Quercetin Alleviates Insulin Resistance and Repairs Intestinal Barrier in db/db Mice by Modulating Gut Microbiota.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease which seriously affects public health. Gut microbiota remains a dynamic balance state in healthy individuals, and its disorder may affect health status and even results in metabolic diseases. Quercetin, a natural flavonoid, has been shown to have biological activities that can be used in the prevention and treatment of metabolic diseases. This study aimed to explore the mechanism of quercetin in alleviating T2DM based on gut microbiota. db/db mice were adopted as the model for T2DM in this study. After 10 weeks of administration, quercetin could significantly decrease the levels of body weight, fasting blood glucose (FBG), serum insulin (INS), the homeostasis model assessment of insulin resistance (HOMA-IR), monocyte chemoattractant protein-1 (MCP-1), D-lactic acid (D-LA), and lipopolysaccharide (LPS) in db/db mice. 16S rRNA gene sequencing and untargeted metabolomics analysis were performed to compare the differences of gut microbiota and metabolites among the groups. The results demonstrated that quercetin decreased the abundance of Proteobacteria, Bacteroides, Escherichia-Shigella and Escherichia_coli. Moreover, metabolomics analysis showed that the levels of L-Dopa and S-Adenosyl-L-methionine (SAM) were significantly increased, but 3-Methoxytyramine (3-MET), L-Aspartic acid, L-Glutamic acid, and Androstenedione were significantly decreased under quercetin intervention. Taken together, quercetin could exert its hypoglycemic effect, alleviate insulin resistance, repair the intestinal barrier, remodel the intestinal microbiota, and alter the metabolites of db/db mice.

A novel yeast-based biosensor for the quick determination of Deoxynivalenol.

Mycotoxins are commonly found in food materials and severely threaten human health. Antibodies play a key role as a part of immunological techniques in detecting mycotoxins. Therefore, highly specific antibodies and detection techniques against mycotoxins need to be developed for advancements in medical research. In this study, we presented a novel strategy for quickly screening highly specific antigen-binding fragment (Fab) antibodies based on yeast surface display (YSD) and detecting small-molecule compounds based on a YSD biosensor. We constructed a yeast surface display Deoxynivalenol (DON)-Fab library with 105 cfu/mL with a galactose-inducible bidirectional promoter. By conducting efficient magnetic-activated cell sorting and fluorescence-activated cell sorting (MACS/FACS), four kinds of DON-selective yeasts were screened. As Fab@YSD C4# showed high sensitivity, we used it to build a one-pot Fab@YSD chemiluminescence biosensor with DON-BSA@Biotin and Streptavidin-alkaline phosphatase (SA-ALP). This method showed a low operational threshold (LOD = 0.166 pg/mL) and a high population range (linear range = 0.001-132.111 ng/mL) within 40 min, which facilitated the detection of DON with high specificity and better recovery in real samples (wheat, corn, flour, and cornmeal). Our results suggested that the Fab@YSD chemiluminescence biosensor is an inexpensive, reproducible, user-friendly, and sensitive method for detecting DON and may be used to quickly detect other small-molecule contaminants in food items.

Vitamin B6 alleviates chronic sleep deprivation-induced hippocampal ferroptosis through CBS/GSH/GPX4 pathway.

Several studies have found that sleep deprivation (SD) can lead to neuronal ferroptosis and affect hippocampal function. However, there are currently no effective interventions. Vitamin B6 is a co-factor for key enzymes in the transsulfuration pathway which is critical for maintaining cell growth in the presence of cysteine deprivation. The results showed that SD inhibited cystine-glutamate antiporter light chain subunit xCT protein expression and caused cysteine deficiency, which reduced the synthesis of the glutathione (GSH) to trigger neuronal ferroptosis. Nissl staining further revealed significant neuronal loss and shrinkage in the CA1 and CA3 regions of the hippocampus in SD mice. Typical ferroptotic indicators characterized by lipid peroxidation and iron accumulation were showed in the hippocampus after sleep deprivation. As expected, vitamin B6 could alleviate hippocampal ferroptosis by upregulating the expression of cystathionine beta-synthase (CBS) in the transsulfuration pathway, thereby replenishing the intracellular deficient GSH and restoring the expression of GPX4. Similar anti-ferroptotic effects of vitamin B6 were demonstrated in HT-22 cells treated with ferroptosis activator erastin. Furthermore, vitamin B6 had no inhibitory effect on erastin-induced ferroptosis in CBS-knockout HT22 cells. Our findings suggested chronic sleep deprivation caused hippocampal ferroptosis by disrupting the cyst(e)ine/GSH/GPX4 axis. Vitamin B6 alleviated sleep deprivation-induced ferroptosis by enhancing CBS expression in the transsulfuration pathway.

A novel universal small-molecule detection platform based on antibody-controlled Cas12a switching.

Molecular diagnostics play an important role in illness detection, prevention, and treatment, and are vital in point-of-care test. In this investigation, a novel CRISPR/Cas12a based small-molecule detection platform was developed using Antibody-Controlled Cas12a Biosensor (ACCBOR), in which antibody would control the trans-cleavage activity of CRISPR/Cas12a. In this system, small-molecule was labeled around the PAM sites of no target sequence(NTS), and antibody would bind on the labeled molecule to prevent the combination of CRISPR/Cas12a, resulting the decrease of trans-cleavage activity. Biotin-, digoxin-, 25-hydroxyvitamin D3 (25-OH-VD3)-labeled NTS and corresponding binding protein were separately used to verify its preformance, showing great universality. Finally, one-pot detection of 25-OH-VD3 was developed, exhibiting high sensitivity and excellent specificity. The limit of detection could be 259.86 pg/mL in serum within 30 min. This assay platform also has the advantages of low cost, easy operation (one-pot method), and fast detection (∼30 min), would be a new possibilities for the highly sensitive detection of other small-molecule targets.

Development of antibody-aptamer sandwich-like immunosensor based on RCA and Nicked-PAM CRISPR/Cas12a system for the ultra-sensitive detection of a biomarker.

Biomarkers are the most sensitive reactants and early indicators of many kinds of diseases. The development of highly sensitive and simple techniques to quantify them is challenging. In this study, based on rolling cycle amplification (RCA) and the Nicked PAM/CRISPR-Cas12a system (RNPC) as a signal reporter, a sandwich-type method was developed using antibody@magnetic beads and aptamer for the high-sensitive detection of the C-reactive protein (CRP). The antibody-antigen (target)-aptamer sandwich-like reaction was coupled to RCA, which can produce hundreds of similar binding sites and are discriminated by CRISPR/Cas12a for signal amplification. The ultrasensitivity is achieved based on the dual-signal enhancing strategy, which involves the special recognition of aptamers, RCA, and trans-cleavage of CRISPR/Cas12a. By incorporating the CRISPR/Cas12a system with cleaved PAM, the nonspecific amplification of the RCA reaction alone was greatly reduced, and the dual signal output of RCA and Cas12a improved the detection sensitivity. Our assay can be performed only in two steps. The first step takes only 20 min of target capture, followed by a one-pot reaction, where the target concentration can be obtained by fluorescence values as long as there are 37 °C reaction conditions. Under optimal conditions, this system detected CRP with high sensitivity. The fabricated biosensor showed detection limits of 0.40 pg/mL in phosphate-buffered saline and 0.73 pg/mL in diluted human serum and a broad linear dynamic range of 1.28 pg/mL to 100 ng/mL within a total readout time of 90 min. The method could be used to perform multi-step signal amplification, which can help in the ultrasensitive detection of other proteins. Overall, the proposed biosensor might be used as an immunosensor biosensor platform.

Nanobody-based magnetic chemiluminescence immunoassay for one-pot detection of ochratoxin A.

Ochratoxin A (OTA) contamination seriously threatens food safety and human health and requires sensitive and rapid tools for monitoring. In this study, a convenient enzyme-linked immunosorbent assay based on Avi-labeled nanobody Nb-2G/streptavidin-alkaline phosphatase and magnetic beads (MBS-ELISA) was established for the sensitive detection of OTA, which could be used for one-pot detection without immobilization. After optimization, the 50% inhibitory concentration (IC50) and the lowest limit of detection value of the MBS-ELISA was 1.17 ng/mL and 0.07 ng/mL and the linear range was 248.8 pg/mL-5.28 ng/mL, respectively, which accords with state criteria for food safety. The developed one-step MBS-ELISA was almost 20-times more sensitive than the classic BA-ELISA and could generate results within 15 min, which was significantly less than the classic BA-ELISA at approximately 3 h. The MBS-ELISA indicated good recovery (86.4-114.3%) in spiked sorghum, buckwheat, and mung bean. Thus, MBS-ELISA represents a very promising strategy for the simple, rapid, and accurate detection of OTA and other toxic and hazardous contaminants.

Sensitive colorimetric detection of antibiotic resistant Staphylococcus aureus on dairy farms using LAMP with pH-responsive polydiacetylene.

Rapidly and accurately detecting antibiotic-resistant pathogens in agriculture and husbandry is important since these represent a major threat to public health. While much attention has been dedicated to detecting now-common resistant bacteria, such as methicillin-resistant Staphylococcus aureus, fewer methods have been developed to assess resistance against macrolides in Staphylococcus aureus (SA). Here, we report a visual on-site detection system for macrolide resistant SA in dairy products. First, metagenomic sequencing in raw milk, cow manure, water and aerosol deposit collected from dairy farms around Tianjin was used to identify the most abundant macrolide resistance gene, which was found to be the macB gene. In parallel, SA housekeeping genes were screened to allow selective identification of SA, which resulted in the selection of the SAOUHSC_01275 gene. Next, LAMP assays targeting the above-mentioned genes were developed and interpreted by agarose gel electrophoresis. For on-site application, different pH-sensitive colorimetric LAMP indicators were compared, which resulted in selection of polydiacetylene (PDA) as the most sensitive candidate. Additionally, a semi-quantitative detection could be realized by analyzing the RGB information via smartphone with a LOD of 1.344 × 10-7 ng/µL of genomic DNA from a milk sample. Finally, the proposed method was successfully carried out at a real farm within 1 h from sample to result by using freeze-dried reagents and portable devices. This is the first instance in which PDA is used to detect LAMP products, and this generic read-out system can be expanded to other antibiotic resistant genes and bacteria.

A novel biosensor based on antibody controlled isothermal strand displacement amplification (ACISDA) system.

The overuse of antibiotics has aroused widespread concern in recent decades. Their residues in food and environment may pose potential risks to human health. Therefore, highly sensitive and rapid detection methods of antibiotics are urgently needed. Inspired by allosteric transcription factors (aTFs), we proposed a novel strategy for small molecules detection based on antibody controlled isothermal chain displacement amplification (ACISDA). A combination of nicking endonuclease, Klenow Fragment polymerase, specific antibody and a pair of antigen-labeled DNA regulate the synthesis of a G-quadruplex by isothermal chain displacement amplification. The presence of a target induces the antibody dissociation from the antigen-labeled DNA, which induces the synthesis of a G-quadruplex, and a fluorescent signal is produced by thioflavine T (ThT) binding to G-quadruplex. To test this notion, norfloxacin-conjugated DNA (named Primer-NOR) was prepared and ACISDA system was established combining with anti-norfloxacin antibody. This system could detect norfloxacin in a linear range of 0.1 ∼ 500 ng/mL with detection limit of 0.04 ng/mL, and this system could be applied to the detection of norfloxacin in real samples with good performance. Meanwhile, this system could also realize washing-free, immobilization-free and "ready-to-use", and could be used for other small molecules quickly by replacing the antigen-labeled DNA and specific antibody.

Bifunctional ligand-mediated amplification of polydiacetylene response to biorecognition of diethylstilbestrol for on-site smartphone detection.

Polydiacetylene (PDA) is very suited for sensitively detecting large biomolecules, and its unique chromatic properties enable visual read-out. However, application to the selective detection of small molecules remains challenging. Here, bifunctional ligands are studied to amplify the color change of PDA for biorecognition of small molecules for the smartphone-based detection of diethylstilbestrol (DES). PDA is decorated with streptavidin (PDA-SA, blue), and biotin-modified DES (bio-DES) is prepared as a bifunctional ligand to couple with PDA-SA and DES antibody. Since multiple bio-DES can bind to a single SA, then multiple SAs on PDA lead to an increased surface coverage of the vesicle. In samples without DES, PDA-SA-bio-DES-DES antibody complexes will form, leading to a color transition (blue to red); this color transition is greatly amplified by antibody-induced aggregation of the complexes. When DES is present, aggregation is inhibited due to competition for the antibody and PDA-SA-bio-DES retains its blue color. A linear relationship (0.4-1250 ng mL-1) is found between the colorimetric response and the logarithmic DES concentration, with adequate selectivity, accuracy (82.24-118.64%), and precision (below 8.24%). Finally, a paper-based DES PDA biosensor is developed with visual and smartphone-based detection limits of 10 ng mL-1 and 0.85 ng mL-1 in water, respectively.

A gold nanoparticle-based lateral flow immunoassay for atrazine point-of-care detection using a handhold scanning device as reader.

A method is described to achieve accurate quantitative detection of atrazine (ATZ) in maize by using lateral flow strips based on gold nanoparticles (GNPs) and a handheld scanning reader. GNPs of 15 nm in diameter were applied as label, and a lateral flow immune assay strip was prepared. The linear range was 5.01-95.86 ng mL-1 with a detection limit of 4.92 ng mL-1 in phosphate buffer, 4 times better than the readout by the naked eye. ATZ-spiked corn samples were also analysed. The accuracy of results of spiked samples was confirmed by ELISA and liquid chromatography-tandem mass spectrometry (HPLC), which proved the reliability of the proposed method. A handhold device with an optical scanning system was designed for on-site quantitative detection. Combined with the pretreatment, the assay could be completed in less than 20 min.

Rapid and ultrasensitive detection of DNA and microRNA-21 using a zirconium porphyrin metal-organic framework-based switch fluorescence biosensor.

Sensitive and accurate detection of nucleic acid biomarkers is critical for early cancer diagnosis, disease monitoring, and clinical treatment. In this study, we developed a switch fluorescence biosensor for simple and high-efficient detection of nucleic acid biomarkers using 6-carboxyfluorescein (FAM)-modified single-stranded DNA (ssDNA) probes (FAM-P1/P2), and zirconium porphyrin metal-organic framework nanoparticles (ZrMOF) acted as fluorescence quencher. FAM-P1/P2 probes were adsorbed on ZrMOF surface because of π-π stacking, hydrogen bonding, and electrostatic interactions. Fluorescence quenching event occurred by fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) processes, thereby achieving the "off" fluorescence status. Once the specific binding was formed between the fluorescence probes and the targets, the rigid double-stranded DNA (dsDNA) structures were released from ZrMOF surface, resulting in the recovery of fluorescence and the "on" status. Because of the superior adsorption ability of ZrMOF toward ssDNA than dsDNA, the switch of fluorescence signals from "off" to "on" allowed rapid and ultrasensitive detection of ssDNA (T1) and microRNA-21 (miR-21) within 30 min. The limit of detection (signal-to-noise ratio = 3) for T1 and miR-21 were 2 fM and 11 aM, respectively. Moreover, the proposed strategy was very simple as it worked by the facile adsorption-quenching-recovery mechanism without difficult and complicated immobilization processes. Also, this biosensor showed an excellent analytical performance in the detection of miR-21 in human serum samples. Therefore, this biosensor might be considered a potential tool for the detection of DNA and miRNA biomarkers in clinical samples.

A highly sensitive and dual-readout immunoassay for norfloxacin in milk based on QDs-FM@ALP-SA and click chemistry.

A dual-readout immunoassay based on QDs-FM@ALP-SA and click chemistry was developed for quick and sensitive detection of norfloxacin (NOR), which is an important fluoroquinolone antibiotic. In the system, the NOR-biotin conjugate (NOR-Biotin) was synthesized by click chemistry for signal transformation, and alkaline phosphatase-labeled streptavidin (ALP-SA) was attached to quantum dot fluorescence microspheres (QDs-FM) by an activated ester method to form QDs-FM@ALP-SA for signal amplification. Here, QDs-FM was a dual-functional carrier: it was used not only as a chemiluminescence signal amplification carrier but also as a fluorescent signal due to its fluorescence character. The NOR antibody was coated on a 96-well chemiluminescence microtiter plate, and NOR-Biotin was bound to the antibody specifically. Then, QDs-FM@ALP-SA was combined with NOR-Biotin to develop a direct competition chemiluminescence/fluorescence immunoassay (dc-CLIA/FIA). The IC50 values were 0.345 and 1.206 ng/mL for dc-CLIA/FIA, respectively. The linear range was 0.013-12.48 ng/mL and 0.042-39.86 ng/mL, respectively. The recovery from the standard fortified blank milk samples was in the range of 86.44%-101.3%. Therefore, this method could be a useful tool for routine screening of NOR residues in milk.

Detection of 4 quinolone antibiotics by chemiluminescence based on a novel Nor-Biotin bifunctional ligand and SA-ALP technology.

A simple and effective direct competitive chemiluminescence immunoassay for the detection of 4 kinds of quinolone antibiotics in milk was established using Nor-Biotin (biotin-modified norfloxacin [NOR]) bifunctional ligand and alkaline phosphatase-conjugated streptavidin signal amplification technology. The polyclonal antibody was obtained after the immunization of New Zealand White rabbits using norfloxacin-derived antigen. "Click chemistry" was used for the rapid and facile synthesis of the Nor-Biotin bifunctional ligand. After the optimization of the incubation time and reaction buffer, the direct competitive chemiluminescence assay method was developed and used for sensitive detection of 4 kinds of quinolone drugs (NOR, pefloxacin, ciprofloxacin, and danofloxacin). The IC50 of the 4 kinds of quinolone drugs ranged from 7.35 to 24.27 ng/mL, and the lowest detection limits ranged from 0.05 to 0.16 ng/mL, which were below their maximum residue levels, approved by the EU for treatment of food-producing animals. To demonstrate the applicability of the assay, artificially contaminated milk samples with the 4 quinolone drugs were analyzed. The mean recovery rates of the drugs ranged from 86.31% to 112.11%.

Simultaneous detection of diethylstilbestrol and estradiol residues with a single immunochromatographic assay strip.

An immunochromatographic assay (ICA) based on competitive format was developed and validated for simultaneously rapid and sensitive detection of diethylstilbestrol (DES) and estradiol (E2) in milk and tissue samples. For this purpose, two monoclonal antibodies raised against those two estrogens were conjugated to gold nanoparticles and were applied to the conjugate pads of the test strip. The competitors of the DES-BSA/E2-BSA conjugates were immobilized onto a nitrocellulose membrane at two detection zones to form T1 and T2, respectively. The immunochromatographic assay had a visual detection limit of DES at 30 ng/g in milk powder, 25 ng/g in liquid milk, and 25 ng/g in shrimp tissue, respectively, and the results can be judged within 7-10 min. The visual detection limit of E2 was 75 ng/g in milk powder, 65 ng/g in liquid milk, and 60 ng/g in shrimp tissue, respectively, and the results can be judged within 3-4 min. It had advantages in easy operation without requiring sophisticated equipment and specialized skills. By testing thirty milk and shrimp tissue samples from the local market, the method was compared with the HPLC-MS / MS method, and there was no statistical difference between the two methods. Furthermore, the immunochromatographic assay had good specificity, simple procedure, and low cost. This protocol was well suited for the food safety monitoring and early warning.

Complete antigen-bridged DNA strand displacement amplification immuno-PCR assay for ultrasensitive detection of salbutamol.

Monitoring of low-level analytes are typical examples for analytical challenges. Salbutamol (SAL), a phenol-ß2-agonist, has a very low residual content in the environment. Here, we present an ultrasensitive complete antigen-bridged PCR assay for detecting salbutamol (SAL). These DNA probes modified SAL complete antigens target recognition SAL antibodies and agglutinate synthetic DNA conjugates, thus enabling ligation of DNA probes to form a full-length DNA amplicon that contained a recognition site for cleavage endonuclease and subsequent quantification by qPCR. Moreover, SAL antibodies were modified with magnetic beads which were used to reduce the background noise and sample matrix effect, and the DNA signals were isothermally amplified by strand displacement amplification technology. Some key parameters which influence assay performance were optimized: the length of the bridge oligonucleotide, the concentration of immunomagnetic beads, SAL probes, and initiation chain, etc. Under the optimum conditions, the signal amplification of proposed Immuno-PCR assay for the detection of SAL was exponential, resulting in high potential sensitivity(~1 fg/mL) and a broad detection dynamic range (> 105 fold). Using this proposed method, we detected SAL in spiked tap water and urine samples with acceptable recoveries ranging from 88.1 to 103.3%. Theoretically, the method developed here has broad applicability and practical utility in immunoassays of a wide variety of analytes.

Effects of bisphenol A and nanoscale and microscale polystyrene plastic exposure on particle uptake and toxicity in human Caco-2 cells.

Microplastics are abundant in oceans, lakes, soils and even air, and can pose potential threats to human health through food or respiratory intake. Moreover, microplastics have synergistic toxicity to the body after absorbing organic pollutants. In this study, laser scanning confocal microscope and flow cytometry were used to observe the intake of colonic cancer Caco-2 cells to polystyrene plastic with five different particlesizes (300 nm, 500 nm, 1 µm, 3 µm, 6 µm). The uptake rates of microplastics with different particle sizes were 73%, 71%, 49%, 43%, and 30%, respectively. Then, High Performance Liquid Chromatography (HPLC) was used to analyze the adsorption differences of polystyrene plastic with different particle sizes to bisphenol A (BPA). Finally, the proliferation toxicity of polystyrene microplastics with different particle sizeson Caco-2 cells before and after adsorption of BPA was compared. MTT experiments confirmed that microplastics caused an increase in cytotoxicity. This result may be related to increased cellular oxidative stress and mitochondrial depolarization. This hypothesis has been confirmed in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) assays because nanoscale microplastics cause a large amount of ROS on Caco-2 cells after microplastic exposure, and micron-scale microplastics cause a significant decrease in MMP. At the same time, nanoscale microplastics can cause further depolarization of mitochondria due to their large specific surface area adsorption of BPA, which leads to enhanced cytotoxicity of microplastics after BPA adsorption. The results of this study are of great significance in the evaluation of the safety of microplastics in the human body.

Development of sandwich chemiluminescent immunoassay based on an anti-staphylococcal enterotoxin B Nanobody-Alkaline phosphatase fusion protein for detection of staphylococcal enterotoxin B.

In this study, sandwich chemiluminescent immunoassay (CLIA) for the detection of Staphylococcal enterotoxin B (SEB) was developed using nanobody-alkaline phosphatase (Nb-ALP) fusion protein. The SEB-binding nanobodies were obtained from a naïve phage-display library and the Nb-ALP fusion protein was constructed and obtained as a thermally stable and potentially effective substance for detecting antibodies in CLIA. The working range of the sandwich CLIA based on anti-SEB monoclonal antibodies (mAbs) and our fusion protein, Nb37-ALP, was 3.12-50.0 ng mL-1 with SC50 = 8.59 ± 0.37 ng mL-1. The limit of detection was 1.44 ng mL-1 according to the blank value plus 3 standard deviations. In order to understand the interaction of SEB and Nb37 in depth, the 3D structure of the SEB-Nb37 complex was constructed and verified by molecular modeling and the docking method. The results showed that the complementary-determining region 3 (CDR3) of Nb37 embedded itself in the opening generated by the major histocompatibility complex (MHC) and T-cell receptor- (TcR) binding sites of SEB, indicating that Nb37 may affect the recognition of SEB by MHC class Ⅱ molecules and the TcR. The arginine residue (Arg) 101, Arg102 and phenylalanine residue (Phe)103 of CDR3 in Nb37 may have contributed to specific binding to form six salt-bridges between these and SEB. In conclusion, in terms of their specificity and sensitivity, the obtained anti-SEB Nb-ALP appears to have the potential to replace chemically labeled probes for the detection of SEB.