A Low-Cost Handheld Centrifugal Microfluidic System for Multiplexed Visual Detection Based on Isothermal Amplification.

A low-cost, handheld centrifugal microfluidic system for multiplexed visual detection based on recombinase polymerase amplification (RPA) was developed. A concise centrifugal microfluidic chip featuring four reaction units was developed to run multiplexed RPA amplification in parallel. Additionally, a significantly shrunk-size and cost-effective handheld companion device was developed, incorporating heating, optical, rotation, and sensing modules, to perform multiplexed amplification and visual detection. After one-time sample loading, the metered sample was equally distributed into four separate reactors with high-speed centrifugation. Non-contact heating was adopted for isothermal amplification. A tiny DC motor on top of the chip was used to drive steel beads inside reactors for active mixing. Another small DC motor, which was controlled by an elaborate locking strategy based on magnetic sensing, was adopted for centrifugation and positioning. Visual fluorescence detection was optimized from different sides, including material, surface properties, excitation light, and optical filters. With fluorescence intensity-based visual detection, the detection results could be directly observed through the eyes or with a smartphone. As a proof of concept, the handheld device could detect multiple targets, e.g., different genes of African swine fever virus (ASFV) with the comparable LOD (limit of detection) of 75 copies/test compared to the tube-based RPA.

A high-throughput microfluidic nanoimmunoassay for detecting anti-SARS-CoV-2 antibodies in serum or ultralow-volume blood samples.

Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti-SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 µL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general.

Triple Emulsion-Based Rapid Microfluidic Production of Core-Shell Hydrogel Microspheres for Programmable Biomolecular Conjugation.

We report a simple and rapid microfluidic approach to produce core-shell hydrogel microspheres in a single step. We exploit triple emulsion drops with sacrificial oil layers that separate two prepolymer phases, forming poly(ethylene glycol)-based core-shell microspheres via photopolymerization followed by spontaneous removal of the oil layer. Our technique enables the production of monodisperse core-shell microspheres with varying dimensions of each compartment by independently and precisely controlled flow rates. This leads to stable and uniform incorporation of functional moieties in the core compartment with negligible cross-contamination into the shell layer. Selective conjugation of biomolecules is enabled through a rapid bioorthogonal reaction with functional groups in the core compartment with minimal non-specific adsorption. Finally, in-depth protein conjugation kinetics studies using microspheres with varying shell porosities highlight the capability to provide tunable size-selective diffusion barriers by simple tuning of prepolymer compositions for the shell layer. Combined, these results illustrate a significant step forward for programmable high-throughput fabrication of multifunctional hydrogel microspheres, which possess substantial potential in a large array of biomedical and biochemical applications.

Rapid and quantitative detection of SARS-CoV-2 specific IgG for convalescent serum evaluation.

Convalescent serum with a high abundance of neutralization IgG is a promising therapeutic agent for rescuing COVID-19 patients in the critical stage. Knowing the concentration of SARS-CoV-2 S1-specific IgG is crucial in selecting appropriate convalescent serum donors. Here, we present a portable microfluidic ELISA technology for rapid (15 min), quantitative, and sensitive detection of anti-SARS-CoV-2 S1 IgG in human serum with only 8 µL sample volume. We first identified a humanized monoclonal IgG that has a high binding affinity and a relatively high specificity towards SARS-CoV-2 S1 protein, which can subsequently serve as the calibration standard of anti-SARS-CoV-2 S1 IgG in serological analyses. We then measured the abundance of anti-SARS-CoV-2 S1 IgG in 16 convalescent COVID-19 patients. Due to the availability of the calibration standard and the large dynamic range of our assay, we were able to identify "qualified donors" for convalescent serum therapy with only one fixed dilution factor (200 ×). Finally, we demonstrated that our technology can sensitively detect SARS-CoV-2 antigens (S1 and N proteins) with pg/mL level sensitivities in 40 min. Overall, our technology can greatly facilitate rapid, sensitive, and quantitative analysis of COVID-19 related markers for therapeutic, diagnostic, epidemiologic, and prognostic purposes.

A single-view field filter device for rare tumor cell filtration and enumeration.

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 µL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.

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.

Reliable and reusable whole polypropylene plastic microfluidic devices for a rapid, low-cost antimicrobial susceptibility test.

Using an antimicrobial susceptibility test (AST) as an example, this work demonstrates a practical method to fabricate microfluidic chips entirely from polypropylene (PP) and the benefits for potential commercial use. Primarily caused by the misuse and abuse of antibiotics, antimicrobial resistance (AMR) is a major threat to modern medicine. The AST is a promising technique to help with the optimal use of antibiotics for reducing AMR. However, current phenotypic ASTs suffer from long turnaround time, while genotypic ASTs suffer from low reliability, and both are unaffordable for routine use. New microfluidics based AST methods are rapid but still unreliable as well as costly due to the PDMS chip material. Herein, we demonstrate a convenient method to fabricate whole PP microfluidic chips with high resolution and fidelity. Unlike PDMS chips, the whole PP chips showed better reliability due to their inertness; they are solvent-compatible and can be conveniently reused and recycled, which largely decreases the cost, and are environmentally friendly. We specially designed 3D chambers that allow for quick cell loading without valving/liquid exchange; this new hydrodynamic design satisfies the shear stress requirement for on-chip bacterial culture, which, compared to reported designs for similar purposes, allows for a simpler, more rapid, and high-throughput operation. Our system allows for reliable tracking of individual cells and acquisition of AST results within 1-3 hours, which is among the group of fastest phenotypic methods. The PP chips are more reliable and affordable than PDMS chips, providing a practical solution to improve current culture-based AST and benefiting the fight against AMR through helping doctors prescribe effective, narrow-spectrum antibiotics; they will also be broadly useful for other applications wherein a reliable, solvent-resistant, anti-fouling, and affordable microfluidic chip is needed.

A rapid and low-cost fabrication and integration scheme to render 3D microfluidic architectures for wearable biofluid sampling, manipulation, and sensing.

The large-scale deployment of wearable bioanalytical devices for general population longitudinal monitoring necessitates rapid and high throughput manufacturing-amenable fabrication schemes that render disposable, low-cost, and mechanically flexible microfluidic modules capable of performing a variety of bioanalytical operations within a compact footprint. The spatial constraints of previously reported wearable bioanalytical devices (with microfluidic operations confined to 2D), their lack of biofluid manipulation capability, and the complex and low-throughput nature of their fabrication process inherently limit the diversity and frequency of end-point assessments and prevent their deployment at large scale. Here, we devise a simple, scalable, and low-cost "CAD-to-3D Device" fabrication and integration scheme, which renders 3D and complex microfluidic architectures capable of performing biofluid sampling, manipulation, and sensing. The devised scheme is based on laser-cutting of tape-based substrates, which can be programmed at the software-level to rapidly define microfluidic features such as a biofluid collection interface, microchannels, and VIAs (vertical interconnect access), followed by the vertical assembly of pre-patterned layers to realize the final device. To inform the utility of our fabrication scheme, we demonstrated three representative devices to perform sweat collection (with visualizable secretion profile), sample filtration, and simultaneous biofluid actuation and sensing (using a sandwiched-interface). Our devised scheme can be adapted for the fabrication and manufacturing of current and future wearable bioanalytical devices, which in turn will catalyze the large-scale production and deployment of such devices for general population health monitoring.

Tumor spheroid-on-a-chip: a standardized microfluidic culture platform for investigating tumor angiogenesis.

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

Three-dimensional paper based platform for automatically running multiple assays in a single step.

Paper based assays are paving the way to automated, simplified, robust and cost-effective point of care testing (POCT). We propose a method for fabricating three dimensional (3D) microfluidic paper based analytical devices (µPADs) via combining thin adhesive films and paper folding, which avoids the use of cellulose powders and the complex folding sequence and simultaneously permits assays in several layers. To demonstrate the effectiveness of this approach, a 3DµPADs was designed to conduct more assays on a small footprint, allowing dual colorimetric and electrochemical detections. More importantly, we further developed a 3D platform for implementing automated and multiplexed ELISA in parallel, since ELISA, a routine and standard laboratory method, has rarely been used in practical analyses outside of the laboratory. In this configuration, complex and multistep diagnostic assays can be carried out with the addition of the sample and buffer in a simple fashion. Using Troponin I as model, the device showed a broad dynamic range of detection with a detection limit of 0.35 ng/mL. Thus, the developed platforms allow for various assays to be cost-effectively carried out on a single 3D device, showing great potential in an academic setting and point of care testing under resource-poor conditions.

A microfluidic based biosensor for rapid detection of Salmonella in food products.

An impedance based microfluidic biosensor for simultaneous and rapid detection of Salmonella serotypes B and D in ready-to-eat (RTE) Turkey matrix has been presented. Detection of Salmonella at a concentration as low as 300 cells/ml with a total detection time of 1 hour has been achieved. The sensor has two sensing regions, with each formed from one interdigitated electrode array (IDE array) consisting of 50 finger pairs. First, Salmonella antibody type B and D were prepared and delivered to the sensor to functionalize each sensing region without causing any cross contamination. Then the RTE Turkey samples spiked with Salmonella types B and D were introduced into the biosensor via the antigen inlet. The response signal resulted from the binding between Salmonella and its specific antibody demonstrated the sensor's ability to detect a single type of pathogen, and multiple pathogens simultaneously. In addition, the biosensor's selectivity was tested using non-specific binding of E. coli O157 and E. coli DH5 Alpha while the IDE array was coated with the Salmonella antibody. The results also showed the sensor is capable to differentiate low concentration of live Salmonella cells from high concentration of dead Salmonella cells, and high concentration of E. coli cells. A detailed study on antibody immobilization that includes antibody concentration, antibody coating time (0.5-3 hours) and use of cross-linker has been performed. The study showed that Salmonella antibody to Salmonella antigen is not a factor of antibody concentration after electrodes were saturated with antibody, while the optimal coating time was found to be 1.5 hours, and the use of cross-linker has improved the signal response by 45-60%.

A low-cost, non-invasive phase velocity and length meter and controller for multiphase lab-in-a-tube devices.

Opportunities for accessible microfluidic device integration have sharply grown with the rise of readily available lab-in-a-tube strategies. Herein, we present a facile, non-invasive, plug-and-play phase velocity and length measuring strategy for rapid deployment onto tube-based microfluidic systems, enabling quick and accurate residence (reaction) time measurement and tuning. Our approach utilizes inexpensive off-the-shelf optical phase sensors and requires no prior knowledge of the fluid composition or physical properties. Compared to camera-based measurements in fluoropolymer tubing, the optical phase sensor-based technique shows mean absolute percentage errors of 1.3% for velocity and 3.3% for length. Utilizing the developed multiphase flow monitoring technique, we screen the accessible parameter space of gas-liquid segmented flows. To further demonstrate the functionality of this process monitoring strategy, we implement two feedback controllers to establish simultaneous setpoint control for phase velocity and length. Next, to showcase the effectiveness and versatility of the developed multiphase flow process controller, we apply it to systematic studies of the effect of liquid slug velocity (controlling precursor mixing timescale) on the colloidal synthesis of cesium lead tribromide nanocrystals. By varying the liquid slug velocity and maintaining constant precursor composition, liquid slug length, and residence time, we observe a bandgap tunability from 2.43 eV (510 nm) to 2.52 eV (494 nm).

A low-cost microfluidic platform for rapid and instrument-free detection of whooping cough.

Whooping cough also called Pertussis is a highly contagious respiratory infection that affects all age populations. Given recent pertussis outbreaks, there is an urgent need for a point-of-care (POC) device for rapid diagnosis of pertussis. Herein, we report a low-cost microfluidic POC device integrated with loop-mediated isothermal amplification (LAMP) technique for the rapid and accurate diagnosis of pertussis. The 3D-printed bioanalyzer housed not only the biochip but also an in-house-developed portable and fully battery-powered heater for rapid POC detection of pertussis, without the need of external electricity. The fluorescence-based results could be rapidly visualized in about one hour by the naked eye without the need for any additional instrumentation. In addition, a simple centrifuge-free sample preparation process was optimized for the efficient lysis of pertussis samples and successfully used for direct detection of bacteria in nasopharyngeal samples. High sensitivity, with a limit of detection (LOD) of 5 DNA copies per LAMP zone, and high specificity were demonstrated. We envision that the microfluidic POC device can be used in various venues such as medical clinics, schools, and other low-resource settings for the fast detection of pertussis.

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.

Comparative Analysis of Droplet-Based Ultra-High-Throughput Single-Cell RNA-Seq Systems.

Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

Low-Cost and Rapid-Production Microfluidic Electrochemical Double-Layer Capacitors for Fast and Sensitive Breast Cancer Diagnosis.

This technical note describes a new microfluidic sensor that combines low-cost (USD $0.97) with rapid fabrication and user-friendly, fast, sensitive, and accurate quantification of a breast cancer biomarker. The electrodes consisted of cost-effective bare stainless-steel capillaries, whose mass production is already well-established. These capillaries were used as received, without any surface modification. Microfluidic chips containing electrical double-layer capillary capacitors (µEDLC) were obtained by a cleanroom-free prototyping that allows the fabrication of dozens to hundreds of chips in 1 h. This sensor provided the successful quantification of CA 15-3, a biomarker protein for breast cancer, in serum samples from cancer patients. Antibody-anchored magnetic beads were utilized for immunocapture of the marker, and then, water was added to dilute the protein. Next, the CA 15-3 detection (<2 min) was made without using redox probes, antibody on electrode (sandwich immunoassay), or signal amplification strategies. In addition, the capacitance tests eliminated external pumping systems and precise volumetric sampling steps, as well as presented low sample volume (5 µL) and high sensitivity using bare capillaries in a new design for double-layer capacitors. The achieved limit-of-detection (92.0 µU mL-1) is lower than that of most methods reported in the literature for CA 15-3, which are based on nanostructured electrodes. The data shown in this technical note support the potential of the µEDLC toward breast cancer diagnosis even at early stages. We believe that accurate analyses using a simple sample pretreatment such as magnetic field-assisted immunocapture and cost-effective bare electrodes can be extended to quantify other cancer biomarkers and even biomolecules by changing the biorecognition element.

Ferrodrop Dose-Optimized Digital Quantification of Biomolecules in Low-Volume Samples.

We present an approach to estimate the concentration of a biomolecule in a solution by sampling several nanoliter-scale volumes and determining if the volumes contain any biomolecules. In this method, varying volume fractions (nanoliter-scale) of a sample of nucleic acids are introduced to an array of uniform volume reaction wells (100 µL), which are then fluorescently imaged to determine if signal is above a threshold after nucleic acid amplification, all without complex instrumentation. The nanoliter volumes are generated and introduced using the simple positioning of a permanent magnet, and imaging is performed with a cellphone-based fluorescence detection scheme, both methods suitable for limited-resource settings. We use the length of time a magnetic field is applied to generate a calibrated number of nanoliter ferrodrops of sample mixed with ferrofluid at a step emulsification microfluidic junction. Each dose of ferrodrops is then transferred into larger microliter scale reaction wells on chip through a simple shift of the external magnet. Nucleic acid amplification is achieved using loop-mediated isothermal amplification (LAMP). By repeating each nanoliter dosage a number of times to calculate the probability of a positive signal at each dosage, we can use a binomial probability distribution to estimate the sample nucleic acid concentration. Using this approach we demonstrate detection of lambda DNA molecules down to 25 copies per microliter. The ability to dose separate nanoliter-scale volumes of a low-volume sample across wells in this platform is suited for multiplexed assays. This platform has the potential to be applied to a range of diseases by mixing a sample with magnetic nanoparticles.

Simple and inexpensive micromachined aluminum microfluidic devices for acoustic focusing of particles and cells.

We introduce a new method to construct microfluidic devices especially useful for bulk acoustic wave (BAW)-based manipulation of cells and microparticles. To obtain efficient acoustic focusing, BAW devices require materials that have high acoustic impedance mismatch relative to the medium in which the cells/microparticles are suspended and materials with a high-quality factor. To date, silicon and glass have been the materials of choice for BAW-based acoustofluidic channel fabrication. Silicon- and glass-based fabrication is typically performed in clean room facilities, generates hazardous waste, and can take several hours to complete the microfabrication. To address some of the drawbacks in fabricating conventional BAW devices, we explored a new approach by micromachining microfluidic channels in aluminum substrates. Additionally, we demonstrate plasma bonding of poly(dimethylsiloxane) (PDMS) onto micromachined aluminum substrates. Our goal was to achieve an approach that is both low cost and effective in BAW applications. To this end, we micromachined aluminum 6061 plates and enclosed the systems with a thin PDMS cover layer. These aluminum/PDMS hybrid microfluidic devices use inexpensive materials and are simply constructed outside a clean room environment. Moreover, these devices demonstrate effectiveness in BAW applications as demonstrated by efficient acoustic focusing of polystyrene microspheres, bovine red blood cells, and Jurkat cells and the generation of multiple focused streams in flow-through systems. Graphical abstract The aluminum acoustofluidic device and the generation of multinode focusing of particles.