Gravity-Oriented Microfluidic Device for Biocompatible End-to-End Fabrication of Cell-Laden Microgels.

Droplet microfluidics are extensively utilized to generate monodisperse cell-laden microgels in biomedical applications. However, maintaining cell viability is still challenging due to overexposure to harsh conditions in subsequent procedures that recover the microgels from the oil phase. Here, a gravity-oriented microfluidic device for end-to-end fabrication of cell-laden microgels is reported, which integrates dispersion, gelation, and extraction into a continuous workflow. This innovative on-chip extraction, driven by native buoyancy and kinetically facilitated by pseudosurfactant, exhibits 100% retrieval efficiency for microgels with a wide range of sizes and stiffnesses. The viability of encapsulated cells is perfectly maintained at ≈98% with minimal variations within and between batches. The end-to-end fabrication remarkably enhances the biocompatibility and practicality of microfluidics-based cell encapsulation and is promising to be compatible with various applications ranging from single-cell analysis to clinical therapy.

Paper-based microfluidic chip for rapid detection of SARS-CoV-2 N protein.

This research has developed a method for rapid detection of SARS-CoV-2 N protein on a paper-based microfluidic chip. The chitosan-glutaraldehyde cross-linking method is used to fix the coated antibody, and the sandwich enzyme-linked immunosorbent method is used to achieve the specific detection of the target antigen. The system studied the influence of coating antibody concentration and enzyme-labeled antibody concentration on target antigen detection. According to the average gray value measured under different N protein concentrations, the standard curve of the method was established and the sensitivity was tested, and its linear regression was obtained. The equation is y = 9.8286x+137.6, R2 = 0.9772 > 0.90, which shows a high degree of fit. When the concentration of coating antibody and enzyme-labeled antibody were 1 µg/mL and 2 µg/mL, P > 0.05, the difference was not statistically significant, so the lower concentration of 1 µg/mL was chosen as the coating antibody concentration. The results show that the minimum concentration of N protein that can be detected by this method is 8 µg/mL, and the minimum concentration of coating antibody and enzyme-labeled antibody is 1 µg/mL, which has the characteristics of high sensitivity and good repeatability.

A millisecond passive micromixer with low flow rate, low sample consumption and easy fabrication.

Fast mixing of small volumes of solutions in microfluidic devices is essential for an accurate control and observation of the dynamics of a reaction in biological or chemical studies. It is often, however, a challenging task, as the Reynolds number (Re) in microscopic devices is typically < 100. In this report, we detail a novel mixer based on the "staggered herring bone" (SHB) pattern and "split-recombination" strategies with an optimized geometry, the periodic rotation of the flow structure can be controlled and recombined in a way that the vortices and phase shifts of the flow induce intertwined lamellar structures, thus increasing the contact surface and enhancing mixing. The optimization improves the mixing while using a low flow rate, hence a small volume for mixing and moderate pressure drops. The performances of the patterns were first simulated using COMSOL Multiphysics under different operating conditions. The simulation indicates that at very low flow rate (1-12 µL·min-1) and Re (3.3-40), as well as a very small working volume (~ 3 nL), a very good mixing (~ 98%) can be achieved in the ms time range (4.5-78 ms). The most promising design was then visualized experimentally, showing results that are consistent with the outcomes of the simulations. Importantly, the devices were fabricated using a classical soft-lithography method, as opposed to additive manufacturing often used to generate complex mixing structures. This new device minimizes the sample consumption and could therefore be applied for studies using precious samples.

Putting Science into Standards workshop on standards for organ-on-chip.

The European Commission Joint Research Centre and the European Standardization Organizations CEN and CENELEC organized the "Putting Science into Standards" workshop, focusing on organ-on-chip technologies. The workshop, held online on 28-29 April, 2021, aimed at identifying needs and priorities for standards development and suggesting possible ways forward.

Designing of various biosensor devices for determination of apoptosis: A comprehensive review.

Apoptosis is a type of cell death caused by the occurrence of both pathological and physiological conditions triggered by ligation of death receptors outside the cell or triggered by DNA damage and/or cytoskeleton disruption. Timely monitoring of apoptosis can effectively help early diagnosis of related diseases and continuous assessment of the effectiveness of drugs. Detecting caspases, a protease family closely related to cellular apoptosis, and its identification as markers of apoptosis is a popular procedure. Biosensors are used for early diagnosis and play a very important role in preventing disease progression in various body sections. Recently, there has been a widespread increase in the desire to use materials made of paper (e.g. nitrocellulose membrane) for Point-of-Care (POC) testing systems since paper and paper-like materials are cheap, abundant and degradable. Microfluidic paper-based analytical devices (µPADs) are highly promising as they are cost-effective, easy to use, fast, precise and sustainable over time and under different environmental conditions. In this review, we focused our efforts on compiling the different approaches on identifying apoptosis pathway while giving brief information about apoptosis and biosensors. This review includes recent advantages in biosensing techniques to simply determine what happened in the cell life and which direction it would continue. As a conclusion, we believed that the review may help to researchers to compare/update the knowledge about diagnosis of the apoptosis pathway while reminding the basic definitions about the apoptosis and biosensor technologies.

The Use of Total Thrombus Formation Analysis System as a Tool to Assess Platelet Function in Bleeding and Thrombosis Risk-A Systematic Review.

BACKGROUND: Today there are many devices that can be used to study blood clotting disorders by identifying abnormalities in blood platelets. The Total Thrombus Formation Analysis System is an automated microchip flow chamber system that is used for the quantitative analysis of clot formation under blood flow conditions. For several years, researchers have been using a tool to analyse various clinical situations of patients to identify the properties and biochemical processes occurring within platelets and their microenvironment. METHODS: An investigation of recent published literature was conducted based on PRISMA. This review includes 52 science papers directly related to the use of the Total Clot Formation Analysis System in relation to bleeding, surgery, platelet function assessment, anticoagulation monitoring, von Willebrand factor and others. CONCLUSION: Most available studies indicate that The Total Thrombus Formation Analysis System may be useful in diagnostic issues, with devices used to monitor therapy or as a significant tool for predicting bleeding events. However, T-TAS not that has the potential for diagnostic indications, but allows the direct observation of the flow and the interactions between blood cells, including the intensity and dynamics of clot formation. The device is expected to be of significant value for basic research to observe the interactions and changes within platelets and their microenvironment.

Prostate cancer and microfluids.

Microfluidic systems aim to detect sample matter quickly with high sensitivity and resolution, on a small scale. With its increased use in medicine, the field is showing significant promise in prostate cancer diagnosis and management due, in part, to its ability to offer point-of-care testing. This review highlights some of the research that has been undertaken in respect of prostate cancer and microfluidics. Firstly, this review considers the diagnosis of prostate cancer through use of microfluidic systems and analyses the detection of prostate specific antigen, proteins, and circulating tumor cells to highlight the scope of current advancements. Secondly, this review analyses progressions in the understanding of prostate cancer physiology and considers techniques used to aid treatment of prostate cancer, such as the creation of a micro-environment. Finally, this review highlights potential future roles of microfluidics in assisting prostate cancer, such as in exosomal analysis. In conclusion, this review shows the vast scope and application of microfluidic systems and how these systems will ensure advancements to future prostate cancer management.

3D Collagen Vascular Tumor-on-a-Chip Mimetics for Dynamic Combinatorial Drug Screening.

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

Flow induces barrier and glycocalyx-related genes and negative surface charge in a lab-on-a-chip human blood-brain barrier model.

Microfluidic lab-on-a-chip (LOC) devices allow the study of blood-brain barrier (BBB) properties in dynamic conditions. We studied a BBB model, consisting of human endothelial cells derived from hematopoietic stem cells in co-culture with brain pericytes, in an LOC device to study fluid flow in the regulation of endothelial, BBB and glycocalyx-related genes and surface charge. The highly negatively charged endothelial surface glycocalyx functions as mechano-sensor detecting shear forces generated by blood flow on the luminal side of brain endothelial cells and contributes to the physical barrier of the BBB. Despite the importance of glycocalyx in the regulation of BBB permeability in physiological conditions and in diseases, the underlying mechanisms remained unclear. The MACE-seq gene expression profiling analysis showed differentially expressed endothelial, BBB and glycocalyx core protein genes after fluid flow, as well as enriched pathways for the extracellular matrix molecules. We observed increased barrier properties, a higher intensity glycocalyx staining and a more negative surface charge of human brain-like endothelial cells (BLECs) in dynamic conditions. Our work is the first study to provide data on BBB properties and glycocalyx of BLECs in an LOC device under dynamic conditions and confirms the importance of fluid flow for BBB culture models.

NemaLife chip: a micropillar-based microfluidic culture device optimized for aging studies in crawling C. elegans.

In this study, we report a microfluidic device for the whole-life culture of the nematode Caenorhabditis elegans that allows the scoring of animal survival and health measures. This device referred to as the NemaLife chip features: (1) an optimized micropillar arena in which animals can crawl, (2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and (3) ports that allow rapid accessibility for feeding the adult-only population and introducing reagents as needed. The pillar arena geometry was optimized to accommodate the growing body size during culture and emulate the body gait and locomotion of animals reared on agar. Likewise, feeding protocols were optimized to recapitulate longevity outcomes typical of standard plate growth. Key benefits of the NemaLife Chip include eliminating the need to perform repeated manual transfers of adults during survival assays, negating the need for progeny-blocking chemical interventions, and avoiding the swim-induced stress across lifespan in animals reared in liquid. We also show that the culture of animals in pillar-less microfluidic chambers reduces lifespan and introduces physiological stress by increasing the occurrence of age-related vulval integrity disorder. We validated our pillar-based device with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan in the NemaLife chip. Overall, the capacity to generate reliable lifespan and physiological data underscores the potential of the NemaLife chip to accelerate healthspan and lifespan investigations in C. elegans.

Lab-on-a-Chip Zika Detection With Reverse Transcription Loop-Mediated Isothermal Amplification-Based Assay for Point-of-Care Settings.

CONTEXT.­: Zika virus (ZIKV) infection, primarily transmitted by mosquitoes, causes various neurologic disorders. To differentiate ZIKV from other arboviruses, such as dengue, chikungunya, and yellow fever viruses, a highly specific, sensitive, and automated detection system is needed for point-of-care (POC) settings. OBJECTIVE.­: To detect ZIKV at POC settings, we have developed a fully automated lab-on-a-chip microfluidic platform for rapid disease detection by using reverse transcription loop-mediated isothermal amplification. DESIGN.­: The developed setup consists of a microfluidic chip, a platform for magnetic actuation, and a heater along with the sensor to precisely control the temperature for the target amplification. The platform accurately controls the movement of the magnetic beads that enable the isolation and purification of the target nucleotides adhered to their surface for the amplification and disease detection on the microfluidic chip. RESULTS.­: Within 40 minutes, change in color due to the presence of ZIKV amplicons was visually observed with the spiked plasma samples in the end point analysis. Also, we have accurately and specifically identified ZIKV in a small number of de-identified clinical samples. CONCLUSIONS.­: All-inclusive, the developed fully automated POC ZIKV diagnostic chip is rapid, simple, easy to use, inexpensive, and suitable for the areas where facilities are limited.

Microfluidic endothelium-on-a-chip development, from in vivo to in vitro experimental models.

In the last years, animal testing in medical research has been a controversial topic because of various reasons, such as ethical considerations and species differences. Therefore, more attention has been given to develop new technologies that can replace animal experiments and create in vitro models. Organ-on-a-chip (OOC) technology is a new and advanced technology based on microfluidic devices that can mimic the structure and function of entire organs and tissues as in vitro models. OOC models are miniature tissues and organs that assign characteristics for three-dimensional (3D) cell culture representation that resemble the original organs, together with their specific microenvironment microfluidic systems and specific biophysical processes, in order to mimic the normal physiological conditions and functionalities of the organs. Existing OOC models, such as liver, pancreas, heart, skin, brain, kidney, vessels, have been developed and designed for a specific function study. This review focuses on the main knowledge concerning OOC research and especially vascular endothelium-on-a-chip (EOC) model, developed in order to offer specific tools for studying vascular functions in physiological and pathological conditions. The field of OOC devices is still at the beginning, but in the future, this technology may have important roles in developing novel therapeutic approaches, offering new therapeutic molecules and providing the first step towards personalized medicine.

Labchip-based diagnosis system for on-site application: Sensitive and easy-to-implement detection of single recoverable Cronobacter in infant formula without post-enrichment treatment.

Microfluidic labchips have achieved much advancement in the molecular diagnosis of foodborne pathogens. Whereas difficulties in the flow control during the transportation of liquid fluids can occur and should be overcome. Manipulations of reaction temperature and the complex procedures from sample pre-treatment to analysis in a single chip device are major obstacles for the on-site application. Thus, the efficient temperature control of samples without any flow of reaction fluids in microfluidic channels of plastic chip and the simplest protocol omitting post-enrichment processing steps may overcome these limitations represented by the stability and the complexity, respectively. This study aims to develop a novel type of labchip and thermocycler specialized for the gene amplification in microfluidic channels and to evaluate the detectability by sensing the minimum recoverable level of Cronobacter in powdered infant formula (PIF). We developed a thermocycling device accelerating reactions through dual heating-blocks optimized to control temperatures of samples in microfluidic-channels by direct contact with labchip sequentially and repetitively. The structural design of microfluidic channels was to eliminate interference factors associated with the optical detection of fluorescent signals (without distortion due to air bubbles in the reaction chamber). To improve the applicability, a portable device and simplified operation to allow direct loading of samples in the chip without post-enrichment procedures were also adopted. Detection performance was evaluated by a sensitivity/specificity tests using 50 isolates of Cronobacter. Cross-reactivity tests for non-Cronobacter organisms and gDNA [human, raw materials of PIF (cow, soybean)] showed that there was no interference-factor causing false-positive results. In terms of the applied research conducted by using PIF, the enrichment of samples without broth medium (distilled water) displayed outstanding performance and 12 h of incubation facilitated detecting target at concentration as low as 1 CFU/300 g PIF (as initial contamination level) without post-enrichment treatment. Validation of the operation conditions using 30 commercial PIF products was also consistent. The present study presents a novel approach of microfluidic technology with perspective to not only the performance and the practicability [easy-to-implement protocol, portable materials, cost-effectiveness (the use of a miniaturized plastic chip requires a minimum level of materials)] for on-site diagnosis.

Development of a flow standard to enable highly reproducible measurements of deformability of stored red blood cells in a microfluidic device.

BACKGROUND: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices. STUDY DESIGN AND METHODS: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C. RESULTS: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average. CONCLUSIONS: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method.

Leukocyte adhesion to P-selectin and the inhibitory role of Crizanlizumab in sickle cell disease: A standardized microfluidic assessment.

Upregulated expression of P-selectin on activated endothelium and platelets significantly contributes to the initiation and progression of vaso-occlusive crises (VOC), a major cause of morbidity in sickle cell disease (SCD). Crizanlizumab (ADAKVEO®), a humanized monoclonal antibody against P-selectin, primarily inhibits the interaction between leukocytes and P-selectin, and has been shown to decrease the frequency of VOCs in clinical trials. However, the lack of reliable in vitro assays that objectively measure leukocyte adhesion to P-selectin remains a critical barrier to evaluating and improving the therapeutic treatment in SCD. Here, we present a standardized microfluidic BioChip whole blood adhesion assay to assess leukocyte adhesion to P-selectin under physiologic flow conditions. Our results demonstrated heterogeneous adhesion by leukocytes to immobilized P-selectin, and dose-dependent inhibition of this adhesion following pre-exposure to Crizanlizumab. Importantly, treatment with Crizanlizumab following adhesion to P-selectin promoted detachment of rolling, but not of firmly adherent leukocytes. Taken together, our results suggest that the microfluidic BioChip system is a promising in vitro assay with which to screen patients, monitor treatment response, and guide current and emerging anti-adhesive therapies in SCD.

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior.

We demonstrate a method for the generation of controlled, dynamic chemical pulses-where localized chemoattractant becomes suddenly available at the microscale-to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert ('caged') by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

Open-source, 3D-printed Peristaltic Pumps for Small Volume Point-of-Care Liquid Handling.

Microfluidic technologies are frequently employed as point-of-care diagnostic tools for improving time-to-diagnosis and improving patient outcomes in clinical settings. These microfluidic devices often are designed to operate with peripheral equipment for liquid handling that increases the cost and complexity of these systems and reduces their potential for widespread adoption in low resource healthcare applications. Here, we present a low-cost (~$120), open-source peristaltic pump constructed with a combination of three dimensional (3D)-printed parts and common hardware, which is amenable to deployment with microfluidic devices for point-of-care diagnostics. This pump accepts commonly available silicone rubber tubing in a range of sizes from 1.5 to 3 mm, and is capable of producing flow rates up to 1.6 mL min-1. This device is programmed with an Arduino microcontroller, allowing for custom flow profiles to fit a wide range of low volume liquid handling applications including precision liquid aliquoting, flow control within microfluidics, and generation of physiologically relevant forces for studying cellular mechanobiology within microfluidic systems.

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips.

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

Lyophilization of chemiluminescent substrate reagents for high-sensitive microchannel-based lateral flow assay (MLFA) in point-of-care (POC) diagnostic system.

Over the last few years, lateral flow assay (LFA) devices have grown to be the most common point-of-care test (POCT) platform facilitating disease diagnostics in low-resource environments. However, the lack of consistency and the limited sensitivity of these devices often lead to misdiagnosis and generates the need for an alternate approach. A chemiluminescence based microchannel-based lateral flow assay (MLFA) in a POCT platform can result in a much higher sensitivity but involves multiple additional steps of liquid reagents for the sequential execution of the signal amplification protocol. One of the best ways to develop a sample-to-answer system with minimum user intervention is to dry reagents on a chip prior to sample addition and to control the flow of the biological fluid through the drying chambers resulting in the reconstitution of the reagents. This work reports the methods for the successful lyophilization of the chemiluminescent substrate and its reconstitution in artificial serum without any significant loss of functionality. The lyophilized reagents were reconstituted and incorporated into the reaction chambers of a designed polymer lab-on-a-chip to implement a sandwich assay for the detection of malarial biomarkers. The results report a limit of detection (LOD) of 5.75 ng mL-1 which is sensitive enough to detect active malarial infection. Successful lyophilization and reconstitution of the chemiluminescent substrate, as reported here, can pave the way towards developing an autonomous POCT system implementing chemiluminescence based sandwich ELISA for enhanced sensitivity, portability, and ease-of-use in resource limited settings.

A Microfluidic Device for Simultaneous Extraction of Plasma, Red Blood Cells, and On-Chip White Blood Cell Trapping.

This study reports a microfluidic device for whole blood processing. The device uses the bifurcation law, cross-flow method, and hydrodynamic flow for simultaneous extraction of plasma, red blood cells, and on-chip white blood cell trapping. The results demonstrate successful plasma and red blood cell collection with a minimum dilution factor (0.76x) and low haemolysis effect. The extracted red blood cells can also be applied for blood type tests. Moreover, the device can trap up to ~1,800 white blood cells in 20 minutes. The three components can be collected simultaneously using only 6 µL of whole blood without any sample preparation processes. Based on these features, the microfluidic device enables low-cost, rapid, and efficient whole blood processing functionality that could potentially be applied for blood analysis in resource-limited environments or point-of-care settings.