Effects of off-axis translocation through nanopores on the determination of shape and volume estimates for individual particles.

Resistive pulses generated by nanoparticles that translocate through a nanopore contain multi-parametric information about the physical properties of those particles. For example, non-spherical particles sample several different orientations during translocation, producing fluctuations in blockade current that relate to their shape. Due to the heterogenous distribution of electric field from the center to the wall of a nanopore while a particle travels through the pore, its radial position influences the blockade current, thereby affecting the quantification of parameters related to the particle's characteristics. Here, we investigate the influence of these off-axis effects on parameters estimated by performing finite element simulations of dielectric particles transiting a cylindrical nanopore. We varied the size, ellipsoidal shape, and radial position of individual particles, as well as the size of the nanopore. As expected, nanoparticles translocating near the nanopore wall produce increase current blockades, resulting in overestimates of particle volume. We demonstrated that off-axis effects also influence estimates of shape determined from resistive pulse analyses, sometimes producing a multiple-fold deviation in ellipsoidal length-to-diameter ratio between estimates and reference values. By using a nanopore with the minimum possible diameter that still allows the particle to rotate while translocating, off-axis effects on the determination of both volume and shape can be minimized. In addition, tethering the nanoparticles to a fluid coating on the nanopore wall makes it possible to determine an accurate particle shape with an overestimated volume. This work provides a framework to select optimal ratios of nanopore to nanoparticle size for experiments targeting free translocations.

Leveraging artificial intelligence and data science techniques in harmonizing, sharing, accessing and analyzing SARS-COV-2/COVID-19 data in Rwanda (LAISDAR Project): study design and rationale.

BACKGROUND: Since the outbreak of COVID-19 pandemic in Rwanda, a vast amount of SARS-COV-2/COVID-19-related data have been collected including COVID-19 testing and hospital routine care data. Unfortunately, those data are fragmented in silos with different data structures or formats and cannot be used to improve understanding of the disease, monitor its progress, and generate evidence to guide prevention measures. The objective of this project is to leverage the artificial intelligence (AI) and data science techniques in harmonizing datasets to support Rwandan government needs in monitoring and predicting the COVID-19 burden, including the hospital admissions and overall infection rates. METHODS: The project will gather the existing data including hospital electronic health records (EHRs), the COVID-19 testing data and will link with longitudinal data from community surveys. The open-source tools from Observational Health Data Sciences and Informatics (OHDSI) will be used to harmonize hospital EHRs through the Observational Medical Outcomes Partnership (OMOP) Common Data Model (CDM). The project will also leverage other OHDSI tools for data analytics and network integration, as well as R Studio and Python. The network will include up to 15 health facilities in Rwanda, whose EHR data will be harmonized to OMOP CDM. EXPECTED RESULTS: This study will yield a technical infrastructure where the 15 participating hospitals and health centres will have EHR data in OMOP CDM format on a local Mac Mini ("data node"), together with a set of OHDSI open-source tools. A central server, or portal, will contain a data catalogue of participating sites, as well as the OHDSI tools that are used to define and manage distributed studies. The central server will also integrate the information from the national Covid-19 registry, as well as the results of the community surveys. The ultimate project outcome is the dynamic prediction modelling for COVID-19 pandemic in Rwanda. DISCUSSION: The project is the first on the African continent leveraging AI and implementation of an OMOP CDM based federated data network for data harmonization. Such infrastructure is scalable for other pandemics monitoring, outcomes predictions, and tailored response planning.

Wafer-scale fabrication of fused silica chips for low-noise recording of resistive pulses through nanopores.

This paper presents a maskless method to manufacture fused silica chips for low-noise resistive-pulse sensing. The fabrication includes wafer-scale density modification of fused silica with a femtosecond-pulsed laser, low-pressure chemical vapor deposition (LPVCD) of silicon nitride (SiN x ) and accelerated chemical wet etching of the laser-exposed regions. This procedure leads to a freestanding SiN x window, which is permanently attached to a fused silica support chip and the resulting chips are robust towards Piranha cleaning at ∼80 °C. After parallel chip manufacturing, we created a single nanopore in each chip by focused helium-ion beam or by controlled breakdown. Compared to silicon chips, the resulting fused silica nanopore chips resulted in a four-fold improvement of both the signal-to-noise ratio and the capture rate for signals from the translocation of IgG1 proteins at a recording bandwidth of 50 kHz. At a bandwidth of ∼1 MHz, the noise from the fused silica nanopore chips was three- to six-fold reduced compared to silicon chips. In contrast to silicon chips, fused silica chips showed no laser-induced current noise-a significant benefit for experiments that strive to combine nanopore-based electrical and optical measurements.

Fluid surface coatings for solid-state nanopores: comparison of phospholipid bilayers and archaea-inspired lipid monolayers.

In the context of sensing and characterizing single proteins with synthetic nanopores, lipid bilayer coatings provide at least four benefits: first, they minimize unwanted protein adhesion to the pore walls by exposing a zwitterionic, fluid surface. Second, they can slow down protein translocation and rotation by the opportunity to tether proteins with a lipid anchor to the fluid bilayer coating. Third, they provide the possibility to impart analyte specificity by including lipid anchors with a specific receptor or ligand in the coating. Fourth, they offer a method for tuning nanopore diameters by choice of the length of the lipid's acyl chains. The work presented here compares four properties of various lipid compositions with regard to their suitability as nanopore coatings for protein sensing experiments: (1) electrical noise during current recordings through solid-state nanopores before and after lipid coating, (2) long-term stability of the recorded current baseline and, by inference, of the coating, (3) viscosity of the coating as quantified by the lateral diffusion coefficient of lipids in the coating, and (4) the success rate of generating a suitable coating for quantitative nanopore-based resistive pulse recordings. We surveyed lipid coatings prepared from bolaamphiphilic, monolayer-forming lipids inspired by extremophile archaea and compared them to typical bilayer-forming phosphatidylcholine lipids containing various fractions of curvature-inducing lipids or cholesterol. We found that coatings from archaea-inspired lipids provide several advantages compared to conventional phospholipids; the stable, low noise baseline qualities and high viscosity make these membranes especially suitable for analysis that estimates physical protein parameters such as the net charge of proteins as they enable translocation events with sufficiently long duration to time-resolve dwell time distributions completely. The work presented here reveals that the ease or difficulty of coating a nanopore with lipid membranes did not depend significantly on the composition of the lipid mixture, but rather on the geometry and surface chemistry of the nanopore in the solid state substrate. In particular, annealing substrates containing the nanopore increased the success rate of generating stable lipid coatings.

Nanopore-Based, Rapid Characterization of Individual Amyloid Particles in Solution: Concepts, Challenges, and Prospects.

Aggregates of misfolded proteins are associated with several devastating neurodegenerative diseases. These so-called amyloids are therefore explored as biomarkers for the diagnosis of dementia and other disorders, as well as for monitoring disease progression and assessment of the efficacy of therapeutic interventions. Quantification and characterization of amyloids as biomarkers is particularly demanding because the same amyloid-forming protein can exist in different states of assembly, ranging from nanometer-sized monomers to micrometer-long fibrils that interchange dynamically both in vivo and in samples from body fluids ex vivo. Soluble oligomeric amyloid aggregates, in particular, are associated with neurotoxic effects, and their molecular organization, size, and shape appear to determine their toxicity. This concept article proposes that the emerging field of nanopore-based analytics on a single molecule and single aggregate level holds the potential to account for the heterogeneity of amyloid samples and to characterize these particles-rapidly, label-free, and in aqueous solution-with regard to their size, shape, and abundance. The article describes the concept of nanopore-based resistive pulse sensing, reviews previous work in amyloid analysis, and discusses limitations and challenges that will need to be overcome to realize the full potential of amyloid characterization on a single-particle level.

Investigation of Reagent Delivery Formats in a Multivalent Malaria Sandwich Immunoassay and Implications for Assay Performance.

Conventional lateral flow tests (LFTs), the current standard bioassay format used in low-resource point-of-care (POC) settings, have limitations that have held back their application in the testing of low concentration analytes requiring high sensitivity and low limits of detection. LFTs use a premix format for a rapid one-step delivery of premixed sample and labeled antibody to the detection region. We have compared the signal characteristics of two types of reagent delivery formats in a model system of a sandwich immunoassay for malarial protein detection. The premix format produced a uniform binding profile within the detection region. In contrast, decoupling the delivery of sample and labeled antibody to the detection region in a sequential format produced a nonuniform binding profile in which the majority of the signal was localized to the upstream edge of the detection region. The assay response was characterized in both the sequential and premix formats. The sequential format had a 4- to 10-fold lower limit of detection than the premix format, depending on assay conjugate concentration. A mathematical model of the assay quantitatively reproduced the experimental binding profiles for a set of rate constants that were consistent with surface plasmon resonance measurements and absorbance measurements of the experimental multivalent malaria system.

ENCoDE - a skin tone and clinical dataset from a prospective trial on acute care patients.

Background: Although hypothesized to be the root cause of the pulse oximetry disparities, skin tone and its use for improving medical therapies have yet to be extensively studied. Studies previously used self-reported race as a proxy variable for skin tone. However, this approach cannot account for skin tone variability within race groups and also risks the potential to be confounded by other non-biological factors when modeling data. Therefore, to better evaluate health disparities associated with pulse oximetry, this study aimed to create a unique baseline dataset that included skin tone and electronic health record (EHR) data. Methods: Patients admitted to Duke University Hospital were eligible if they had at least one pulse oximetry value recorded within 5 minutes before an arterial blood gas (ABG) value. We collected skin tone data at 16 different body locations using multiple devices, including administered visual scales, colorimetric, spectrophotometric, and photography via mobile phone cameras. All patients' data were linked in Duke's Protected Analytics Computational Environment (PACE), converted into a common data model, and then de-identified before publication in PhysioNet. Results: Skin tone data were collected from 128 patients. We assessed 167 features per skin location on each patient. We also collected over 2000 images from mobile phones measured in the same controlled environment. Skin tone data are linked with patients' EHR data, such as laboratory data, vital sign recordings, and demographic information. Conclusions: Measuring different aspects of skin tone for each of the sixteen body locations and linking them with patients' EHR data could assist in the development of a more equitable AI model to combat disparities in healthcare associated with skin tone. A common data model format enables easy data federation with similar data from other sources, facilitating multicenter research on skin tone in healthcare. Description: A prospectively collected EHR-linked skin tone measurements database in a common data model with emphasis on pulse oximetry disparities.

Harmonizing Norwegian registries onto OMOP common data model: Mapping challenges and opportunities for pregnancy and COVID-19 research.

OBJECTIVE: Norwegian health registries covering entire population are used for administration, research, and emergency preparedness. We harmonized these data onto the Observational Medical Outcomes Partnership common data model (OMOP CDM) and enrich real-world data in OMOP format with COVID-19 related data. METHODS: Data from six registries (2018-2021) covering birth registrations, selected primary and secondary care events, vaccinations, and communicable disease notifications were mapped onto the OMOP CDM v5.3. An Extract-Transform-Load (ETL) pipeline was developed on simulated data using data characterization documents and scanning tools. We ran dashboard quality checks, cohort generations, investigated differences between source and mapped data, and refined the ETL accordingly. RESULTS: We mapped 1.5 billion rows of data of 5,673,845 individuals. Among these, there were 804,277 pregnancies, 483,585 mothers together with 792,477 children, and 472,948 fathers. We identified 382,516 positive tests for COVID-19 in 380,794 patients. These figures are consistent with results from source data. In addition to 11 million source codes mapped automatically, we mapped 237 non-standard codes to standard concepts and introduced 38 custom concepts to accommodate pregnancy-related terminologies that were not supported by OMOP CDM vocabularies. A total of 3,700/3,705 (99.8%) checks passed. The 5 failed checks could be explained by the nature of the data and only represent a small number of records. DISCUSSION AND CONCLUSION: Norwegian registry data were successfully harmonized onto OMOP CDM with high level of concordance and provides valuable source for federated COVID-19 related research. Our mapping experience is highly valuable for data partners with Nordic health registries.

Dissolvable bridges for manipulating fluid volumes in paper networks.

A capability that is key to increasing the performance of paper microfluidic devices is control of fluid transport in the devices. We present dissolvable bridges as a novel method of manipulating fluid volumes within paper-based devices. We demonstrate and characterize the operation of the bridges, including tunability of the volumes passed from 10 µL to 80 µL, using parameters such as geometry and composition. We further demonstrate the utility of dissolvable bridges in the important context of automated delivery of different volumes of a fluid from a common source to multiple locations in a device for simple device loading and activation.

Two-dimensional paper network format that enables simple multistep assays for use in low-resource settings in the context of malaria antigen detection.

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

Enhanced sensitivity of lateral flow tests using a two-dimensional paper network format.

Point-of-care diagnostic assays that are rapid, easy-to-use, and low-cost are needed for use in low-resource settings; the lateral flow test has become the standard bioassay format in such settings because it meets those criteria. However, for a number of analytes, conventional lateral flow tests lack the sensitivity needed to have clinical utility. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions. The two-dimensional structures allow incorporation of multistep processes for improved sensitivity, while still retaining the positive aspects of conventional lateral flow tests. Here we create an easy-to-use, signal-amplified immunoassay based on a modified commercial strip test for human chorionic gonadotropin, the hormone used to detect pregnancy, and demonstrate an improved limit of detection compared to a conventional lateral flow assay. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore.

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.

It's Not a Bug, It's a Feature: Functional Materials in Insects.

Over the course of their wildly successful proliferation across the earth, the insects as a taxon have evolved enviable adaptations to their diverse habitats, which include adhesives, locomotor systems, hydrophobic surfaces, and sensors and actuators that transduce mechanical, acoustic, optical, thermal, and chemical signals. Insect-inspired designs currently appear in a range of contexts, including antireflective coatings, optical displays, and computing algorithms. However, as over one million distinct and highly specialized species of insects have colonized nearly all habitable regions on the planet, they still provide a largely untapped pool of unique problem-solving strategies. With the intent of providing materials scientists and engineers with a muse for the next generation of bioinspired materials, here, a selection of some of the most spectacular adaptations that insects have evolved is assembled and organized by function. The insects presented display dazzling optical properties as a result of natural photonic crystals, precise hierarchical patterns that span length scales from nanometers to millimeters, and formidable defense mechanisms that deploy an arsenal of chemical weaponry. Successful mimicry of these adaptations may facilitate technological solutions to as wide a range of problems as they solve in the insects that originated them.

Bioinspired, nanoscale approaches in contemporary bioanalytics (Review).

The genesis for this topical review stems from the interdisciplinary Biointerfaces International conference 2016 (BI 2016) in Zurich, Switzerland, wherein the need for advances in analytical tools was both expressed and addressed. Pushing the limits of detection for characterizing individual components, such as single proteins, single drug-delivery vehicles, or probing single living cells in a more natural environment, will contribute to the understanding of the complex biomolecular systems central to a number of applications including medical diagnostics, tissue engineering, and drug screening and delivery. Accordingly, the authors begin with an overview of single nanoparticle analytics highlighting two emerging techniques and how they compare with existing techniques. The first is based on single particle tracking of nanoparticles tethered to a mobile supported lipid bilayer, enabling the simultaneous characterization of both size and composition of individual nanoparticles. The second technique is based on probing variations in the ionic conduction across nanoscale apertures for detection of not only nanoparticles but also membrane-tethered proteins, thereby allowing a multiparameter characterization of individual nanoscopic objects, addressing their size, shape, charge, and dipole moment. Subsequently, the authors lead into an example of an area of application that stands to benefit from such advances in bioanalytics, namely, the development of biomimetic lipid- and polymer-based assemblies as stimuli-responsive artificial organelles and nanocarriers designed to optimize delivery of next generation high-molecular-weight biological drugs. This in turn motivates the need for additional advanced techniques for investigating the cellular response to drug delivery, and so the review returns again to bioanalytics, in this case single-cell analysis, while highlighting a technique capable of probing and manipulating the content of individual living cells via fluidic force microscopy. In presenting a concerted movement in the field of bioinspired bioanalytics, positioned in the context of drug delivery, while also noting the critical role of surface modifications, it is the authors' aim to evaluate progress in the field of single component bioanalytics and to emphasize the impact of initiating and maintaining a fruitful dialogue among scientists, together with clinicians and industry, to guide future directions in this area and to steer innovation to successful translation.

Formation of Single Nanopores with Diameters of 20-50 nm in Silicon Nitride Membranes Using Laser-Assisted Controlled Breakdown.

Nanopores with diameters from 20 to 50 nm in silicon nitride (SiN x) windows are useful for single-molecule studies of globular macromolecules. While controlled breakdown (CBD) is gaining popularity as a method for fabricating nanopores with reproducible size control and broad accessibility, attempts to fabricate large nanopores with diameters exceeding ∼20 nm via breakdown often result in undesirable formation of multiple nanopores in SiN x membranes. To reduce the probability of producing multiple pores, we combined two strategies: laser-assisted breakdown and controlled pore enlargement by limiting the applied voltage. Based on laser power-dependent increases in nanopore conductance upon illumination and on the absence of an effect of ionic strength on the ratio between the nanopore conductance before and after laser illumination, we suggest that the increased rate of controlled breakdown results from laser-induced heating. Moreover, we demonstrate that conductance values before and after coating the nanopores with a fluid lipid bilayer can indicate fabrication of a single nanopore versus multiple nanopores. Complementary flux measurements of Ca2+ through the nanopore typically confirmed assessments of single or multiple nanopores that we obtained using the coating method. Finally, we show that thermal annealing of CBD pores significantly increased the success rate of coating and reduced the current noise before and after lipid coating. We characterize the geometry of these nanopores by analyzing individual resistive pulses produced by translocations of spherical proteins and demonstrate the usefulness of these nanopores for estimating the approximate molecular shape of IgG proteins.

Real-time shape approximation and fingerprinting of single proteins using a nanopore.

Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.