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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.

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.

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

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. However, decontamination efficacy across N95s is poorly understood, due to the dependence on received UV-C dose, which varies across the complex three-dimensional N95 shape. 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 couple optical simulation with in-situ PCI dosimetry to relate UV-C dose and viral inactivation at specific on-N95 locations, informing the design of safe and effective decontamination protocols.

COVID-19-induced personal protective equipment shortages necessitate effective, accessible N95 decontamination, especially in low-resource settings. UV-C irradiation inactivates SARS-CoV-2 analogs on N95 respirators when ≥1.0 J/cm^2 is delivered[1]. However, lack of robust and scalable UV-C dose validation tools impedes effective, widespread adoption. Tailored for field-deployment in settings lacking specialized UV-C radiometers, we design workflows to quantify photochromic UV-C dose indicators and extend dynamic range using low-cost filters. We demonstrate that photochromic quantification meets specifications required for robust UV-C measurements. Previously infeasible on-N95 measurements show ~20X dose variation within a UV-C system, highlighting how photochromics can underpin new validation strategies. Reference: 1. Brian Heimbuch and Del Harnish. Research to Mitigate a Shortage of Respiratory Protection Devices During Public Health Emergencies. (2019).

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.

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 (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.

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/cm2 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 (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.

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.

We report a microfluidic platform that maps target protein and mRNA expression with inter- and intraembryonic resolution., 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.

We demonstrate a highly selective and sensitive aptamer-based “capture-release” fluorescence detection of a malarial biomarker, i.e., Plasmodium lactose dehydrogenase (pLDH) protein, using single-layer two-dimensional MoS2 nanosheets. The detection of the target pLDH protein utilizing the aptamer-nanosheet sensing platform is rapid and can be easily completed within 10 min. In addition, the developed biomolecular sensor is capable of detecting the target malarial biomarker in homogeneous protein solutions as well as in heterogeneous mixtures of proteins. We anticipate that the ultrathin MoS2 nanosheet-based biosensor will facilitate the further development of biomolecular nanosensors for the detection of a wide range of biomarkers for malaria and other diseases.

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.

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 MoS

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. NRF (Natl Research Foundation, S’pore) MOE (Min. of Education, S’pore) Accepted version

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.

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 (R(2) > 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, β-TUB, CK8, STAT3, ER-α), with

Carbon nanomaterials are some of the most versatile nanomaterials. Along with increasing explorations into their utilization in a plethora of biological and biomedical applications, there have been emerging interests and needs in understanding the molecular hemocompatibility of these engineered nanomaterials when coming into contact with blood. Here, we evaluate the nano-bio interactions of one-dimensional (1D) and two-dimensional (2D) carbon nanomaterials with blood plasma proteins. Different facets of the nanomaterial–protein interactions, specifically, the adsorption, equilibrium binding and conformational stability of proteins upon association with carbon nanomaterials are established, based on the quantification of various parameters, such as association constant, binding cooperativity and protein secondary structural change. In light of our data, we demonstrate that the carbon nanomaterial–plasma protein interactions may be significantly influenced by the density of the oxygenated functionalities of the nanomaterials and to a certain extent, their dimensionality and surface area. This work offers a broad insight into the nano-bio interactions between carbon nanomaterials and blood plasma proteins and provides a strong basis for the design and use of 1D and 2D carbon nanomaterials for a wide variety of bioapplications. Oxygenated functional groups on one- and two-dimensional carbon nanomaterials play important roles in determining their interactions with blood plasma proteins. Chwee Teck Lim from the National University of Singapore and colleagues investigated how albumin, globulin and fibrinogen proteins found in blood plasma formed complexes with three types of tiny carbon compounds — short nanotubes containing carboxyl groups, thin graphene nanoplatelets and Swiss cheese—like sheets of porous graphene oxide. Using ultraviolet-visible and fluorescence spectroscopy techniques to probe biomolecular loading, the team found that porous graphene oxide had a greater affinity for proteins than nanotubes or nanoplatelets, in correlation with the density of oxygenated groups on each structure. Polarized-light measurement indicated that at a concentration of 40 micrograms per milliliter, porous graphene oxide induced conformational changes to fibrinogen that include a possible helix unraveling and protein denaturing. The nano-bio interactions of 1D and 2D carbon nanomaterials (COOH-functionalized carbon nanotube (CNT-COOH), graphene nanoplatelet (GNP) and porous graphene oxide (PGO)) with blood plasma proteins (albumin, globulin and fibrinogen) are evaluated in this work. It is demonstrated that these associations may be significantly influenced by the density of the oxygenated functionalities of the nanomaterials and to a certain extent, their dimensionality and surface area. This work offers a broad insight into the carbon nanomaterial–plasma protein interactions and provides a strong basis for the design and use of low-dimensional carbon nanomaterials for a wide variety of biological and biomedical applications.