Search Results (396)

Recent investigations into C-reactive protein (CRP) dynamics have shown that by evaluating the change in CRP level in a patient over time, it is possible to distinguish between bacterial and viral infections more accurately thereby guiding antimicrobial prescription practices. Consequently, a biosensor targeted towards CRP was developed using a nano- and microfiber mesh-based transducer. The produced transducers were functionalized with streptavidin, after which a biorecognition element, anti-CRP antibodies, could be bound to the sensor. Confirmation of the sensor production phases was obtained using fluorescence microscopy. The sensors were evaluated using Electrochemical Impedance Spectroscopy (EIS) and showed increasing changes in the impedance modulus corresponding to increasing concentrations of CRP in solution following a parabolic trend line. Full article

Giardiasis is a globally prevalent waterborne zoonosis. Rapid enrichment and detection technologies for this disease are essential. Cyst outer wall proteins are ideal targets for the enrichment and detection of cysts in the environment, but there are few available targets with suboptimal effectiveness. In this study, Giardia duodenalis (G. duodenalis) cysts were purified, and outer wall proteins were biotinylated, followed by streptavidin magnetic bead purification and mass spectrometry. Sixty-three novel cyst wall proteins were identified, and their functions were annotated through Gene Ontology (GO) and KEGG analyses. The β-giardin and α-1 giardin were among the newly identified and predicted to be located on the outer wall of G. duodenalis cysts. For the characterization of these two targets, we applied sequence analysis, prokaryotic expression, preparation of polyclonal antibodies, and determination of subcellular localization. Finally, based on β-giardin immunomagnetic beads were prepared using the polyclonal antibodies and tested for their enrichment efficiency. Immunomagnetic beads targeting β-giardin achieved 65% cyst enrichment efficiency in fecal samples, comparable to conventional methods. Clinical evaluation across 163 multi-host fecal samples (ferrets, Siberian tigers, red-crowned cranes) demonstrated concordance with nested PCR, successfully enriching cysts from PCR-positive specimens. The immunomagnetic beads method targeting β-giardin demonstrated effective G. duodenalis cyst enrichment in multi-host fecal samples. These results provide a proteomic framework for the cyst wall proteins of G. duodenalis, expanding the detection targets for G. duodenalis cysts. It also establishes a theoretical foundation for subsequent research on the composition and function of G. duodenalis cysts. Full article

Carbyne-containing materials offer significant potential for biosensor applications due to their unique chemical and mechanical properties. In this study, carbyne-enriched carbon coatings deposited on SiO₂/Si chips using ion-assisted pulse-plasma deposition were evaluated for the first time as substrates for optical biosensing. At first, the carbyne-enriched coatings were characterized by X-ray photoelectron spectroscopy, Raman spectroscopy, Atomic Force Microscopy, and the sessile drop method to assess their composition, structure, and wettability. After that, chips with carbyne-enriched coatings were modified with biomolecules through physical absorption or covalent bonding, and the respective biomolecular interactions were monitored in real-time by White Light Reflectance Spectroscopy (WLRS). In both cases, SiO₂/Si chips modified with an aminosilane were used as reference substrates. Physical adsorption was tested through immobilization of an antibody against C-reactive protein (CRP) to enable its immunochemical detection, whereas covalent bonding was tested through coupling of biotin and monitoring its reaction with streptavidin. It was found that the carbyne-enriched carbon-coated chips retained both their antibody adsorption capability and their covalent bonding ability for over 18 months, while the modified with aminosilane SiO₂/Si chips lost 90% of their antibody adsorption capacity and covalent bonding ability after two months of storage. These findings highlight the strong potential of carbyne-enriched carbon-coated chips as robust biosensing substrates, with applications extending beyond WLRS. Full article

Proximity biotinylation, which utilizes various biotin ligating enzymes (BioID, TurboID, etc.), is widely used as a powerful tool for identifying novel protein–protein interactions. However, this method has a significant limitation: the use of streptavidin on beads for enriching biotinylated proteins often results in a high background of peptides from streptavidin itself, which interferes with identification by peptide mass fingerprinting. This limitation makes it practically impossible to study samples containing a small amount of material, such as individual insect tissues. In this study, we compared different precipitation and elution conditions for the purification of biotinylated proteins from protein extracts of Drosophila melanogaster S2 cells. We found that biotinylated proteins can be purified using anti-biotin antibodies, although with lower efficiency than streptavidin-based resin. We also demonstrated that protease-resistant streptavidin (prS), previously tested in mammalian cells, can be used effectively to purify biotinylated proteins from tissues of D. melanogaster. In our experiments, prS showed precipitation efficiency comparable to regular streptavidin but generated a lower background in peptide fingerprinting. To further demonstrate the applicability of prS for studying protein–protein interactions in D. melanogaster tissues, we carried out experiments to identify interaction partners of the ecdysone receptor (EcR) in D. melanogaster ovarian tissue using TurboID-based proximity biotinylation. As a result, EcR was found to interact with both previously described and novel protein partners in Drosophila ovaries. Full article

We report herein the development of a polyclonal antibody against ochratoxin A (OTA) using a specially designed synthetic OTA derivative as the immunizing hapten. This OTA derivative contains a tetrapeptide linker (glycyl-glycyl-glycyl-lysine, GGGK), through which it can be linked to a carrier protein and form an immunogenic conjugate. The OTA derivative (OTA-glycyl-glycyl-glycyl-lysine, OTA-GGGK) has been synthesized on a commercially available resin via the well-established Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS) strategy; overall, this approach has allowed us to avoid tedious liquid-phase synthesis protocols, which are often characterized by multiple steps, several intermediate products and low overall yield. Subsequently, OTA-GGGK was conjugated to bovine thyroglobulin through glutaraldehyde, and the conjugate was used in an immunization protocol. The antiserum obtained was evaluated with a simple-format ELISA in terms of its titer and capability of recognizing the natural free hapten; the anti-OTA antibody, as a whole IgG fragment, was successfully applied to three different immunoanalytical systems for determining OTA in various food materials and wine samples, i.e., a multi-mycotoxin microarray bio-platform, an optical immunosensor, and a biotin–streptavidin ELISA, which has proved the analytical effectiveness and versatility of the anti-OTA antibody developed. The same approach may be followed for developing antibodies against other low-molecular-weight toxins and hazardous substances. Full article

The human Tau protein stands for one of the most conspicuous and crucial hallmarks of Alzheimer’s disease (AD) diagnosis, along with other tauopathies. However, the assay for direct detection of tiny Tau protein concentrations in human samples continues to pose a significant challenge for the early diagnosis of AD. Thus, an amplification-based strategy is required. In this proposed work, we established an impedimetric immunosensor to detect human Tau-441 protein in PBS buffer using a sandwich approach, wherein we employed two distinct monoclonal antibodies (HT7 and BT2) that specifically recognize the amino acids 159–198 of the target protein. Through this strategy, we were able to detect as low as 0.08 pg/mL. These findings were attributed to the use of a biotinylated antibody (BT2)-streptavidin complex, which facilitated the amplification of the normalized signal, resulting in a lower limit of detection in comparison to the directly based immunosensors. Subsequently, we investigated the designed immunosensor to assess the assay’s selectivity in the presence of different off-targets, and no cross-interaction was recorded. The outcomes of our study provide valuable new insights into the application of sandwich-based assay as a highly sensitive and selective immunosensor for the detection of small protein. Full article

Background: Cow’s milk allergy (CMA) is the most common food allergy in children, typically resolving by adolescence. In contrast, the clinical spectrum of allergies to non-cow mammalian milk and their patterns of IgE cross-reactivity are less well documented. Nutritional differences between various mammalian milks may also impact dietary management in milk-allergic patients. Objectives: To characterize clinical features, onset age, and IgE cross-reactivity patterns of non-cow mammalian milk allergies in adult patients seen at a tertiary allergy center, and to compare these findings with published cases. Methods: A retrospective analysis of patients included in the “Extended Laboratory Investigation for Rare Causes of Anaphylaxis study” with mammalian milk allergy was performed using clinical history, skin testing, and serum-specific IgE measurements. Cross-reactivity patterns were assessed in selected cases using immunoblotting, specific IgE inhibition, and basophil activation testing, and compared with published reports of non-cow mammalian milk allergy. Results: In our case series of 22 patients with mammalian milk allergy and 10 healthy control subjects, 3 patients were identified with isolated adult-onset non-cow mammalian milk allergy (n = 1 buffalo milk; n = 2 mare milk), confirmed via immunoblotting and basophil activation testing. Streptavidin-based specific IgE measurement for buffalo cheese was positive in the buffalo milk allergic patient. The literature review identified 82 cases of non-cow mammalian milk allergy. These cases typically showed late onset (mean age 8.6 years; range 1–70 years), severe reactions (CoFAR (Consortium for Food Allergy Research) grade 3 or 4 in 66%, and one fatality), and selective sensitization (affecting sheep and/or goat, camel, mare, buffalo, donkey, or combinations thereof in 56, 10, 5, 5, 4, and 2 cases, respectively). Conclusions: Non-cow mammalian milk allergies are rare but generally present later in life with selective IgE cross-reactivity, differing from the broader cross-reactivity observed in CMA. This selectivity may allow for safe dietary alternatives. These findings underscore the need for improved diagnostics and personalized dietary management in this patient population. Full article

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

The rapid and accurate detection of foodborne pathogens is essential for ensuring food safety. Escherichia coli O157:H7 (E. coli O157:H7) and Salmonella Typhimurium (S. Typhimurium) are major foodborne pathogenic bacteria that pose significant public health risks, highlighting the need for effective detection methods. In this study, highly sensitive double-antibody sandwich-based enzyme-linked immunosorbent assays (ELISAs) were developed for the rapid detection of E. coli O157:H7 and S. Typhimurium, utilizing a streptavidin-polymerized horseradish peroxidase (SA-PolyHRP)-based signal enhancement system. Systematic optimization was performed on key parameters, including the capture antibody concentration, detection antibody, and blocking agent. Compared to the method using SA-HRP, substitution with SA-PolyHRP significantly improved detection sensitivity, achieving limits of detection (LODs) of 1.4 × 10⁴ CFU/mL for E. coli O157:H7 and 6.0 × 10³ CFU/mL for S. Typhimurium, with sensitivity enhancements of 7.86-fold and 1.83-fold, respectively. Specificity tests confirmed no cross-reactivity with non-target or closely related pathogenic strains. The matrix effect was effectively mitigated through 10-fold and 100-fold dilutions for E. coli O157:H7 and S. Typhimurium, respectively. Both pathogens were successfully detected in beef samples spiked with 5 CFU after 5 h of incubation. This study demonstrates the effectiveness of PolyHRP-based signal enhancement for the highly sensitive and specific detection of foodborne pathogens, offering a promising approach for rapid food safety monitoring and public health protection. Full article

Aflatoxin M1 (AFM1) appears in the milk of animals that have consumed feed contaminated with aflatoxin B1. AFM1 presence in milk is regulated by the European Commission, which has set the maximum allowable limits for adult and infant consumption to 50 and 25 pg/mL, respectively. Here, a rapid and sensitive method for detecting AFM1 in milk based on an immersible silicon photonic chip is presented. The chip features two U-shaped silicon nitride waveguides formed as Mach–Zehnder interferometers. One interferometer is functionalized with AFM1–bovine serum albumin conjugate and the other with BSA to serve as a blank. The chip is connected to a broad-band white LED and a spectrophotometer by a bifurcated optical fiber and an assay is performed by immersing the chip in a mixture of milk with the anti-AFM1 antibody. Then, the chip is sequentially immersed in biotinylated anti-rabbit IgG antibody and streptavidin solutions for signal enhancement. The assay is completed in 20 min and the detection limit for AFM1 in undiluted milk is 20 pg/mL. Given its analytical performance and the absence of pumps and fluidics that lead to a compact instrument design, the proposed immunosensor is ideal for the on-site detection of AFM1 in milk samples. Full article

3D-printed microdevices have become increasingly important to the advancement of point-of-care (POC) immunoassays. Despite its great potential, using 3D-printed surfaces on the solid support for immunorecognition has been limited due to the non-ideal adsorption properties for many photocurable resins. In this work, we report a simple surface modification protocol that works for diverse commercial photocurable resins, improving ELISAs performed directly on 3D-printed devices. This surface modification strategy involves surface activation via air plasma followed by the one-step incubation of GLYMO-labeled streptavidin. We successfully immobilized biotinylated anti-activin A antibodies on the 3D-printed surfaces and performed the complete ELISA protocol on the 3D-printed surfaces. We demonstrated that this protocol achieved an improved performance over passive adsorption for ELISAs. The present method is also compatible with diverse commercial resins and works with both microwells and microchannels. Finally, this method demonstrated a comparable limit of detection to the ELISA performed using commercial microwells. We believe the simplicity and broad compatibility of the present surface modification strategy will facilitate the development of 3D-printed POC ELISA devices. Full article

Quantitative nucleic acid detection is widely used in molecular diagnostics for infectious diseases. Here, we demonstrate that the previously developed MLFIA (magnetically localized fluorescent immunoassay) has the potential to detect Polymerase Chain Reaction (PCR) and loop-mediated isothermal amplification (LAMP) products using biotinylated and fluorescent primers and streptavidin-coated magnetic nanoparticles. The functionalized nanoparticles separate amplified DNA from non-incorporated primers in situ, allowing the quantification of DNA products. We compare magnetically localized fluorescence detection to commercial technologies based on the DNA intercalation of fluorescent dyes. Our system allows the detection of PCR and LAMP products but is approximately 10 times less sensitive than standard commercial assays. Future optimizations, such as enhancing the signal-to-noise ratio and improving nanoparticle functionalization, could significantly increase sensitivity and bring it closer to current diagnostic standards. This work highlights the potential of magnetically localized fluorescence detection to detect DNA. Full article