RESUMEN
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-1 increase 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.
Asunto(s)
Nanoporos , ADN , Nanotecnología/métodosRESUMEN
Drop-casting is frequently used to deliver a sample for surface-enhanced Raman spectroscopy (SERS) and can result in inhomogeneous sample distribution during solvent evaporation. While soaking can provide better analyte homogeneity, it may require more sample than is available. Failure to optically sample analyte-rich substrate locations can compromise measurement outcomes. We developed and tested 3D printed SERS substrate holders that provided spatial registry of the dried sample droplet center for subsequent optical measurements. We found that deliberate and controlled spatial offsets (0-900 µm) between the analyte drop center and the laser excitation prevented signal intensity drops of as much as â¼3× and improved reproducibility. Thus, the use of offset-controlled 3D printed holders provided a quick and inexpensive way to improve the reliability of SERS measurements when using the convenient and popular choice of sample drop-casting.
RESUMEN
Solid-state nanopore (SSN)-based analytical methods have found abundant use in genomics and proteomics with fledgling contributions to virology - a clinically critical field with emphasis on both infectious and designer-drug carriers. Here we demonstrate the ability of SSN to successfully discriminate adeno-associated viruses (AAVs) based on their genetic cargo [double-stranded DNA (AAVdsDNA), single-stranded DNA (AAVssDNA) or none (AAVempty)], devoid of digestion steps, through nanopore-induced electro-deformation (characterized by relative current change; ΔI/I0). The deformation order was found to be AAVempty > AAVssDNA > AAVdsDNA. A deep learning algorithm was developed by integrating support vector machine with an existing neural network, which successfully classified AAVs from SSN resistive-pulses (characteristic of genetic cargo) with >95% accuracy - a potential tool for clinical and biomedical applications. Subsequently, the presence of AAVempty in spiked AAVdsDNA was flagged using the ΔI/I0 distribution characteristics of the two types for mixtures composed of â¼75 : 25% and â¼40 : 60% (in concentration) AAVempty : AAVdsDNA.
Asunto(s)
Nanoporos , Algoritmos , ADN , ADN de Cadena Simple , Dependovirus/genéticaRESUMEN
Vesicles perform many essential functions in all living organisms. They respond like a transducer to mechanical stress in converting the applied force into mechanical and biological responses. At the same time, both biochemical and biophysical signals influence the vesicular response in bearing mechanical loads. In recent years, liposomes, artificial lipid vesicles, have gained substantial attention from the pharmaceutical industry as a prospective drug carrier which can also serve as an artificial cell-mimetic system. The ability of these vesicles to enter through pores of even smaller size makes them ideal candidates for therapeutic agents to reach the infected sites effectively. Engineering of vesicles with desired mechanical properties that can encapsulate drugs and release as required is the prime challenge in this field. This requirement has led to the modifications of the composition of the bilayer membrane by adding cholesterol, sphingomyelin, etc. In this article, we review the manufacturing and characterization techniques of various artificial/synthetic vesicles. We particularly focus on the electric field-driven characterization techniques to determine different properties of vesicle and its membranes, such as bending rigidity, viscosity, capacitance, conductance, etc., which are indicators of their content and mobility. Similarities and differences between artificial vesicles, natural vesicles, and cells are highlighted throughout the manuscript since most of these artificial vesicles are intended for cell mimetic functions.
Asunto(s)
Células Artificiales , Exosomas , Liposomas , Células Cultivadas , Portadores de Fármacos , Capacidad Eléctrica , Humanos , Membrana Dobles de Lípidos , Ensayo de Materiales , ViscosidadRESUMEN
In this study, we investigated the voltage and pH responsiveness of human serum transferrin (hSTf) protein using silicon nitride (Si xN y) nanopores. The Fe(III)-rich holo form of hSTf was dominant when pH > pI, while the Fe(III)-free apo form was dominant when pH < pI. The translocations of hSTf were purely in an electrophoretic sense, thus depended on its pI and the solution pH. With increasing voltage, voltage driven protein unfolding became prominent which was indicated by the trends associated with change in conductance, due to hSTf translocation, and in the excluded electrolyte volume. Additionally, analysis of the translocation events of the pure apo form of hSTf showed a clear difference in the event population compared to that of the holo form. The results obtained demonstrate the successful application of nanopore devices to distinguish between the holo and apo forms of hSTf in a mixture and to analyze its folding and unfolding phenomenon over a range of pH and applied voltages.
Asunto(s)
Nanoporos , Transferrina/química , Electricidad , Electroforesis/instrumentación , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Nanoporos/ultraestructura , Conformación Proteica , Pliegue de Proteína , Compuestos de Silicona/químicaRESUMEN
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/I0Ì) 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.
Asunto(s)
Elasticidad , Liposomas/ultraestructura , Nanotecnología/métodos , Automatización/métodos , Electricidad , Microesferas , Nanoporos , Poliestirenos/normasRESUMEN
Polysaccharides have key biological functions and can be harnessed for therapeutic roles, such as the anticoagulant heparin. Their complexity-e.g., >100 monosaccharides with variety in linkage and branching structure-significantly complicates analysis compared to other biopolymers such as DNA and proteins. More, and improved, analysis tools have been called for, and here we demonstrate that solid-state silicon nitride nanopore sensors and tuned sensing conditions can be used to reliably detect native polysaccharides and enzymatic digestion products, differentiate between different polysaccharides in straightforward assays, provide new experimental insights into nanopore electrokinetics, and uncover polysaccharide properties. We show that nanopore sensing allows us to easily differentiate between a clinical heparin sample and one spiked with the contaminant that caused deaths in 2008 when its presence went undetected by conventional assays. The work reported here lays a foundation to further explore polysaccharide characterization and develop assays using thin-film solid-state nanopore sensors.
Asunto(s)
Glicómica/métodos , Heparina/química , Nanoporos , Compuestos de Silicona/química , Alginatos/química , Calibración , Sulfatos de Condroitina/químicaRESUMEN
Silicon nitride fabricated by low-pressure chemical vapor deposition (LPCVD) to be silicon-rich (SiNx), is a ubiquitous insulating thin film in the microelectronics industry, and an exceptional structural material for nanofabrication. Free-standing <100 nm thick SiNx membranes are especially compelling, particularly when used to deliver forefront molecular sensing capabilities in nanofluidic devices. We developed an accessible, gentle, and solution-based photodirected surface metallization approach well-suited to forming patterned metal films as integral structural and functional features in thin-membrane-based SiNx devices-for use as electrodes or surface chemical functionalization platforms, for example-augmenting existing device capabilities and properties for a wide range of applications.
RESUMEN
We describe a method for simply characterizing the size and shape of a nanopore during solution-based fabrication and surface modification, using only low-overhead approaches native to conventional nanopore measurements. Solution-based nanopore fabrication methods are democratizing nanopore science by supplanting the traditional use of charged-particle microscopes for fabrication, but nanopore profiling has customarily depended on microscopic examination. Our approach exploits the dependence of nanopore conductance in solution on nanopore size, shape, and surface chemistry in order to characterize nanopores. Measurements of the changing nanopore conductance during formation by etching or deposition can be analyzed using our method to characterize the nascent nanopore size and shape, beyond the typical cylindrical approximation, in real-time. Our approach thus accords with ongoing efforts to broaden the accessibility of nanopore science from fabrication through use: it is compatible with conventional instrumentation and offers straightforward nanoscale characterization of the core tool of the field.
RESUMEN
A method to directly electrolessly plate silicon-rich silicon nitride with thin gold films was developed and characterized. Films with thicknesses <100 nm were grown at 3 and 10 °C between 0.5 and 3 h, with mean grain sizes between â¼20 and 30 nm. The method is compatible with plating free-standing ultrathin silicon nitride membranes, and we successfully plated the interior walls of micropore arrays in 200 nm thick silicon nitride membranes. The method is thus amenable to coating planar, curved, and line-of-sight-obscured silicon nitride surfaces.