A dual-mode lateral flow immunoassay by ultrahigh signal-to background ratio SERS probes for nitrofurazone metabolites ultrasensitive detection.

In this work, an ultra-sensitive lateral flow immunoassay (LFIA) with SERS/colorimetric dual signal mode was constructed for the detection of nitrofurazone metabolites, an antibiotic prohibited in animal-origin foods. Au@4-MBN@AgNRs nano-sandwich structural signal tag integrates the unique advantages of high signal-to-background ratio and anti-matrix interference through geometric control of SERS tag and nanoengineering adjustment of chemical composition. Under the optimal conditions, the detection limits of nitrofurazone metabolites by SERS/colorimetric dual-mode LFIA were 20 pg/mL (colorimetric mode) and 0.08 pg/mL (SERS mode). Excitingly, the vLOD of the colorimetric signal improved by a factor of 100 compared to Au NPs-based LFIA. In this study, the proposed dual-mode LFIA was successfully applied to the on-site real-time detection of honey, milk powder, and chicken. It is anticipated that with low background interference and anti-matrix interference output signal, our proposed dual-mode strategy can pave an innovative pathway for the fabrication of a powerful biosensor.

IL-33/ST2L signaling alleviates diabetic nephropathy by regulating endoplasmic reticulum stress and apoptosis.

OBJECTIVE: Diabetic nephropathy (DN) is a serious chronic complication of diabetes mellitus (DM). Endoplasmic reticulum (ER) stress is an important factor in the regulation of pathological processes in DN, and excessive ER stress can lead to apoptosis. Although the IL-33/ST2 axis is known to be involved in diabetic kidney disease or related nephropathy, its role and molecular mechanisms remain poorly understood in terms of DN. The purpose of this study was to investigate the effects of IL-33/ST2 signaling on DN and to characterize the roles that ER stress and apoptosis play in DN. METHODS: To investigate this study, mice were randomly assigned into DN (induced by 0.1% STZ) and Control groups. Biochemical indices (FBG, BUN, UPR, UCE) were measured in serum and urine samples to reflect blood glucose and kidney damage. Quantitative real-time PCR, western blot, and immunofluorescence were used to assess gene and protein expression of the IL-33/ST2 axis and ER stress relative signaling molecule. Apoptosis was analyzed by flow cytometry. RESULTS: IL-33 levels are significantly increased in the kidneys of patients and mice with DN. Double immunofluorescence staining showed that IL-33 colocalized with CD31-positive endothelial cells. Treatment with IL-33 attenuated kidney injury in Streptozotocin (STZ)-treated mice. In vitro, we showed that IL-33 attenuated ER stress and apoptosis in glomerular endothelial cells. However, sST2 treatment significantly reversed these effects of IL-33. CONCLUSION: Together, these data suggest that IL-33/ST2 signaling mitigates STZ-induced renal damage, partly at least, by suppressing ER stress and apoptosis. Therefore, IL-33 may be an effective therapeutic target in DN.

Nanosheet antibody mimics based label-free and dual-readout lateral flow immunoassay for Salmonella enteritidis rapid detection.

Portable devices for on-site foodborne pathogens detection are urgently desirable. Lateral flow immunoassay (LFIA) provides an efficient strategy for pathogens detection, however, antibody labeling independence and detection reliability, are still challenging. Here, we report the development of a label-free LFIA with dual-readout using glucan-functionalized two-dimensional (2D) transition metal dichalcogenides (TMDs) tungsten disulfide (WS2) as detection probes for sensitive detection of Salmonella enteritidis (S. enteritidis). In particular, glucan-functionalized WS2, synthesized via liquid exfoliation, are reliable detection antibody candidates which served as antibody mimics for bacteria capturing. This LFIA has not only eliminated the intricate antibody labeling process and screening of paired antibodies in conventional LFIAs, but also promised dual-readout (colorimetric/Raman) for flexible detection. Under optimized conditions, this LFIA achieves selective detection of S. enteritidis with a low visual detection limit of 103 CFU/mL and a broad linear range of 103-108 CFU/mL. Additionally, the LFIA could be successfully applied in drinking water and milk with recoveries of 85%-109%. This work is desirable to expand the application of 2D TMDs in biosensors and offers a brand-new alternative protocol of detection antibodies in foodborne pathogens detection.

"Potential Scalpel": A Bioassisted Ultrafast Staining Lateral Flow Immunoassay from De Novo to Results.

It is of great importance to overcome potential incompatibility problems between dyestuffs and antibodies (mAbs) for extensive commercial application of a dyestuff-chemistry-based ultrafast colorimetric lateral flow immunoassay (cLFIA). Herein, inspired by traditional staining technologies, a basic dyestuff gallocyanine (GC)-assisted biogenic "potential scalpel"-based cLFIA (GC-ABPS-based cLFIA) by employing clenbuterol (CLE) as proof-of-concept was proposed to solve a high degree of incompatibility between the same potential dyestuffs and mAbs. Goat antimouse immunoglobulin (Ab2) could serve as the "potential scalpel" to form the positive potential value biomolecular network self-assemblers (BNSA) with anti-CLE mAbs (AbCLE) by noncovalent force. The cLFIA completed the entire detection process from de novo to detection results within 30 min thanks to the easy availability and ideal marking efficiency (≤1 min, saving 0.4-10 h) of GC. Encouragingly, the proposed ultrafast GC-ABPS-based cLFIA has also exhibited high sensitivity (0.411 ng mL-1) and low cost (300 times) compared with other cLFIAs. Also, the feasibility of the proposed cLFIA was demonstrated by detecting CLE in beef, pork ham, and skim milk. Finally, the proposed GC-ABPS-based cLFIA has broadened the application range of dyestuffs and provided an effective reference strategy for the application of dyestuffs in food safety monitoring.

Schiff-Base Chemistry-Coupled Catechol Oxidase-Like Nanozyme Reaction as a Universal Sensing Mode for Ultrasensitive Biosensing.

Expanding sensing modes and improving catalytic performance of nanozyme-based analytical chemistry are beneficial to realizing the desired biosensing of analytes. Herein, Schiff-base chemistry coupled with a novel catechol oxidase-like nanozyme (CHzyme) is designed and constructed, exhibiting two main advantages, including (1) improving catalytic performance by nearly 2-fold compared with only the oxidase-like role of CHzyme; (2) increasing the designability of the output signal by signal transduction of cascade reaction. Thereafter, the substrate sensing modes based on a cascade reaction between the CHzyme-catalyzed reaction and Schiff-base chemistry are proposed and comprehensively studied, containing catalytic substrate sensing mode, competitive substrate sensing mode, and generated substrate sensing mode, expecting to be employed in environmental monitoring, food analyses, and clinical diagnoses, respectively. More meaningfully, the generated substrate sensing mode is successfully applied to construct a cascade reaction coupling ratiometric fluorescent immunoassay for the detection of clenbuterol, increasing 15-fold in detection sensitivity compared with the traditional enzyme-linked immunosorbent assay. It is expected that the expanded universal substrate sensing modes and the Schiff-base chemistry-enhanced nanozyme can enlighten the exploration of innovative biosensors.

A lateral flow immunoassay based on chemisorbed probes in virtue of hydrogen bond receptors on the Bi2S3 NPs.

Improving detection sensitivity is still a major research emphasis for lateral flow immunoassay (LFIA). Increasing the binding efficiency and stability of the probe is an achievable and effective solution. In this work, we developed a highly sensitive lateral flow immunoassay for clenbuterol detection by using bismuth sulfide nanoparticle (Bi2S3) nanoparticles (NPs) as a novel marker. Here, Bi2S3 NPs can link with the antibody by hydrogen bonding to improve the performance of the probe, e.g., stability and sensitivity. Benefiting from the direct hydrogen bonding between Bi2S3 NPs and the monoclonal antibody (mAb), high sensitivity is obtained by the proposed LFIA with a lower visible detection limit of 0.1 ng mL-1 and a cut-off value of 4 ng·mL-1 for CLE detection, which is 5-fold and 7.5-fold improved than the conventional Au NPs based LFIA. In addition, the encouraging practical application results in milk, pork, and beef show that the bismuth sulfide nanoparticle has a great popularizing potential in the performance promotion of LFIAs for food safety monitoring.

Chemical staining enhanced Enzyme-linked immunosorbent assay for sensitive determination of Clenbuterol in food.

Exploring a novel strategy for strengthening the catalytic activity of enzyme facilitates the development of a sensitive enzyme-linked immunosorbent assay (ELISA). Herein, a chemical staining (CS) strategy was firstly discovered to possess the ability to directly improve the catalytic activity of horseradish peroxidase. Based on this discovery, coomassie brilliant blue was introduced into ELISA to establish a CS enhanced ELISA (CS-ELISA) to detect clenbuterol (CL) by simply staining monoclonal antibodies. Satisfactorily, the most important analytical parameters of CS-ELISA, including sensitivity (0.074 ng mL-1) and linear range (0.2-2 ng mL-1) were all improving 2-folds compared with conventional ELISA. Moreover, the CS-ELISA shows good applicability in the detection of CL in pork tenderloin samples. The proposed CS-ELISA shows various advantages, such as cost-effective, easily accessible, enhanced catalytic activity of enzyme, higher sensitivity, and broader linear range, providing a new insight into enhanced ELISA for food safety.

Highly photothermal and biodegradable nanotags-embedded immunochromatographic assay for the rapid monitoring of nitrofurazone.

Immunochromatographic assay platforms are up-and-coming detection tools for disease diagnosis and harmful substances monitoring in food. Herein, by combining photothermal imaging and immunochromatographic analysis, a photothermal immunoassay is developed for the rapid and ultrasensitive detection of nitrofurazone. The nickel disulfide nanosphere with excellent biocompatibility, biodegradability, and high photothermal conversion efficiency, is introduced to offer straightforward readout by color and temperature based on the nature of the crystal, without advanced equipment. It is demonstrated that the nitrofurazone metabolite of semicarbazide can be qualitatively detected by colorimetric signals with a visual limit of 2 µg kg-1. And the quantitative detection limit of photothermal signals is 0.01 µg kg-1, improving the detection sensitivity by about 200 times. Furthermore, recovery rates of the proposed method in food samples were 93 %-120 %. This photothermal immunoassay not merely provides straightforward, rapid, simultaneous qualitative/quantitative detection tactics but may also be valuable in automated and portable diagnostic applications.

Effects of photodynamic therapy on Streptococcus mutans and enamel remineralization of multifunctional TiO2-HAP composite nanomaterials.

BACKGROUND: As photosensitizer and photocatalyst, titanium dioxide (TiO2) can produce a photodynamic reaction for antibacterial treatment. This study aims to explore a Titanium dioxide/nano-hydroxyapatite (TiO2-HAP) composite combined with the dental curing lamp (385-515 nm) in clinical which could inhibit the dental plaque biofilm formed by Streptococcus mutans (S. mutans) and promote the enamel surface remineralization simultaneously. METHODS: X-ray Diffraction (XRD) and high resolution transmission electron microscope (HRTEM) were used to detect the characterization of TiO2-HAP composite nanomaterials. Photodynamic properties of TiO2-HAP were detected by Diffuse reflectance spectrum (DRS) and fluorescence spectroscopy. Bacterial growth was measured by reading the absorbance of bacterial cultures and confocal microscope was used to observe the biofilm removal ability of nanomaterials. The ability of TiO2-HAP to promote enamel remineralization was measured by Scanning electron microscope (SEM). RESULTS: The OD 600 of S. mutans was 0.76 in the control group and 0.13 in group of TiO2-HAP with exposure to light-emitting diode (LED) (150 mW/cm2) for 5 min, suggesting its sustained antibacterial potency and inhibition of the metabolic activity of dental plaque microcosm biofilm. Also, the release of calcium and phosphorus ions in TiO2-HAP can promote enamel mineralization simultaneously. After 15 days of remineralization, the Ca/P ratio of demineralized enamel surface increased from 1.28 to 1.67, which was similar to that of normal enamel. CONCLUSIONS: The TiO2-HAP exhibit a promising anti-bacterial activity and remineralization capacity which can prevent the occurrence of caries to the greatest extent and promote the biomimetic mineralization of dental tissues.

Nature-inspired nanozymes as signal markers for in-situ signal amplification strategy: A portable dual-colorimetric immunochromatographic analysis based on smartphone.

Owing to the lack of a universal descriptor to predict the nanozymes as signal markers (SM) of immunochromatographic analysis (ICA), the present exploitation of nanozymes as SM heavily relies on trial-and-error strategies, which obstructs the rational design of nanozymes with monoclonal antibodies (mAbs) recognition activity. Herein, inspired by the structure of the active center of natural multi-iron peroxidases and polyphenol-protein interactions, a rational design of an artificial peroxidase with mAbs recognition activity by utilizing gallic acid (GA) to chelate with multivalent iron was successfully proposed by utilizing gallic acid (GA) to chelate with multivalent iron. The most essential features of peroxidase-like Fe-GA nanozymes (FGN) were investigated, showing high catalytic performance and good stability. Subsequently, FGN was employed as SM for mAbs in ICA, which played the following triple roles in the ICA sensor: (i) the direct recognizer of mAbs; (ii) the generator of original colorimetric signal; (iii) the generator of catalytic in-suit amplification colorimetric signal. To make the ICA more portable, we have employed a smartphone and principal component analysis (PCA) to assist this on-site detection. As a proof-of-concept, clenbuterol (CLL) was analyzed by the proposed nanozymes-mediated dual-colorimetric ICA based on a smartphone. Notably, the proposed dual-colorimetric ICA exhibits high analytical performance for the quantification of CLL in the detection range of 0-6 ng mL-1 with a limit of detection (LOD) as low as 0.172 ng mL-1. Meanwhile, the proposed dual-colorimetric ICA exhibits remarkable feasibility and was successfully employed for the detection of CLL in pork and chicken matrixes.

A fungal microRNA-like RNA subverts host immunity and facilitates pathogen infection by silencing two host receptor-like kinase genes.

Small RNAs (sRNAs) play important roles in various biological processes by silencing their corresponding target genes in most eukaryotes. However, cross-kingdom regulation mediated by fungal microRNA-like RNAs (milRNAs) in plant-pathogen interactions is still largely unknown. Using molecular, genetic, histological, and biochemical approaches, we found that the apple tree Valsa canker pathogen Valsa mali milRNA Vm-milR1 could suppress the host immunity by silencing two host receptor-like kinase genes, MdRLKT1 and MdRLKT2. Vm-milR1 was highly induced during V. mali infection. Deletion of Vm-milR1 precursor abolished the generation of Vm-milR1 and reduced the virulence of V. mali. Inoculation of Vm-milR1 deletion mutants induced the host defence responses, including reactive oxygen species (ROS) accumulation, callose deposition, and high expression of defence-related genes. Furthermore, Vm-milR1 was confirmed to be able to suppress the expression of MdRLKT1 and MdRLKT2 in a sequence-specific manner. Moreover, overexpression of either MdRLKT1 or MdRLKT2 enhanced apple resistance to V. mali by activating the host defence responses. Furthermore, knockdown of MdRLKT1 or MdRLKT2 compromised the host resistance to V. mali. Our study revealed that V. mali was equipped with Vm-milR1 as an sRNA effector to silence host receptor-like kinase genes, suppress the host defence responses, and facilitate pathogen infection.

Highly Sensitive Colorimetric/Surface-Enhanced Raman Spectroscopy Immunoassay Relying on a Metallic Core-Shell Au/Au Nanostar with Clenbuterol as a Target Analyte.

Lateral flow immunoassay (LFIA) has emerged as an effective technique in the field of food safety and environmental monitoring. However, sensitive and quantitative detection is still challenging for LFIAs in complex environments. In this work, a dual-model colorimetric/SERS lateral flow immunoassay for ultrasensitive determination of clenbuterol was constructed based on a metallic core-shell Au/Au nanostar acting as a multifunction tag. Raman reporter molecules are located between the core (AuNP) and shell (Au nanostar) to form a sandwich structure, which contributes to eliminate the environmental interference and improve the detection stability. In addition, the Au/Au nanostar provides a much higher Raman enhancement due to the presence of sharp tips and larger surface roughness in comparison with gold nanoparticles (AuNPs). Thus, on the basis of the antibody-antigen interaction, the dual-model immunoassay can produce strong colorimetric and surface-enhanced Raman spectroscopy (SERS) signals for highly sensitive detection of the target analyte, clenbuterol. Under optimal conditions, clenbuterol could be detected by the colorimetric model with a visual detection limit of 5 ng/mL. Meanwhile, the SERS signal of the Au/Au nanostar was accumulated on the test line for the SERS model detection with a quantitative detection limit as low as 0.05 ng/mL, which is at least 200-fold lower than that of the traditional AuNPs-based immunoassay. Furthermore, recovery rates of the proposed method in food samples were 86-110%. This dual-model immunoassay provides an effective tool for antibiotic residues analysis and demonstrates a broad potential for future applications in food safety monitoring.

A secreted catalase contributes to Puccinia striiformis resistance to host-derived oxidative stress.

Plants can produce reactive oxygen species (ROS) to counteract pathogen invasion, and pathogens have also evolved corresponding ROS scavenging strategies to promote infection and pathogenicity. Catalases (CATs) have been found to play pivotal roles in detoxifying H2O2 formed by superoxide anion catalyzed by superoxide dismutases (SODs). However, few studies have addressed H2O2 removing during rust fungi infection of wheat. In this study, we cloned a CAT gene PsCAT1 from Puccinia striiformis f. sp. tritici (Pst), which encodes a monofunctional heme-containing catalase. PsCAT1 exhibited a high degree of tolerance to pH and temperature, and forms high homopolymers.Heterologous complementation assays in Saccharomyces cerevisiae reveal that the signal peptide of PsCAT1 is functional. Overexpression of PsCAT1 enhanced S. cerevisiae resistance to H2O2. Transient expression of PsCAT1 in Nicotiana benthamiana suppressed Bax-induced cell death. Knockdown of PsCAT1 using a host-induced gene silencing (HIGS) system led to the reduced virulence of Pst, which was correlated to H2O2 accumulation in HIGS plants. These results indicate that PsCAT1 acts as an important pathogenicity factor that facilitates Pst infection by scavenging host-derived H2O2.

Adaptive regulation of virulence genes by microRNA-like RNAs in Valsa mali.

MicroRNAs play important roles in the regulation of gene expression in plants and animals. However, little information is known about the action mechanism and function of fungal microRNA-like RNAs (milRNAs). In this study, combining deep sequencing, molecular and histological assays, milRNAs and their targets in the phytopathogenic fungus Valsa mali were isolated and identified. A critical milRNA, Vm-milR16, was identified to adaptively regulate the expression of virulence genes. Fourteen isolated milRNAs showed high expression abundance. Based on the assessment of a pathogenicity function of these milRNAs, Vm-milR16 was found to be a critical milRNA in V. mali by regulating sucrose non-fermenting 1 (VmSNF1), 4,5-DOPA dioxygenase extradiol (VmDODA), and a hypothetical protein (VmHy1). During V. mali infection, Vm-milR16 is downregulated, while its targets are upregulated. Overexpression of Vm-milR16, but not mutated Vm-milR16, significantly reduces the expression of targets and virulence of V. mali. Furthermore, deletion of VmSNF1, VmDODA and VmHy1 significantly reduce virulence of V. mali. All three targets seem to be essential for oxidative stress response and VmSNF1 is required for expression of pectinase genes during V. mali-host interaction. Our results demonstrate Vm-milRNAs contributing to the infection of V. mali on apple trees by adaptively regulating virulence genes.