RESUMO
The tapered geometry of nanopipettes offers a unique perspective on protein transport through nanopores since both a gradual and fast confinement are possible depending on the translocation direction. The protein capture rate, unfolding, speed of translocation, and clogging probability are studied by toggling the LiCl concentration between 2 and 4 M. Interestingly, the proteins in this study could be transported with or against electrophoresis and offer vastly different attributes of sensing. Herein, a ruleset for studying proteins is developed that prevents irreversible pore clogging and yields upward of >100,000 events/nanopore. The extended duration of experiments further revealed that the capture rate takes â¼2 h to reach a steady state, emphasizing the importance of reaching equilibrated transport for studying the energetics and kinetics 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 electroosmotic-dominant transport, whereas electrophoretic-dominant events show no evidence of unfolding. Thus, our findings showcase the optimal conditions for protein translocations and the impact on studying protein unfolding, transporting energetics, and acquiring high bandwidth data.
Assuntos
Cloreto de Lítio , Nanoporos , Desdobramento de Proteína , Proteínas , Eletro-Osmose , Cinética , Transporte ProteicoRESUMO
Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the electrical sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concentrations can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irrespective of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelectric point (pI) of Cas9 was found to be correlated and a critical factor in our experiments. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be critical for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Nanoporos , Eletro-Osmose/instrumentação , Eletro-Osmose/métodos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Conformação Proteica , Desdobramento de ProteínaRESUMO
Nanopores are ideally suited for the analysis of long DNA fragments including chromosomal DNA and synthetic DNA with applications in genome sequencing and DNA data storage, respectively. Hydrodynamic fluid flow has been shown to slow down DNA transit time within the pore, however other influences of hydrodynamic forces have yet to be explored. In this report, a broad analysis of pressure-biased nanopores and the impact of hydrodynamics on DNA transit time, capture rate, current blockade depth, and DNA folding are conducted. Using a 10 nm pore, it is shown that hydrodynamic flow inhibits the early stages of linearization of DNA and produces predominately folded events which are initiated by folded DNA (2-strands) entering the pore. Furthermore, utilizing larger pores (30 nm) leads to unique DNA gating behavior in which DNA events can be switched on and off with the application of pressure. A computational model, based on combining electrophoretic drift velocities with fluid velocities, accurately predicts the pore size required to observe DNA gating. Hydrodynamic fluid flow generated by a pressure bias, or potentially more generally by other mechanisms like electroosmotic flow, is shown to have significant effects on DNA sensing and can be useful for DNA sensing technologies.
Assuntos
Nanoporos , Sequência de Bases , DNA/genética , Eletroforese , HidrodinâmicaRESUMO
Sensing via analyte passage through a constricted aperture is a powerful and robust technology which is being utilized broadly, from DNA sequencing to single virus and cell characterization. Micro- and nanoscale structures typically translocate a constricted aperture, or pore, using electrophoretic force. In the present work, we explore the advances in metrology which can be achieved through rapid directional switching of hydrodynamic forces. Interestingly, multipass measurements of microscale and nanoscale structures achieve cell discrimination. We explore this cell-discrimination phenomenon as well as other features of hydrodynamic focusing such as dynamic trapping and discrete interval sensing.
Assuntos
Cinética , Eletroforese , Análise de Sequência de DNARESUMO
Modern diagnostics strive to be accurate, fast, and inexpensive in addition to properly identifying the presence of a disease, infection, or illness. Early diagnosis is key; catching a disease in its early stages can be the difference between fatality and treatment. The challenge with many diseases is that detectability of the disease scales with disease progression. Since single molecule sensors, e.g., nanopores, can sense biomolecules at low concentrations, they have the potential to become clinically relevant in many of today's medical settings. With nanopore-based sensing, lower volumes and concentrations are required for detection, enabling it to be clinically beneficial. Other advantages to using nanopores include that they are tunable to an enormous variety of molecules and boast low costs, and fabrication is scalable for manufacturing. We discuss previous reports and the potential for incorporating nanopores into the medical field for early diagnostics, therapeutic monitoring, and identifying relapse/recurrence.
Assuntos
Técnicas Biossensoriais , Diagnóstico Precoce , Nanomedicina/tendências , Nanotecnologia/tendências , Humanos , NanoporosRESUMO
The use of atomically thin graphene for molecular sensing has attracted tremendous attention over the years and, in some instances, could displace the use of classical thin films. For nanopore sensing, graphene must be suspended over an aperture so that a single pore can be formed in the free-standing region. Nanopores are typically drilled using an electron beam (e-beam) which is tightly focused until a desired pore size is obtained. E-beam sculpting of graphene however is not just dependent on the ability to displace atoms but also the ability to hinder the migration of ad-atoms on the surface of graphene. Using relatively lower e-beam fluxes from a thermionic electron source, the C-atom knockout rate seems to be comparable to the rate of carbon ad-atom attraction and accumulation at the e-beam/graphene interface (i.e., Rknockout ≈ Raccumulation). Working at this unique regime has allowed the study of carbon ad-atom migration as well as the influence of various substrate materials on e-beam sculpting of graphene. We also show that this information was pivotal to fabricating functional graphene nanopores for studying DNA with increased spatial resolution which is attributed to atomically thin membranes.
Assuntos
Nanoporos , DNA , Elétrons , GrafiteRESUMO
This paper describes the use of gold nanoparticles to study particle translocation dynamics through silicon nitride solid-state nanopores. Gold nanoparticles were dispersed in 20 mM KCl solution containing nonionic surfactant Triton X-100 and their translocation was studied at different applied voltages. The use of low electrolyte concentration resulted in current enhancement upon particle translocation. The counterion cloud around the nanoparticles is proposed to be the reason for current enhancement phenomena because associated counterion cloud is believed to increase the ion density inside the pore during particle translocation. Further, single particle diffusion events were also recorded at 0 mV voltage bias and 0 pA background ionic current with high signal-to-noise ratio as the particles moved down their concentration gradient. The ability of nanopore sensors to detect single particle diffusion can be extended to field-free analysis of biomolecules in their native state and at or near physiological salt concentrations.
Assuntos
Técnicas Eletroquímicas/métodos , Ouro/análise , Nanopartículas Metálicas/análise , Nanoporos , Difusão , Ouro/metabolismo , Tamanho da PartículaRESUMO
Partially or fully disordered proteins are instrumental for signal-transduction pathways; however, many mechanistic aspects of these proteins are not well-understood. For example, the number and nature of intermediate states along the binding pathway is still a topic of intense debate. To shed light on the conformational heterogeneity of disordered protein domains and their complexes, we performed single-molecule experiments by translocating disordered proteins through a nanopore embedded within a thin dielectric membrane. This platform allows for single-molecule statistics to be generated without the need of fluorescent labels or other modification groups. These studies were performed on two different intrinsically disordered protein domains, a binding domain from activator of thyroid hormone and retinoid receptors (ACTR) and the nuclear coactivator binding domain of CREB-binding protein (NCBD), along with their bimolecular complex. Our results demonstrate that both ACTR and NCBD populate distinct conformations upon translocation through the nanopore. The folded complex of the two disordered domains, on the other hand, translocated as one conformation. Somewhat surprisingly, we found that NCBD undergoes a charge reversal under high salt concentrations. This was verified by both translocation statistics as well as by measuring the ζ-potential. Electrostatic interactions have been previously suggested to play a key role in the association of intrinsically disordered proteins, and the observed behavior adds further complexity to their binding reactions.
Assuntos
Proteína de Ligação a CREB/metabolismo , Luz , Nanoporos , Coativador 3 de Receptor Nuclear/metabolismo , Espalhamento de Radiação , Hormônios Tireóideos/metabolismo , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/genética , Humanos , Coativador 3 de Receptor Nuclear/química , Coativador 3 de Receptor Nuclear/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sais/química , Transdução de Sinais , Eletricidade Estática , Hormônios Tireóideos/químicaRESUMO
Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid-state nanopores for the detection of gp120-associated complexes are investigated. They exhibit monovalent and multivalent binding to anti-gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein-specific detection schemes, which are shown to be feasible in the realm of a 'real-world' sample and an automated multidimensional method of detecting events.
Assuntos
Nanoporos , Nanotecnologia/métodos , Proteínas/química , CinéticaRESUMO
Bacterial flagella are particularly attractive bio-templates for nanotubes due to their tubular structures and small inner and outer diameters. In this work, flagella isolated from Salmonella typhimurium were used as templates for silica-mineralized nanotubes. The process involved pretreatment of flagella with aminopropyltriethoxysilane (APTES), followed by the addition of tetraethoxysilane (TEOS). By controlling the concentration of TEOS and the reaction time, we developed a simple and precise method for creating silica-mineralized flagella nanotubes (SMFNs) with various thicknesses of the silica layer. It is demonstrated that flagella can be utilized for the fabrication of SMFNs with tunable thickness. A thicker silica layer was obtained as the concentration ratio of TEOS and reaction time was increased. The present experimental evidence has shown the feasibility of using such fabrication techniques to manufacture nanotubes without genetic modification of flagella which retain the original morphology.
Assuntos
Flagelos , Nanotecnologia/métodos , Nanotubos/química , Dióxido de Silício/química , Microscopia Eletrônica de Varredura , Nanotubos/ultraestrutura , Tamanho da Partícula , Propilaminas , Salmonella typhimurium , Silanos/químicaRESUMO
Fast protein translocations often lead to bandwidth-limited amplitude-attenuated event signatures. In this study, we developed a protein- and electrolyte chemistry-centric pathway to construct a readily executable decision tree for the detection of non-attenuated protein translocations using conventional electronics. Each optimization encompasses increasing capture rate (CR), signal-to-noise ratio (SNR), and minimizing irreversible analyte clogging to collect >104 events/pipette spanning a host of electric fields. This was demonstrated using 11 proteins ranging from â¼12 kDa to â¼720 kDa. Moreover, both symmetric and asymmetric electrolyte conditions (cis and trans chamber electrolyte concentration ratios <> 1) were explored. As a result, asymmetric electrolyte conditions were favorable on the extreme ends of the size spectrum (i.e., larger, and smaller proteins) and while the remainder of proteins were best sensed under symmetric electrolyte conditions. Under these optimal conditions, only â²10% of events were attenuated at 500 mV (â² 5% for most proteins at 500 mV with only â²1-5% of the population faster than â¼7 µs, which is the theoretical attenuation threshold for 100 kHz bandwidth). Finally, applied voltage (Vapp), peak current drop (ΔIp), electrolyte conductivity (K), and open-pore conductance (G0) were used to generate a linear relationship to evaluate the molecular weight of the protein (Mw) using plots of (dΔIp)/(dVapp) vs Mw/(G0/K).
Assuntos
Nanoporos , Condutividade Elétrica , Eletrônica , Peso Molecular , Razão Sinal-RuídoRESUMO
Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concentrations. Contrarily, conductive pulses are observed under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a molecule-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).
Assuntos
Nanoporos , Condutividade Elétrica , Eletro-Osmose , Eletroforese , ÍonsRESUMO
Nanopores are a promising single-molecule sensing device class that captures molecular-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a molecule. A systematic study was conducted here to study the CPS-based molecular discrimination and its voltage-dependent characteristics. CPS was observed when the cation movement along both electrical and chemical gradients was favored, which led to an â¼3× improvement in SNR (i.e., signal-to-noise ratio) and an â¼8× increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asymmetric salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other molecular systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10aâa protein with broad utilities in genetic engineering and immunologyâbound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically observed for proteins.
Assuntos
Nanoporos , DNA/química , Nanotecnologia , Razão Sinal-Ruído , Cloreto de SódioRESUMO
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.
Assuntos
Eletricidade , Nanoporos , Desnaturação Proteica , Temperatura , Proteínas/químicaRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Microscopia Eletrônica de Varredura/métodos , Nanoporos/ultraestrutura , Bacteriófago lambda/genética , DNA Viral/análise , Condutividade Elétrica , Porosidade , Sensibilidade e EspecificidadeRESUMO
Nanopore sensing has been widely used in applications ranging from DNA sequencing to disease diagnosis. To improve these capabilities, pressure-biased nanopores have been explored in the past to-primarily-increase the residence time of the analyte inside the pore. Here, we studied the effect of pressure on the ability to accurately quantify the excluded volume which depends on the current drop magnitude produced by a single entity. Using the calibration standard, the inverse current drop (1/ΔI) decreases linearly with increasing pressure, while the dwell drop reduces exponentially. We therefore had to derive a pressure-corrected excluded volume equation to accurately assess the volume of translocating species under applied pressure. Moreover, a method to probe deformation in nanoliposomes and a single cell is developed as a result. We show that the soft nanoliposomes and even cells deform significantly under applied pressure which can be probed in terms of the shape factor which was introduced in the excluded volume equation. The proposed work has practical applications in mechanobiology, namely, assessing the stiffness and mechanical rigidity of liposomal drug carriers. Pressure-biased pores also enabled multiple observations of cell-cell aggregates as well as their subsequent rupture, potentially allowing for the study of microbial symbioses or pathogen recognition by the human immune system.
Assuntos
Nanoporos , Humanos , Fenômenos Biomecânicos , Análise de Sequência de DNA , Biofísica , LipídeosRESUMO
Nanopore sensing is a promising tool with widespread application in single-molecule detection. Borosilicate glass nanopores are a viable alternative to other solid-state nanopores due to low noise and cost-efficient fabrication. For dielectric materials, including borosilicate glass, the capacitive noise is one of the major contributors to noise, which depends on the wall thickness and the surface area submerged in an ionic solution. Here, we investigated the root mean square (IRMS) noise and ionic conductance for borosilicate nanopores in different depths (i.e., tip submersion depth) ranging from the solution surface (assumed to be zero) to 5000 µm. Our findings demonstrate a decrease in IRMS noise as the pipette moves toward the surface. We further demonstrate that borosilicate nanopores can detect single lambda DNA (λ-DNA) molecules with a high signal-to noise ratio close to the liquid-air interface. Specifically, our results indicate a higher signal to noise ratio as the submersion depth is reduced owing to the reduced surface area and thus capacitive noise. Further, our experimental results show higher DNA capture frequency at the air-water interface due to a combined effect of evaporation and an evaporation-induced thermal gradient at the surface. Therefore, our findings demonstrate that borosilicate glass nanopores are suitable for studying interfacial concentration gradients of molecules, specifically DNA, with a higher signal to noise.
Assuntos
Nanoporos , DNA , Íons , Nanotecnologia , Razão Sinal-RuídoRESUMO
Bacteria are microscopic, single-celled organisms that utilize a variety of nanofluidic structures. One of the best known and widely used nanofluidic structures that are derived from bacteria is the alpha-hemolysin pore. This pore, which self-assembles in lipid bilayers, has been used for a wide variety of sensing applications, most notably, DNA sensing. Synthetic pores drilled in a wide variety of materials, such as silicon nitride and polymers have been developed that use inspiration from the alpha-hemolysin pore. Higher-aspect-ratio nanofluidic structures, akin to nanotubes, are also synthesized by bacteria. Examples of such structures include those that are associated with bacterial transport apparatus, such as pili, and are used by bacteria to facilitate the transfer of genetic material from one bacterium to another. Flagella, and its associated structures, such as the rod and hook, are also tubular nanostructures, through which the protein, flagellin, travels to assemble the flagellum. Genetic engineering allows for the creation of modified bacterial nanopores and nanotubes that can be used for a variety of medical and engineering purposes.
Assuntos
Bactérias/química , Engenharia , Medicina , Microfluídica/métodos , Nanoestruturas/química , Nanotubos/química , PorosidadeRESUMO
Single-molecule techniques are being developed with the exciting prospect of revolutionizing the healthcare industry by generating vast amounts of genetic and proteomic data. One exceptionally promising route is in the use of nanopore sensors. However, a well-known complexity is that detection and capture is predominantly diffusion limited. This problem is compounded when taking into account the capture volume of a nanopore, typically 10(8)-10(10) times smaller than the sample volume. To rectify this disproportionate ratio, we demonstrate a simple, yet powerful, method based on coupling single-molecule dielectrophoretic trapping to nanopore sensing. We show that DNA can be captured from a controllable, but typically much larger, volume and concentrated at the tip of a metallic nanopore. This enables the detection of single molecules at concentrations as low as 5 fM, which is approximately a 10(3) reduction in the limit of detection compared with existing methods, while still maintaining efficient throughput.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Técnicas Eletroquímicas , Membranas Artificiais , NanoporosRESUMO
A simple and versatile method for the direct fabrication of tunneling electrodes with controllable gap distance by using electron-beam-induced deposition (EBID) is presented. We show that tunneling nanogaps smaller than the minimum feature size realizable by conventional EBID can be achieved with a standard scanning electron microscope. These gaps can easily be embedded in nanopores with high accuracy. The controllability of this fabrication method and the nanogap geometry was verified by SEM and TEM imaging. Furthermore, tunneling spectroscopy in a group of solvents with different barrier heights was used to determine the nanogap functionality. Ultimately, the presented fabrication method can be further applied for the fabrication of arrays of nanogap/nanopores or nanogap electrodes with tunable electrode materials. Additionally, this method can also offer direct fabrication of nanoscale electrode systems with tunable spacing for redox cycling and plasmonic applications, which represents an important step in the development of tunneling nanopore structures and in enhancing the capabilities of nanopore sensors.