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The study was carried out in a training establishment for technical officers, the total population being 21,000. There were 27 messes employing 465 food handlers of which 304 were included. The messes were inspected every month with regard to environmental sanitation and personal hygiene of the personnel working in the kitchens. Of the 27 the hygiene conditions of 20 was found good, 7 satisfactory and none of them was found unsatisfactory. 1.6% workers were suffering form skin infections, 2.6% with intestinal parasites, 1.3% respiratory disease and 13.4% had hypertension. All were protected against enteric groups of diseases and tetanus.

Serial macromolecular X-ray crystallography plays an important role in elucidating protein structures and consequently progressing the field of targeted therapeutics. The use of pulsed beams at different repetition frequencies requires the development of various sample-conserving injection strategies to minimize sample wastage between X-ray exposures. Fixed-target sample delivery methods that use solid support to hold the crystals in the X-ray beam path are gaining interest as a sample-conserving delivery system for X-ray crystallography with high crystal hit rates. Here, we present a novel fixed-target microfluidic system for delivering protein microcrystals to X-ray beams for diffraction data collection and structure determination. The fixed-target design consists of 3 symmetric sections arranged in an area of 1 in. × 1 in. with up to 18,000 crystal traps per device. Each trap is targeted to hold one crystal up to 50 μm in size in the largest dimension. The device has been fabricated using cyclic olefin copolymer (COC) for high-quality diffraction data collection with low background scattering induced through the fixed-target material. The newly developed fixed-target device is designed for vacuum compatibility which will enable the use in vacuum experimental chambers of X-ray radiation sources including the newly developed, first-of-its-kind compact X-ray light source (CXLS), which is currently in commissioning at Arizona State University. To assess the validity of the COC device, serial crystallography experiments were performed on the model protein lysozyme at the new European Synchrotron Radiation Facility-Extremely Brilliant Source (ESRF-EBS) beamline ID29. A 1.6 Å crystal structure of the protein was solved, demonstrating that, in general, the COC device can be used to generate high-quality data from macromolecular crystals at the CXLS and synchrotron radiation sources, which holds enormous potential for advancing the field of protein structure determination by fixed-target X-ray crystallography.

Polymer beads, especially polystyrene particles, have been extensively used as model species in insulator-based dielectrophoresis (iDEP) studies. Their use in alternating current iDEP (AC-iDEP) is less explored; however, an assessment in the low-frequency regime (≤10 kHz) allows to link surface conduction effects with the surface properties of polymer particles. Here, we provide a case study for various experimental conditions assessing sub-micrometer polystyrene particles with AC-iDEP and link to accepted surface conduction theory to predict and experimentally verify the observed AC-iDEP trapping behavior based on apparent zeta potential and solution conductivity. We find excellent agreement with the theoretical predictions, but also the occurrence of concentration polarization electroosmotic flow under the studied conditions, which have the potential to confound acting dielectrophoresis conditions. Furthermore, we study a case relevant to the assessment of microplastics in human and animal body fluids by mimicking the protein adsorption of high abundant proteins in blood by coating polystyrene beads with bovine serum albumin, a highly abundant protein in blood. Theoretical predictions and experimental observations confirm a difference in observed AC-iDEP behavior between coated and non-coated particles, which might be exploited for future studies of microplastics in blood to assess their exposure to humans and animals., (© 2024 Wiley‐VCH GmbH.)

Organelle size varies with normal and abnormal cell function. Thus, size-based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator-based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub-micrometer-sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size-based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low-frequency DC-biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters-improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283-1296)-demonstrating that fine-tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes., (© 2023 Wiley-VCH GmbH.)

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

In tissue engineering, polyurethane-based implants have gained significant traction because of their high compatibility and inertness. The implants therefore show fewer side effects and lasts longer. Also, the mechanical properties can be tuned and morphed into a particular shape, owing to which polyurethanes show immense versatility. In the last 3 years, scientists have devised methods to enhance the strength of and induce dynamic properties in polyurethanes, and these developments offer an immense opportunity to use them in tissue engineering. The focus of this review is on applications of polyurethane implants for biomedical application with detailed analysis of hard tissue implants like bone tissues and soft tissues like cartilage, muscles, skeletal tissues, and blood vessels. The synthetic routes for the preparation of scaffolds have been discussed to gain a better understanding of the issues that arise regarding toxicity. The focus here is also on concerns regarding the biocompatibility of the implants, given that the precursors and byproducts are poisonous.

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Ltd.)

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time., Competing Interests: A.E.G., J.C.V., and A.R. hold a patent on electrical droplet stimulation in a 3D-printed device., (© 2022 The Authors.)

Heterogeneity in organelle size has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing the size-based subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. Here, we demonstrated a ratchet migration mechanism using insulator-based dielectrophoresis in conjunction with a continuous flow component that allows the size-based separation of submicrometer particles. The ratchet mechanism was realized in a microfluidic device exhibiting an array of insulating posts, tailoring electrokinetic and dielectrophoretic transport. A numerical model was developed to elucidate the particle migration and the size-based separation in various conditions. Experimentally, the size-based separation of a mixture of polystyrene beads (0.28 and 0.87 μ $\umu $ m) was accomplished demonstrating good agreement with the numerical model. Furthermore, the size-based separation of mitochondria was investigated using a mitochondria mixture isolated from HepG2 cells and HepG2 cells carrying the gene Mfn-1 knocked out, indicating distinct size-related migration behavior. With the presented continuous flow separation device, larger amounts of fractionated organelles can be collected in the future allowing access to the biomolecular signature of mitochondria subpopulations differing in size., (© 2022 Wiley-VCH GmbH.)

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macro-molecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX., (© Diandra Doppler et al. 2022.)

Using hydrogels for delivering cancer therapeutics is advantageous in pharmaceutical usage as they have an edge over traditional delivery, which is tainted due to the risk of toxicity that it imbues. Hydrogel usage leads to the development of a more controlled drug release system owing to its amenability for structural metamorphosis, its higher porosity to seat the drug molecules, and its ability to shield the drug from denaturation. The thing that makes its utility even more enhanced is that they make themselves more recognizable to the body tissues and hence can stay inside the body for a longer time, enhancing the efficiency of the delivery, which otherwise is negatively affected since the drug is identified by the human immunity as a foreign substance, and thus, an attack of the immunity begins on the drug injected. A variety of hydrogels such as thermosensitive, pH-sensitive, and magnetism-responsive hydrogels have been included and their potential usage in drug delivery has been discussed in this review that aims to present recent studies on hydrogels that respond to alterations under a variety of circumstances in "reducing" situations that mimic the microenvironment of cancerous cells.

Cancer cannot be controlled by the usage of drugs alone, and thus, nanotechnology is an important technique that can provide the drug with an impetus to act more effectively. There is adequate availability of anticancer drugs that are classified as alkylating agents, hormones, or antimetabolites. Nanoparticle (NP) carriers increase the residence time of the drug, thereby enhancing the survival rate of the drug, which otherwise gets washed off owing to the small size of the drug particles by the excretory system. For example, for enhancing the circulation, a coating of nonfouling polymers like PEG and dextran is done. Famous drugs such as doxorubicin (DOX) are commonly encapsulated inside the nanocomposite. The various classes of nanoparticles are used to enhance drug delivery by aiding it to fight against the tumor. Targeted therapy aims to attack the cells with features common to the cancer cells while minimizing damage to the normal cell, and these therapies work in one in four ways. Some block the cancer cells from reproducing newer cells, others release toxic substances to kill the cancer cells, some stimulate the immune system to destroy the cancer cells, and some block the growth of more blood vessels around cancer cells, which starve the cells of the nutrients, which is needed for their growth. This review aims to testify the advancements nanotechnology has brought in cancer therapy, and its statements are supported with recent research findings and clinical trial results.

Carbon nanotubes (CNTs) are one of the most extensively studied nanomaterials in the 21st century. Since their discovery in 1991, many studies have been reported advancing our knowledge in terms of their structure, properties, synthesis, and applications. CNTs exhibit unique electrothermal and conductive properties which, combined with their mechanical strength, have led to tremendous attention of CNTs as a nanoscale material in the past two decades. To introduce the various types of CNTs, we first provide basic information on their structure followed by some intriguing properties and a brief overview of synthesis methods. Although impressive advances have been demonstrated with CNTs, critical applications require purification, positioning, and separation to yield desired properties and functional elements. Here, we review a versatile technique to manipulate CNTs based on their dielectric properties, namely dielectrophoresis (DEP). A detailed discussion on the DEP aspects of CNTs including the theory and various technical microfluidic realizations is provided. Various advancements in DEP-based manipulations of single-walled and multiwalled CNTs are also discussed with special emphasis on applications involving separation, purification, sensing, and nanofabrication., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Preterm birth (PTB) related health problems take over one million lives each year, and currently, no clinical analysis is available to determine if a fetus is at risk for PTB. Here, we describe the preparation of a key PTB risk biomarker, thrombin-antithrombin (TAT), and characterize it using dot blots, MS, and microchip electrophoresis (µCE). The pH for fluorescently labeling TAT was also optimized using spectrofluorometry and spectrophotometry. The LOD of TAT was measured in µCE. Lastly, TAT was combined with six other PTB risk biomarkers and separated in µCE. The ability to make and characterize TAT is an important step toward the development of an integrated microfluidic diagnostic for PTB risk., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) is an emerging field for structural biology. One of its major impacts lies in the ability to reveal the structure of complex proteins previously inaccessible with synchrotron-based crystallography techniques and allowing time-resolved studies from femtoseconds to seconds. The nature of this serial technique requires new approaches for crystallization, data analysis, and sample delivery. With continued advancements in microfabrication techniques, various developments have been reported in the past decade for innovative and efficient microfluidic sample delivery for crystallography experiments using XFELs. This article summarizes the recent developments in microfluidic sample delivery with liquid injection and fixed-target approaches, which allow exciting new research with XFELs. Graphical abstract.

Preterm birth (PTB) is defined as birth before the 37th week of pregnancy and results in 15 million early deliveries worldwide every year. Presently, there is no clinical test to determine PTB risk; however, a panel of nine biomarkers found in maternal blood serum has predictive power for a subsequent PTB. A significant step in creating a clinical diagnostic for PTB is designing an automated method to extract and purify these biomarkers from blood serum. Here, microfluidic devices with 45 μm × 50 μm cross-section channels were 3D printed with a built-in polymerization window to allow a glycidyl methacrylate monolith to be site-specifically polymerized within the channel. This monolith was then used as a solid support to attach antibodies for PTB biomarker extraction. Using these functionalized monoliths, it was possible to selectively extract a PTB biomarker, ferritin, from buffer and a human blood serum matrix. This is the first demonstration of monolith formation in a 3D printed microfluidic device for immunoaffinity extraction. Notably, this work is a crucial first step toward developing a 3D printed microfluidic clinical diagnostic for PTB risk.

Separations of bioanalytes require robust, effective, and selective migration phenomena. However, due to the complexity of biological matrices such as body fluids or tissue, these requirements are difficult to achieve. The separations field is thus constantly evolving to develop suitable methods to separate biomarkers and fractionate biospecimens for further interrogation of biomolecular content. Advances in the field of microfabrication allow the tailored generation of micro- and nanofluidic environments. These can be exploited to induce interactions and dynamics of biological species with the corresponding geometrical features, which in turn can be capitalized for novel separation approaches. This review provides an overview of several unique separation applications demonstrated in recent years in tailored micro- and nanofluidic environments. These include electrokinetic methods such as dielectrophoresis and electrophoresis, but also rather nonintuitive ratchet separation mechanisms, continuous flow separations, and fractionations such as deterministic lateral displacement, as well as methods employing entropic forces for separation.

Preterm birth (PTB) is responsible for over one million infant deaths annually worldwide. Often, the first and only indication of PTB risk is the onset of early labor. Thus, there is an urgent need for an early PTB risk diagnostic that is inexpensive, reliable, and robust. Here, we describe the development of a microchip electrophoresis (μCE) method for separating a mixture of six PTB protein and peptide biomarkers present in maternal blood serum. μCE devices were photografted with a poly(ethylene glycol) diacrylate surface coating to regulate EOF and reduce nonspecific analyte adsorption. Separation conditions including buffer pH, buffer concentration, and applied electric field were varied to improve biomarker peak resolution while minimizing deleterious effects like Joule heating. In this way, it was possible to separate six PTB biomarkers, the first μCE separation of this biomarker panel. LODs were also measured for each of the six PTB biomarkers. In the future, this μCE separation can be integrated with upstream maternal blood serum sample preparation steps to yield a complete PTB risk diagnosis microdevice., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (μCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a μCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined μCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and μCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

Biomarkers are often present in complex biological fluids like blood, requiring multiple, slow sample preparation steps that pose limitations in simplifying analysis. Here we report integrated immunoaffinity extraction and separation devices for analysis of preterm birth biomarkers in a human blood serum matrix. A reactive polymer monolith was used for immobilization of antibodies for selective extraction of target preterm birth biomarkers. Microfluidic immunoaffinity extraction protocols were optimized and then integrated with microchip electrophoresis for separation. Using these integrated devices, a ∼30 min analysis was carried out on low nanomolar concentrations of two preterm birth biomarkers spiked in a human serum matrix. This work is a promising step towards the development of an automated, integrated platform for determination of preterm birth risk.

Microfluidics is a vibrant and expanding field that has the potential for solving many analytical challenges. Microfluidics show promise to provide rapid, inexpensive, efficient, and portable diagnostic solutions that can be used in resource-limited settings. Researchers have recently reported various microfluidic platforms for biomarker analysis applications. Sample preparation processes like purification, preconcentration and labeling have been characterized on-chip. Additionally, improvements in microfluidic separation techniques have been reported for molecular biomarkers. This review critically evaluates microfluidic sample preparation platforms and separation methods for biomarker analysis reported in the last two years. Key advances in device operation and ability to process different sample matrices in a variety of device materials are highlighted. Finally, current needs and potential future directions for microfluidic device development to realize its full diagnostic potential are discussed., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low-abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH-mediated solid-phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed-phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH-mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50-fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15-fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

On-chip preconcentration, purification, and fluorescent labeling are desirable sample preparation steps to achieve complete automation in integrated microfluidic systems. In this work, we developed electrokinetically operated microfluidic devices for solid-phase extraction and fluorescent labeling of preterm birth (PTB) biomarkers. Reversed-phase monoliths based on different acrylate monomers were photopolymerized in cyclic olefin copolymer microdevices and studied for the selective retention and elution of a fluorescent dye and PTB biomarkers. Octyl methacrylate-based monoliths with desirable retention and elution characteristics were chosen and used for on-chip fluorescent labeling of three PTB biomarkers. Purification of on-chip labeled samples was done by selective elution of unreacted dye prior to sample. Automated and rapid on-chip fluorescent labeling was achieved with similar efficiency to that obtained for samples labeled off chip. Additionally, protocols for microchip electrophoresis of several off-chip-labeled PTB biomarkers were demonstrated in poly(methyl methacrylate) microfluidic devices. This study is an important step toward the development of integrated on-chip labeling and separation microfluidic devices for PTB biomarkers.

In recent years, the use of monolithic porous polymers has seen significant growth. These materials present a highly useful support for various analytical and biochemical applications. Since their introduction, various approaches have been introduced to produce monoliths in a broad range of materials. Simple preparation has enabled their easy implementation in microchannels, extending the range of applications where microfluidics can be successfully utilized. This review summarizes progress regarding monoliths and related porous materials in the field of microfluidics between 2010 and 2015. Recent developments in monolith preparation, solid-phase extraction, separations, and catalysis are critically discussed. Finally, a brief overview of the use of these porous materials for analysis of subcellular and larger structures is given.