Search Results (407)

Membrane proteins remain the most challenging targets for structural characterization, yet their elucidation provides valuable insights into protein function, disease mechanisms, and drug specificity. Structural biology platforms have advanced rapidly in recent years, notably through the development and implementation of nanodiscs—discoidal lipid–protein complexes that encapsulate and solubilize membrane proteins within a controlled, native-like environment. While nanodiscs have become powerful tools for studying membrane proteins, faithfully reconstituting the compositional asymmetry intrinsic to nearly all biological membranes has not yet been achieved. Proper membrane leaflet lipid distribution is critical for accurate protein folding, stability, and insertion. Here, we share a protocol for reconstituting tailored compositional asymmetry within nanodiscs through membrane extraction from giant unilamellar vesicles (GUVs) treated with a leaflet-specific methyl-β-cyclodextrin (mβCD) lipid exchange. Nanodisc asymmetry is verified through a geometric approach: biotin-DPPE-preloaded mβCD engages in lipid exchange with the outer leaflet of POPC GUVs solubilized by the lipid-free membrane scaffold protein (MSP) Δ49ApoA-I to form nanodisc structures. Once isolated, nanodiscs are introduced to the biotin-binding bacterial protein streptavidin. High-speed atomic force microscopy imaging depicts nanodisc–dimer complexes, indicating that biotin-DPPE was successfully reconstituted into a single leaflet of the nanodiscs. This finding outlines the first step toward engineering tailored nanodisc asymmetry and mimicking the native environment of integral proteins—a potentially powerful tool for accurately reconstituting and structurally analyzing integral membrane proteins whose functions are modulated by lipid asymmetry. Full article

Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a’s trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV. Full article

Severe dengue virus (DENV) infections are associated with circulating non-neutralizing antibodies generated during heterotypic infections. Although antibodies are key mediators of both protection and pathogenesis, the specific dynamics of B cells (Bc) and their antibody responses remain insufficiently characterized due to limited methods of identifying DENV-specific Bc (DENV-Bc) and the absence of animal models resembling the human disease. Here, we developed a spectral flow cytometry assay employing biotinylated virus-like particles (VLPs) to detect DENV-Bc in C57BL/6 mice and children hospitalized with dengue. DENV-1 and DENV-2 VLPs were biotinylated, and the efficiency of biotin incorporation was assessed with an HABA-avidin assay and ELISA. Serotype specificity and optimal binding conditions were confirmed using hybridomas 4G2 (pan-flavivirus) and 3H5-1 (DENV-2 specific). Fluorescent agglutimers were subsequently generated by coupling biotinylated VLPs to streptavidin–fluorochrome complexes. Splenocytes from intraperitoneally DENV-infected mice and peripheral blood mononuclear cells (PBMCs) from naturally infected pediatric patients were stained with these VLPs and Bc-lineage markers. Biotinylated VLPs bound specifically to hybridomas, and this binding was competitively inhibited by unlabeled VLPs. After secondary DENV challenge, VLPs identified DENV-specific class-switched plasmablasts in mice. Circulating DENV-specific plasmablasts were also detected in children, with agglutimers enabling the discrimination of serotype-specific and cross-reactive responses in primary and secondary infections. This VLP-based approach represents a scalable platform to investigate the protective and pathogenic roles of DENV-Bc in infection and vaccination. Full article

Background: Detecting low-frequency mutations is crucial for predicting prognosis and monitoring minimal residual disease (MRD) in acute myeloid leukemia (AML). However, the presence of abundant wild-type sequences hinders the detection of rare mutant alleles. We present a highly sensitive method called ACE (Amplifying–Cleaving–Enriching) to selectively enrich mutant sequences. Methods: ACE includes three steps: (1) initial PCR amplification using biotin-labeled primers, (2) cleavage of wild-type sequences with a specific restriction enzyme, and (3) enrichment of undigested mutant alleles via streptavidin-labeled magnetic beads. Results: Using two rounds of ACE, we achieved over 80,000-fold enrichment of mutant sequences carrying FLT3-TKD, enabling the detection of mutant alleles at levels as low as 0.0001% in AML patient blood samples. Additionally, the ACE method can be adapted to nearly any driver mutation by introducing wild-type-specific restriction sites through PCR with mismatched primers, which has been validated in the IDH1 mutation. Furthermore, the ACE method can be flexibly integrated into conventional detection techniques including Sanger sequencing, quantitative real-time PCR, allele-specific PCR, and even with advanced techniques like droplet digital PCR. Conclusions: ACE significantly enhances the sensitivity of existing techniques for rare mutation detection and holds potential for broad clinical applications. Full article

This paper proposes a method for E. coli detection in a microplate format using low-molecular-weight compounds that specifically interact with the lipopolysaccharides (LPSs) of E. coli cell walls. These compounds can amplify analytical signals by binding to multiple repeating cell surface structures, while the selectivity for E. coli is ensured by preliminary cultivation on selective media, such as Endo or MacConkey agar. 3-Aminophenylboronic acid (APBA) was selected as the binding reagent for detecting E. coli LPSs. Conjugates of streptavidin (STP) and bovine serum albumin (BSA) with APBA and conjugates of biotin and soybean trypsin inhibitor (STI) and BSA were synthesized. The conditions for the sequential formation of “sandwich” type complexes (BSA-APBA conjugate/E. coli/STP-APBA/STI–biotin/STP–peroxidase) and their colorimetric detection using chromogenic peroxidase substrate were determined. The detection limit was 3 × 10² cells/mL, and the range of quantitative determination covered five orders of magnitude—from 10³ to 10⁸ cells/mL. The developed assay was successfully tested using inactivated cells of pathogenic E. coli strains, confirming its potential for application. The assay was demonstrated to have universality, with the ability to detect E. coli, other bacterial pathogens, and LPS alone. This method could be adopted for the quantitative determination of different specific bacterial species using different selective media. Full article

The epidemic of infectious diseases, such as influenza A, has imposed a severe health burden on the population. Early detection, diagnosis, reporting, isolation, and treatment are crucial for the prevention, control, and management of infectious diseases. Nucleic acid testing represents a vital approach for the rapid diagnosis of pathogenic microorganism types. However, current nucleic acid detection methods face notable bottlenecks: traditional CRISPR fluorescence assays require time-consuming pre-amplification of target nucleic acids, while existing carbon-nanotube field-effect transistor (FET)-based platforms, though amplification-free, often necessitate complex chip surface modification and probe immobilization, and suffer from non-reusable chips, all limiting their utility in point-of-care testing (POCT) and large-scale screening. This study reports a CRISPR-based amplification-free RNA detection platform (CRISPR-FET) for the rapid identification of influenza A virus. The CRISPR-FET platform described herein enables the detection of viral RNA without amplification within 20 min, with a limit of detection as low as 1 copy/μL. Secondly, a reporter RNA conjugated with gold particles is used to achieve signal amplification in FET detection; meanwhile, the method eliminates probe immobilization, thereby omitting this step and simplifying chip modification to reduce complex work-flows and pre-treatment costs. The chip’s reusability further enhances cost-effectiveness. Additionally, streptavidin-modified magnetic bead adsorption minimizes background errors from excessive reporter RNA and non-target nucleic acids. Finally, validation with 24 clinical samples confirmed the platform’s efficacy. By integrating rapidity, simplicity, and high sensitivity, alongside cost advantages from reusable chips, this CRISPR-FET platform meets the critical need for early influenza A diagnosis and holds promise for advancing POCT and large-scale epidemiological screening. Full article

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a critical biomarker for the early diagnosis of acute kidney injury (AKI). The development of novel detection platforms that combine rapid analysis with high sensitivity is essential for improving clinical outcomes. In this study, we established an antibody-based detection system for NGAL using biolayer interferometry (BLI), a label-free optical biosensing technique that monitors real-time interference patterns generated by white light reflected from biomolecular binding events on a biosensor surface. A panel of six anti-NGAL monoclonal antibodies was generated and characterized for its binding properties, identifying candidates with high specificity for NGAL. For robust sensor functionalization, selected monoclonal antibodies were biotinylated and immobilized onto streptavidin-coated biosensor tips, establishing a stable and efficient detection interface. The optimized BLI platform demonstrated a limit of detection (LOD) of 46.1 ng/mL with wild dynamic range of 19 to 40,000 ng/mL. The platform’s accuracy was validated using human serum samples, with spike-and-recovery experiments yielding recovery rates of 96.6–104.6%. This demonstrates the capability to accurately quantify NGAL under physiologically relevant conditions with minimal matrix interference. Furthermore, the real-time kinetic measurements enabled rapid analysis, with the entire assay completed in less than half an hour. These findings establish a proof-of-concept for a BLI-based biosensor for NGAL detection, demonstrating sensitivity and specificity that show potential for clinical applications. Full article

In this work, we investigated the influence of silver nanoparticle (AgNP) size (diameters of 20, 50, and 100 nm) and magnetic bead (MB) size (diameters from 100 to 4500 nm) on silver-gold galvanic exchange signal generation in magnetic electrochemical assays. Two conjugation strategies, including biotin-streptavidin interaction and a streptavidin-specific aptamer interaction, were compared to assess differences in binding chemistry and conjugation efficiency. Calibration studies showed that 50 nm diameter AgNPs provided the best sensitivity and galvanic exchange efficiency, yielding the lowest detection limits across both conjugation strategies. Larger AgNPs produced stronger signals but reached saturation rapidly, whereas smaller particles required higher concentrations to achieve equivalent silver content. Among MBs, 1000 nm beads consistently gave the highest galvanic exchange efficiency, offering sufficient surface area for AgNP loading while minimizing steric hindrance and electrode obstruction. These findings were confirmed by complementary electrochemical impedance spectroscopy, UV-Vis absorbance, and SEM imaging, which collectively demonstrated the strong influence of bead size on charge transfer resistance and conjugation efficiency. Overall, the combination of 50 nm AgNPs with 1000 nm MBs emerged as the optimal configuration, providing improved sensitivity and reproducibility. We believe these results offer valuable design guidelines for the development of next-generation aptamer-based electrochemical biosensors for biomarker detection. Full article

We report the first analytical application of albumin nanoparticles loaded with luminescent europium complexes for immunoassay development. These nanoparticles, synthesized via a desolvation method, exhibited a uniform spherical morphology with a hydrodynamic diameter of 263 nm and strong, long-lived luminescence at 615 nm (λex = 360 nm). Surface functionalization with streptavidin enabled specific binding to biotinylated proteins. The nanoparticles were applied as labels in a sandwich time-resolved solid-phase immunoassay for human IgG detection in black 96-well plates. Unlike commercial DELFIA assays, the method eliminates the need for signal enhancement steps, as the nanoparticles intrinsically contain high concentrations of europium complexes. Optimization studies revealed that the sharp emission peaks of europium can compromise assay reproducibility; however, employing surface scanning and increasing measurement replicates per well partially mitigated this effect. Time-resolved detection reduced background by two orders of magnitude and increased signal intensity nearly tenfold in IgG-positive samples. The assay demonstrated minimal cross-reactivity with IgA and IgM (~2%) and enabled IgG detection at serum dilutions up to 1:100,000. Comparative analysis showed strong concordance with commercial immunoassays and no concentration-dependent bias. The primary limitation observed was suboptimal intra-assay reproducibility (CV > 20% in four of six tested sera). Full article

Transcription factors orthodenticle homeobox 2 gene (OTX2), paired box 6 gene (PAX6), and SRY-box transcription factor 2 gene (SOX2) are key regulators of ocular morphogenesis; however, their comparative embryonic localization across species—and the correspondence between transcript and protein distributions—remain incompletely defined. Chromogenic in situ hybridization (CISH) was employed to detect OTX2, PAX6, and SOX2 transcripts, while biotin–streptavidin immunohistochemistry (IHC) was used to assess Otx2, Pax6, and Sox2 protein expression. A semi-quantitative scoring system was applied to evaluate positive structures across ocular compartments. Transcripts were predominantly localized to the retina in both species, with occasional low-level expression in the optic nerve, sclera, and eyelid. Proteins displayed broader distributions: Otx2 was abundant in the retina and eyelid, while Pax6 and Sox2 were detected in multiple tissues, including cornea and extraocular muscles. OTX2, PAX6, and SOX2 show retina-predominant transcription and wider protein expression across ocular tissues. These findings highlight spatial differences between transcript and protein localization, supporting a complex regulatory framework underlying vertebrate eye development. Full article

Multiple-mode immunoassays have the advantages of self-correction, self-validation, and high accuracy and reliability. In this work, we developed a strategy for the design of triple-mode immunoassays with the self-assemblies of three-in-one small molecules as signal reporters. Pyrroloquinoline quinone (PQQ), with a well-defined redox peak and excellent spectroscopic and fluorescent signals, was chosen as the signaling molecule. PQQ was coordinated with Cu²⁺ to form metal–organic nanoparticle as the signal label. Hexahistidine (His₆)-tagged recognition element (recombinant streptavidin) was attached to the Cu-PQQ surface through metal coordination interaction between the His₆ tag and the unsaturated metal site. The captured Cu-PQQ nanoparticle released a large number of PQQ molecules under an acidic condition, which could be simultaneously monitoring by electrochemical, UV-vis, and fluorescent techniques, thereby allowing for the development of triple-model immunoassays. The three methods were used to determine the concentration of carcinoembryonic antigen (CEA) with the detection limits of 0.01, 0.1, and 0.1 ng/mL, respectively. This strategy opens up a universal route for the preparation of multiple-model signal labels and the oriented immobilization of bioreceptors for molecular recognition. Full article

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