Fully integrated and automated centrifugal microfluidic chip for point-of-care multiplexed molecular diagnostics.

Public health events caused by pathogens have imposed significant economic and societal burdens. However, conventional methods still face challenges including complex operations, the need for trained operators, and sophisticated instruments. Here, we proposed a fully integrated and automated centrifugal microfluidic chip, also termed IACMC, for point-of-care multiplexed molecular diagnostics by harnessing the advantages of active and passive valves. The IACMC incorporates multiple essential components including a pneumatic balance module for sequential release of multiple reagents, a pneumatic centrifugation-assisted module for on-demand solution release, an on-chip silicon membrane module for nucleic acid extraction, a Coriolis force-mediated fluid switching module, and an amplification module. Numerical simulation and visual validation were employed to iterate and optimize the chip's structure. Upon sample loading, the chip automatically executes the entire process of bacterial sample lysis, nucleic acid capture, elution quantification, and isothermal LAMP amplification. By optimizing crucial parameters including centrifugation speed, direction of rotation, and silicone membrane thickness, the chip achieves exceptional sensitivity (twenty-five Salmonella or forty Escherichia coli) and specificity in detecting Escherichia coli and Salmonella within 40 min. The development of IACMC will drive advancements in centrifugal microfluidics for point-of-care testing and holds potential for broader applications in precision medicine including high-throughput biochemical analysis immune diagnostics, and drug susceptibility testing.

Controlled-diffusion centrifugal microfluidic for rapid antibiotic susceptibility testing.

The abuse of antibiotics has become a global public safety issue, leading to the development of antimicrobial resistance (AMR). The development of antimicrobial susceptibility testing (AST) is crucial in reducing the growth of AMR. However, traditional AST methods are time-consuming (e.g., 24-72 h), labor-intensive, and costly. Here, we propose a controlled-diffusion centrifugal microfluidic platform (CCM) for rapid AST to obtain highly precise minimum inhibitory concentration (MIC) values. Antibiotic concentration gradients are generated by controlled moving and diffusing of antibiotic and buffer solution along the main microchannel within 3 min. The solution and bacterial suspension are then injected into the outermost reaction chamber by simple centrifugation. The CCM successfully determined the MIC for three commonly used antibiotics in clinical settings within 4-9 h. To further enhance practicality, reduce costs, and meet point-of-care testing demands, we have developed an integrated mobile detection platform for automated MIC value acquisition. The proposed CCM is a simple, low-cost, and portable method for rapid AST with broad clinical and in vitro applications.

A smartphone-based centrifugal mHealth platform implementing hollow daisy-shaped quick response chip for hematocrit measurement.

Due to the ever-increasing challenge of emerging and reemerging infections on global health, the development of POCT tools has been propelled. However, conventional point-of-care testing methods suffer from several limitations, including cumbersome operation, long detection times, and low accuracy, which hamper their widespread application. Compared to traditional disease diagnostic equipment, mobile health platforms offer several advantages, including portability, ease of operation, and automated analysis of detection results through recognition algorithms. Consequently, they hold great promise for the future. Here, we developed a smartphone-based centrifugal mHealth platform implementing daisy-shaped quick response chip for hematocrit measurement. The centrifugal microfluidic chip is combined with a smartphone through a back-clip-on mobile phone adapter whose control circuit is designed with low power consumption to enable the platform to operate without requiring a high-power source that is inconvenient to carry, thereby achieving the goal of portability. Concurrently, we designed a quick response chip featuring a unique hollow daisy structure that is in line with the properties of hematocrit detection. The distinctive configuration of the chip enables adequate centrifugal force to be supplied for hematocrit detection. Additionally, our customized quick response code recognition algorithm is able to recognize this chip, facilitating non-experts in performing hematocrit intelligent recognition with their smartphones.

Programmable Gravity Self-Driven Microfluidic Chip for Point-of-Care Multiplied Immunoassays.

Point-of-care testing (POCT) is experiencing a groundbreaking transformation with microfluidic chips, which offer precise fluid control and manipulation at the microscale. Nevertheless, chip design or operation for existing platforms is rather cumbersome, with some even heavily depending on external drivers or devices, impeding their broader utilization. This study develops a unique programmable gravity self-driven microfluidic chip (PGSMC) capable of simultaneous multi-reagent sequential release, multi-target analysis, and multi-chip operation. All necessary reagents are introduced in a single step, and the process is initiated simply by flipping the PGSMC vertically, eliminating the need for additional steps or devices. Additionally, it demonstrates successful immunoassays in less than 60 min for antinuclear antibodies testing, compared to more than 120 min by traditional methods. Assessment using 25 clinically diagnosed cases showcases remarkable sensitivity (96%), specificity (100%), and accuracy (99%). These outcomes underscored its potential as a promising platform for POCT with high accuracy, speed, and reliability, highlighting its capability for automated fluid control.

On-Line Dual-Active Valves Based Centrifugal Microfluidic Chip for Fully Automated Point-of-Care Immunoassay.

There remains an unmet need for a fully integrated microfluidic platform that can automatically perform multistep and multireagent immunoassays. Here, we proposed a novel online dual-active valve-based centrifugal microfluidic chip, termed DAVM, for fully automatic point-of-care immunoassay. Practically, the puncture valve, one of the dual active valves, is capable of achieving precise, on-demand, sequential release of prestored reagents, while the other valve-reversible active valve enables controlled retention and drainage of the reaction solutions. Thereby, our technology mitigates the challenges of hydrophilic/hydrophobic modifications and unstable valve control performance commonly observed in passive valve controls. As a proof of concept, the indirect enzymatic immunoblotting technique was employed on DAVM for fully automated immunological analysis of eight targets, yielding outcomes within an hour. Furthermore, we conducted a comparative analysis of 28 clinical samples with autoimmune diseases. According to 224 clinical data, the sample testing concordance rate between DAVM and the traditional instrument was 82%, with a target compliance rate of 97%. Therefore, our DAVM system has powerful potential for fully automated immunoassays.

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae.

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.

Handyfuge Microfluidic for On-Site Antibiotic Susceptibility Testing.

Low-cost, rapid, and accurate acquisition of minimum inhibitory concentrations (MICs) is key to limiting the development of antimicrobial resistance (AMR). Until now, conventional antibiotic susceptibility testing (AST) methods are typically time-consuming, high-cost, and labor-intensive, making them difficult to accomplish this task. Herein, an electricity-free, portable, and robust handyfuge microfluidic chip was developed for on-site AST, termed handyfuge-AST. With simply handheld centrifugation, the bacterial-antibiotic mixtures with accurate antibiotic concentration gradients could be generated in less than 5 min. The accurate MIC values of single antibiotics (including ampicillin, kanamycin, and chloramphenicol) or their combinations against Escherichia coli could be obtained within 5 h. To further meet the growing demands of point-of-care testing, we upgraded our handyfuge-AST with a pH-based colorimetric strategy, enabling naked eye recognition or intelligent recognition with a homemade mobile app. Through a comparative study of 60 clinical data (10 clinical samples corresponding to six commonly used antibiotics), the accurate MICs by handyfuge-AST with 100% categorical agreements were achieved compared to clinical standard methods (area under curves, AUCs = 1.00). The handyfuge-AST could be used as a low-cost, portable, and robust point-of-care device to rapidly obtain accurate MIC values, which significantly limit the progress of AMR.

Hand-powered centrifugal micropipette-tip with distance-based quantification for on-site testing of SARS-CoV-2 virus.

This paper proposed a hand-powered centrifugal micropipette-tip strategy, termed HCM, for all-in-one immunoassay combined with a distance-based readout for portable quantitative detection of SARS-CoV-2. The target SARS-CoV-2 virus antigen triggers the binding of multiple monoclonal antibody-coated red latex nanobeads, forming larger complexes. Following incubation and centrifugation, the formed aggregated complexes settle at the bottom of the tip, while free red nanobeads remain suspended in the solution. The HCM enables sensitive (1 ng/mL) and reliable quantification of SARS-CoV-2 within 25 min. With the advantages of free washing, free fabrication, free instrument, and without the optical device, the proposed low-cost and easy-to-use HCM immunoassay shows great potential for quantitative POC diagnostics for SARS-CoV-2.

Rapid Assembly of Cellulose Microfibers into Translucent and Flexible Microfluidic Paper-Based Analytical Devices via Wettability Patterning.

Microfluidic paper-based analytical devices (µPADs) are emerging as powerful analytical platforms in clinical diagnostics, food safety, and environmental protection because of their low cost and favorable substrate properties for biosensing. However, the existing top-down fabrication methods of paper-based chips suffer from low resolution (>200 µm). Additionally, papers have limitations in their physical properties (e.g., thickness, transmittance, and mechanical flexibility). Here, we demonstrate a bottom-up approach for the rapid fabrication of heterogeneously controlled paper-based chip arrays. We simply print a wax-patterned microchip with wettability contrasts, enabling automatic and selective assembly of cellulose microfibers to construct predefined paper-based microchip arrays with controllable thickness. This paper-based microchip printing technology is feasible for various substrate materials ranging from inorganic glass to organic polymers, providing a versatile platform for the full range of applications including transparent devices and flexible health monitoring. Our bottom-up printing technology using cellulose microfibers as the starting material provides a lateral resolution down to 42 ± 3 µm and achieves the narrowest channel barrier down to 33 ± 2 µm. As a proof-of-concept demonstration, a flexible paper-based glucose monitor is built for human health care, requiring only 0.3 µL of sample for testing.

Defining the HIV Capsid Binding Site of Nucleoporin 153.

The interaction between the HIV-1 capsid and human nucleoporin 153 (NUP153) is vital for delivering the HIV-1 preintegration complex into the nucleus via the nuclear pore complex. The interaction with the capsid requires a phenylalanine/glycine-containing motif in the C-terminus of NUP153 (NUP153C). This study used molecular modeling and biochemical assays to comprehensively determine the amino acids in NUP153 that are important for capsid interaction. Molecular dynamics, FoldX, and PyRosetta simulations delineated the minimal capsid binding motif of NUP153 based on the known structure of NUP153 bound to the HIV-1 capsid hexamer. Computational predictions were experimentally validated by testing the interaction of NUP153 with capsid using an in vitro binding assay and a cell-based TRIM-NUP153C restriction assay. This work identified eight amino acids from P1411 to G1418 that stably engage with capsid, with significant correlations between the interactions predicted by molecular models and empirical experiments. This validated the usefulness of this multidisciplinary approach to rapidly characterize the interaction between human proteins and the HIV-1 capsid. IMPORTANCE The human immunodeficiency virus (HIV) can infect nondividing cells by interacting with the host nuclear pore complex. The host nuclear pore protein NUP153 directly interacts with the HIV capsid to promote viral nuclear entry. This study used a multidisciplinary approach combining computational and experimental techniques to comprehensively map the effect of mutating the amino acids of NUP153 on HIV capsid interaction. This work showed a significant correlation between computational and empirical data sets, revealing that the HIV capsid interacted specifically with only six amino acids of NUP153. The simplicity of the interaction motif suggested other FG-containing motifs could also interact with the HIV-1 capsid. Furthermore, it was predicted that naturally occurring polymorphisms in human and nonhuman primates would disrupt NUP153 interaction with capsid, potentially protecting certain populations from HIV-1 infection.

Multi-reagents dispensing centrifugal microfluidics for point-of-care testing.

Point-of-care testing (POCT) has shown great advantages for public health monitoring in resource-limited settings. However, developing of POCT tools with automated and accurate quantitative dispensing of multiple reagents and samples is challenging. Here, we demonstrate a novel multi-reagents dispensing centrifugal microfluidics (MDCM) that allows rapid and automated dispensing of multiple reagents and samples with high throughput and accuracy. The MDCM was designed with multiple aliquoting units with the hydrophobic valve at different radial positions. All reagents and samples were loaded simultaneously, dispensed in parallel by centrifugation at low speed, and then introduced into the reaction chamber sequentially by centrifugation at high speed. Two MDCM chips are demonstrated, including a uniform concentration generator and a gradient concentration generator. The concentration coefficient of variation (CV) among the independent reaction chambers was lower than 0.56%, and the theoretical quantitative concentration gradient was strongly correlated with the actual concentration gradient (R2 = 0.9938). We have successfully applied the MDCM to loop-mediated isothermal amplification (LAMP)-based nucleic acid detection for multiple infectious pathogens and antimicrobial susceptibility testing (AST) for kanamycin sulfate against E. coli. To further extend the applications, the MDCM has also been applied to bicinchoninic acid (BCA) protein assays with online calibration, reducing the detection time from 2 h to 10 min with a twenty-fold reduction in reagent consumption. These results indicated that the MDCM is a high potential platform for POCT.

Hand-powered vacuum-driven microfluidic gradient generator for high-throughput antimicrobial susceptibility testing.

The growth of bacterial resistance to antimicrobials is a serious problem attracting much attention nowadays. To prevent the misuse and abuse of antimicrobials, it is important to carry out antibiotic susceptibility testing (AST) before clinical use. However, conventional AST methods are relatively laborious and time-consuming (18-24 h). Here, we present a hand-powered vacuum-driven microfluidic (HVM) device, in which a syringe is used as the only vacuum source for rapid generating concentration gradient of antibiotics in different chambers. The HVM device can be preassembled with various amounts of antibiotics, lyophilized, and stored for ready-to-use. Bacterial samples can be loaded into the HVM device through a simple suction step. With the assistance of Alamar Blue, the AST assay and the minimum inhibitory concentration (MIC) of different antibiotics can be investigated by comparing the growth results of bacteria in different culture chambers. In addition, a parallel HVM device was proposed, in which eight AST assays can be performed simultaneously. The results of MIC of three commonly used antibiotics against E. coli K-12 in our HVM device were consistent with those obtained by traditional method while the detection time was shortened to less than 8 h. We believe that our platform is high-throughput, cost-efficient, easy to use, and suitable for POCT applications.

Rapid Determination of Phase Diagrams for Biomolecular Liquid-Liquid Phase Separation with Microfluidics.

Biomolecular phase separation is currently emerging in both the medical and life science fields. Meanwhile, the application of liquid-liquid phase separation has been extended to many fields including drug discovery, fibrous material fabrication, 3D printing, and polymer design. Although more than 8600 proteins and other synthetic macromolecules are capable of phase separation as recently reported, there is still a lack of a high-throughput approach to quantitatively characterize its phase behaviors. To meet this requirement, here, we proposed fast and high-resolution acquisition of biomolecular phase diagrams using microfluidic chips. Using this platform, we demonstrated the phase behavior of polyU/RRASLRRASLRRASL in a quantitative manner. Up to 1750 concentration conditions can be generated in 140 min. The detection limitation of our device to capture the saturation concentration for phase separation is about 5 times lower than that of the traditional turbidity method. Thus, our results provide a basis for the rapid acquisition of phase diagrams with high-throughput and pave the way for its wide application.

Vaginal Isolates of Candida glabrata Are Uniquely Susceptible to Ionophoric Killer Toxins Produced by Saccharomyces cerevisiae.

Compared to other species of Candida yeasts, the growth of Candida glabrata is inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins are broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

Integrated and finger-actuated microfluidic chip for point-of-care testing of multiple pathogens.

The integration of gel-based loop-mediated isothermal amplification (gLAMP) and finger-actuated microfluidic chip (µFAchip) was developed for the simultaneous detection of various different types of bacterial pathogens. The developed µFAchip consisted of three PDMS layers attached together by two adhesive tapes. Multiple chambers in the top PDMS layer were used for sample preparation, and the corresponding chambers in the bottom PDMS layer was used for long-term storage of LAMP reagents without DNA templates. The thin PDMS layer in the middle contained cross-shaped cuts as finger-actuated valves for fluid control. To reduce operation steps on the chip, such as pipetting and manipulation of samples, Whatman CloneSaver card was pre-embedded in the top chambers for on-chip DNA extraction and purification. Upon a simple press on the top layer, the finger-actuated valve was opened up, allowing DNA samples on the top layer flow into the bottom reaction chambers for gLAMP reaction. For POCT applications, on-chip LAMP reaction and imaging were conducted on a miniaturized peltier heater and a portable fluorescence imaging system respectively. Under the optimized condition, multiple pathogens were detected simultaneously with high selectivity and sensitivity (as low as 1.6 cells). The developed µFAchip provided a rapid and easy-to-operate platform for gLAMP-based pathogen detection, with the potential for in-field detection, especially in areas with limited resources.

Multichannel Synchronous Hydrodynamic Gating Coupling with Concentration Gradient Generator for High-Throughput Probing Dynamic Signaling of Single Cells.

Cell signaling greatly affected by complicated and temporally dynamic extracellular microenvironments controls most of the physiological functions in vivo. To reconstruct or simulate such microenvironments in vitro represents a fundamental approach for revealing the underlying mechanisms of those sophisticated processes. Recent advances in microfluidics have added a new dimension to cell signaling analysis, for example, concentration gradient generators (amplitude aspect) or hydrodynamic gating strategy (frequency aspect), but it is still challengeable to capture single-cell dynamic signaling in response to a mimicked extracellular microenvironment with varied stimuli waveforms of different amplitude and frequency in a high-throughput manner. In this article, we proposed a novel microfluidic strategy coupling multichannel synchronous hydrodynamic gating with microfluidic concentration gradient generators (µMHG-CGG) to probe dynamic signaling of single cells with high throughput. The µMHG-CGG allows rapid delivery of dynamic chemical signals in both high frequency (as high as 670 mHz) and multiple amplitude domains at the same time and simultaneously high-throughput probing cell dynamics at single-cell resolution in real time. By applying the proposed system, the mechanisms for encoding/decoding systems (termed "frequency coding" or "amplitude coding") via GPCRs-mediated signaling pathways responding to histamine (HA) and adenosine triphosphate (ATP) in single HeLa cells were investigated. The optimal drug concentrations of single cells responses to HA and ATP individually or in combination were also successfully discussed, allowing us to obtain both single-cell heterogeneity and statistics from the cell population.

A review on microfluidics manipulation of the extracellular chemical microenvironment and its emerging application to cell analysis.

Spatiotemporal manipulation of extracellular chemical environments with simultaneous monitoring of cellular responses plays an essential role in exploring fundamental biological processes and expands our understanding of underlying mechanisms. Despite the rapid progress and promising successes in manipulation strategies, many challenges remain due to the small size of cells and the rapid diffusion of chemical molecules. Fortunately, emerging microfluidic technology has become a powerful approach for precisely controlling the extracellular chemical microenvironment, which benefits from its integration capacity, automation, and high-throughput capability, as well as its high resolution down to submicron. Here, we summarize recent advances in microfluidics manipulation of the extracellular chemical microenvironment, including the following aspects: i) Spatial manipulation of chemical microenvironments realized by convection flow-, diffusion-, and droplet-based microfluidics, and surface chemical modification; ii) Temporal manipulation of chemical microenvironments enabled by flow switching/shifting, moving/flowing cells across laminar flows, integrated microvalves/pumps, and droplet manipulation; iii) Spatiotemporal manipulation of chemical microenvironments implemented by a coupling strategy and open-space microfluidics; and iv) High-throughput manipulation of chemical microenvironments. Finally, we briefly present typical applications of the above-mentioned technical advances in cell-based analyses including cell migration, cell signaling, cell differentiation, multicellular analysis, and drug screening. We further discuss the future improvement of microfluidics manipulation of extracellular chemical microenvironments to fulfill the needs of biological and biomedical research and applications.

Microfluidic chip electrophoresis for biochemical analysis.

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics-based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.

Imperfection works: Survival, transmission and persistence in the system of Heliothis virescens ascovirus 3h (HvAV-3h), Microplitis similis and Spodoptera exigua.

Ascoviruses are insect-specific large DNA viruses that mainly infect noctuid larvae, and are transmitted by parasitoids in the fields. Heliothis virescens ascovirus 3h (HvAV-3h) has been recently isolated from Spodoptera exigua, without parasitoid vector identified previously. Here we report that Microplitis similis, a solitary endoparasitoid wasp, could transmit HvAV-3h between S. exigua larvae in the laboratory. When the female parasitoid wasp acquired the virus and served as a vector, the period of virion viability on the ovipositor was 4.1 ± 1.4 days. Infected host larvae were still acceptable for egg laying by parasitoids, and the parasitoids thereafter transmitted virus to healthy hosts. Virus acquisition occurred only from donor hosts between 3 and 9 days post infection. The peak of virus acquisition (80.9 ± 6.3%) was found when M. similis wasps oviposited in larvae that had been inoculated with the virus 7 days previously. When virus infection of the host took place during the life cycle of the parasitoid wasp, it caused 1- to 4-day-old immature parasitoids death in the host, whilst a small proportion of 5- to 6-day-old and the majority of 7-day-old parasitoids larvae survived from the virus-infected hosts. Viral contamination did not reduce the life span or fecundity of female M. similis.

Genome sequence and organization analysis of Heliothis virescens ascovirus 3f isolated from a Helicoverpa zea larva.

The complete genome sequence of Heliothis virescens ascovirus 3f (HvAV-3f) was obtained. The HvAV-3f genome has a circular genome of 198,157bp with a G+C content of 46.0%, and encodes 190 open reading frames (ORFs) longer than 69 amino acids. Two major homologous regions (hrs) and 29 'baculovirus repeat ORFs' (bro) were found in the genome. BLAST analyses revealed that three HvAV-3f genes were homologous to that of lepidopteran insects. Nine ORFs were unique to HvAV-3f, in which two ORFs showed significant levels of similarity to genes that have not been previously described for ascoviruses in the Genbank database.