Search Results (79)

A fluorescent sandwich assay was devised to quantify CK-MB. In a typical immunoassay, antibodies bind to the target, and the detected signal is quantified according to the target’s concentration. We innovated a unique fluorescence assay known as the “enzyme-linked aptamer assay” (ELAA) by substituting antibodies with a pair of high-affinity aptamers labelled with biotin, namely apt. A1 and apt. A2. Avidin-labelled ALP binds to biotin-labelled aptamers, hydrolyzing its substrate, 2-phosphoascorbic acid trisodium salt, resulting in the formation of ascorbic acid. The catalytic hydrolysate functions as a reducing agent, causing the deterioration of MoS₂ nanosheets. This results in the transformation of MoS₂ nanosheets into nanoribbons, leading to the release of quenched AGQDs. The reestablishment of fluorescence is triggered by Förster Resonance Energy Transfer (FRET) between the MoS₂ nanoribbons and AGQDs, enhancing the sensitivity of disease biomarker detection. The working range for detection falls between 2.5 nM and 160 nM, and the limit of detection (LOD) for CK-MB is verified at 0.20 nM. Full article

The accurate and sensitive detection of tumor-derived exosomes holds significant promise for the early diagnosis of cancer. In this study, an electrochemical aptasensor was developed for the high-performance detection of exosomes by integrating dual-probe recognition and hybridization chain reaction (HCR). A dual-probe recognition unit composed of a MUC1 aptamer (MUC1-Apt) probe and cholesterol probe was designed for capturing target exosomes and reducing the interference from free proteins, significantly improving the accuracy of exosome detection. It should be noted that the dual-probe recognition unit was formed in conjunction with the HCR. Moreover, a large number of biotins were also assembled on the HCR product, which were used to capture avidin–horseradish peroxidase (SA-HRP) for signal amplification. The CD63 aptamer (CD63-Apt) was immobilized on the surface of a gold electrode for specifically capturing exosomes to construct a classical sandwiched structure. The loaded SA-HRP can efficiently catalyze the reaction of 3, 3′, 5, 5′ tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) to generate a large electrochemical signal. According to this phenomenon, a linear relationship of this proposed aptasensor was achieved between the electrochemical response and 1 × 10²–1 × 10⁷ particles/mL exosomes, with a detection limit of 45 particles/mL. Moreover, the aptasensor exhibited accepted stability and potential clinical applicability. All results proved that this aptasensor has a promising application in exosome-based disease diagnostics. Full article

Many countries have established regulatory frameworks to monitor and mitigate Salmonella contamination in poultry products. The ability to rapidly quantify Salmonella is critical for poultry processors to facilitate early detection, implement corrective measures, and enhance product safety. This study aimed to develop an Immunomagnetic Chemiluminescent Assay (IMCA) for the quantification of Salmonella Typhimurium in ground chicken. Immunomagnetic microbeads functionalized with monoclonal antibodies were employed to selectively capture and concentrate Salmonella from ground chicken samples. A biotin-labeled monoclonal antibody, followed by an avidin-horseradish peroxidase conjugate, was used to bind the captured bacteria and initiate a chemiluminescent reaction catalyzed by peroxidase. Light emission was quantified in relative light units (RLUs) using two luminometers. Ground chicken samples were inoculated with a four-strain S. Typhimurium cocktail ranging from 0 to 3.5 Log CFU/g. Bacterial concentrations were confirmed using the Most Probable Number (MPN) method. Samples underwent enrichment in Buffered Peptone Water (BPW) supplemented with BAX MP Supplement at 42 °C for 6 and 8 h before analysis via IMCA. A linear regression analysis demonstrated that the optimal quantification of Salmonella was achieved at the 8 h enrichment period (R² ≥ 0.89), as compared to the 6 h enrichment. The limit of quantification (LOQ) was determined to be below 1 CFU/g. A strong positive correlation (R² ≥ 0.88) was observed between IMCA and MPN results, indicating methodological consistency. These findings support the application of IMCA as a rapid and reliable method for the detection and quantification of Salmonella in ground chicken. Full article

Detecting multiple tumor markers is of great importance. It helps in early cancer detection, accurate diagnosis, and monitoring treatment. In this work, gold nanoparticles–toluidine blue–graphene oxide (AuNPs-TB–GO) and gold nanoparticles–carboxyl ferrocene–tungsten disulfide (AuNPs–FMC–WS₂) nanocomposites were prepared for labeling Carcinoembryonic antigen (CEA) antibody and Carbohydrate antigen 72–4 (CA72-4) antibody, respectively, and used as two kinds of probes with different electrochemical signals. With the excellent magnetic performance of biotin immune magnetic beads (IMBs), the biofunctional IMBs were firmly deposited on the magnetic glassy carbon electrode (MGCE) surface by applying a constant magnetic field, and then the CEA and CA72-4 antibody were immobilized on the IMBs by the avidin–biotin conjugation. The assay was based on the change in the detection peak current. Under the optimum experimental conditions, the linear range of detection of CEA is of the two-component immunosensor is from 0.01 to 120 ng/mL, with a low detection limit of 0.003 ng/mL, and the linear range of detection of CA72-4 is from 0.05 to 35 U/mL, with a detection limit of 0.016 U/mL. The results showed that the proposed immunosensor enabled simultaneous monitoring of CEA and CA72-4 and exhibited good reproducibility, excellent high selectivity, and sensitivity. In particular, the proposed multiplexed immunoassay approach does not require sophisticated fabrication and is well-suited for high-throughput biosensing and application to other areas. Full article

Background/Objectives: The focus of this study was to prepare and characterize docetaxel (DCX)-loaded lipid/polymer hybrid nanoparticles (LPHNps) functionalized with the monoclonal antibody (mAb) Chi-Tn for a potential active targeting approach in lung cancer treatment. Methods: We synthesized DOTAP-PLGA hybrid nanoparticles loaded with DCX and functionalized them with Chi-Tn mAb through a biotin–avidin approach. The physicochemical characterization involved dynamic light scattering, transmission electron microscopy, Raman spectroscopy, and atomic force microscopy. The in vitro and in vivo evaluations encompassed uptake studies, cell viability tests, and the assessment of tumor growth control in a lung cancer model. Results: The nanoparticles featured a hydrophobic PLGA core with 99.9% DCX encapsulation efficiency, surrounded by a DOTAP lipid shell ensuring colloidal stability with a high positive surface charge. The incorporation of PEGylated lipids on their surface helps evade the immune system and facilitate Chi-Tn mAb attachment. The resulting nanoparticles exhibit a spherical shape with monodisperse particle sizes averaging 250 nm, and demonstrate sustained drug release. In vitro uptake studies and viability assays conducted in A549 cancer cells show that the Chi-Tn mAb enhances nanoparticle internalization and significantly reduces cell viability. In vivo studies demonstrate a notable reduction in tumor volume and an increased survival rate in the A549 tumor xenograft mice model when DCX was encapsulated in nanoparticles and targeted with Chi-Tn mAb in comparison to the free drug. Conclusions: Therefore, Chi-Tn-functionalized LPHNps hold promise as carriers for actively targeting DCX to Tn-expressing carcinomas. Full article

Biotin (vitamin B7, or vitamin H) is a water-soluble B-vitamin that functions as a cofactor for carboxylases, i.e., enzymes involved in the cellular metabolism of fatty acids and amino acids and in gluconeogenesis; moreover, as reported, biotin may be involved in gene regulation. Biotin is not synthesized by human cells, but it is found in food and is also produced by intestinal bacteria. Biotin status/homeostasis in human individuals depends on several factors, including efficiency/deficiency of the enzymes involved in biotin recycling within the human organism (biotinidase, holocarboxylase synthetase), and/or effectiveness of intestinal uptake, which is mainly accomplished through the sodium-dependent multivitamin transporter. In the last years, administration of biotin at high/“pharmacological” doses has been proposed to treat specific defects/deficiencies and human disorders, exhibiting mainly neurological and/or dermatological symptoms and including biotinidase deficiency, holocarboxylase synthetase deficiency, and biotin–thiamine-responsive basal ganglia disease. On the other hand, according to warnings of the Food and Drug Administration, USA, high biotin levels can affect clinical biotin-(strept)avidin assays and thus lead to false results during quantification of critical biomarkers. In this review article, recent findings/advancements that may offer new insight in the abovementioned research fields concerning biotin will be presented and briefly discussed. Full article

Therapeutic microbubbles (thMBs) contain drug-filled liposomes linked to microbubbles and targeted to vascular proteins. Upon the application of a destructive ultrasound trigger, drug uptake to tumour is improved. However, the structure of thMBs currently uses powerful non-covalent bonding of biotin with avidin-based proteins to link both the liposome to the microbubble (MB) and to bind the targeting antibody to the liposome–MB complex. This linkage is not currently FDA-approved, and therefore, an alternative, maleimide–thiol linkage, that is currently used in antibody–drug conjugates was examined. In a systematic manner, vascular endothelial growth factor receptor 2 (VEGFR2)-targeted MBs and thMBs using both types of linkages were examined for their ability to specifically bind to VEGFR2 in vitro and for their ultrasound imaging properties in vivo. Both showed equivalence in the production of the thMB structure, in vitro specificity of binding and safety profiles. In vivo imaging showed subtle differences for thMBs where biotin thMBs had a faster wash-in rate than thiol thMBs, but thiol thMBs were longer-lived. The drug delivery to tumours was also equivalent, but interestingly, thiol thMBs altered the biodistribution of delivery away from the lungs and towards the liver compared to biotin thMBs, which is an improvement in biosafety. Full article

Background: Idiopathic pulmonary fibrosis (IPF) is a fatal and chronic interstitial lung disease. Intricate pathogenesis of pulmonary fibrosis and only two approved medications with side effects and high cost bring us the challenge of fully understanding this lethal disease and urgency to find more safe and low-cost therapeutic alternatives. Purpose: Demethyleneberberine (DMB) has been demonstrated to have various anti-inflammatory, antioxidant, antifibrosis and anti-cancer bioactivities. The objective of this study was to evaluate the effect of DMB on pulmonary fibrosis and investigate the mechanism. Methods: Bleomycin (BLM)-induced pulmonary fibrosis was established in mice to evaluate the antifibrotic effect of DMB in vivo. A549 and MRC5 cells were used to evaluate the effect of DMB on epithelial–mesenchymal transition (EMT) and fibroblast–myofibroblast transition (FMT) in vitro. High throughput sequencing, biotin–avidin system and site-directed mutagenesis were applied to explore the mechanism of DMB in alleviating pulmonary fibrosis. Results: DMB alleviated BLM-induced pulmonary fibrosis in vivo by improving the survival state of mice, significantly reducing pulmonary collagen deposition and oxidative stress and improving lung tissue morphology. Meanwhile, DMB was demonstrated to inhibit epithelial–mesenchymal transition (EMT) and fibroblast–myofibroblast transition (FMT) in vitro. High throughput sequencing analysis indicated that GREM1, a highly upregulated profibrotic mediator in IPF and BLM-induced pulmonary fibrosis, was significantly downregulated by DMB. Furthermore, USP11 was revealed to be involved in the deubiquitination of GREM1 in this study and DMB promoted the ubiquitination and degradation of GREM1 by inhibiting USP11. Remarkably, DMB was demonstrated to selectively bind to the Met776 residue of USP11, leading to disruption of USP11 deubiquitinating GREM1. In addition, DMB presented an equivalent antifibrotic effect at a lower dose compared with pirfenidone and showed no obvious toxicity or side effects. Conclusions: This study revealed that USP11/GREM1 could be a potential target for IPF management and identified that DMB could promote GREM1 degradation by inhibiting USP11, thereby alleviating pulmonary fibrosis. Full article

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin–avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidin^TM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to Neutravidin^TM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications. Full article

Biotin, or vitamin B7, is essential for metabolic reactions. It must be obtained from external sources such as food and biotin/vitamin supplements because it is not biosynthesized by mammals. Therefore, there is a need to monitor its levels in supplements. However, biotin detection methods, which include chromatographic, immune, enzymatic, and microbial assays, are tedious, time-consuming, and expensive. Thus, we synthesized a product called biotin-naphthoquinone, which produces chemiluminescence upon its redox cycle reaction with dithiothreitol and luminol; then it was used as a chemiluminescence sensor for biotin–avidin interaction. When a quinone biotinylated compound binds avidin, the chemiluminescence decreases noticeably due to the proximity between quinone and avidin, and when free biotin is added in a competitive assay, the chemiluminescence returns. The chemiluminescence is regained as the free biotin displaces biotinylated quinone in its complex with avidin, freeing biotin-naphthoquinone. Many experiments, including the use of a biotin-free quinone, proved the competitive nature of the assay. The competitive assay method used in this study was linear in the range of 1.0–100 µM with a detection limit of 0.58 µM. The competitive chemiluminescence assay could detect biotin in vitamin B7 tablets with good recovery of 91.3 to 110% and respectable precision (RSD < 8.7%). Full article

An immunoassay is an analytical test method in which analyte quantitation is based on signal responses generated as a consequence of an antibody–antigen interaction. They are the method of choice for the measurement of a large panel of diagnostic markers. Not only are they fully automated, allowing for a short turnaround time and high throughput, but offer high sensitivity and specificity with low limits of detection for a wide range of analytes. Many immunoassay manufacturers exploit the extremely high affinity of biotin for streptavidin in their assay design architectures as a means to immobilize and detect analytes of interest. The biotin–(strept)avidin system is, however, vulnerable to interference with high levels of supplemental biotin that may cause elevated or suppressed test results. Since this system is heavily applied in clinical diagnostics, biotin interference has become a serious concern, prompting the FDA to issue a safety report alerting healthcare workers and the public about the potential harm of ingesting high levels of supplemental biotin contributing toward erroneous diagnostic test results. This review includes a general background and historical prospective of immunoassays with a focus on the biotin–streptavidin system, interferences within the system, and what mitigations are applied to minimize false diagnostic results. Full article

The use of antimicrobial growth promoters (AGPs) is banned because of problems associated with drug residues in animal products and increased bacterial resistance. The immunization of chickens with specific antigens is a promising strategy for generating specific antibodies that can target a wide range of antibiotic-resistant bacteria and can be used as an alternative to antibiotics. Immunoglobulin Y (IgY) antibodies in a polyclonal antibody (pAb) format, when administered orally, modulate the ruminal microbiome and maintain animal health and performance; however, there are concerns pertaining to protein impurities and biotin concentrations in the samples. Signal amplification strategies involving the noncovalent interaction of biotin with streptavidin is extensively used in diagnosis and scientific research, particularly in enzyme-linked immunosorbent assays (ELISAs). However, the high concentrations of biotin in samples, especially in those derived from rich sources such as egg yolk, can pose challenges and potentially harm the accuracy of diagnostic tests and protein concentration measurements. This study aimed to evaluate the influence of biotin on the measurement of IgY in freeze-dried egg yolk samples obtained from immunized laying hens using immunoassays with biotin–avidin/streptavidin. The detection of IgY in yolk samples using ELISA with streptavidin–biotin binding could lead to misdiagnosis due to biotin interference; the level of interference varies with the specific assay conditions and the concentration of biotin in the yolk samples. An ELISA without streptavidin–biotin binding is advisable to avoid interactions between biotin and target proteins, prevent biotin interference with the results, and achieve more reliable and accurate results. Full article

An important biomarker of oxidative damage in cellular DNA is the formation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). Although several methods are available for the biochemical analysis of this molecule, its determination at the single cell level may provide significant advantages when investigating the influence of cell heterogeneity and cell type in the DNA damage response. to. For this purpose, antibodies recognizing 8-oxodG are available; however, detection with the glycoprotein avidin has also been proposed because of a structural similarity between its natural ligand biotin and 8-oxodG. Whether the two procedures are equivalent in terms of reliability and sensitivity is not clear. In this study, we compared the immunofluorescence determination of 8-oxodG in cellular DNA using the monoclonal antibody N45.1 and labeling using avidin conjugated with the fluorochrome Alexa Fluor488 (AF488). Oxidative DNA damage was induced in different cell types by treatment with potassium bromate (KBrO₃), a chemical inducer of reactive oxygen species (ROS). By using increasing concentrations of KBrO₃, as well as different reaction conditions, our results indicate that the monoclonal antibody N45.1 provides a specificity of 8-oxodG labeling greater than that attained with avidin-AF488. These findings suggest that immunofluorescence techniques are best suited to the in situ analysis of 8-oxodG as a biomarker of oxidative DNA damage. Full article

Molecular immobilization and recognition are two key events for the development of biosensors. The general ways for the immobilization and recognition of biomolecules include covalent coupling reactions and non-covalent interactions of antigen–antibody, aptamer–target, glycan–lectin, avidin–biotin and boronic acid–diol. Tetradentate nitrilotriacetic acid (NTA) is one of the most common commercial ligands for chelating metal ions. The NTA–metal complexes show high and specific affinity toward hexahistidine tags. Such metal complexes have been widely utilized in protein separation and immobilization for diagnostic applications since most of commercialized proteins have been integrated with hexahistidine tags by synthetic or recombinant techniques. This review focused on the development of biosensors with NTA–metal complexes as the binding units, mainly including surface plasmon resonance, electrochemistry, fluorescence, colorimetry, surface-enhanced Raman scattering spectroscopy, chemiluminescence and so on. Full article

The H9N2 subtype of low-pathogenic avian influenza viruses (LPAIV) is a widespread pathogen of poultry that can also infect humans. The characterization of viral infections is a complex process, involving clinical, pathological, and virological investigations. The aim of this study was to adapt and optimize an immunohistochemical (IHC) technique developed for LPAIVs specifically for the detection of H9N2 virus antigens in infected tissues. Twenty-one-day-old broiler chickens were inoculated with three different strains of H9N2 virus by different infection routes (i.e., intranasal-intratracheal and intravenous) or co-infected with infectious bronchitis virus (IBV) and observed for 11 days post infection. The suggested IHC protocol was modified: (i) DAB (diamino-benzidine) was substituted with AEC (3-amino-9-ethyl carbazole) as chromogen; and (ii) indirect two-step immune reactions of monoclonal primary and peroxidase-labeled anti-mouse secondary antibodies were used instead of avidin–biotin complexes. Avian influenza virus antigen appears as a red precipitate in the nuclei of affected cells but can also be identified in the cytoplasm. Mild hyperemia and congestion were observed in the trachea, air sac, and lungs of the challenged birds, and fibrinous exudate was found at the bifurcation in a few cases. Neither gross pathological nor IHC lesions were found in the control group. Using the optimized protocol and an associated scoring scheme, it was demonstrated that the H9N2 strains tested exhibited respiratory and urinary tract tropism irrespective of the route of inoculation. On day 5, viral antigen was detected in the respiratory tract and kidney in 30–50% of the samples. On day 11, no IHC signal was observed, indicating the lack of viral replication. Slight differences in viral antigen expression were found between the different H9N2 virus strains, but, in contrast to highly pathogenic avian influenza (HPAI), no viral antigen was detected in the brain and pancreas. Thus, IHC can be considered as an informative, visual addition to the toolkit for the characterization of H9N2 LPAIV infections. Full article