Automatic Microfluidic Harmonized RAA-CRISPR Diagnostic System for Rapid and Accurate Identification of Bacterial Respiratory Tract Infections.

Respiratory tract infections (RTIs) pose a grave threat to human health, with bacterial pathogens being the primary culprits behind severe illness and mortality. In response to the pressing issue, we developed a centrifugal microfluidic chip integrated with a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats (CRISPR) system to achieve rapid detection of respiratory pathogens. The limitations of conventional two-step CRISPR-mediated systems were effectively addressed by employing the all-in-one RAA-CRISPR detection method, thereby enhancing the accuracy and sensitivity of bacterial detection. Moreover, the integration of a centrifugal microfluidic chip led to reduced sample consumption and significantly improved the detection throughput, enabling the simultaneous detection of multiple respiratory pathogens. Furthermore, the incorporation of Chelex-100 in the sample pretreatment enabled a sample-to-answer capability. This pivotal addition facilitated the deployment of the system in real clinical sample testing, enabling the accurate detection of 12 common respiratory bacteria within a set of 60 clinical samples. The system offers rapid and reliable results that are crucial for clinical diagnosis, enabling healthcare professionals to administer timely and accurate treatment interventions to patients.

Portable Biosensor with Bimetallic Metal-Organic Frameworks for Visual Detection and Elimination of Bacteria.

A multifunctional platform that meets the demands of both bacterial detection and elimination is urgently needed because of their harm to human health. Herein, a "sense-and-treat" biosensor was developed by using immunomagnetic beads (IMBs) and AgPt nanoparticle-decorated PCN-223-Fe (AgPt/PCN-223-Fe, PCN stands for porous coordination network) metal-organic frameworks (MOFs). The synthesized AgPt/PCN-223-Fe not only exhibited excellent peroxidase-like activity but also could efficiently kill bacteria under near infrared (NIR) irradiation. This biosensor enabled the colorimetric detection of E. coli O157:H7 in the range of 103-108 CFU/mL with a limit of detection of 276 CFU/mL, accompanied with high selectivity, good reproducibility, and wide applicability in diverse real samples. Furthermore, the biosensor possessed a highly effective antibacterial rate of 99.94% against E. coli O157:H7 under 808 nm light irradiation for 20 min. This strategy can provide a reference for the design of novel versatile biosensors for bacterial discrimination and antibacterial applications.

Microfluidic Biosensor Integrated with Signal Transduction and Enhancement Mechanism for Ultrasensitive Noncompetitive Assay of Multiple Mycotoxins.

To achieve high-throughput ultrasensitive detection of mycotoxins in food, a functional DNA-guided transition-state CRISPR/Cas12a microfluidic biosensor (named FTMB) was successfully constructed. The signal transduction CRISPR/Cas12a strategy in FTMB has utilized DNA sequences with a specific recognition function and activators to form trigger switches. Meanwhile, the transition-state CRISPR/Cas12a system was constructed by adjusting the composition ratio of crRNA and activator to achieve a high response for low concentrations of target mycotoxins. On the other hand, the signal enhancement of FTMB has efficiently integrated the signal output of quantum dots (QDs) with the fluorescence enhancement effect of photonic crystals (PCs). The construction of universal QDs for the CRISPR/Cas12a system and PC films matching the photonic bandgap produced a significant signal enhancement by a factor of 45.6. Overall, FTMB exhibited a wide analytic range (10-5-101 ng·mL-1), low detection of limit (fg·mL-1), short detection period (∼40 min), high specificity, good precision (coefficients of variation <5%), and satisfactory practical sample analysis capacity (the consistency with HPLC at 88.76%-109.99%). It would provide a new and reliable solution for the rapid detection of multiple small molecules in the fields of clinical diagnosis and food safety.

Nanozyme-Mediated Catalytic Signal Amplification for Microfluidic Biosensing of Foodborne Bacteria.

Early detection of foodborne bacteria is urgently needed to ensure food quality and to avoid the outbreak of foodborne bacterial diseases. Here, a kind of metal-organic framework (Zr-MOF) modified with Pt nanoparticles (Pt-PCN-224) was designed as a peroxidase-like signal amplifier for microfluidic biosensing of foodborne bacteria. Taking Escherichia coli (E. coli) O157:H7 as a model, a linear range from 2.93 × 102 to 2.93 × 108 CFU/mL and a limit of detection of 2 CFU/mL were obtained. The whole detection procedure was integrated into a single microfluidic chip. Water, milk, and cabbage samples were successfully detected, showing consistency with the results of the standard culture method. Recoveries were in the range from 90 to 110% in spiked testing. The proposed microfluidic biosensor realized the specific and sensitive detection of E. coli O157:H7 within 1 h, implying broad prospects of MOF with biomimetic enzyme activities for biosensing.

Fully Integrated Microfluidic Biosensor with Finger Actuation for the Ultrasensitive Detection of Escherichia coli O157:H7.

A portable microfluidic biosensor was developed for the detection of E. coli O157:H7 using finger actuation. The chip was assembled with three functional zones, immunomagnetic separation, nucleic acid extraction and purification, and signal detection. First, antibody-modified magnetic nanoparticles (MNPs) were used to separate the target bacteria from the sample. The captured bacteria were then lysed and silica-coated MNPs were used to absorb DNA, followed by washing and eluting to obtain purified DNA. The obtained DNA was subjected to amplification and fluorescence detection based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeat-associated protein/Cas12a reaction. The fluorescence images were collected and analyzed using a smartphone app under a 3D-printed detection device. It could quantitatively detect E. coli O157:H7 from 102 to 108 CFU/mL in 2.5 h with a limit of detection (LOD) of 10 CFU/mL. The recovery rate ranged from 104 to 120%. Overall, the biosensor realizes "sample-in and answer-out" assay for E. coli O157:H7 and eliminates the need for external pumps and skilled personnel.

Loop-mediated isothermal amplification-based microfluidic chip for pathogen detection.

Due to the significant growth of food production, the potential likelihood of food contamination is increasing. Foodborne illness caused by bacterial pathogens has considerably increased over the past decades, while at the same time, the species of harmful microorganisms also varied. Conventional bacterial culturing methods have been unable to satisfy the growing requirement for food safety inspections and food quality assurance. Therefore, rapid and simple detection methods are urgently needed. The loop-mediated isothermal amplification (LAMP) technology is a highly promising approach for the rapid and sensitive detection of pathogens, which allows nucleic acid amplification under isothermal conditions. The integration of the LAMP assay onto a microfluidic chip is highly compatible with point-of-care or resource-limited settings, as it offers the capability to perform experiments in combination with high screening efficiency. Here, we provide an overview of recent advances in LAMP-based microfluidic chip technology for detecting pathogens, based on real-time or endpoint determination mechanisms. We also discuss the promoting aspects of using the LAMP technique in a microfluidic platform, to supply a guideline for further molecular diagnosis and genetic analysis.

An ESIPT-based fluorescent probe for the determination of hypochlorous acid (HClO): mechanism study and its application in cell imaging.

A highly selective and sensitive probe for the detection of hypochlorous acid (HClO) in real samples was designed and synthesized by using the specific reaction between HClO and phenyl azo group. Upon reaction with HClO, the nonfluorescent probe generated a highly fluorescent 2-(2-hydroxy-4-chlorophenyl)benzimidazole (HBI-Cl) fluorophore, which underwent the excited state intramolecular proton transfer process to give strong fluorescence turn-on. The sensing mechanism, conversion of the nonfluorescent azo moiety into the fluorescent derivative of HBI upon reaction with HClO, was verified by independent synthesis of HBI-Cl (ϕfl ≈ 0.75). The theoretical computing results were in agreement with the experimental results that the azo moiety was the reactive site to realize fluorescence detection for HClO. Additionally, the probe was successfully utilized to determine HClO in tap water, exogenous HClO in HeLa cells, and endogenous HClO in MCF-7 cells with a low detection limit and cytotoxicity. Graphical abstract ᅟ.

Hydrogen sulphide improves adaptation of Zea mays seedlings to iron deficiency.

Hydrogen sulphide (H2S) is emerging as a potential molecule involved in physiological regulation in plants. However, whether H2S regulates iron-shortage responses in plants is largely unknown. Here, the role of H2S in modulating iron availability in maize (Zea mays L. cv Canner) seedlings grown in iron-deficient culture solution is reported. The main results are as follows: Firstly, NaHS, a donor of H2S, completely prevented leaf interveinal chlorosis in maize seedlings grown in iron-deficient culture solution. Secondly, electron micrographs of mesophyll cells from iron-deficient maize seedlings revealed plastids with few photosynthetic lamellae and rudimentary grana. On the contrary, mesophyll chloroplasts appeared completely developed in H2S-treated maize seedlings. Thirdly, H2S treatment increased iron accumulation in maize seedlings by changing the expression levels of iron homeostasis- and sulphur metabolism-related genes. Fourthly, phytosiderophore (PS) accumulation and secretion were enhanced by H2S treatment in seedlings grown in iron-deficient solution. Indeed, the gene expression of ferric-phytosiderophore transporter (ZmYS1) was specifically induced by iron deficiency in maize leaves and roots, whereas their abundance was decreased by NaHS treatment. Lastly, H2S significantly enhanced photosynthesis through promoting the protein expression of ribulose-1,5-bisphosphate carboxylase large subunit (RuBISCO LSU) and phosphoenolpyruvate carboxylase (PEPC) and the expression of genes encoding RuBISCO large subunit (RBCL), small subunit (RBCS), D1 protein (psbA), and PEPC in maize seedlings grown in iron-deficient solution. These results indicate that H2S is closely related to iron uptake, transport, and accumulation, and consequently increases chlorophyll biosynthesis, chloroplast development, and photosynthesis in plants.

A SARS-CoV-2 Nanobody Displayed on the Surface of Human Ferritin with High Neutralization Activity.

Purpose: COVID-19 is rampant throughout the world, which has caused great damage to human lives and seriously hindered the development of the global economy. Aiming at the treatment of SARS-CoV-2, in this study, we proposed a novel fenobody strategy based on ferritin (Fe) self-assembly technology. Methods: The neutralizing nanobody H11-D4 of SARS-CoV-2 fused to the C-terminus of end-modified human ferritin was expressed in E. coli and silkworm baculovirus expression systems. A large number of nanoparticles were successfully self-assembled in silkworms, while relatively few nanoparticles can be observed in the treated products from E. coli by electron microscopy. Subsequently, the fenobody's expression level and neutralizing activity were then evaluated. Results: The results showed that the IC50 of H11-D4 and fenobody Fe-H11-D4 expressed in E. coli were 171.1 nmol L-1 and 20.87 nmol L-1, respectively. However, the IC50 of Fe-HD11-D4 expressed in silkworms was 1.46 nmol L-1 showing better neutralization activity. Conclusion: Therefore, fenobodies can be well self-assembled in silkworm baculovirus expression system, and ferritin self-assembly technology can effectively improve nanobody neutralization activity.

High-throughput identification of meat ingredients in adulterated foods based on centrifugal integrated purification-CRISPR array.

Rapid, accurate, and sensitive analytical methods for the detection of food fraud are now an urgent requirement in the global food industry to ensure food quality. In response to this demand, a centrifugal integrated purification-CRISPR array for meat adulteration (CIPAM) was established. In detail, CIPAM system combines microneedles for DNA extraction and RAA-CRISPR/Cas12a integrated into a centrifugal microfluidic chip for the detection of meat adulteration. The RAA-CRISPR/Cas12a reaction reagents were pre-embedded into the different reaction chambers on the microfluidic chip to achieve the streamline of operations, markedly simplifying the detection process. The whole reaction was completed within 30 min with a detection limit of 0.1 % (w/w) in pig, chicken, duck, and lamb products. Referring to the results of the standard method, CIPAM system achieved 100 % accuracy. The automatic multiplex detection process implemented in the developed CIPAM system met the needs of food regulatory authorities.

Selenium-decorated biocompatible honeycomb films with redox-switchable surface for controlling cell adhesion/detachment.

HYPOTHESIS: Selenium (Se)-containing compound is sensitive to redox stimulation, showing hydrophobic-hydrophilic reversible transition. Introduction of such compound into honeycomb film could confer on it redox-switchable surface wettability, which is expected to control cell adhesion/detachment behavior. EXPERIMENTS: Didodecyl selenide was designed and mixed with polystyrene to prepare honeycomb films using "breath figure" method. The film microstructures were characterized by scanning electron microscope and atomic force microscopy, and the arrangement of Se atoms in honeycomb film was determined by X-ray photoelectron spectroscopy and energy dispersive spectrometry. The variation of film wettability upon the alternating stimulation of H2O2 and Vc was examined. Then the cell adhesion, proliferation, and controlled detachment on honeycomb films were conducted. FINDINGS: The introduction of didodecyl selenide helps to form ordered honeycomb film, and Se atoms were found to located on the bottom, pore walls, and top surface of the film. The presence of didodecyl selenide not only greatly improves film biocompatibility by enhancing cell thioredoxin reductase activity, but also imparts the film with H2O2-/vitamin C-regulated tunable wettability that controls cell adhesion and detachment. H2O2 treatment produces a hydrophilic surface for cell adhesion and proliferation, whereas the addition of vitamin C generates hydrophobic surfaces and allows cells to detach while remaining alive with high activity.

Multiplexed detection of foodborne pathogens using one-pot CRISPR/Cas12a combined with recombinase aided amplification on a finger-actuated microfluidic biosensor.

Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings.

Microfluidic biosensor for one-step detection of multiplex foodborne bacteria ssDNA simultaneously by smartphone.

As a major threat to food safety due to their pathogenicity, foodborne bacteria have received much attention. In this paper, we present a one-step and wash-free microfluidic biosensor platform by smartphone for simultaneous multiple foodborne bacteria target single-stranded DNA (ssDNA) detection. This technology is based on the fluorescence resonance energy transfer (FRET) between the graphene oxide (GO) and fluorescence molecules modified capture ssDNA of the target bacteria ssDNA (ctDNA) which were coated on the microfluidic chips. The fluorescence recovery was recorded by a smartphone fluorescent detector. With an optimal analytical performance, the platform realized the detection of four kinds of bacteria ssDNA simultaneously within 5 min, with the limits of detection (LODs) of 0.17, 0.18, 0.27, and 0.17 nM, respectively. And the throughput analysis of trace amounts of foodborne bacteria ssDNA in milk and water samples were successfully detected. This one-step and wash-free microfluidic biosensor can be used as a tool for food safety analysis.

Molecular Docking Revealed the Potential Anti-Oxidative Stress Mechanism of the Walnut Polypeptide on HT22 Cells.

The preparation of novel antioxidant peptides from food raw materials is one of the research focuses, but there are fewer studies on the preparation of antioxidant peptides from walnut meal, a by-product of processing walnuts. This study analyzed the antioxidant properties and protective effects of walnut protein hydrolyzed by alkaline protease and trypsin on the oxidative stress of HT22 cells. The peptides were identified by UPLC-MS/MS, and the anti-oxidative peptides were screened based on virtual computer tools. The potential anti-oxidative stress mechanism of the walnut polypeptide on HT22 cells was explored by molecular docking. The results revealed that walnut protein hydrolysates (WPH) with molecular weights of less than 1 kDa had good antioxidant properties and inhibited oxidative damage of HT22 cells by regulating the levels of reactive oxygen species (ROS) and antioxidant enzyme catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Six of the ninety identified new peptides showed good solubility, non-toxicity, and bioactivity. The molecular docking results showed that the six peptides could dock with Keap1 successfully, and EYWNR and FQLPR (single-letter forms of peptide writing) could interact with the binding site of Nrf2 in the Keap1-Kelch structural domain through hydrogen bonds with strong binding forces. The results of this study provided important information on the antioxidant molecular mechanism of the walnut polypeptide and provided a basis for further development of walnut antioxidant polypeptide products.

Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.

Baculovirus-expressed self-assembling SARS-CoV-2 nanoparticle vaccines targeting the S protein induce protective immunity in mice.

Spike (S) protein, a homotrimeric glycoprotein, is the most important antigen target for SARS-CoV-2 vaccines. A complete simulation of the advanced structure of this homotrimer during subunit vaccine development is the most likely method to improve its immunoprotective effects. In this study, preparation strategies for the S protein receptor-binding domain, S1 region, and ectodomain trimer nanoparticles were designed using ferritin nanoparticle self-assembly technology. The Bombyx mori baculovirus expression system was used to prepare three nanoparticle vaccines with high expression levels recorded in silkworms. The results in mice showed that the nanoparticle vaccine prepared using this strategy could induce immune responses when administered via both the subcutaneous administration and oral routes. Given the stability of these ferritin-based nanoparticle vaccines, an easy-to-use and low-cost oral immunization strategy can be employed in vaccine blind areas attributed to shortages of ultralow-temperature equipment and medical resources in underdeveloped areas. Oral vaccines are also promising candidates for limiting the spread of SARS-CoV-2 in domestic and farmed animals, especially in stray and wild animals.

Rapid quantitative detection of Vibrio parahaemolyticus via high-fidelity target-based microfluidic identification.

With the rapid development of logistics, a growing number of pathogenic microorganisms has the means to spread worldwide using food as a carrier; thus, there is an urgent need to develop effective detection strategies to ensure food safety. By combining novel markers identified by pan-genome analysis and a digital recombinase-aided amplification (RAA) detection method based on a microfluidic chip, a strategy of high-fidelity target-based microfluidic identification (HFTMI) has been developed. Herein, a proof-of-concept study of HFTMI for rapid pathogen detection of V. parahaemolyticus was investigated. Specific primers designed for the gene group_41170 identified in the pan-genome analysis showed high sensitivity and a broad spectrum for the detection of V. parahaemolyticus. Different power systems were investigated to increase the partition rate on specifically designed chamber-based digital chips. The performance of HFTMI was greatly improved compared with qPCR. Collectively, this novel HFTMI system provides more reliable guidance for food safety testing.

Novel multiplex PCR assays for rapid identification of Salmonella serogroups B, C1, C2, D, E, S. enteritidis, and S. typhimurium.

Foodborne illnesses caused by Salmonella represent a significant public health problem worldwide. The aim of this study was to establish multiplex PCR (mPCR) for the rapid identification of Salmonella serogroups B, C1, C2, D, and E as well as for the serovars enteritidis and typhimurium. Employing pan-genome analysis and PCR verification, B-rfbJ, C1-9679, C2-pimB, D-rfbJ, E-rfbC, and four genes (SE18636, SE16574, SE2599, and SE13329) were identified as specific target genes for Salmonella serogroups B, C1, C2, D, E, and S. enteritidis, respectively. Thereafter, three novel mPCR assays (one of 3-mPCR and two of 2-mPCR) were successfully developed to identify these bacteria based on the target genes and another S. typhimurium-specific STM4495 gene. The primers targeting C1-9679, C2-pimB, and E-rfbC genes specific to the serogroups C1, C2, and E, respectively, constituted a 3-mPCR, while the other two 2-mPCRs, respectively, consisting primers specific to serogroup D and S. enteritidis (D-rfbJ and SE16574), and serogroup B and S. typhimurium-specific primers (B-rfbJ and STM4495), were also designed. The specificity of each mPCR was further evaluated by using non-target strains. The detection limits of mPCRs were approximately 103-104 CFU mL-1 in pure culture and 104-105 CFU g-1 in spiked chicken meat. In addition, mPCR assays could correctly detect target Salmonella in food samples. These results suggest that specific targets could be mined efficiently through a pan-genome analysis tool, and the novel mPCR assays developed in this study offer a promising technique for rapid and accurate detection of five serogroups of Salmonella (B, C1, C2, D, and E) and two serovars (S. enteritidis and S. typhimurium).

Transcriptome analysis of pre-immune state induced by interferon gamma inhibiting the replication of H9N2 avian influenza viruses in chicken embryo fibroblasts.

Interferon (IFN), a critical antiviral cytokine produced by pathogens-induced cells, plays an important role in host innate immune system. In this study, to investigate the inhibition effect of IFN on avian influenza virus (AIV), Chicken Embryo Fibroblasts (CEFs) was infected by H9N2 AIV. The pre-immune state and transcriptome analysis have been observed and performed. The result showed chicken interferon gamma (chIFN-γ) have the most inhibitory effect on H9N2 virus among three types of chicken interferons (chIFNs). Inhibition of chIFN-γ on H9N2 virus was verified by indirect immunofluorescence, RT-qPCR and western blot. The possible signaling pathways induced by chIFN-γ with or without virus were analyzed by transcriptome. The transcriptome data were compared among H9N2-infected, chIFN-γ-treated, chIFN-γ + H9N2-treated, and Control groups. In summary, RNA-sequencing (RNA-seq) data suggested that H9N2 virus infection resulted in corresponding response of certain defensive, inflammatory and metabolism pathways to the virus replication in CEFs. Furthermore, while CEFs were treated with chIFN-γ, many immune-related signaling pathways in cells are affected and altered. Antiviral genes involved in these immune pathways such as interferon regulatory factors, chemokines, interferon-stimulated genes (ISGs) and transcription factors were significantly up-regulated, and showed significant antiviral responses. Compared with virus infected CEFs alone, pretreatment with IFN induced the expression of antiviral genes and activated related antiviral pathways, inhibited the viral replication as result. Our study provided functional annotations for antiviral genes and the basis for studying the mechanism of chIFN-γ mediated response against H9N2 AIV.

A microfluidic genoserotyping strategy for fast and objective identification of common Salmonella serotypes isolated from retail food samples in China.

Strict monitoring of Salmonella serotypes is the most effective way to limit the risk of its transmission to humans. In this study, a microfluidic genoserotyping strategy was developed for the rapid and cost-effective detection of 11 common Salmonella serotypes from retail food samples, i.e. Typhimurium, Derby, Indiana, Agona, Rissen, Braenderup, Hadar, Enteritidis, Weltevreden, Meleagridis, and Pomona. The programmable LAMP in the strategy can complete the whole detection process within 40 min and avoid false positive results. The limit of detection was 102 or 103 CFU/mL. Referring to the results of standard culture method, the LAMP-chip was verified with 100% accuracy on testing 688 Salmonella and 22 non-Salmonella strains. This simple, portable and accurate microfluidic genoserotyping strategy is fully adapted to the needs of food regulatory authorities and has wide application prospect in food safety field.