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The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 μL sample with a pipette. The device, which we call the μSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2-3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the sample for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus (

Cellular analyses are increasingly used to diagnose diseases at point-of-care and global healthcare settings. Some analyses are simple as they rely on chromogenic stains (blood counts, malaria) but others often require higher multiplexing to define and quantitate cell populations (cancer diagnosis, immunoprofiling). Simplifying the latter with inexpensive solutions represents a current bottleneck in designing start-end pipelines. Based on the hypothesis that novel film adhesives could be used to create inexpensive disposable devices, we tested a number of different designs and materials, to rapidly perform 12–15 channel single-cell imaging. Using an optimized passive pumping layer-stack microfluidic (PLASMIC) device (< 1$ in supplies) we show that rapid, inexpensive cellular analysis is feasible.

The ability to observe cells in live organisms is essential for understanding their function in complex in vivo milieus. A major challenge today has been the limited ability to perform higher multiplexing beyond four to six colors to define cell subtypes in vivo. Here, a click chemistry-based strategy is presented for higher multiplexed in vivo imaging in mouse models. The method uses a scission-accelerated fluorophore exchange (SAFE), which exploits a highly efficient bioorthogonal mechanism to completely remove fluorescent signal from antibody-labeled cells in vivo. It is shown that the SAFE-intravital microscopy imaging method allows 1) in vivo staining of specific cell types in dorsal and cranial window chambers of mice, 2) complete un-staining in minutes, 3) in vivo click chemistries at lower (µm) and thus non-toxic concentrations, and 4) the ability to perform in vivo cyclic imaging. The potential utility of the method is demonstrated by 12 color imaging of immune cells in live mice.

Desulfovibrio alaskensis G20 biofilms were cultivated on 316 steel, 1018 steel, or borosilicate glass under steady-state conditions in electron-acceptor limiting (EAL) and electron-donor limiting (EDL) conditions with lactate and sulfate in a defined medium. Increased corrosion was observed on 1018 steel under EDL conditions compared to 316 steel, and biofilms on 1018 carbon steel under the EDL condition had at least twofold higher corrosion rates compared to the EAL condition. Protecting the 1018 metal coupon from biofilm colonization significantly reduced corrosion, suggesting that the corrosion mechanism was enhanced through attachment between the material and the biofilm. Metabolomic mass spectrometry analyses demonstrated an increase in a flavin-like molecule under the 1018 EDL condition and sulfonates under the 1018 EAL condition. These data indicate the importance of S-cycling under the EAL condition, and that the EDL is associated with increased biocorrosion via indirect extracellular electron transfer mediated by endogenously produced flavin-like molecules.

New tools in the field of biocompatible chemistries are enabling researchers to probe immunological and genetic information with highly multiplexed capabilities. These bioorthogonal click chemistry reactions provide a platform for tumor and immune cell profiling for dozens of markers on the same cell sample simultaneously, providing a more complete snapshot of the disease.

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level., Riazanski et al describe a platform to capture extracellular vesicles (EVs) using a nanoporous silicon nitride membrane, investigate the expression of NHE1 protein on the surface of EVs and monitor the transport of Na+ and H+ at the single EV level. The authors report a mechanistic function of the proteins found in EVs and specifically identify NHE1 on a single EV, where it maintains pH neutrality within single vesicles.

Localized residual strain develops within the metallurgical texture of 1018 carbon steel from metallurgical processes, such as fabrication, annealing, and shaping. This residual strain results in accelerated localized pitting due to the formation of anodic sites at these locations. Once initiated, micron-sized corrosion pits can coalesce to form sites of potential catastrophic failure. In this contribution, we focus on the localized biocorrosion which initiates and grows in areas of localized strain such as the interfaces between manganese sulfide (MnS) inclusions and ferrite grains in the steel, at grain boundaries between ferrite grains with different crystallographic orientations and at pearlite grains (intergrown cementite (Fe3C) and ferrite), which are readily found in 1018 carbon steel. Here we hypothesize and show experimentally that accelerated biocorrosion in 1018 carbon steel finds its roots in the electrochemical potential difference (micro galvanic cells) generated between the unstrained ferrite iron (α - Fe) and the lattice defects, dislocations and mismatches found at interfaces formed between α - Fe and secondary phases i.e. MnS inclusions, cementite lamellar structures and grain boundaries distributed throughout the 3D network of the carbon steel. This hypothesis is supported by results from multiple micro- and nanoscale imaging and analytical methods obtained from field emission scanning electron microscopy, energy dispersive spectroscopy, electron backscattered diffraction and Auger nanoprobe electron spectroscopy. The morphology and composition of grains in the steel coupons were characterized before and after exposure to suboxic and sulfidogenic environments dominated by aerobic and anaerobic marine organisms. Corrosion processes are demonstrated to initiate in localized areas of high residual strain.

Beyond their use as separation tools, nanoporous membranes can be used for the capture and analysis of biological nanoparticles such as virus or small extracellular vesicles (sEVs). While the challenges of imaging sub-optical particles can be addressed with high resolution imaging techniques, separation challenges remain. We have recently reported on a method of capturing and isolating sEVs, including diagnostically valuable exosomes, in the pores of ultrathin (100 nm thick) nanoporous silicon nitride (NPN) membranes in a microfluidic flow scheme we termed tangential flow for analyte capture (TFAC). Here we present static analytical and dynamic computational models of nanoparticle capture on NPN and compare them to a range of experiments using 60 nm gold nanoparticles (AuNPs) as surrogates for biological nanoparticles. In the presence of 1 mg/mL bovine serum albumin to prevent the formation of gold nanoparticle aggregates, we find that the dynamic model is highly predictive of particle capture over a range of practical flow conditions (~ 10% error at low ultrafiltration rates, ~ 1% error at high ultrafiltration rates). Despite the small size of nanoparticles, we find that convective transport dominates diffusive transport during capture (Peclet number > 100), enabling the simple rule-of-thumb that the fraction of nanoparticles captured from solution roughly equals the fraction of the input sample that is ultrafiltered (

INTRODUCTION: The pathophysiological increase in microvascular permeability plays a well-known role in the onset and progression of diseases like sepsis and atherosclerosis. However, how interactions between neutrophils and the endothelium alter vessel permeability is often debated. METHODS: In this study, we introduce a microfluidic, silicon-membrane enabled vascular mimetic (μSiM-MVM) for investigating the role of neutrophils in inflammation-associated microvascular permeability. In utilizing optically transparent silicon nanomembrane technology, we build on previous microvascular models by enabling in situ observations of neutrophil-endothelium interactions. To evaluate the effects of neutrophil transmigration on microvascular model permeability, we established and validated electrical (transendothelial electrical resistance and impedance) and small molecule permeability assays that allow for the in situ quantification of temporal changes in endothelium junctional integrity. RESULTS: Analysis of neutrophil-expressed β(1) integrins revealed a prominent role of neutrophil transmigration and basement membrane interactions in increased microvascular permeability. By utilizing blocking antibodies specific to the β(1) subunit, we found that the observed increase in microvascular permeability due to neutrophil transmigration is constrained when neutrophil-basement membrane interactions are blocked. Having demonstrated the value of in situ measurements of small molecule permeability, we then developed and validated a quantitative framework that can be used to interpret barrier permeability for comparisons to conventional Transwell™ values. CONCLUSIONS: Overall, our results demonstrate the potential of the μSiM-MVM in elucidating mechanisms involved in the pathogenesis of inflammatory disease, and provide evidence for a role for neutrophils in inflammation-associated endothelial barrier disruption. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12195-020-00611-6) contains supplementary material, which is available to authorized users.

Critical flux defines the ideal operating conditions in tangential flow filtration (TFF). Below this value a TFF process can, in principle, proceed indefinitely without membrane fouling. The critical flux behavior of ultrathin membranes, which are particularly valuable for small volume processing, has not yet been explored. Here we investigate the critical flux behavior of ultrathin nanoporous silicon nitride (NPN) membranes in concentrated protein solutions using a microfluidic TFF device. We find that NPN (~19% porosity; 60 nm average pore size; 100 nm thick) has the ability to process bovine serum albumin (BSA) solutions as concentrated as 10 mg/mL at 30 μL/min without exceeding a critical flux threshold. At 30 mg/mL and 60 mg/mL, NPN enables 40% and 20% of the supply to be filtered without fouling. In these experiments, NPN membranes achieve critical flux values as high as ~1200 LMH, which is 5x larger than 0.2 μm polyethersulfone (PES) membranes processing 5 mg/mL BSA and an order of magnitude higher than track-etch polymeric membranes (10 μm thick; 80 nm pores) in direct comparisons. The effects of nanoparticle capture on critical flux are also studied here, simulating the capture of small extracellular vesicles (sEVs) in forthcoming diagnostic applications. Our work not only demonstrates the utility of ultrathin membranes for processing concentrated protein solutions, it illustrates the value of applying membrane science principles developed for macroscale systems (e.g. critical flux, TFF) to the micro/nano scale.

A practical method has been developed for rapidly determining the depth of a corrosion micro-pit from the path lengths of X rays passing through the walls of the pit on their way to an X-ray detector. The method takes advantage of the attenuation of the Bremsstrahlung and characteristic X-ray radiation accompanying each X-ray spectrum, and the results are verified independently using AFM and the special pit geometry surrounding MnS inclusions in 1018 carbon steel. The method has general validity and is especially valuable in those cases where the pit depth-to-width ratio is too steep to measure using the conventional methods.

Membranes have been used extensively for the purification and separation of biological species. A persistent challenge is the purification of species from concentrated feed solutions such as extracellular vesicles (EVs) from biological fluids. We investigated a new method to isolate micro- and nano-scale species termed tangential flow for analyte capture (TFAC), which is an extension of traditional tangential flow filtration (TFF). Initially, EV purification from plasma on ultrathin nanomembranes was compared between both normal flow filtration (NFF) and TFF. NFF resulted in rapid formation of a protein cake which completely obscured any captured EVs and also prevented further transport across the membrane. On the other hand, TFF showed capture of CD63 positive EVs with minimal contamination. We explored the use of TFF to capture target species over membrane pores, wash and then release in a physical process that does not rely upon affinity or chemical interactions. This process of TFAC was studied with model particles on both ultrathin nanomembranes and conventional thickness membranes (polycarbonate track-etch). Successful capture and release of model particles was observed using both membranes. Ultrathin nanomembranes showed higher efficiency of capture and release with significantly lower pressures indicating that ultrathin nanomembranes are well-suited for TFAC of delicate nanoscale particles such as EVs.

In a saline anaerobic sulfidogenic environment, pitting on 1018 carbon steel was initiated within a 20–30 nm zone at the MnS inclusion boundary. Nanoscale analysis was performed using scanning electron microscopy and a scanning Auger nanoprobe. The pitting was more pronounced in the presence of a biofilm of sulfate-reducing bacteria than in abiotic sulfide medium. It is proposed that initiation of an anodic reaction leading to dissolution of Fe matrix and subsequent pitting of steel in MnS inclusion boundary regions is due to disorder and strain exerted on the Fe matrix by MnS contamination of the interface from metallurgical processes.

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.