however, there is a great void in the literature for single‐molecule protein sensing and this, to the best of our knowledge, is the first time where h‐BN—a material with large band‐gap, low dielectric constant, reduced parasitic capacitance and minimal charge transfer induced noise—is used for protein profiling. The corresponding ΔG(change in pore conductance due to analyte translocation) profiles showed a bimodal Gaussian distribution where the lower and higher ΔGdistributions were attributed to (pseudo‐) folded and unfolded conformations respectively. With increasing voltage, the voltage induced unfolding increased (evident by decrease in ΔG) and plateaued after ∼400 mV of applied voltage. From the ΔGversus voltage profile corresponding to the pseudo‐folded state, we calculated the molecular radius of hSTf, and was found to be ∼3.1 nm which is in close concordance with the literature reported value of ∼3.25 nm.

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