A cost-effective and facile technique for realizing fabric based microfluidic channels using beeswax and PVC stencils.

Microfluidic channels fabricated over fabrics or papers have the potential to find substantial application in the next generation of wearable healthcare monitoring systems. The present work focuses on the fabrication procedures that can be used to obtain practically realizable fabric-based microfluidic channels (µFADs) utilizing patterning masks and wax, unlike conventional printing techniques. In this study, comparative analysis was used to differentiate channels obtained using different masking tools for channel patterning as well as different wax materials as hydrophobic barriers. Drawbacks of the conventional tape and candle wax technique were noted and a novel approach was used to create microfluidic channels through a facile and simple masking technique using PVC clear sheets as channel stencils and beeswax as the channel barriers. The resulting fabric based microfluidic channels with varying widths as well as complex microchannel, microwell, and micromixer designs were investigated and a minimum channel width resolution of 500 µm was successfully obtained over cotton based fabrics. Thereafter, the PVC clear sheet-beeswax based microwells were successfully tested to confine various organic and inorganic samples indicating vivid applicability of the technique. Finally, the microwells were used to make a simple and facile colorimetric assay for glucose detection and demonstrated effective detection of glucose levels from 10 mM to 50 mM with significant color variation using potassium iodide as the coloring agent. The above findings clearly suggest the potential of this alternative technique for making low-cost and practically realizable fabric based diagnostic devices (µFADs) in contrast to the other approaches that are currently in use.

High-throughput microbead assay system with a portable, cost-effective Wi-Fi imaging module, and disposable multi-layered microfluidic cartridges for virus and microparticle detection, and tracking.

In recent years biomedical scientific community has been working towards the development of high-throughput devices that allow a reliable, rapid and parallel detection of several strains of virus or microparticles simultaneously. One of the complexities of this problem lies on the rapid prototyping of new devices and wireless rapid detection of small particles and virus alike. By reducing the complexity of microfluidics microfabrication and using economic materials along with makerspace tools (Kundu et al. 2018) it is possible to provide an affordable solution to both the problems of high-throughput devices and detection technologies. We present the development of a wireless, standalone device and disposable microfluidics chips that rapidly generate parallel readouts for selected, possible virus variants from a nasal or saliva sample, based on motorized and non-motorized microbeads detection, and imaging processing of the motion tracks of these beads in micrometers. Microbeads and SARS-CoV-2 COVID-19 Delta variant were tested as proof-of-concept for testing the microfluidic cartridges and wireless imaging module. The Microbead Assay (MA) system kit consists of a Wi-Fi readout module, a microfluidic chip, and a sample collection/processing sub-system. Here, we focus on the fabrication and characterization of the microfluidic chip to multiplex various micrometer-sized beads for economic, disposable, and simultaneous detection of up to six different viruses, microparticles or variants in a single test, and data collection using a commercially available, Wi-Fi-capable, and camera integrated device (Fig. 1).

A low-cost self-dispersing method of droplet array generation enabled by a simple reusable mask for bioanalysis and bioassays.

Discontinuous dewetting is an attractive technique that can produce droplet array of specific volume, geometry and at predefined location on a substrate. Droplet array has great potential in bioanalysis such as high-throughput live cell screening, digital PCR, and drug candidates. Here, we propose a self-dispersing droplet array generation method, which has advantages of low cost, simple operation, and easy large-area production ability. Droplet array of specific volumes was generated on a polymethyl methacrylate (PMMA) substrate using a simple reusable polyimide (PI) adhesive mask. Experiment shows that the generated droplet array can be used to successfully capture single particles which obeys Poisson distribution in a high-throughput manner. Furthermore, a droplet-array sandwiching chip was created based on the self-dispersion method for rapid detection of human serum albumin (HSA) at wide range of 183-11,712 µg/mL with low reagent consumption of 2.2 µL, demonstrating its potential applications in convenient high-throughput bioanalysis and bioassays.

Emerging design strategies for constructing multiplex lateral flow test strip sensors.

Conventional lateral flow test strip (LFTS) sensors are insufficiently accurate and reliable due to their single-target detection with limited sample information in a single test. The increasing demand for the simultaneous determination of multiple analytes has recently been accelerating the rapid development of high-throughput and multiplexed LFTS sensing technologies. In this contribution, we systematically summarize the recent achievements on the design, development, and application of multiplexed LFTS sensors for improved rapid on-site diagnostics. The discussion focuses on emerging design strategies to increase multiplexing capacity for enhancing analytical efficiency and precision. As a proof-of-concept, several typical examples are presented. The advantages and disadvantages of such approaches are critically analyzed. Finally, we briefly discuss the current challenges and future perspectives.

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.

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Strong rotating flow in stationary droplets in low power budget using wire electrode configuration.

We explore a simple strategy of generating strong rotating flow in a stationary surface-droplet, using an intricate interplay of local electrical and thermal fields. Wire electrodes are employed to generate on-spot heating without necessitating any elaborate micro-fabrication, which causes strong local gradients in electrical properties to induce mobile charges into the droplet. Applying a low voltage (∼10 V), strong rotational velocity of the order of mm/s can be achieved in the system, within the standard operating ranges of operating and geometrical parameters. Further, altering the diameter of the electrode, vortices can be tuned locally or globally in low power budget, without incurring any droplet oscillations. These results may turn out to be of immense consequence in enhancing micromixing in a plethora of droplet based applications ranging from thermal management to medical diagnostics to be potentially employed in resource-limited settings.

Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing.

In the face of looming threats from multi-drug resistant microorganisms, there is a growing need for technologies that will enable rapid identification and drug susceptibility profiling of these pathogens in health care settings. In particular, recent progress in microfluidics and nucleic acid amplification is pushing the boundaries of timescale for diagnosing bacterial infections. With a diverse range of techniques and parallel developments in the field of analytical chemistry, an integrative perspective is needed to understand the significance of these developments. This review examines the scope of new developments in assay technologies grouped by key enabling domains of research. First, we examine recent development in nucleic acid amplification assays for rapid identification and drug susceptibility testing in bacterial infections. Next, we examine advances in microfluidics that facilitate acceleration of diagnostic assays via integration and scale. Lastly, recentdevelopments in biosensor technologies are reviewed. We conclude this review with perspectives on the use of emerging concepts to develop paradigm-changing assays.

Signal amplification strategies for paper-based analytical devices.

Paper-based analytical devices (PADs) are very popular for point-of-care diagnostics, which provide a fast, cost-effective and possible multiplexed detection of a spectrum of molecules. They have been matching forward proudly to contribute to the modern analytical science and life science. Accompanying with their advantages and huge potentials, low detection sensitivity is continuing to challenge the application of PADs from bench to bedside. In order to improve the sensitivity and enhance the signal readout, variable signal amplification strategies have been investigated and applied for PADs. In this review, we have firstly classified formats of PADs according to the engineering design. Advances for improving sensitivity of PADs in recent five years are then summarised according to three popular types of signal amplification strategies (nanomaterial based, nucleic acid based, and engineering of PADs based). Pros and cons of each signal amplification approach have been discussed accordingly. Finally, the future perspectives of PADs are proposed.

Recent advances in thread-based microfluidics for diagnostic applications.

Over the past decades, researchers have been seeking attractive substrate materials to keep microfluidics improving to outbalance the drawbacks and issues. Cellulose substrates, including thread, paper and hydrogels are alternatives due to their distinct structural and mechanical properties for a number of applications. Thread have gained considerable attention and become promising powerful tool due to its advantages over paper-based systems thus finds numerous applications in the development of diagnostic systems, smart bandages and tissue engineering. To the best of our knowledge, no comprehensive review articles on the topic of thread-based microfluidics have been published and it is of significance for many scientific communities working on Microfluidics, Biosensors and Lab-on-Chip. This review gives an overview of the advances of thread-based microfluidic diagnostic devices in a variety of applications. It begins with an overall introduction of the fabrication followed by an in-depth review on the detection techniques in such devices and various applications with respect to effort and performance to date. A few perspective directions of thread-based microfluidics in its development are also discussed. Thread-based microfluidics are still at an early development stage and further improvements in terms of fabrication, analytical strategies, and function to become low-cost, low-volume and easy-to-use point-of-care (POC) diagnostic devices that can be adapted or commercialized for real world applications.

Microfluidic assemblies designed for assessment of drug effects on deformability of human erythrocytes.

Extreme deformability of human erythrocytes is a prerequisite for their ability to squeeze through narrow capillaries of the blood microcirculation system. Various drugs can modify this deformability and consequently provoke circulation problems. We demonstrate that microfluidic assemblies are very convenient platforms for in vitro study of the associated processes. Two types of microfluidic channels were designed to quantitatively investigate modifications of erythrocyte deformability induced by hydrogen peroxide, ethanol and pentoxifylline based on transit velocity measurements. With a high sensitivity our microfluidic assemblies show that hydrogen peroxide decreases erythrocyte deformability in a dose-dependent manner. Then, results on ethanol resolve a biphasic nature of this reactant on the deformability of single erythrocyte cells. Results on pentoxifylline provide evidence that, similar to ethanol, also this medical drug has a double-sided effect on the erythrocyte deformability, i.e. increasing the deformability at low concentrations, while decreasing it at higher ones. Taken together, our microfluidic designs propose a potent measurement method for the erythrocyte deformability, as well as providing a perspective to evaluate effects of drugs on it.

Computer-aided design of microfluidic resistive network using circuit partition and CFD-based optimization and application in microalgae assessment for marine ecological toxicity.

We present an automatic design process for microfluidic dilution network towards marine ecological toxicity assessment on microalgae. Based on the hydraulic-electric circuit analogy, we defined an abstract specification using computer-aided designing system. Several approaches, especially circuit partition, were applied to minimize design effort. Computational fluid dynamics (CFD) simulation was exploited to convert the electrics specification to fabrication model. We automatically designed the combinational-mixing-serial dilution microfluidics to generate parallel stepwise gradients for mixing chemicals (binary/ternary/quaternary mixture) using the present algorithm. We critically discussed design rules and evaluated the microfluidic performance by colorimetric analysis. To examine whether these microfluidic chips can be used for toxicity test on microalgae, single and joint toxic effects of heavy metals (copper, mercury, zinc, and cadmium) were examined on line. In all cases, dose-related toxic responses were successfully detected. These results provided a solution for designing resistive network using circuit partition and CFD-based optimization and a route to develop a promising user-friendly alternative for microalgae bioassays as well as cell-based screening experiments in risk assessment.

High-content, cell-by-cell assessment of HER2 overexpression and amplification: a tool for intratumoral heterogeneity detection in breast cancer.

Immunohistochemistry and fluorescence in situ hybridization are the two standard methods for human epidermal growth factor receptor 2 (HER2) assessment. However, they have severe limitations to assess quantitatively intratumoral heterogeneity (ITH) when multiple subclones of tumor cells co-exist. We develop here a high-content, quantitative analysis of breast cancer tissues based on microfluidic experimentation and image processing, to characterize both HER2 protein overexpression and HER2 gene amplification at the cellular level. The technique consists of performing sequential steps on the same tissue slide: an immunofluorescence (IF) assay using a microfluidic protocol, an elution step for removing the IF staining agents, a standard FISH staining protocol, followed by automated quantitative cell-by-cell image processing. Moreover, ITH is accurately detected in both cluster and mosaic form using an analysis of spatial association and a mathematical model that allows discriminating true heterogeneity from artifacts due to the use of thin tissue sections. This study paves the way to evaluate ITH with high accuracy and content while requiring standard staining methods.

Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests.

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges ('chips') that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed.

Rapid detection and subtyping of multiple influenza viruses on a microfluidic chip integrated with controllable micro-magnetic field.

Influenza viruses have threatened animals and public health systems continuously. Moreover, there are many subtypes of influenza viruses, which have brought great difficulties to the classification of influenza viruses during any influenza outbreak. So it is crucial to develop a rapid and accurate method for detecting and subtyping influenza viruses. In this work, we reported a rapid method for simultaneously detecting and subtyping multiple influenza viruses (H1N1, H3N2 and H9N2) based on nucleic acid hybridization on a microfluidic chip integrated with controllable micro-magnetic field. H1N1, H3N2 and H9N2 could be simultaneously detected in 80min with detection limits about 0.21nM, 0.16nM, 0.12nM in order. Moreover, the sample and reagent consumption was as low as only 3µL. The results indicated that this approach possessed fast analysis and high specificity. Therefore, it is expected to be used to simultaneously subtype and detect multiple targets, and may provide a powerful technique platform for the rapid detection and subtyping analysis of influenza viruses.

High-Speed Melting Analysis: The Effect of Melting Rate on Small Amplicon Microfluidic Genotyping.

BACKGROUND: High-resolution DNA melting analysis of small amplicons is a simple and inexpensive technique for genotyping. Microfluidics allows precise and rapid control of temperature during melting. METHODS: Using a microfluidic platform for serial PCR and melting analysis, 4 targets containing single nucleotide variants were amplified and then melted at different rates over a 250-fold range from 0.13 to 32 °C/s. Genotypes (n = 1728) were determined manually by visual inspection after background removal, normalization, and conversion to negative derivative plots. Differences between genotypes were quantified by a genotype discrimination ratio on the basis of inter- and intragenotype differences using the absolute value of the maximum vertical difference between curves as a metric. RESULTS: Different homozygous curves were genotyped by melting temperature and heterozygous curves were identified by shape. Technical artifacts preventing analysis (0.3%), incorrect (0.06%), and indeterminate (0.4%) results were minimal, occurring mostly at slow melting rates (0.13-0.5 °C/s). Genotype discrimination was maximal at around 8 °C/s (2-8 °C/s for homozygotes and 8-16 °C/s for heterozygotes), and no genotyping errors were made at rates >0.5 °C/s. PCR was completed in 10-12.2 min, followed by melting curve acquisition in 4 min down to <1 s. CONCLUSIONS: Microfluidics enables genotyping by melting analysis at rates up to 32 °C/s, requiring <1 s to acquire an entire melting curve. High-speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons. Combined with extreme PCR, high-speed melting promises nucleic acid amplification and genotyping in < 1 min.

Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes.

In vitro models that better reflect in vivo epithelial barrier (patho-)physiology are urgently required to predict adverse drug effects. Here we introduce extracellular matrix-supported intestinal tubules in perfused microfluidic devices, exhibiting tissue polarization and transporter expression. Forty leak-tight tubules are cultured in parallel on a single plate and their response to pharmacological stimuli is recorded over 125 h using automated imaging techniques. A study comprising 357 gut tubes is performed, of which 93% are leak tight before exposure. EC50-time curves could be extracted that provide insight into both concentration and exposure time response. Full compatibility with standard equipment and user-friendly operation make this Organ-on-a-Chip platform readily applicable in routine laboratories.Efforts to determine the effects of drugs on epithelial barriers could benefit from better in vitro models. Here the authors develop a microfluidic device supporting the growth and function of extracellular matrix-supported intestinal tubules, and evaluate the effect of staurosporine and acetylsalicylic acid on barrier integrity.

Fast, Sensitive, and Quantitative Point-of-Care Platform for the Assessment of Drugs of Abuse in Urine, Serum, and Whole Blood.

Drug abuse is a major public health problem in many countries in Europe and North America. Currently available platforms for drug abuse assessment are facing technical challenges of nonquantitation, inaccuracy, low throughput, incompatibility with diverse complex specimens, long assay times, and requirement of instrument and/or expertise for readout. Here, we report an integrated competitive volumetric-bar-chart chip (CV-Chip) to assay multiple drug targets at the point-of-care (POC). To the best of our knowledge, it is the first time that a POC platform has been demonstrated to fully address the above-mentioned limitations. We applied this integrated CV-chip platform to assay multiple drugs in 38 patient urine and serum samples and validated the on-chip results with an LC-MS/MS method, indicating a clinical sensitivity and specificity of 0.94 and 1.00, respectively. We further demonstrated that the combination of an on-chip blood separator with the CV-Chip enabled the platform to directly assay finger-prick whole blood samples, which have always been recognized as an ideal biospecimen for POC detections. In summary, this integrated CV-Chip is able to serve as a sensitive, accurate, fast, portable, readout visible, and minimally invasive platform for drug abuse assessment.