Ultrasensitive and Highly Selective Detection of Staphylococcus aureus at the Single-Cell Level Using Bacteria-Imprinted Polymer and Vancomycin-Conjugated MnO2 Nanozyme.

Pathogenic bacterial infections, even at extremely low concentrations, pose significant threats to human health. However, the challenge persists in achieving high-sensitivity bacterial detection, particularly in complex samples. Herein, we present a novel sandwich-type electrochemical sensor utilizing bacteria-imprinted polymer (BIP) coupled with vancomycin-conjugated MnO2 nanozyme (Van@BSA-MnO2) for the ultrasensitive detection of pathogenic bacteria, exemplified by Staphylococcus aureus (S. aureus). The BIP, in situ prepared on the electrode surface, acts as a highly specific capture probe by replicating the surface features of S. aureus. Vancomycin (Van), known for its affinity to bacterial cell walls, is conjugated with a Bovine serum albumin (BSA)-templated MnO2 nanozyme through EDC/NHS chemistry. The resulting Van@BSA-MnO2 complex, serving as a detection probe, provides an efficient catalytic platform for signal amplification. Upon binding with the captured S. aureus, the Van@BSA-MnO2 complex catalyzes a substrate reaction, generating a current signal proportional to the target bacterial concentration. The sensor displays remarkable sensitivity, capable of detecting a single bacterial cell in a phosphate buffer solution. Even in complex milk matrices, it maintains outstanding performance, identifying S. aureus at concentrations as low as 10 CFU mL-1 without requiring intricate sample pretreatment. Moreover, the sensor demonstrates excellent selectivity, particularly in distinguishing target S. aureus from interfering bacteria of the same genus at concentrations 100-fold higher. This innovative method, employing entirely synthetic materials, provides a versatile and low-cost detection platform for Gram-positive bacteria. In comparison to existing nanozyme-based bacterial sensors with biological recognition materials, our assay offers distinct advantages, including enhanced sensitivity, ease of preparation, and cost-effectiveness, thereby holding significant promise for applications in food safety and environmental monitoring.

A Novel DNA Synthesis Platform Design with High-Throughput Paralleled Addressability and High-Density Static Droplet Confinement.

Using DNA as the next-generation medium for data storage offers unparalleled advantages in terms of data density, storage duration, and power consumption as compared to existing data storage technologies. To meet the high-speed data writing requirements in DNA data storage, this paper proposes a novel design for an ultra-high-density and high-throughput DNA synthesis platform. The presented design mainly leverages two functional modules: a dynamic random-access memory (DRAM)-like integrated circuit (IC) responsible for electrode addressing and voltage supply, and the static droplet array (SDA)-based microfluidic structure to eliminate any reaction species diffusion concern in electrochemical DNA synthesis. Through theoretical analysis and simulation studies, we validate the effective addressing of 10 million electrodes and stable, adjustable voltage supply by the integrated circuit. We also demonstrate a reaction unit size down to 3.16 × 3.16 µm2, equivalent to 10 million/cm2, that can rapidly and stably generate static droplets at each site, effectively constraining proton diffusion. Finally, we conducted a synthesis cycle experiment by incorporating fluorescent beacons on a microfabricated electrode array to examine the feasibility of our design.

A Novel Microfluidic Strategy for Efficient Exosome Separation via Thermally Oxidized Non-Uniform Deterministic Lateral Displacement (DLD) Arrays and Dielectrophoresis (DEP) Synergy.

Exosomes, with diameters ranging from 30 to 150 nm, are saucer-shaped extracellular vesicles (EVs) secreted by various type of human cells. They are present in virtually all bodily fluids. Owing to their abundant nucleic acid and protein content, exosomes have emerged as promising biomarkers for noninvasive molecular diagnostics. However, the need for exosome separation purification presents tremendous technical challenges due to their minuscule size. In recent years, microfluidic technology has garnered substantial interest as a promising alternative capable of excellent separation performance, reduced reagent consumption, and lower overall device and operation costs. In this context, we hereby propose a novel microfluidic strategy based on thermally oxidized deterministic lateral displacement (DLD) arrays with tapered shapes to enhance separation performance. We have achieved more than 90% purity in both polystyrene nanoparticle and exosome experiments. The use of thermal oxidation also significantly reduces fabrication complexity by avoiding the use of high-precision lithography. Furthermore, in a simulation model, we attempt to integrate the use of dielectrophoresis (DEP) to overcome the size-based nature of DLD and distinguish particles that are close in size but differ in biochemical compositions (e.g., lipoproteins, exomeres, retroviruses). We believe the proposed strategy heralds a versatile and innovative platform poised to enhance exosome analysis across a spectrum of biochemical applications.

Nematicidal glycosylated resorcylic acid lactones from the fungus Pochonia chlamydosporia PC-170 and their key biosynthetic genes.

Chemical study of the nematicidal biocontrol fungus Pochonia chlamydosporia PC-170 led to discovery of six resorcylic acid lactones (RALs), including three nematicidal glycosylated RALs, monocillin VI glycoside (1), colletogloeolactone A (2) and monocillin II glycoside (3), and three antibacterial non-glycosylated RALs, monocillin VI (4), monocillin IV (5) and monocillin II (6). The planar structure of the new compound monocillin VI glycoside (1) was elucidated using HRESIMS and NMR data, and its monosaccharide configuration was further determined through sugar hydrolysis experiment and GC-MS analysis method. Furthermore, their two biosynthetic-related PKS genes, pchE and pchI, were identified through the gene knockout experiment. The glycosylated RALs 1-3 exhibited nematicidal activity against Meloidogyne incognita, with LC50 values of 94, 152 and 64 µg/mL, respectively, and thus had great potential in the development of new nematicidal natural products to control M. incognita in the future.

NRPS-like ATRR in Plant-Parasitic Nematodes Involved in Glycine Betaine Metabolism to Promote Parasitism.

Plant-parasitic nematodes (PPNs) are among the most serious phytopathogens and cause widespread and serious damage in major crops. In this study, using a genome mining method, we identified nonribosomal peptide synthetase (NRPS)-like enzymes in genomes of plant-parasitic nematodes, which are conserved with two consecutive reducing domains at the N-terminus (A-T-R1-R2) and homologous to fungal NRPS-like ATRR. We experimentally investigated the roles of the NRPS-like enzyme (MiATRR) in nematode (Meloidogyne incognita) parasitism. Heterologous expression of Miatrr in Saccharomyces cerevisiae can overcome the growth inhibition caused by high concentrations of glycine betaine. RT-qPCR detection shows that Miatrr is significantly upregulated at the early parasitic life stage (J2s in plants) of M. incognita. Host-derived Miatrr RNA interference (RNAi) in Arabidopsis thaliana can significantly decrease the number of galls and egg masses of M. incognita, as well as retard development and reduce the body size of the nematode. Although exogenous glycine betaine and choline have no obvious impact on the survival of free-living M. incognita J2s (pre-parasitic J2s), they impact the performance of the nematode in planta, especially in Miatrr-RNAi plants. Following application of exogenous glycine betaine and choline in the rhizosphere soil of A. thaliana, the numbers of galls and egg masses were obviously reduced by glycine betaine but increased by choline. Based on the knowledge about the function of fungal NRPS-like ATRR and the roles of glycine betaine in host plants and nematodes, we suggest that MiATRR is involved in nematode-plant interaction by acting as a glycine betaine reductase, converting glycine betaine to choline. This may be a universal strategy in plant-parasitic nematodes utilizing NRPS-like ATRR to promote their parasitism on host plants.

Development of sensorimotor-visual connectome gradient at birth predicts neurocognitive outcomes at 2 years of age.

Functional connectome gradients represent fundamental organizing principles of the brain. Here, we report the development of the connectome gradients in preterm and term babies aged 31-42 postmenstrual weeks using task-free functional MRI and its association with postnatal cognitive growth. We show that the principal sensorimotor-to-visual gradient is present during the late preterm period and continuously evolves toward a term-like pattern. The global measurements of this gradient, characterized by explanation ratio, gradient range, and gradient variation, increased with age (p < 0.05, corrected). Focal gradient development mainly occurs in the sensorimotor, lateral, and medial parietal regions, and visual regions (p < 0.05, corrected). The connectome gradient at birth predicts cognitive and language outcomes at 2-year follow-up (p < 0.005). These results are replicated using an independent dataset from the Developing Human Connectome Project. Our findings highlight early emergent rules of the brain connectome gradient and their implications for later cognitive growth.

Capacitive Bionic Magnetic Sensors Based on One-Step Biointerface Preparation.

Magnetic biomolecule-based bionic magnetic field sensors are anticipated to open up novel pathways for magnetic field detection. The detection range and accuracy of current bionic magnetic field sensors are limited, and little work is based on the capacitive response principle. We successfully developed a biochemical interface with an extralarge target-receptor size ratio, which can be manufactured in a single step for weak magnetic field detection across a wide frequency range, and we used electrochemical capacitance as a magnetic field change conduction strategy. The thickness-controllable nanoscale bovine serum albumin/graphene layer on an indium tin oxide working electrode combines with the one-step preparation method to immobilize the MagR/Cry4 complex. This capacitive bionic magnetic sensor can achieve the detection range of 0-120 mT. This biointerface design strategy obtains the further improvement of the performance of this bionic magnetic field sensor. Furthermore, the biointerface construction and optimization methodology in this proposal has potential applications in the design of other medical biosensors.

Genome-wide transcriptome profiling reveals molecular response pathways of Trichoderma harzianum in response to salt stress.

Trichoderma harzianum exhibits a strong biological control effect on many important plant pathogens, such as Fusarium oxysporum, Botrytis cinerea, and Meloidogyne. However, its biocontrol effectiveness is weakened or reduced under salt stress. The aim of this study was to investigate the molecular response of T. harzianum to salt stress at the whole-genome level. Here, we present a 44.47 Mb near-complete genome assembly of the T. harzianum qt40003 strain for the first time, which was assembled de novo with 7.59 Gb Nanopore sequencing long reads (~170-fold) and 5.2 Gb Illumina short reads (~116-fold). The assembled qt40003 genome contains 12 contigs, with a contig N50 of 4.81 Mb, in which four of the 12 contigs were entirely reconstructed in a single chromosome from telomere to telomere. The qt40003 genome contains 4.27 Mb of repeat sequences and 12,238 protein-coding genes with a BUSCO completeness of 97.5%, indicating the high accuracy and completeness of our gene annotations. Genome-wide transcriptomic analysis was used to investigate gene expression changes related to salt stress in qt40003 at 0, 2% (T2), and 4% (T4) sodium chloride concentrations. A total of 2,937 and 3,527 differentially expressed genes (DEGs) were obtained under T2 and T4 conditions, respectively. GO enrichment analysis showed that the T2-treatment DEGs were highly enriched in detoxification (p < 0.001), while the T4 DEGs were mainly enriched in cell components, mostly in cellular detoxification, cell surface, and cell wall. KEGG metabolic pathway analysis showed that 91 and 173 DEGs were significantly enriched in the T2 and T4 treatments, respectively (p < 0.01), mainly in the glutathione metabolism pathway. We further experimentally analyzed the differentially expressed glutathione transferase genes in the glutathione metabolic pathway, most of which were downregulated (13/15). In addition, we screened 13 genes related to active oxygen clearance, including six upregulated and seven downregulated genes, alongside five fungal hydrophobic proteins, of which two genes were highly expressed. Our study provides high-quality genome information for the use of T. harzianum for biological control and offers significant insights into the molecular responses of T. harzianum under salt-stress conditions.

The root-knot nematode effector Mi2G02 hijacks a host plant trihelix transcription factor to promote nematode parasitism.

Root-knot nematodes (RKNs) cause huge agricultural losses every year. They secrete a repertoire of effectors to facilitate parasitism through the induction of plant-derived giant feeding cells, which serve as their sole source of nutrients. However, the mode of action of these effectors and their targeted host proteins remain largely unknown. In this study, we investigated the role of the effector Mi2G02 in Meloidogyne incognita parasitism. Host-derived Mi2G02 RNA interference in Arabidopsis thaliana affected giant cell development, whereas ectopic expression of Mi2G02 promoted root growth and increased plant susceptibility to M. incognita. We used various combinations of approaches to study the specific interactions between Mi2G02 and A. thaliana GT-3a, a trihelix transcription factor. GT-3a knockout in A. thaliana affected feeding-site development, resulting in production of fewer egg masses, whereas GT-3a overexpression in A. thaliana increased susceptibility to M. incognita and also root growth. Moreover, we demonstrated that Mi2G02 plays a role in maintaining GT-3a protein stabilization by inhibiting the 26S proteasome-dependent pathway, leading to suppression of TOZ and RAD23C expression and thus promoting nematode parasitism. This work enhances our understanding of how a pathogen effector manipulates the role and regulation of a transcription factor by interfering with a proteolysis pathway to reprogram gene expression for development of nematode feeding cells.

In pursuit of degenerative brain disease diagnosis: Dementia biomarkers detected by DNA aptamer-attached portable graphene biosensor.

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aß), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aß, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aß, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.

Meloidogyne enterolobii MeMSP1 effector targets the glutathione-S-transferase phi GSTF family in Arabidopsis to manipulate host metabolism and promote nematode parasitism.

Meloidogyne enterolobii is an emerging root-knot nematode species that overcomes most of the nematode resistance genes in crops. Nematode effector proteins secreted in planta are key elements in the molecular dialogue of parasitism. Here, we show the MeMSP1 effector is secreted into giant cells and promotes M. enterolobii parasitism. Using co-immunoprecipitation and bimolecular fluorescent complementation assays, we identified glutathione-S-transferase phi GSTFs as host targets of the MeMSP1 effector. This protein family plays important roles in plant responses to abiotic and biotic stresses. We demonstrate that MeMSP1 interacts with all Arabidopsis GSTF. Moreover, we confirmed that the N-terminal region of AtGSTF9 is critical for its interaction, and atgstf9 mutant lines are more susceptible to root-knot nematode infection. Combined transcriptome and metabolome analyses showed that MeMSP1 affects the metabolic pathways of Arabidopsis thaliana, resulting in the accumulation of amino acids, nucleic acids, and their metabolites, and organic acids and the downregulation of flavonoids. Our study has shed light on a novel effector mechanism that targets plant metabolism, reducing the production of plant defence-related compounds while favouring the accumulation of metabolites beneficial to the nematode, and thereby promoting parasitism.

A root-knot nematode effector manipulates the rhizosphere microbiome for establishing parasitism relationship with hosts.

Introduction: Root-knot nematode (RKN; Meloidogyne spp.) is one of the most infamous soilborne plant diseases, causing severe crop losses every year. Effector proteins secreted by RKNs play crucial roles during plant-nematode interaction. However, less is known about whether RKN effector proteins can impact the rhizosphere microbial environment. Methods: In this study, we investigated the rhizosphere microbiome community of MiMIF-2 (a plant immunity-modulating effector) transgenic Arabidopsis thaliana with or without nematode infection using the Illumina high-throughput sequencing analysis. Results and discussion: The results showed that the bacterial species richness index increased, while the fungi species richness index decreased in M. incognita-infected MiMIF-2 transgenic A. thaliana plants. The relative abundance of genera such as Clitopilus, Komagataeibacter, Lactobacillus, Prevotella, Moritella, Vibrio, Escherichia-Shigella, and Pseudomonas was reduced in MiMIF-2 transgenic A. thaliana plants compared to wild type, but was significantly increased after inoculation with M. incognita. The Cluster of Orthologous Genes (COG) function classification analysis revealed a decrease in the relative abundance of defense mechanisms, secondary metabolite biosynthesis, transport, and nematode infection catabolism-related functions in MiMIF-2 lines compared to the wild type. These differences may be the reason for the increased susceptibility of MiMIF-2 transgenic A. thaliana to nematode infection. Our results provide a new insight into RKN effector proteins and their association with the microbial community, host, and plant pathogens, which will lead to the exploration of new innovative ideas for future biological control of RKNs.

Synergistic Modulation of a Tunable Microenvironment to Fabricate a Liver Fibrosis Chip for Drug Testing.

Liver fibrosis is a progressive physiological change that occurs after liver injury and seriously endangers human health. The lack of reliable and physiologically relevant pathological models of liver fibrosis leads to a longer drug development period and sizeable economic investment. The fabrication of a biomimetic liver-on-a-chip is significant for liver disease treatment and drug development. Here, a sandwich chip with a microwell array structure in its bottom layer was fabricated to simulate the Disse space structure of hepatic sinusoids in vitro. By synergistic modulation of the cross-linking degree of gelatin-methacryloyl (GelMA) hydrogels and the induction of transforming growth factor-beta (TGF-ß), the early and late stages of liver fibrosis were designed in the chip. Owing to its three-dimensional-mixed-culture strategy, it was possible to construct a liver sinusoid model in vitro to allow for faithful physiological emulation. The model was further subjected to drug treatment, and it presented a significant difference in treatment response in early and late fibrosis progression. Our system provides a unique method for emulating liver function through a vitro liver fibrosis-on-a-chip, potentially paving the way for investigating human liver fibrosis and related drug development.

A mosquito mouthpart-like bionic neural probe.

Advancements in microscale electrode technology have revolutionized the field of neuroscience and clinical applications by offering high temporal and spatial resolution of recording and stimulation. Flexible neural probes, with their mechanical compliance to brain tissue, have been shown to be superior to rigid devices in terms of stability and longevity in chronic recordings. Shuttle devices are commonly used to assist flexible probe implantation; however, the protective membrane of the brain still makes penetration difficult. Hidden damage to brain vessels during implantation is a significant risk. Inspired by the anatomy of the mosquito mouthparts, we present a biomimetic neuroprobe system that integrates high-sensitivity sensors with a high-fidelity multichannel flexible electrode array. This customizable system achieves distributed and minimally invasive implantation across brain regions. Most importantly, the system's nonvisual monitoring capability provides an early warning detection for intracranial soft tissues, such as vessels, reducing the potential for injury during implantation. The neural probe system demonstrates exceptional sensitivity and adaptability to environmental stimuli, as well as outstanding performance in postoperative and chronic recordings. These findings suggest that our biomimetic neural-probe device offers promising potential for future applications in neuroscience and brain-machine interfaces. A mosquito mouthpart-like bionic neural probe consisting of a highly sensitive tactile sensor module, a flexible microelectrode array, and implanted modules that mimic the structure of mosquito mouthparts. The system enables distributed implantation of electrode arrays across multiple brain regions while making the implantation minimally invasive and avoiding additional dural removal. The tactile sensor array can monitor the implantation process to achieve early warning of vascular damage. The excellent postoperative short-term recording performance and long-term neural activity tracking ability demonstrate that the system is a promising tool in the field of brain-computer interfaces.

Magnetic Bead Manipulation in Microfluidic Chips for Biological Application.

Magnetic beads manipulation in microfluidic chips is a promising research field for biological application, especially in the detection of biological targets. In this review, we intend to present a thorough and in-depth overview of recent magnetic beads manipulation in microfluidic chips and its biological application. First, we introduce the mechanism of magnetic manipulation in microfluidic chip, including force analysis, particle properties, and surface modification. Then, we compare some existing methods of magnetic manipulation in microfluidic chip and list their biological application. Besides, the suggestions and outlook for future developments in the magnetic manipulation system are also discussed and summarized.

Droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction and amplification for the detection of pathogens and tumor mutation sites.

Traditional nucleic acid extraction and detection is based on open operation, which may cause cross-contamination and aerosol formation. This study developed a droplet magnetic-controlled microfluidic chip integrated nucleic acid extraction, purification and amplification. The reagent is sealed in oil to form a droplet, and the nucleic acid is extracted and purified by controlling the movement of the magnetic beads (MBs) through a permanent magnet, ensuring a closed environment. This chip can automatically extract nucleic acid from multiple samples within 20 min, and can be directly placed in the in situ amplification instrument for amplification without further transfer of nucleic acid, characterized by simple, fast, time-saving and labor-saving. The results showed that the chip was able to detect <10 copies/test SARS-CoV-2 RNA, and EGFR exon 21 L858R mutations were detected in H1975 cells as low as 4 cells. In addition, on the basis of the droplet magnetic-controlled microfluidic chip, we further developed a multi-target detection chip, which used MBs to divide the nucleic acid of the sample into three parts. And the macrolides resistance mutations A2063G and A2064G, and the P1 gene of mycoplasma pneumoniae (MP) were successfully detected in clinical samples by the multi-target detection chip, providing the possibility for future application in the detection of multiple pathogens.

Microfluidic based single cell or droplet manipulation: Methods and applications.

The isolation of single cell or droplet is first and crucial step to single-cell analysis, which is important for cancer research and diagnostic methods. This review provides an overview of technologies that are currently used or in development to realize the isolation. Microfluidic based manipulation is an emerging technology with the distinct advantages of miniaturization and low cost. Therefore, recent developments in microfluidic isolated methods have attracted extensive attention. We introduced herein five strategies based on microfluid: trap, microfluidic discrete manipulation, bioprinter, capillary and inertial force. For every technology, their basic principles and features were discussed firstly. Then some modified approaches and applications were listed as the extension. Finally, we compared the advantages and drawbacks of these methods, and analyzed the trend of the manipulation based on microfluidics.

Variation and stability of rhizosphere bacterial communities of Cucumis crops in association with root-knot nematodes infestation.

Introduction: Root-knot nematodes (RKN) disease is a devastating disease in Cucumis crops production. Existing studies have shown that resistant and susceptible crops are enriched with different rhizosphere microorganisms, and microorganisms enriched in resistant crops can antagonize pathogenic bacteria. However, the characteristics of rhizosphere microbial communities of Cucumis crops after RKN infestation remain largely unknown. Methods: In this study, we compared the changes in rhizosphere bacterial communities between highly RKN-resistant Cucumis metuliferus (cm3) and highly RKN-susceptible Cucumis sativus (cuc) after RKN infection through a pot experiment. Results: The results showed that the strongest response of rhizosphere bacterial communities of Cucumis crops to RKN infestation occurred during early growth, as evidenced by changes in species diversity and community composition. However, the more stable structure of the rhizosphere bacterial community in cm3 was reflected in less changes in species diversity and community composition after RKN infestation, forming a more complex and positively co-occurrence network than cuc. Moreover, we observed that both cm3 and cuc recruited bacteria after RKN infestation, but the bacteria enriched in cm3 were more abundant including beneficial bacteria Acidobacteria, Nocardioidaceae and Sphingomonadales. In addition, the cuc was enriched with beneficial bacteria Actinobacteria, Bacilli and Cyanobacteria. We also found that more antagonistic bacteria than cuc were screened in cm3 after RKN infestation and most of them were Pseudomonas (Proteobacteria, Pseudomonadaceae), and Proteobacteria were also enriched in cm3 after RKN infestation. We hypothesized that the cooperation between Pseudomonas and the beneficial bacteria in cm3 could inhibit the infestation of RKN. Discussion: Thus, our results provide valuable insights into the role of rhizosphere bacterial communities on RKN diseases of Cucumis crops, and further studies are needed to clarify the bacterial communities that suppress RKN in Cucumis crops rhizosphere.

An adaptive three-dimensional hydrodynamic focusing microfluidic impedance flow cytometer.

Microfluidic impedance cytometry is emerging as a label-free, low-cost and portable solution for cell analysis. Impedance-based cell or particle characterization is provided by microfluidic and electronic devices. We report the design and characterization of a miniaturized flow cytometer based on a 3-dimensional (3D) hydrodynamic focusing mechanism. A sheath adaptively concentrated the sample laterally and vertically at the bottom of the microchannel, reducing the variance of particle translocation height and increasing the signal-to-noise ratio of the particle impedance pulse. Through simulation and confocal microscopy experiments, it has been verified that an increase in the ratio of sheath to sample decreased the cross-sectional area of the concentrated stream, which can be reduced to 26.50% of the pre-focusing value. The appropriate sheath flow settings increased the impedance pulse amplitude for different particles, and the coefficient of variation reduced by at least 35.85%, contributing to a more accurate representation of the particle impedance characteristic distribution. The system displayed the difference of HepG2 cell impedance before and after drug treatment, which is consistent with the results of flow cytometry, providing a convenient and inexpensive solution for monitoring cell status.

On-Chip Nucleic Acid Purification Followed by ddPCR for SARS-CoV-2 Detection.

We developed a microfluidic chip integrated with nucleic acid purification and droplet-based digital polymerase chain reaction (ddPCR) modules to realize a 'sample-in, result-out' infectious virus diagnosis. The whole process involved pulling magnetic beads through drops in an oil-enclosed environment. The purified nucleic acids were dispensed into microdroplets by a concentric-ring, oil-water-mixing, flow-focusing droplets generator driven under negative pressure conditions. Microdroplets were generated with good uniformity (CV = 5.8%), adjustable diameters (50-200 µm), and controllable flow rates (0-0.3 µL/s). Further verification was provided by quantitative detection of plasmids. We observed a linear correlation of R2 = 0.9998 in the concentration range from 10 to 105 copies/µL. Finally, this chip was applied to quantify the nucleic acid concentrations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The measured nucleic acid recovery rate of 75 ± 8.8% and detection limit of 10 copies/µL proved its on-chip purification and accurate detection abilities. This chip can potentially be a valuable tool in point-of-care testing.