Nanowire Array Breath Acetone Sensor for Diabetes Monitoring.

Diabetic ketoacidosis (DKA) is a life-threatening acute complication of diabetes characterized by the accumulation of ketone bodies in the blood. Breath acetone, a ketone, directly correlates with blood ketones. Therefore, monitoring breath acetone can significantly enhance the safety and efficacy of diabetes care. In this work, the design and fabrication of an InP/Pt/chitosan nanowire array-based chemiresistive acetone sensor is reported. By incorporation of chitosan as a surface-functional layer and a Pt Schottky contact for efficient charge transfer processes and photovoltaic effect, self-powered, highly selective acetone sensing is achieved. The sensor has exhibited an ultra-wide acetone detection range from sub-ppb to >100 000 ppm level at room temperature, covering those in the exhaled breath from healthy individuals (300-800 ppb) to people at high risk of DKA (>75 ppm). The nanowire sensor has also been successfully integrated into a handheld breath testing prototype, the Ketowhistle, which can successfully detect different ranges of acetone concentrations in simulated breath samples. The Ketowhistle demonstrates the immediate potential for non-invasive ketone monitoring for people living with diabetes, in particular for DKA prevention.

Nanopore Fabrication Made Easy: A Portable, Affordable Microcontroller-Assisted Approach for Tailored Pore Formation via Controlled Breakdown.

With growing interest in solid-state nanopore sensing─a single-molecule technique capable of profiling a host of analyte classes─establishing facile and scalable approaches for fabricating molecular-size pores is becoming increasingly important. The introduction of nanopore fabrication by controlled breakdown (CBD) has transformed the economics and accessibility of nanopore fabrication. Here, we introduce the design of an Arduino-based, portable USB-powered CBD device, with an estimated cost of <150 USD, which is ≈10-100× cheaper than most commercial solutions, capable of fabricating single nanopores conducive for single molecule sensing experiments. We demonstrate the facile fabrication of 60 tailored nanopores (∼2.6-12.6 nm) with ∼80% of the pores within 1 nm of the target diameter. Selected pores were then tested with double-stranded DNA, the canonical molecular ruler, demonstrating their performance for single-molecule sensing applications. The device is constructed with off-the-shelf readily available components and controlled using a highly customizable MATLAB application, which has capabilities encompassing pore fabrication, pore enlargement, and current-voltage acquisition for pore size estimation. When combined with a portable amplifier, this device also provides a fully portable sensing platform, an important step toward portable solid-state nanopore sensing applications.

High Accuracy Protein Identification: Fusion of Solid-State Nanopore Sensing and Machine Learning.

Proteins are arguably one of the most important class of biomarkers for health diagnostic purposes. Label-free solid-state nanopore sensing is a versatile technique for sensing and analyzing biomolecules such as proteins at single-molecule level. While molecular-level information on size, shape, and charge of proteins can be assessed by nanopores, the identification of proteins with comparable sizes remains a challenge. Here, solid-state nanopore sensing is combined with machine learning to address this challenge. The translocations of four similarly sized proteins is assessed using amplifiers with bandwidths (BWs) of 100 kHz and 10 MHz, the highest bandwidth reported for protein sensing, using nanopores fabricated in <10 nm thick silicon nitride membranes. F-values of up to 65.9% and 83.2% (without clustering of the protein signals) are achieved with 100 kHz and 10 MHz BW measurements, respectively, for identification of the four proteins. The accuracy of protein identification is further enhanced by classifying the signals into different clusters based on signal attributes, with F-value and specificity of up to 88.7% and 96.4%, respectively, for combinations of four proteins. The combined use of high bandwidth instruments, advanced clustering and machine learning methods allows label-free identification of proteins with high accuracy.

Over One Million DNA and Protein Events Through Ultra-Stable Chemically-Tuned Solid-State Nanopores.

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.

Ultrathin, High-Lifetime Silicon Nitride Membranes for Nanopore Sensing.

Thin membranes are highly sought-after for nanopore-based single-molecule sensing, and fabrication of such membranes becomes challenging in the ≲10 nm thickness regime where a plethora of useful molecule information can be acquired by nanopore sensing. In this work, we present a scalable and controllable method to fabricate silicon nitride (SixNy) membranes with effective thickness down to ∼1.5 nm using standard silicon processing and chemical etching using hydrofluoric acid (HF). Nanopores were fabricated using the controlled breakdown method with estimated pore diameters down to ∼1.8 nm yielding events >500,000 and >1,800,000 from dsDNA and bovine serum albumin (BSA) protein, respectively, demonstrating the high-performance and extended lifetime of the pores fabricated through our membranes. We used two different compositions of SixNy for membrane fabrication (near-stoichiometric and silicon-rich SixNy) and compared them against commercial membranes. The final thicknesses of the membranes were measured using ellipsometry and were in good agreement with the values calculated from the bulk etch rates and DNA translocation characteristics. The stoichiometry and the density of the membrane layers were characterized with Rutherford backscattering spectrometry while the nanopores were characterized using pH-conductance, conductivity-conductance, and power spectral density (PSD) graphs.

Surface Functionalization and Texturing of Optical Metasurfaces for Sensing Applications.

Optical metasurfaces are planar metamaterials that can mediate highly precise light-matter interactions. Because of their unique optical properties, both plasmonic and dielectric metasurfaces have found common use in sensing applications, enabling label-free, nondestructive, and miniaturized sensors with ultralow limits of detection. However, because bare metasurfaces inherently lack target specificity, their applications have driven the development of surface modification techniques that provide selectivity. Both chemical functionalization and physical texturing methodologies can modify and enhance metasurface properties by selectively capturing analytes at the surface and altering the transduction of light-matter interactions into optical signals. This review summarizes recent advances in material-specific surface functionalization and texturing as applied to representative optical metasurfaces. We also present an overview of the underlying chemistry driving functionalization and texturing processes, including detailed directions for their broad implementation. Overall, this review provides a concise and centralized guide for the modification of metasurfaces with a focus toward sensing applications.

Modulation of electrophoresis, electroosmosis and diffusion for electrical transport of proteins through a solid-state nanopore.

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.

Nanopore Data Analysis: Baseline Construction and Abrupt Change-Based Multilevel Fitting.

Solid-state nanopore technology delivers single-molecule resolution information, and the quality of the deliverables hinges on the capability of the analysis platform to extract maximum possible events and fit them appropriately. In this work, we present an analysis platform with four baseline fitting methods adaptive to a wide range of nanopore traces (including those with a step or abrupt changes where pre-existing platforms fail) to maximize extractable events (2× improvement in some cases) and multilevel event fitting capability. The baseline fitting methods, in the increasing order of robustness and computational cost, include arithmetic mean, linear fit, Gaussian smoothing, and Gaussian smoothing and regressed mixing. The performance was tested with ultra-stable to vigorously fluctuating current profiles, and the event count increased with increasing fitting robustness prominently for vigorously fluctuating profiles. Turning points of events were clustered using the dbscan method, followed by segmentation into preliminary levels based on abrupt changes in the signal level, which were then iteratively refined to deduce the final levels of the event. Finally, we show the utility of clustering for multilevel DNA data analysis, followed by the assessment of protein translocation profiles.

Targeting improved reproducibility in surface-enhanced Raman spectroscopy with planar substrates using 3D printed alignment holders.

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 ∼3× 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.

Assessment of 1/f noise associated with nanopores fabricated through chemically tuned controlled dielectric breakdown.

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/f noise 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. Theαs of CT-CDB nanopores was ∼5 orders of magnitude greater than theirαb , which suggests that the surface contribution plays a dominant role in 1/f noise. 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/f noise was also measured for several electrolytes and LiCl was found to outperform NaCl, KCl, RbCl, and CsCl. The 1/f noise 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.

Adeno-associated virus characterization for cargo discrimination through nanopore responsiveness.

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 ∼75 : 25% and ∼40 : 60% (in concentration) AAVempty : AAVdsDNA.

Chemically tailoring nanopores for single-molecule sensing and glycomics.

A nanopore can be fairly-but uncharitably-described as simply a nanofluidic channel through a thin membrane. Even this simple structural description holds utility and underpins a range of applications. Yet significant excitement for nanopore science is more readily ignited by the role of nanopores as enabling tools for biomedical science. Nanopore techniques offer single-molecule sensing without the need for chemical labelling, since in most nanopore implementations, matter is its own label through its size, charge, and chemical functionality. Nanopores have achieved considerable prominence for single-molecule DNA sequencing. The predominance of this application, though, can overshadow their established use for nanoparticle characterization and burgeoning use for protein analysis, among other application areas. Analyte scope continues to be expanded, and with increasing analyte complexity, success will increasingly hinge on control over nanopore surface chemistry to tune the nanopore, itself, and to moderate analyte transport. Carbohydrates are emerging as the latest high-profile target of nanopore science. Their tremendous chemical and structural complexity means that they challenge conventional chemical analysis methods and thus present a compelling target for unique nanopore characterization capabilities. Furthermore, they offer molecular diversity for probing nanopore operation and sensing mechanisms. This article thus focuses on two roles of chemistry in nanopore science: its use to provide exquisite control over nanopore performance, and how analyte properties can place stringent demands on nanopore chemistry. Expanding the horizons of nanopore science requires increasing consideration of the role of chemistry and increasing sophistication in the realm of chemical control over this nanoscale milieu.

Beyond nanopore sizing: improving solid-state single-molecule sensing performance, lifetime, and analyte scope for omics by targeting surface chemistry during fabrication.

Solid-state nanopores (SSNs) are single-molecule resolution sensors with a growing footprint in real-time bio-polymer profiling-most prominently, but far from exclusively, DNA sequencing. SSNs accessibility has increased with the advent of controlled dielectric breakdown (CDB), but severe fundamental challenges remain: drifts in open-pore current and (irreversible) analyte sticking. These behaviors impede basic research and device development for commercial applications and can be dramatically exacerbated by the chemical complexity and physical property diversity of different analytes. We demonstrate a SSN fabrication approach attentive to nanopore surface chemistry during pore formation, and thus create nanopores in silicon nitride (SiNx) capable of sensing a wide analyte scope-nucleic acid (double-stranded DNA), protein (holo-human serum transferrin) and glycan (maltodextrin). In contrast to SiNx pores fabricated without this comprehensive approach, the pores are Ohmic in electrolyte, have extremely stable open-pore current during analyte translocation (>1 h) over a broad range of pore diameters ([Formula: see text]3- ∼30 nm) with spontaneous current correction (if current deviation occurs), and higher responsiveness (i.e. inter-event frequency) to negatively charged analytes (∼6.5 × in case of DNA). These pores were fabricated by modifying CDB with a chemical additive-sodium hypochlorite-that resulted in dramatically different nanopore surface chemistry including ∼3 orders of magnitude weaker Ka (acid dissociation constant of the surface chargeable head-groups) compared to CDB pores which is inextricably linked with significant improvements in nanopore performance with respect to CDB pores.

Mechanical characterization of vesicles and cells: A review.

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.

Push-Button Method To Create Nanopores Using a Tesla-Coil Lighter.

Controlled dielectric breakdown (CDB) of silicon nitride thin films immersed in electrolyte solution has been used to fabricate single nanofluidic channels with ∼10 nm and smaller diameters, nanopores, useful in single-molecule sensing and ionic circuit construction. A hand-held Tesla-coil lighter was used to form nanofluidic ionic conductors through a ∼10 nm thick silicon nitride membrane. Modifications to the conventional approach were required by the low-overhead Tesla-coil-assisted method (TCAM): increased circuit resistance by including water in place of electrolyte and discrete rather than continuous voltage applications. The resulting ionic conductance could be tuned with the number of voltage applications. TCAM and conventional CDB produced nanopores with different conductance versus pH curves, suggesting different surface chemistry. Nevertheless, sensing experiments using the canonical test molecule, λ-DNA, produced signals comparable to translocation results through solid-state nanopores fabricated by other methods. Thus, the TCAM method offers flexibility in fabrication and in the properties and function of the nanoscale ionic conductors that it can generate.

Chemically Functionalizing Controlled Dielectric Breakdown Silicon Nitride Nanopores by Direct Photohydrosilylation.

Nanopores are a prominent enabling tool for single-molecule applications such as DNA sequencing, protein profiling, and glycomics, and the construction of ionic circuit elements. Silicon nitride (SiNx) is a leading scaffold for these <100 nm-diameter nanofluidic ion-conducting channels, but frequently challenging surface chemistry remains an obstacle to their use. We functionalized more than 100 SiNx nanopores with different surface terminations-acidic (Si-R-OH, Si-R-CO2H), basic (Si-R-NH2), and nonionizable (Si-R-C6H3(CF3)2)-to chemically tune the nanopore size, surface charge polarity, and subsequent chemical reactivity and to change their conductance by changes of solution pH. The initial one-reaction-step covalent chemical film formation was by hydrosilylation and could be followed by straightforward condensation and click reactions. The hydrosilylation reaction step used neat reagents with no special precautions such as guarding against water content. A key feature of the approach was to combine controlled dielectric breakdown (CDB) with hydrosilylation to create and functionalize SiNx nanopores. CDB thus replaced the detrimental but conventionally necessary surface pretreatment with hydrofluoric acid. Proof-of-principle detection of the canonical test molecule, λ-DNA, yielded signals that showed that the functionalized pores were not obstructed by chemical treatments but could translocate the biopolymer. The characteristics were tuned by the surface coating character. This robust and flexible surface coating method, freed by CDB from HF etching, portends the development of nanopores with surface chemistry tuned to match the application, extending even to the creation of biomimetic nanopores.

Molecular-Level Profiling of Human Serum Transferrin Protein through Assessment of Nanopore-Based Electrical and Chemical Responsiveness.

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.