Search Results (203)

Salmonella is a rapidly spreading and widespread zoonotic infectious disease that poses a serious threat to the safety of both poultry and human lives. Therefore, the timely detection of Salmonella in foods and animals has become an urgent need for food safety. This work describes the construction of an aptamer-based sensor for Salmonella detection, using Fe₃O₄ magnetic beads and Ag@Au core–shell nanoparticles-embedded 4-mercaptobenzoic acid (4MBA). Leveraging the high affinity between biotin and streptavidin, aptamers were conjugated to Fe₃O₄ magnetic beads. These beads were then combined with Ag@4MBA@Au nanoparticles functionalized with complementary aptamers through hydrogen bonding and π-π stacking interactions, yielding a SERS-based aptamer sensor with optimized Raman signals from 4MBA. When target bacteria are present, aptamer-conjugated magnetic beads exhibit preferential binding to the bacteria, leading to a decrease in the surface-enhanced Raman scattering (SERS) signal. And it was used for the detection of five different serotypes of Salmonella, respectively, and the results showed that the aptamer sensor exhibited a good linear relationship between the concentration range of 10²–10⁸ CFU/mL and LOD is 35.51 CFU/mL. The SERS aptasensor was utilized for the detection of spiked authentic samples with recoveries between 94.0 and 100.4%, which proved the usability of the method and helped to achieve food safety detection. Full article

Hydrocortisone is a typical glucocorticoid commonly used in livestock production; however, its overuse can result in hormone residues in milk. Long-term consumption of such milk may lead to a series of health issues. Therefore, the timely and rapid detection of hydrocortisone in milk is crucial for protecting human health. In this study, a magnetic particle-based direct chemiluminescence immunoassay (MP-DCLIA) incorporating a streptavidin–biotin signal amplification system was developed for the rapid and high-throughput detection of hydrocortisone in milk. Automated operations reduce human error and enhance the accuracy and repeatability of tests. The assay can be completed in 12 min with a linear detection range of 13.09–261.71 μg/L, a limit of detection (LOD) of 4.94 μg/L, a limit of quantification (LOQ) of 14.84 μg/L, and intra- and inter-batch variations of less than 5%. The method demonstrated stability and exhibited no cross-reactivity with structural analogues. Spiked recoveries of milk samples ranged from 85.85% to 100.30%, with results strongly correlating with those obtained from LC-MS/MS. The MP-DCLIA offers rapidity, high efficiency, stability, and precision, making it a promising tool for practical testing applications. Full article

Hedgehog (HH) signaling plays important roles in the development of the nervous system (Sonic hedgehog), bone, cartilage (Indian Hedgehog) and testis (Desert Hedgehog). Research on HH and testes has mostly been conducted in HH-knockout mice and rats, etc. The relationship between HH and cellular junctions has mostly been found in the nervous system and intestine. However, few research studies concerning the link between HH signaling and cell junctions in testis function have been reported. We identified the members of HH signaling that are involved in Eriocheir sinensis testes: HH, Smoothen, Patched, Kif27 and Ci. HH has only one homolog in E. sinensis and is expressed in several types of germ cells in the testes. We found that Kif27 colocalized with Ci in the testes. The knockdown of HH induced enlarged interstitial spaces of the seminiferous tubules. A biotin–streptavidin immunofluorescence experiment indicated that the hemolymph–testis barrier (HTB) was disrupted. Western blot results showed that pinin, HH signaling and cell proliferation- and apoptosis-related protein levels were downregulated. Further immunofluorescent results showed the dislocation of several junction proteins, the abnormality of F-actin and the slowdown of germ cell proliferation and apoptosis. While β-catenin entered the spermatocyte nucleus, it did not activate Wnt-β-catenin signaling, which indicated that the disturbance of the cell cycle in germ cells was not caused by Wnt-β-catenin signaling. In summary, HH signaling plays some roles beyond our understanding in the regulation of the HTB and the germ cell cycle in E. sinensis testes. Full article

The assurance of food safety requires sensitive monitoring of multiple mycotoxins due to their severe impacts on the food industry and high health risks posed to consumers. Herein, we proposed a chemiluminescent/colorimetric dual-signal readout microfluidic method, incorporating a streptavidin-biotin-alkaline phosphatase (SA-Biotin-ALP) signal amplification system for the highly sensitive detection of Deoxynivalenol (DON), Ochratoxin A (OTA), and Aflatoxin B1 (AFB1). The indirect competitive enzyme-linked immunoassay (ic-ELISA) was integrated into microfluidic chip, resulting in sensitive detection ranges of DON in the range of 4–128 ng/mL, 2–64 ng/mL for OTA, and 0.2–6.4 ng/mL for AFB1, with the limit of detection (LOD) being 2.636 ng/mL, 1.492 ng/mL, and 0.131 ng/mL, respectively. Recovery rates in beer samples ranged from 91.93% to 109.31%. Furthermore, a dual-mode microfluidic workstation (DMMW) was developed to facilitate rapid, automated detection for these mycotoxins, simplifying the detection procedure, enhancing the detection efficiency, and reducing the requirement for specialized personnel, thus confirming significant potential for the rapid detection of mycotoxins in complex matrices such as beer. Full article

In this study, we developed a low-cost, high-sensitivity chemiluminescence competitive aptamer sensor for the detection of aflatoxin B₁ (AFB₁) in wheat samples. The optical fiber sensor was self-made, and it utilized biotin and streptavidin (SA) link aptamer and horseradish peroxidase (HRP) for the chemiluminescence detection, achieving competitive assay between the AFB₁ and AFB₁ antigen. We adjusted the experimental conditions of the sensor base on the date of optimization of the experimental conditions and chose coated antigens on the surface of carboxyl magnetic particles. Under conditions optimized by testing key parameters, the assay results showed that the chemiluminescence intensity and AFB₁ concentration demonstrated a strong linear relationship (R² = 0.995), the dynamic range was from 0.1 to 10 ng/mL with a detection limit of 0.09 ng/mL, and the aptamer exhibited good specificity and anti-interference ability. Testing the wheat samples showed that the spiked recovery rate ranged from 79.19% to 113.21%. The sensor possesses characteristics of low detection limits, simple manufacturing methods, and affordability, providing a novel solution for the development of low-cost and high-sensitivity AFB₁ detection equipment. Full article

Rapidly growing industrial biotechnology and bio-manufacturing require simple and cost-effective bioseparation tools. A novel strategy of bioseparation based on the streptavidin-decorated polyhydroxyalkanoate (PHA) nano-granules was developed in this study. By fusing to the N-terminus of PHA-associated phasin protein, the streptavidin was one-step immobilized on the surface of PHA nano-granules simultaneously with the accumulation of PHA in recombinant Escherichia coli. About 1.95 g/L of PHA nano-granules (54.51 wt% of cell dry weight) were produced after 48 h bacterial cultivation. The following qualitative and quantitative characterizations demonstrated that the streptavidin accounted for approximately 6.78% of the total weight of the purified PHA nano-granules and confirmed a considerable biotin affinity of 0.1 ng biotin/μg surface protein. As a proof of concept, the nano-granules were further functionalized with biotinylated oligo(dT) for mRNA isolation and about 1.26 μg of mRNA (occupied 2.59%) was purified from 48.45 μg of total RNA, achieving good integrity and high purity with few DNA and rRNA contaminations. Moreover, the nano-granules retained more than 80% of their initial mRNA recovery efficiency after ten cycles of repeated use. The PHA-SAP nano-granules were also functionalized with biotinylated magnetic beads, allowing magnetic recovery of the PHA nano-granules from cell lysates that still needs optimization. Our study provides a novel and expandable platform of PHA nano-granules that can be further functionalized with various biological groups for bioseparation applications. The functional PHA nano-granules have a great potential to serve as bioseparation resin for large-scale purification processes after suitable optimizations for “bench-to-factory” translation, contributing to scalable and sustainable bioprocessing. Full article

The association between microRNAs and various diseases, especially cancer, has been established in recent years, indicating that miRNAs can potentially serve as biomarkers for these diseases. Determining miRNA concentrations in biological samples is crucial for disease diagnosis. Nevertheless, the stem-loop reverse transcription quantitative PCR method, the gold standard for detecting miRNA, has great challenges in terms of high costs and enzyme limitations when applied to clinical biological samples. In this study, an isothermal signal amplification method based on a duplex-specific nuclease (DSN) enzyme-driven DNA walker and an improved catalytic hairpin assembly (CHA) was designed for miRNA detection. First, biotin–triethylene glycol-modified trigger-releasable DNA probes were conjugated to the streptavidin-coated magnetic beads for recognizing the target miRNA. The DSN enzyme specifically hydrolyzes DNA strands when the DNA probe hybridizes with the targeted miRNA. This recycling process converts the input miRNA into short trigger fragments (catalysts). Finally, three hairpins of improved CHA are driven by this catalyst, resulting in the three-armed CHA products and a fluorescence signal as the output. This dual-cycle biosensor shows a good linear relationship in the detection of miR-21 and miR-141 over the final concentration range of 250 fM to 50 nM, presenting an excellent limit of detection (2.95 amol). This system was used to detect miR-21 and miR-141 in MCF-7 and 22RV1 cells, as well as in 1% human serum. This system can be used to evaluate the expression levels of miRNAs in different biological matrices for the clinical diagnosis and prognosis of different cancers. Full article

Necroptosis is a regulated form of cell death implicated in several pathological conditions, including viral infections. In this study, we investigated the expression and correlation of necroptosis markers MLKL, RIP1 and RIP3 in human liver tissue from fatal cases of yellow fever (YF) using immunohistochemistry (IHC). The liver samples were obtained from 21 YF-positive individuals and five flavivirus-negative controls with preserved liver parenchymal architecture. The cases underwent histopathological analysis, followed by tissue immunostaining with the immunohistochemical method of streptavidin–biotin peroxidase. Using the in situ method, we evaluated the centrilobular zone (Z3), midzonal zone (Z2), periportal zone and portal tract (PT) of human liver parenchyma with markers for necroptosis, RIPK1, RIPK3 and MLKL. A quantitative analysis revealed a significantly higher expression of MLKL, RIP1 and RIP3 in the liver parenchyma of YF cases compared to controls in different zones (Z3, Z2, Z1) and portal tracts (PTs) of the liver, especially in zone 2. Immunostaining confirmed the localization of MLKL, RIP1 and RIP3 in hepatocytes and inflammatory infiltrates, highlighting their involvement in the pathogenesis of YF. A Pearson correlation analysis demonstrated significant correlations among necroptosis markers, which indicates their coordinated regulation during YF-induced liver injury. Full article

Cystathionine γ-lyase (CSE) is the second enzyme in the trans-sulfuration pathway that converts cystathionine to cysteine. It is also one of three major enzymes responsible for the biosynthesis of hydrogen sulfide (H₂S). CSE is believed to be the major source of endogenous H₂S in the cardiovascular system, and the CSE/H₂S system plays a crucial role in a variety of physiological and pathological processes. However, the regulatory mechanism of the CSE/H₂S system is less well understood, especially at the post-translational level. Here, we demonstrated that the persulfidation of CSE inhibits its activity by ~2-fold in vitro. The loss of this post-translational modification in the presence of dithiothreitol (DTT) results in a reversal of basal activity. Cys137 was identified as the site for persulfidation by combining mass spectrometry, mutagenesis, activity analysis and streptavidin–biotin pull-down assays. To test the physiological relevance of the persulfidation regulation of CSE, human aortic vascular smooth muscle cells (HA-VSMCs) were incubated with vascular endothelial growth factor (VEGF), which is known to enhance endogenous H₂S levels. Under these conditions, consistent with the change tendency of the cellular H₂S level, the CSE persulfidation levels increased transiently and then gradually decreased to the basal level. Collectively, our study revealed a negative feedback regulation mechanism of the CSE/H₂S system via the persulfidation of CSE and demonstrated the potential for maintaining cellular H₂S homeostasis under oxidative stress conditions, particularly in tissues where CSE is a major source of H₂S. Full article

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that causes extensive damage to multiple organs and tissues and has no known cure. This study introduces a microfluidic detection platform that combines a microfluidic reaction chip with a micro-spectrometer to accurately detect the anti-cyclic citrullinated peptide antibody (anti-CCP Ab) biomarker, commonly associated with arthritis. The surface of the microfluidic reaction chip is functionalized using streptavidin to enable the subsequent immobilization of biotinylated-labeled cyclic citrullinated peptide (biotin–CCP) molecules through a streptavidin–biotin reaction. The modified chip is then exposed to anti-CCP Ab, second antibody conjugated with horseradish peroxidase (HRP) (2nd Ab-HRP), 3,3′,5,5′-tetramethylbenzidine (TMB), and a stop solution. Finally, the concentration of the anti-CCP Ab biomarker is determined by analyzing the optical density (OD) of the colorimetric reaction product at 450 nm using a micro-spectrometer. The detection platform demonstrated a strong correlation (R² = 0.9966) between OD and anti-CCP Ab concentration. This was based on seven control samples with anti-CCP Ab concentrations ranging from 0.625 to 100 ng/mL. Moreover, for 30 artificial serum samples with unknown anti-CCP Ab concentrations, the biosensor achieves a correlation coefficient of (R² = 0.9650). The proposed microfluidic detection platform offers a fast and effective method for accurately identifying and quantifying the anti-CCP Ab biomarker. Thus, it offers a valuable tool for the early diagnosis and monitoring of RA and its progression in point-of-care settings. Full article

Aflatoxin B₁ (AFB₁) is highly toxic and difficult to prevent. It is mainly produced by fungi and exists in plants and animals and is classified by the World Health Organization as a class I carcinogen, posing a serious threat to human and animal health. Therefore, it is important to establish an efficient, sensitive, and on-site detection method for AFB₁ to protect human health. The immunochromatographic test strip method is simple, sensitive, and can achieve real-time detection. However, traditional immunochromatographic test strips have low sensitivity due to their relatively weak optical properties. In this study, Nb-G8 was biotinylated using a chemical method. Two sizes of gold nanoflowers (AuNFs) were prepared and combined with biotinylated G8 and streptavidin to form two types of probes. These probes were sprayed on gold standard pads and expanded pads, respectively, to enhance the signals through the high affinity interaction between streptavidin and biotin. Under the optimal experimental conditions, the half maximal inhibitory concentration (IC₅₀) of this method was 5.0 ng/mL and the limit of detection (IC₁₀) was 0.03 ng/mL, which increased the sensitivity of the test strip by four-fold compared with that of the traditional biotinylated nanoantibody immunochromatography test strip and had a wider detection range. In conclusion, the use of a high-affinity amplification signal between biotin and streptavidin is a valuable method for the detection of aflatoxin. Full article

Bacillus cereus, a foodborne pathogen, produces resilient endospores that are challenging to detect with conventional methods. This study presents a novel Flower-Shaped PCR Scaffold-based Lateral Flow Biosensor (FSPCRS-LFB), which employs an aptamer-integrated PCR scaffold as capture probes, replacing the traditional streptavidin-biotin (SA-Bio) approach. The FSPCRS-LFB demonstrates high sensitivity and cost-efficiency in detecting B. cereus endospores, with a limit of detection (LOD) of 4.57 endospores/mL a visual LOD of 10² endospores/mL, and a LOD of 6.78 CFU/mL for endospore-cell mixtures. In chicken and tea samples, the platform achieved LODs of 74.5 and 52.8 endospores/mL, respectively, with recovery rates of 82.19% to 97.88%. Compared to existing methods, the FSPCRS-LFB offers a 3.7-fold increase in sensitivity while reducing costs by 26% over the SA-Bio strategy and 87.5% over rolling circle amplification (RCA). This biosensor provides a rapid, sensitive and cost-effective solution for point-of-care testing (POCT) of B. cereus endospores, expanding detection capabilities and offering novel approaches for pathogen detection. Full article

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems. Full article

Laser-induced graphene (LIG) possesses characteristics of easy handling, miniaturization, and unique electrical properties. We modified the surface of LIG by electropolymerizing β-cyclodextrin (β-CD), which was used to immobilize antibodies on the electrode surface for highly sensitive detection of targets. N6-methyladenosine (m6A) is the most prevalent reversible modification in mammalian messenger RNA and noncoding RNA, influencing the development of various cancers. Here, β-CD was electropolymerized to immobilize the anti-m6A antibody, which subsequently recognized the target m6A. This was integrated into the catalytic hydrogen peroxide–hydroquinone (H₂O₂-HQ) redox system using phos-tag-biotin to generate electrochemical signals from streptavidin-modified horseradish peroxidase (SA-HRP). Under optimal conditions, the biosensor exhibited a linear range from 0.1 to 100 nM with a minimum detection limit of 96 pM. The method was successfully applied to the recovery analysis of m6A from HeLa cells through spiking experiments and aims to inspire strategies for point-of-care testing (POCT). Full article

The role of autophagy goes far beyond the elimination of damaged cellular components and the quality control of proteins. It also cleanses cells from inclusions, including pathogenic viruses, and provides energy-forming components. The liver, which is an organ with increased metabolism, is made up of cells that are particularly vulnerable to damage. Therefore, detoxification of liver cells in the process of autophagy has become a very important issue clinically. The aim of this study was an immunohistochemical evaluation of proteins activated in rat liver cells at different stages of hyperbaric autophagy. The rats used for the study were randomly divided into six equivalent groups—three control groups and three experimental groups. Animals from the experimental groups were subjected to hyperbaric treatment in a hyperbaric chamber, with a pressure of 1.6 ATA for 120 min. They breathed atmospheric air. Rats were decapitated within 5 or 10 days after removal from the chamber. Immunohistochemical reactions with beclin 1, LC3B, RAB7, and HSC73 proteins were carried out on preparations made from liver slices. A three-step labeled streptavidin–biotin detection method of paraffin blocks (LSAB three-step) was used for immunohistochemical research. The results were evaluated using computer programs for morphometric analysis of microscopic images by calculating the mean surface areas occupied by a positive immunohistochemical reaction in individual groups for all antibodies tested. Increased closure of substrates in the autophagosome (beclin 1) induced late endosome transport and accelerated autophagosome maturation process (RAB7). Furthermore, a larger number of autophagosomes (LC3B) was observed in liver cells immediately after the cessation of hyperbaric activity; however, this decreased after 5 days. During this time, chaperone-mediated autophagy (HSC73) was observed on a larger scale. This means that increased macroautophagy induced by hyperbaric treatment weakens with time that has elapsed since the cessation of high pressure, whereas similarly induced chaperone-mediated autophagy intensifies over time. Full article