Emerging trends in metal oxide-based electronic noses for healthcare applications: a review.

An electronic nose (E-nose) is a technology fundamentally inspired by the human nose, designed to detect, recognize, and differentiate specific odors or volatile components in complex and chaotic environments. Comprising an array of sensors with meticulously designed nanostructured architectures, E-noses translate the chemical information captured by these sensors into useful metrics using complex pattern recognition algorithms. E-noses can significantly enhance the quality of life by offering preventive point-of-care devices for medical diagnostics through breath analysis, and by monitoring and tracking hazardous and toxic gases in the environment. They are increasingly being used in defense and surveillance, medical diagnostics, agriculture, environmental monitoring, and product validation and authentication. The major challenge in developing a reliable E-nose involves miniaturization and low power consumption. Various sensing materials are employed to address these issues. This review presents the key advancements over the last decade in E-nose technology, specifically focusing on chemiresistive metal oxide sensing materials. It discusses their sensing mechanisms, integration into portable E-noses, and various data analysis techniques. Additionally, we review the primary metal oxide-based E-noses for disease detection through breath analysis. Finally, we address the major challenges and issues in developing and implementing a portable metal oxide-based E-nose.

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.

Lithium Chloride Effects Field-Induced Protein Unfolding and the Transport Energetics Inside a Nanopipette.

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 ∼2 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.

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.

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.

Outcome of chronic inflammatory enteropathy in cats: 65 cases (2011-2021).

OBJECTIVES: Feline chronic inflammatory enteropathy is an idiopathic disease with limited information on variables that might affect treatment outcome and survival. The aim of this study was to determine if clinicopathological variables were associated with death due to gastrointestinal disease in cats with chronic inflammatory enteropathy. MATERIALS AND METHODS: Three medical records databases were retrospectively searched for cats diagnosed with chronic inflammatory enteropathy at the Royal Veterinary College between June 2008 and November 2021. Intestinal biopsy specimens of eligible cases were re-reviewed by one board-certified veterinary pathologist. Outcome information was obtained by contact with the referring veterinary surgeon. Two univariable binary logistic regression models and a Fisher's exact test were performed to assess the association between the outcome of death due to gastrointestinal disease or its short-term survival (≤ versus >1 year) with clinicopathological variables and the attainment of clinical remission. RESULTS: Sixty-five cats diagnosed with chronic inflammatory enteropathy between September 2011 and August 2021 were included in the study with follow-up information available for 54 cats (83%). Of these 54 cats, 20 (37%) were euthanised due to gastrointestinal disease (median 129.5 days; range 8 to 2970 days). Twenty-five (46%) cats were alive and in clinical remission (median 916 days; range 78 to 2113 days) with 16 (64%) diagnosed with food-responsive enteropathy. Attaining clinical remission reduced the likelihood of subsequent death due to gastrointestinal disease. CLINICAL SIGNIFICANCE: Measured physical and laboratory variables at the time of histopathological diagnosis of chronic inflammatory enteropathy were not predictors of death. Alternative diagnostic measures are required to definitively investigate outcome and survival in cats with chronic inflammatory enteropathy.

Enhanced Signal to Noise Ratio Enables High Bandwidth Nanopore Recordings and Molecular Weight Profiling of Proteins.

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 ∼12 kDa to ∼720 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 ≲10% of events were attenuated at 500 mV (≲ 5% for most proteins at 500 mV with only ≲1-5% of the population faster than ∼7 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).

Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance.

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 ∼3× improvement in SNR (i.e., signal-to-noise ratio) and an ∼8× 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, Cas9d10a─a protein with broad utilities in genetic engineering and immunology─bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically observed for proteins.

On the origins of conductive pulse sensing inside a nanopore.

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).

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.

Nanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein's Isoelectric Point.

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.

Investigating protein translocation in the presence of an electrolyte concentration gradient across a solid-state nanopore.

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). The cis side concentration was fixed at 4 M for positive chemical gradients and at 0.5 M LiCl for negative chemical gradients, while the trans side 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 Cl- ions 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.

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.

Histopathological frequency of canine hepatobiliary disease in the United Kingdom.

OBJECTIVES: Canine hepatobiliary disease is common; however, data determining disease frequency and breed predispositions are lacking. The primary objective was to identify the frequency of different hepatobiliary disease in a United Kingdom population of dogs and consequently determine breeds at both an increased and decreased risk of hepatobiliary disease. MATERIALS AND METHODS: Anonymised histopathology reports from a commercial veterinary diagnostic laboratory, which were submitted between August 2013 and February 2018, were analysed. Data were retrospectively categorised into hepatobiliary diseases according to World Small Animal Veterinary Association Standards and the breed, age and genders recorded. Cases with incomplete data or no definitive diagnosis were excluded. Breed predisposition was calculated using odds ratios and 95% confidence intervals against a United Kingdom-based control population of micro-chipped dogs. RESULTS: Histopathology results from 4584 cases met inclusion criteria. The most frequent histological diagnoses were reactive hepatitis (n=770); chronic hepatitis (n=735) and reversible hepatocellular injury (n=589). A number of breeds were shown to be at an increased or decreased risk of individual liver diseases. CLINICAL SIGNIFICANCE: This is the first study to document the histopathological frequency of hepatobiliary diseases in a large cohort of dogs in the United Kingdom, as well as novel possible breed and age predispositions. Despite multivariable analysis not being performed to account for confounding factors, this information hopes to inform and support future investigations for hepatic disease in particular breeds and potential predispositions.

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.