Best Practices for Germicidal Ultraviolet-C Dose Measurement for N95 Respirator Decontamination.

Ultraviolet-C (UV-C) decontamination holds promise in combating the coronavirus disease 2019 pandemic, particularly with its potential to mitigate the N95 respirator shortage. Safe, effective, and reproducible decontamination depends critically on UV-C dose, yet dose is frequently measured and reported incorrectly, which results in misleading and potentially harmful protocols. Understanding best practices in UV-C dose measurement for N95 respirator decontamination is essential to the safety of medical professionals, researchers, and the public. Here, we outline the fundamental optical principles governing UV-C irradiation and detection, as well as the key metrics of UV-C wavelength and dose. In particular, we discuss the technical and regulatory distinctions between UV-C N95 respirator decontamination and other applications of germicidal UV-C, and we highlight the unique considerations required for UV-C N95 respirator decontamination. Together, this discussion will inform best practices for UV-C dose measurement for N95 respirator decontamination during crisis-capacity conditions.

Microparticle Delivery of Protein Markers for Single-Cell Western Blotting from Microwells.

Immunoblotting confers protein identification specificity beyond that of immunoassays by prepending protein electrophoresis (sizing) to immunoprobing. To accurately size protein targets, sample analysis includes concurrent analysis of protein markers with known molecular masses. To incorporate protein markers in single-cell western blotting, microwells are used to isolate individual cells and protein marker-coated microparticles. A magnetic field directs protein-coated microparticles to >75% of microwells, so as to 1) deliver a quantum of protein marker to each cell-laden microwell and 2) synchronize protein marker solubilization with cell lysis. Nickel-coated microparticles are designed, fabricated, and characterized, each conjugated with a mixture of histidine-tagged proteins (42.3-100 kDa). Imidazole in the cell lysis buffer solubilizes protein markers during a 30 s cell lysis step, with an observed protein marker release half-life of 4.46 s. Across hundreds of individual microwells and different microdevices, robust log-linear regression fits (R2 > 0.97) of protein molecular mass and electrophoretic mobility are observed. The protein marker and microparticle system is applied to determine the molecular masses of five endogenous proteins in breast cancer cells (GAPDH, ß-TUB, CK8, STAT3, ER-α), with <20% mass error. Microparticle-delivered protein standards underpin robust, reproducible electrophoretic cytometry that complements single-cell genomics and transcriptomics.

Single-Layer Ternary Chalcogenide Nanosheet as a Fluorescence-Based "Capture-Release" Biomolecular Nanosensor.

The novel application of two-dimensional (2D) single-layer ternary chalcogenide nanosheets as "capture-release" fluorescence-based biomolecular nanosensors is demonstrated. Fluorescently labeled biomolecular probe is first captured by the ultrathin Ta2 NiS5 nanosheets and then released upon adding analyte containing a target biomolecule due to the higher probe-target affinity. Here, the authors use a nucleic acid probe for the model target biomolecule Plasmodium lactate dehydrogenase, which is an important malarial biomarker. The ultrathin Ta2 NiS5 nanosheet serves as a highly efficient fluorescence quencher and the nanosensor developed from the nanosheet is highly sensitive and specific toward the target biomolecule. Apart from the specificity toward the target biomolecule in homogeneous solutions, the developed nanosensor is capable of detecting and differentiating the target in heterogeneous solutions consisting of either a mixture of biomolecules or serum, with exceptional specificity. The simplicity of the "capture-release" method, by eliminating the need for preincubation of the probe with the test sample, may facilitate further development of portable and rapid biosensors. The authors anticipate that this ternary chalcogenide nanosheet-based biomolecular nanosensor will be useful for the rapid detection and differentiation of a wide range of chemical and biological species.

Enhancing the sensing specificity of a MoS2 nanosheet-based FRET aptasensor using a surface blocking strategy.

Aptamer-based biosensing, which uses short, single-stranded nucleic acid segments to bind to a target, can be advantageous over antibody-based diagnostics due to the ease of synthesis and high stability of aptamers. However, the development of most aptamer-based sensors (aptasensors) is still in its initial stages and many factors affecting their performance have not been studied in great detail. Here, we enhance the sensing specificity of a fluorescence resonance energy transfer (FRET)-based MoS2 nanosheet aptasensor in detecting the malarial biomarker Plasmodium lactate dehydrogenase (pLDH). In this sensing scheme, the presence of target is signaled by an increase in fluorescence when fluorescently-labeled aptamers bind to pLDH and release from a quenching material. Interestingly, unlike most of the reported literature on aptasensors, we observe that non-target proteins also cause a considerable increase in the detected fluorescence. This may be due to the nonspecific adsorption of proteins onto the fluorescence quencher, leading to the displacement of aptamers from the quencher surface. To reduce this nonspecific association and to enhance the sensor specificity, we propose the application of a surface blocking agent to the quenching material. Importantly, we demonstrate that the sensing specificity of the MoS2 nanosheet-based aptasensor towards target pLDH biomolecules can be significantly enhanced through surface passivation, thus contributing to the development of highly selective and robust point-of-care malaria diagnostics.

Optical Attenuators Extend Dynamic Range but Alter Angular Response of Planar Ultraviolet-C Dosimeters.

Effective ultraviolet-C (UV-C) decontamination protocols of N95 respirators require validation that the entire N95 surface receives sufficient dose. Photochromic indicators (PCIs) can accurately measure UV-C dose on nonplanar surfaces, but often saturate below doses required to decontaminate porous, multilayered textiles like N95s. Here, we investigate the use of optical attenuators to extend PCI dynamic range while maintaining a near-ideal angular response-critical for accurate measurements of uncollimated UV-C. We show analytically that tuning attenuator refractive index, attenuation coefficient, and thickness can extend dynamic range, but compromises angular response unless the attenuator is an ideal diffuser. To investigate this tradeoff empirically, we stack PCIs behind model specular (floated borosilicate) and diffuse (polytetrafluoroethylene) attenuators, characterize the angular response, and evaluate on-N95 UV-C measurement accuracy within a decontamination system. Both attenuators increase PCI dynamic range >4×, but simultaneously introduce angle-dependent transmittance, which causes location-dependent underestimation of UV-C dose. PCI-borosilicate and PCI-polytetrafluoroethylene stacks underreport true on-N95 dose by (1) 14.7% and 3.6%, respectively, when near-normal to the source lamp array, and (2) 40.8% and 19.8%, respectively, in a steeply sloped location. Overall, we demonstrate that while planar attenuators can increase PCI dynamic range, verifying near-ideal angular response is critical for accurate UV-C measurements.

Current Understanding of Ultraviolet-C Decontamination of N95 Filtering Facepiece Respirators.

Introduction: The COVID-19 pandemic has led to critical shortages of single-use N95 filtering facepiece respirators. The US Centers for Disease Control and Prevention has identified ultraviolet-C (UV-C) irradiation as one of the most promising decontamination methods during crisis-capacity surges; however, understanding the mechanism of pathogen inactivation and post-treatment respirator performance is central to effective UV-C decontamination. Objective: We summarize the UV-C N95 decontamination evidence and identify key metrics. Methods: We evaluate the peer-reviewed literature on UV-C decontamination to inactivate SARS-CoV-2, viral analogues, and other microorganisms inoculated on N95s, as well as the resulting effect on respirator fit and filtration. Where peer-reviewed studies are absent, we discuss outstanding questions and ongoing work. Key Findings: Evidence supports that UV-C exposure of ≥1.0 J/cm2 inactivates SARS-CoV-2 analogues (≥3-log reduction) on the majority of tested N95 models. The literature cautions that (1) viral inactivation is N95 model-dependent and impeded by shadowing, (2) N95 straps require secondary decontamination, (3) higher doses may be necessary to inactivate other pathogens (e.g., some bacterial spores), and (4) while N95 fit and filtration appear to be preserved for 10-20 cycles of 1.0 J/cm2, donning and doffing may degrade fit to unacceptable levels within fewer cycles. Results and Discussion: Effective N95 UV-C treatment for emergency reuse requires both (1) inactivation of the SARS-CoV-2 virus, achieved through application of UV-C irradiation at an appropriate wavelength and effective dose, and (2) maintenance of the fit and filtration efficiency of the N95. Conclusions: UV-C treatment is a risk-mitigation process that should be implemented only under crisis-capacity conditions and with proper engineering, industrial hygiene, and biosafety controls.

Multimodal detection of protein isoforms and nucleic acids from mouse pre-implantation embryos.

Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, 'gel pallets' containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2-3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos.

Quantitative UV-C dose validation with photochromic indicators for informed N95 emergency decontamination.

With COVID-19 N95 shortages, frontline medical personnel are forced to reuse this disposable-but sophisticated-multilayer respirator. Widely used to decontaminate nonporous surfaces, UV-C light has demonstrated germicidal efficacy on porous, non-planar N95 respirators when all surfaces receive ≥1.0 J/cm2 dose. Of utmost importance across disciplines, translation of empirical evidence to implementation relies upon UV-C measurements frequently confounded by radiometer complexities. To enable rigorous on-respirator measurements, we introduce a photochromic indicator dose quantification technique for: (1) UV-C treatment design and (2) in-process UV-C dose validation. While addressing outstanding indicator limitations of qualitative readout and insufficient dynamic range, our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%) required for UV-C dose measurements. In a measurement infeasible with radiometers, we observe a striking ~20× dose variation over N95s within one decontamination system. Furthermore, we adapt consumer electronics for accessible quantitative readout and use optical attenuators to extend indicator dynamic range >10× to quantify doses relevant for N95 decontamination. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical considerations for both photochromic indicators themselves and UV-C decontamination processes.

Multimodal detection of protein isoforms and nucleic acids from low starting cell numbers.

Protein isoforms play a key role in disease progression and arise from mechanisms involving multiple molecular subtypes, including DNA, mRNA and protein. Recently introduced multimodal assays successfully link genomes and transcriptomes to protein expression landscapes. However, the specificity of the protein measurement relies on antibodies alone, leading to major challenges when measuring different isoforms of the same protein. Here we utilize microfluidic design to perform same-cell profiling of DNA, mRNA and protein isoforms (triBlot) on low starting cell numbers (1-100 s of cells). After fractionation lysis, cytoplasmic proteins are resolved by molecular mass during polyacrylamide gel electrophoresis (PAGE), adding a degree of specificity to the protein measurement, while nuclei are excised from the device in sections termed "gel pallets" for subsequent off-chip nucleic acid analysis. By assaying TurboGFP-transduced glioblastoma cells, we observe a strong correlation between protein expression prior to lysis and immunoprobed protein. We measure both mRNA and DNA from retrieved nuclei, and find that mRNA levels correlate with protein abundance in TurboGFP-expressing cells. Furthermore, we detect the presence of TurboGFP isoforms differing by an estimated <1 kDa in molecular mass, demonstrating the ability to discern different proteoforms with the same antibody probe. By directly relating nucleic acid modifications to protein isoform expression in 1-100 s of cells, the triBlot assay holds potential as a screening tool for novel biomarkers in diseases driven by protein isoform expression.

Mapping of UV-C dose and SARS-CoV-2 viral inactivation across N95 respirators during decontamination.

During public health crises like the COVID-19 pandemic, ultraviolet-C (UV-C) decontamination of N95 respirators for emergency reuse has been implemented to mitigate shortages. Pathogen photoinactivation efficacy depends critically on UV-C dose, which is distance- and angle-dependent and thus varies substantially across N95 surfaces within a decontamination system. Due to nonuniform and system-dependent UV-C dose distributions, characterizing UV-C dose and resulting pathogen inactivation with sufficient spatial resolution on-N95 is key to designing and validating UV-C decontamination protocols. However, robust quantification of UV-C dose across N95 facepieces presents challenges, as few UV-C measurement tools have sufficient (1) small, flexible form factor, and (2) angular response. To address this gap, we combine optical modeling and quantitative photochromic indicator (PCI) dosimetry with viral inactivation assays to generate high-resolution maps of "on-N95" UV-C dose and concomitant SARS-CoV-2 viral inactivation across N95 facepieces within a commercial decontamination chamber. Using modeling to rapidly identify on-N95 locations of interest, in-situ measurements report a 17.4 ± 5.0-fold dose difference across N95 facepieces in the chamber, yielding 2.9 ± 0.2-log variation in SARS-CoV-2 inactivation. UV-C dose at several on-N95 locations was lower than the lowest-dose locations on the chamber floor, highlighting the importance of on-N95 dose validation. Overall, we integrate optical simulation with in-situ PCI dosimetry to relate UV-C dose and viral inactivation at specific on-N95 locations, establishing a versatile approach to characterize UV-C photoinactivation of pathogens contaminating complex substrates such as N95s.

Probe-target hybridization depends on spatial uniformity of initial concentration condition across large-format chips.

Diverse assays spanning from immunohistochemistry (IHC), to microarrays (protein, DNA), to high-throughput screens rely on probe-target hybridization to detect analytes. These large-format 'chips' array numerous hybridization sites across centimeter-scale areas. However, the reactions are prone to intra-assay spatial variation in hybridization efficiency. The mechanism of spatial bias in hybridization efficiency is poorly understood, particularly in IHC and in-gel immunoassays, where immobilized targets are heterogeneously distributed throughout a tissue or hydrogel network. In these systems, antibody probe hybridization to a target protein antigen depends on the interplay of dilution, thermodynamic partitioning, diffusion, and reaction. Here, we investigate parameters governing antibody probe transport and reaction (i.e., immunoprobing) in a large-format hydrogel immunoassay. Using transport and bimolecular binding theory, we identify a regime in which immunoprobing efficiency (η) is sensitive to the local concentration of applied antibody probe solution, despite the antibody probe being in excess compared to antigen. Sandwiching antibody probe solution against the hydrogel surface yields spatially nonuniform dilution. Using photopatterned fluorescent protein targets and a single-cell immunoassay, we identify regimes in which nonuniformly distributed antibody probe solution causes intra-assay variation in background and η. Understanding the physicochemical factors affecting probe-target hybridization reduces technical variation in large-format chips, improving measurement precision.

Assessing heterogeneity among single embryos and single blastomeres using open microfluidic design.

The process by which a zygote develops from a single cell into a multicellular organism is poorly understood. Advances are hindered by detection specificity and sensitivity limitations of single-cell protein tools and by challenges in integrating multimodal data. We introduce an open microfluidic tool expressly designed for same-cell phenotypic, protein, and mRNA profiling. We examine difficult-to-study-yet critically important-murine preimplantation embryo stages. In blastomeres dissociated from less well-studied two-cell embryos, we observe no significant GADD45a protein expression heterogeneity, apparent at the four-cell stage. In oocytes, we detect differences in full-length versus truncated DICER-1 mRNA and protein, which are insignificant by the two-cell stage. Single-embryo analyses reveal intraembryonic heterogeneity, differences between embryos of the same fertilization event and between donors, and reductions in the burden of animal sacrifice. Open microfluidic design integrates with existing workflows and opens new avenues for assessing the cellular-to-molecular heterogeneity inherent to preimplantation embryo development.

UV-C decontamination for N95 emergency reuse: Quantitative dose validation with photochromic indicators.

With COVID-19 N95 respirator shortages, frontline medical personnel are forced to reuse this disposable - but sophisticated - multilayer textile respirator. Widely used for decontamination of nonporous surfaces, UV-C light has germicidal efficacy on porous, non-planar N95 respirators when ≥1.0 J/cm^2 dose is applied across all surfaces. Here, we address outstanding limitations of photochromic indicators (qualitative readout and insufficient dynamic range) and introduce a photochromic UV-C dose quantification technique for: (1) design of UV-C treatments and (2) in-process UV-C dose validation. Our methodology establishes that color-changing dosimetry can achieve the necessary accuracy (>90%), uncertainty (<10%), and UV-C specificity (>95%). Furthermore, we adapt consumer electronics for accessible quantitative readout and extend the dynamic range >10× using optical attenuators. In a measurement infeasible with radiometers, we observe striking 20× dose variation over 3D N95 facepieces. By transforming photochromic indicators into quantitative dosimeters, we illuminate critical design considerations for both photochromic indicators and UV-C decontamination.

Paper-based MoS2 nanosheet-mediated FRET aptasensor for rapid malaria diagnosis.

There has been growing interest in the development of paper-based biosensors because their simplicity and low cost are attractive for point-of-care diagnosis, especially in low-resource areas. However, only a limited range of paper materials - primarily chromatography papers - have been incorporated into diagnostics thus far. Here, we investigate the performance of different types of paper in order to develop an aptamer- and MoS2 nanosheet-based sensor relying on fluorescence resonance energy transfer (FRET) to signal the presence of a target protein. An aptamer which binds to a malarial biomarker, Plasmodium lactate dehydrogenase (pLDH), is chosen for this study, as point-of-care diagnostics would be especially advantageous in low-resource areas, such as those where malaria is prevalent. We observe that of all papers tested, a measurable and specific fluorescence recovery can only be produced on the sensor created with printer paper, while no significant fluorescence recovery is generated on sensors made from other types of paper, including chromatography, lens, and filter papers. Therefore, our findings demonstrate the importance of careful material selection for the development of a paper-based diagnostic test, and suggest that commercially-available products such as printer paper may serve as viable materials to develop cost-effective and simple diagnostics.