An integrated microfluidic chip for nucleic acid extraction and continued cdPCR detection of pathogens.

This paper introduces an enclosed microfluidic chip that integrates sample preparation and the chamber-based digital polymerase chain reaction (cdPCR). The sample preparation of the chip includes nucleic acid extraction and purification based on magnetic beads, which adsorb nucleic acids by moving around the reaction chambers to complete the reactions including lysis, washing, and elution. The cdPCR area of the chip consists of tens of thousands of regularly arranged microchambers. After the sample preparation processes are completed, the purified nucleic acid can be directly introduced into the microchambers for amplification and detection on the chip. The nucleic acid extraction performance and digital quantification performance of the system were examined using synthetic SARS-CoV-2 plasmid templates at concentrations ranging from 101-105 copies per µL. Further on, a simulated clinical sample was used to test the system, and the integrated chip was able to accurately detect SARS-CoV-2 virus particle samples doped with interference (saliva) with a detection limit of 10 copies per µL. This integrated system could provide a promising tool for point-of-care testing of pathogenic infections.

High-throughput microfluidic droplets in biomolecular analytical system: A review.

Droplet microfluidic technology has revolutionized biomolecular analytical research, as it has the capability to reserve the genotype-to-phenotype linkage and assist for revealing the heterogeneity. Massive and uniform picolitre droplets feature dividing solution to the level that single cell and single molecule in each droplet can be visualized, barcoded, and analyzed. Then, the droplet assays can unfold intensive genomic data, offer high sensitivity, and screen and sort from a large number of combinations or phenotypes. Based on these unique advantages, this review focuses on up-to-date research concerning diverse screening applications utilizing droplet microfluidic technology. The emerging progress of droplet microfluidic technology is first introduced, including efficient and scaling-up in droplets encapsulation, and prevalent batch operations. Then the new implementations of droplet-based digital detection assays and single-cell muti-omics sequencing are briefly examined, along with related applications such as drug susceptibility testing, multiplexing for cancer subtype identification, interactions of virus-to-host, and multimodal and spatiotemporal analysis. Meanwhile, we specialize in droplet-based large-scale combinational screening regarding desired phenotypes, with an emphasis on sorting for immune cells, antibodies, enzymatic properties, and proteins produced by directed evolution methods. Finally, some challenges, deployment and future perspective of droplet microfluidics technology in practice are also discussed.

Microfluidic-based technologies for diagnosis, prevention, and treatment of COVID-19: recent advances and future directions.

The COVID-19 pandemic has posed significant challenges to existing healthcare systems around the world. The urgent need for the development of diagnostic and therapeutic strategies for COVID-19 has boomed the demand for new technologies that can improve current healthcare approaches, moving towards more advanced, digitalized, personalized, and patient-oriented systems. Microfluidic-based technologies involve the miniaturization of large-scale devices and laboratory-based procedures, enabling complex chemical and biological operations that are conventionally performed at the macro-scale to be carried out on the microscale or less. The advantages microfluidic systems offer such as rapid, low-cost, accurate, and on-site solutions make these tools extremely useful and effective in the fight against COVID-19. In particular, microfluidic-assisted systems are of great interest in different COVID-19-related domains, varying from direct and indirect detection of COVID-19 infections to drug and vaccine discovery and their targeted delivery. Here, we review recent advances in the use of microfluidic platforms to diagnose, treat or prevent COVID-19. We start by summarizing recent microfluidic-based diagnostic solutions applicable to COVID-19. We then highlight the key roles microfluidics play in developing COVID-19 vaccines and testing how vaccine candidates perform, with a focus on RNA-delivery technologies and nano-carriers. Next, microfluidic-based efforts devoted to assessing the efficacy of potential COVID-19 drugs, either repurposed or new, and their targeted delivery to infected sites are summarized. We conclude by providing future perspectives and research directions that are critical to effectively prevent or respond to future pandemics.

Microfluidic Organ-on-A-chip: A Guide to Biomaterial Choice and Fabrication.

Organ-on-A-chip (OoAC) devices are miniaturized, functional, in vitro constructs that aim to recapitulate the in vivo physiology of an organ using different cell types and extracellular matrix, while maintaining the chemical and mechanical properties of the surrounding microenvironments. From an end-point perspective, the success of a microfluidic OoAC relies mainly on the type of biomaterial and the fabrication strategy employed. Certain biomaterials, such as PDMS (polydimethylsiloxane), are preferred over others due to their ease of fabrication and proven success in modelling complex organ systems. However, the inherent nature of human microtissues to respond differently to surrounding stimulations has led to the combination of biomaterials ranging from simple PDMS chips to 3D-printed polymers coated with natural and synthetic materials, including hydrogels. In addition, recent advances in 3D printing and bioprinting techniques have led to the powerful combination of utilizing these materials to develop microfluidic OoAC devices. In this narrative review, we evaluate the different materials used to fabricate microfluidic OoAC devices while outlining their pros and cons in different organ systems. A note on combining the advances made in additive manufacturing (AM) techniques for the microfabrication of these complex systems is also discussed.

FLUIDOT: A Modular Microfluidic Platform for Single-Cell Study and Retrieval, with Applications in Drug Tolerance Screening and Antibody Mining.

Advancements in lab-on-a-chip technologies have revolutionized the single-cell analysis field. However, an accessible platform for in-depth screening and specific retrieval of single cells, which moreover enables studying diverse cell types and performing various downstream analyses, is still lacking. As a solution, FLUIDOT is introduced, a versatile microfluidic platform incorporating customizable microwells, optical tweezers and an interchangeable cell-retrieval system. Thanks to its smart microfluidic design, FLUIDOT is straightforward to fabricate and operate, rendering the technology widely accessible. The performance of FLUIDOT is validated and its versatility is subsequently demonstrated in two applications. First, drug tolerance in yeast cells is studied, resulting in the discovery of two treatment-tolerant populations. Second, B cells from convalescent COVID-19 patients are screened, leading to the discovery of highly affine, in vitro neutralizing monoclonal antibodies against SARS-CoV-2. Owing to its performance, flexibility, and accessibility, it is foreseen that FLUIDOT will enable phenotypic and genotypic analysis of diverse cell samples and thus elucidate unexplored biological questions.

Comparative analysis of loop-mediated isothermal amplification combined with microfluidic chip technology and q-PCR in the detection of clinical infectious pathogens.

BACKGROUND: Rapid diagnosis of infectious pathogens at an early stage is crucial to stabilize the patient's condition, reduce medical costs, and shorten hospital stays. Currently, some point-of-care tests have their own shortcomings. Therefore, we built a microfluidic chip based on loop-mediated isothermal amplification to can quickly and sensitively detect infectious pathogens. METHODS: We extracted the DNA of S. aureus, MRSA, Shigella and Klebsiella pneumoniae. Then, the DNA samples were diluted by 10-fold and examined by two methods: LAMP-microfluidic chip and q-PCR, the sensitivity of whom was also compared. In addition, the specificity of the two was also examined by detecting the target bacteria and other microorganisms using the same methods. Finally, we extracted and tested the DNA of clinically infected humoral samples to determine the coincidence rate between the two methods and the bacterial culture method. RESULTS: For S. aureus, MRSA, Shigella, and Klebsiella pneumoniae, the detection limits of the chip were 2.25 × 103 copies/µl, 5.32 × 103 copies/µl, 2.89 × 103 copies/µl, 6.53 × 102 copies/µl, and the detection limits of q-PCR were 2.25 × 102 copies/µl, 5.32 × 101 copies/µl, 2.89 × 102 copies/µl, 6.53 × 101 copies/µl, respectively. In terms of detection specificity, neither method cross-reacted with other strains. For the detection of infectious humoral samples, the total coincidence rate between the q-PCR and bacterial culture method was 85.7%, 95%, 95%, and 95.5%, and the total coincidence rate between the chip and bacterial culture method was 81%, 95%, 90%, and 86.4%, respectively. CONCLUSION: LAMP-microfluidic chip provides a simple, sensitive, specific, convenient, and rapid pathogen detection method for clinically infected humoral samples without relying on expensive equipment or technical personnels.

Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

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.

Microfluidic characterisation reveals broad range of SARS-CoV-2 antibody affinity in human plasma.

The clinical outcome of SARS-CoV-2 infections, which can range from asymptomatic to lethal, is crucially shaped by the concentration of antiviral antibodies and by their affinity to their targets. However, the affinity of polyclonal antibody responses in plasma is difficult to measure. Here we used microfluidic antibody affinity profiling (MAAP) to determine the aggregate affinities and concentrations of anti-SARS-CoV-2 antibodies in plasma samples of 42 seropositive individuals, 19 of which were healthy donors, 20 displayed mild symptoms, and 3 were critically ill. We found that dissociation constants, K d, of anti-receptor-binding domain antibodies spanned 2.5 orders of magnitude from sub-nanomolar to 43 nM. Using MAAP we found that antibodies of seropositive individuals induced the dissociation of pre-formed spike-ACE2 receptor complexes, which indicates that MAAP can be adapted as a complementary receptor competition assay. By comparison with cytopathic effect-based neutralisation assays, we show that MAAP can reliably predict the cellular neutralisation ability of sera, which may be an important consideration when selecting the most effective samples for therapeutic plasmapheresis and tracking the success of vaccinations.

Rapid differential diagnosis of the B.1.617.2 (delta) variant of SARS-CoV-2 using an automated Cas12a-microfluidic system.

An automated Cas12a-microfluidic system was constructed to distinguish the B.1.617.2 (delta) variant of SARS-CoV-2 from the wild-type virus rapidly and was validated using 30 clinical samples, showing 100% consistency with next-generation sequencing. It will be a potential tool for the rapid differential diagnosis of the delta variant of SARS-CoV-2.

Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS-CoV-2 Spike protein binding to the human angiotensin-converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS-CoV-2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient-specific-vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS-CoV-2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome-hACE2 and anti-Interleukin (IL) 6 antibodies.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

Development of a Promising Method for Producing Oligomeric Mixture of Branched Alkylene Guanidines to Improve Substance Quality and Evaluate Their Antiviral Activity against SARS-CoV-2.

This paper reports the synthesis of branched alkylene guanidines using microfluidic technologies. We describe the preparation of guanidine derivatives at lower temperatures, and with significantly less time than that required in the previously applicable method. Furthermore, the use of microfluidics allows the attainment of high-purity products with a low residual monomer content, which can expand the range of applications of this class of compounds. For all the samples obtained, the molecular-weight characteristics are calculated, based on which the optimal condensation conditions are established. Additionally, in this work, the antiviral activity of the alkylene guanidine salt against the SARS-CoV-2 virus is confirmed.

Thin-film-transistor digital microfluidics for high value in vitro diagnostics at the point of need.

The latest developments in thin-film-transistor digital-microfluidics (TFT-DMF, also known by the commercial name aQdrop™) are reported, and proof of concept application to molecular diagnostics (e.g. for coronavirus disease, COVID-19) at the point-of-need demonstrated. The TFT-DMF array has 41 thousand independently addressable electrodes that are capable of manipulating large numbers of droplets of any size and shape, along any pathway to perform multiple parallel reactions. Droplets are continually tracked and adjusted through closed-loop feedback enabled by TFT based sensors at each array element. The sample-to-answer molecular in vitro diagnostic (IVD) test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) includes nucleic acid extractions from saliva, removal of dsDNA and quantitative reverse transcription polymerase chain reaction (RT-PCR). This proof of concept illustrates how the highly configurable TFT-DMF technology can perform many reactions in parallel and thus support the processing of a range of sample types followed by multiple complex multi-step assays.

Microfluidic detection of human diseases: From liquid biopsy to COVID-19 diagnosis.

Microfluidic devices can be thought of as comprising interconnected miniaturized compartments performing multiple experimental tasks individually or in parallel in an integrated fashion. Due to its small size, portability, and low cost, attempts have been made to incorporate detection assays into microfluidic platforms for diseases such as cancer and infection. Some of these technologies have served as point-of-care and sample-to-answer devices. The methods for detecting biomarkers in different diseases usually share similar principles and can conveniently be adapted to cope with arising health challenges. The COVID-19 pandemic is one such challenge that is testing the performance of both our conventional and newly-developed disease diagnostic technologies. In this mini-review, we will first look at the progress made in the past few years in applying microfluidics for liquid biopsy and infectious disease detection. Following that, we will use the current pandemic as an example to discuss how such technological advancements can help in the current health challenge and better prepare us for future ones.

Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2.

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In one basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex becomes activated only when it specifically binds to target DNA and cleaves it. The activated complex thereafter nonspecifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We discovered that electric field gradients can be used to control and accelerate this CRISPR assay by cofocusing Cas12-gRNA, reporters, and target within a microfluidic chip. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab samples. We here combine this ITP purification with loop-mediated isothermal amplification and the ITP-enhanced CRISPR assay to achieve detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA (from raw sample to result) in about 35 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables an alternate modality for a suite of microfluidic CRISPR-based diagnostic assays.