RESUMEN
Ultrathin nanopore sensors allow single-molecule and polymer measurements at sub-microsecond time resolution enabled by high current signals (â¼10-30 nA). We demonstrate for the first time the experimental probing of the ultrafast translocation and folded dynamics of double-stranded DNA (dsDNA) through a nanopore at 10 MHz bandwidth with acquisition of data points per 25 ns (150 MB/s). By introducing a rigorous algorithm, we are able to accurately identify each current level present within translocation events and elucidate the dynamic folded and unfolded behaviors. The remarkable sensitivity of this system reveals distortions of short-lived folded states at a lower bandwidth. This work revisits probing of dsDNA as a model polymer and develops broadly applicable methods. The combined improvements in sensor signals, instrumentation, and large data analysis methods uncover biomolecular dynamics at unprecedentedly small time scales.
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Nanoporos , Polímeros , Nanotecnología/métodos , ADN/análisisRESUMEN
We demonstrate DNA translocations through silicon nitride pores formed by simple chemical etching on glass substrates using microscopic amounts of hydrofluoric acid. DNA translocations and transmission electron microscopy (TEM) prove the fabrication of nanopores and allow their characterization. From ionic measurements on 318 chips, we report the effective pore diameters ranging from zero (pristine membranes) and sub-nm to over 100 nm, within 50 µm diameter membranes. The combination of ionic conductance, DNA current blockades, TEM imaging, and electron energy loss spectroscopy (EELS) provides comprehensive information about the pore area and number, from single to few pores, and pore structure. We also show the formation of thinned membrane regions as precursors of pores. The average pore density, about 5 × 10-4 pores/µm2, allows pore number adjustment statistically (0, 1, or more). This simple and affordable chemical method for making solid-state nanopores accelerates their adoption for DNA sensing and characterization applications.
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Nanoporos , Compuestos de Silicona/química , ADN/química , Microscopía Electrónica de Transmisión , IonesRESUMEN
Life detection on Mars is an important topic that includes a direct search for biomarkers. This requires instruments for in situ biomarker detection that are compact, lightweight, and able to withstand operations in space. Solid-state nanopores are excellent candidates that allow fast single-molecule detection. They can withstand high temperatures and be sterilized to minimize planetary contamination. The instruments are portable with low-power requirements. We demonstrate a few key results in advancing the use of nanopores for in-space applications. First, we developed modified deoxyribonucleic acid (DNA) extraction protocols to extract DNA from Mars analog soils. Second, we used silicon nitride nanopores to demonstrate the detection of extracted DNA and corresponding current characteristics. The yields and properties of extracted DNA (e.g., estimated diameters) varied somewhat by soil types, extraction methods, and nanopores used. The yields varied from a minimum of 0.9 ng DNA/g soil for a magnesium carbonate sample from Lake Salda to a maximum of 210 ng DNA/g soil for a calcium carbonate sample from Trona Pinnacles. For a given soil type, yields from different methods varied by a factor of up to 50. These observations motivate future studies with a broader range of Mars-like soils and improved instruments to increase signal-to-noise-ratio at higher measurement bandwidths.
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Marte , Nanoporos , ADN , SueloRESUMEN
Enabled by proteins, we present an all-electrical method for rapid detection of small pharmaceuticals (ibuprofen and sulfamethoxazole [SMZ]) in aqueous media using silicon nitride pores. Specifically, we use carrier proteins, bovine serum albumin (BSA), and take advantage of their interactions with two small drug molecules to form BSA-drug complexes which can be detected by nm-diameter pores, thereby confirming the presence of small pharmaceuticals. We demonstrate detection of ibuprofen and SMZ at concentrations down to 100 nM (â¼21 µg/L) and 48.5 nM (12 µg/L), respectively. We observe changes in electrical signal characteristics (reflected in event durations, rates, current magnitudes, and estimated particle diameters) of BSA-drug complexes compared to BSA-only, and differences between these two small pharmaceuticals, possibly paving a path toward developing selective sensors by identifying "electrical fingerprints" of these molecules in the future. These distinct electrical signals are likely a combined result of diffusion, electrophoretic and electroosmotic effects, interactions between the pore and particles, which depend on pore diameters, pH, and the resulting surface charges. The use of single-molecule-counting nanopores allows sensing of small pharmaceuticals, studies of protein conformational changes, and may aid in efforts to evaluate the impact of small drug molecules on aquatic and human life.
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Nanoporos , Humanos , Ibuprofeno/química , Nanotecnología , Albúmina Sérica Bovina/química , SulfametoxazolRESUMEN
Nanopore sensing is a powerful tool for the detection of biomolecules. Solid-state nanopores act as single-molecule sensors that can function in harsh conditions. Their resilient nature makes them attractive candidates for taking this technology into the field to measure environmental samples for life detection in space and water quality monitoring. Here, we discuss the fabrication of silicon nitride pores from â¼1.6 to 20 nm in diameter in 20-nm-thick silicon nitride membranes suspended on glass chips and their performance. We detect pure laboratory samples containing a single analyte including DNA, BSA, microRNA, TAT, and poly-D-lys-hydrobromide. We also measured an environmental (mixed-analyte) sample, containing Antarctic dirt provided by NASA Ames. For DNA measurements, in addition to using KCl and NaCl solutions, we used the artificial (synthetic) seawater, which is a mixture of different salts mimicking the composition of natural seawater. These samples were spiked with double-stranded DNA (dsDNA) fragments at different concentrations to establish the limits of nanopore sensitivity in candidate environment conditions. Nanopore chips were cleaned and reused for successive measurements. A stand-alone, 1-MHz-bandwidth Chimera amplifier was used to determine the DNA concentration in artificial seawater that we can detect in a practical time scale of a few minutes. We also designed and developed a new compact nanopore reader, a portable read-out device with miniaturized fluidic cells, which can obtain translocation data at bandwidths up to 100 kHz. Using this new instrument, we record translocations of 400 bp, 1000 bp, and 15000 bp dsDNA fragments and show discrimination by analysis of current amplitude and event duration histograms.
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ADN/análisis , Membranas Artificiales , Nanoporos , Agua de Mar/análisis , Compuestos de Silicona/química , Regiones Antárticas , ADN/química , Agua de Mar/químicaRESUMEN
Rational optimization of nanoparticle (NP) surfaces is essential for successful conjugation of proteins to NPs for numerous applications. Using surface-roughened NPs (SRNPs) and quasi-spherical NPs (QSNPs) as two model nanostructures, we examined the effects of local surface curvature on protein conformation and interfacial behaviors by circular dichroism (CD) spectroscopy, fluorescence emission spectroscopy (FES), and isothermal titration calorimetry (ITC). The surface of SRNPs consisted of a mixture of undercoordinated and close-packed surface atoms at the highly curved and locally flat surface regions, respectively, whereas QSNPs were primarily enclosed by {100} and {111} facets covered with close-packed surface atoms. Our findings demonstrated that: 1) SRNPs possess higher tendency to denature BSA and accommodate a higher number of BSA molecules on the surface and 2) the aggregation of AuNP-BSA complexes, likely induced by either denatured BSA or reduced electrostatic repulsion between complexes, is dependent on both the BSA concentration and the NP surface curvature. This study also indicated that NP local surface curvature could potentially be used as a design strategy to preserve the biological function of proteins.
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Oro/química , Nanopartículas del Metal/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Tamaño de la Partícula , Conformación Proteica , Propiedades de SuperficieRESUMEN
To maintain osmotic balance, cells usually produce neutral solutes (i.e., osmolytes), together with charged species to cope with salinity stress. Osmolytes are known to be important in stabilizing/destabilizing macromolecules (e.g., proteins) via depletion /accumulation around their surfaces. To better understand the physiological fate of nanoparticles (NPs), we investigated the effect of osmolytes [(urea and trimethylamine N-oxide (TMAO)] and specific anions (NO3- and F-) on the interactions between NPs and supported lipid bilayers (SLBs). Carboxylated polystyrene NPs (60 nm) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were chosen as model NPs and lipid. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to quantify NP deposition dynamics. Microscale thermophoresis (MST) was used to characterize the affinity between DOPC vesicles (or NPs) and osmolytes. Our results show that osmolytes are capable of protecting SLBs from NP-induced disruption. Upon NP deposition onto supported vesicle layers (SVLs), the leakage of encapsulated dyes decreased with the addition of osmolytes. The combination of kosmotropes (TMAO and F-) are more efficient than that of chaotropes (urea and NO3-) in weakening the hydrophobic interaction between NPs and SLBs by preferential binding to NPs and/or SLBs.