Bioconjugate Chemistry最新文献

The extensive utilization of nanomaterials and aptamers in biosensing has positioned the construction of efficient, stable, and user-friendly nanoprobe systems as a pivotal strategy for rapid small-molecule detection. This study developed a dual-probe fluorescence detection system utilizing gold nanoparticles (AuNPs) and quantum dots (QDs) for ochratoxin A (OTA) analysis. The AuNP probes were functionalized via an innovative freeze-driven asymmetric conjugation approach, while a distinct freeze-driven labeling strategy was applied to QDs. Compared to conventional labeling protocols, the freeze-driven methodology reduced conjugation duration from several hours to merely 20 min and was successfully implemented for QD-aptamer conjugation for the first time, facilitating efficient dual-probe assembly. The complete process, spanning from probe preparation to OTA quantification, was accomplished within 1.5 h. Under optimized parameters, the biosensor demonstrated a linear detection range of 0.5-50 ng/mL, achieved a detection limit of 0.183 ng/mL, and exhibited satisfied specificity, reproducibility, and matrix interference resistance. Validation experiments across various herbal specimens confirmed its accuracy and practical applicability. This research not only establishes a rapid and efficient platform for OTA monitoring but also provides a generalized framework for developing nanomaterial-based biosensing systems.

This work addresses the need for cost-effective, stable, and sensitive detection of tetracycline, which is a widely used antibiotic molecule, with environmental and health risks associated with its incorrect use. We explored the use of N-substituted polyglycine oligomers (peptoids) as molecules capable of tetracycline conjugation. Peptoids are readily synthesized by solid-phase synthesis, offering great chemical diversity and enhanced stability. Inspired by a short aptamer that selectively binds tetracycline, we designed helical peptoids containing side chains selected for their ability to promote π-π stacking interactions and hydrogen bonding. Five peptoid sequences were selected, differing in monomer order, and their complexation capacity was investigated by using different mass spectrometric techniques and circular dichroism. Tandem mass spectrometry and ion mobility spectrometry investigations confirmed the complex formation between peptoids and tetracycline. Additionally, circular dichroism revealed that the peptoid sequence influences the complexation. Our findings demonstrate the potential for rationally designed peptoids as capture probes for sensing applications.

The drug release performance of polymer-based nanoscale drug delivery systems (nano-DDSs) is determined by the carrier configurations and the drug-loading modes. Here, to integrate the merits of the polyprodrugs and cross-linked copolymer nanoparticles, pH/glutathione (GSH) dual-responsive core-cross-linked copolyprodrug nanoparticles (PEG-cPMN) were designed as a drug self-delivery system for precise tumor chemotherapy, by facile cross-linking-induced self-assembly of diblock copolymer PEG-PMN with a pH/GSH dual-triggered doxorubicin (DOX)-based dimeric prodrug as a cross-linker, via acid-labile acylhydrazone bond. The optimized copolyprodrug nanoparticles, possessing a DOX content of 24.1% and average hydrodynamic diameter (Dh) of 159 nm, exhibited an excellent pH/GSH dual-triggered drug release, with accumulative DOX release of 45.5% in 105 h in the simulated tumor intracellular microenvironment, while a negligible premature drug leakage of 2.5% in the simulated normal physiological medium. This feature endowed the proposed core-cross-linked copolyprodrug nanoparticles an outstanding tumor-specific on-demand DOX release without obvious cytotoxicity on the normal cells in the in vitro experiments.

Gene therapy has emerged as a powerful approach for treating diverse diseases, including genetic disorders, retinal diseases, and certain cancers. Real-time, noninvasive in vivo tracking of gene expression is essential for evaluating therapeutic efficacy. β-galactosidase (β-gal), a hydrolase encoded by the Escherichia coli lacZ gene or the human GLB1 gene, is widely used as a reporter of gene expression. In humans, β-gal deficiency underlies several fatal neurodegenerative disorders, including GM1 gangliosidosis. Here, we report the development of a β-gal-activated, human serum albumin (HSA)-binding gadolinium(III)-based MR contrast agent for noninvasive assessment of adeno-associated virus (AAV) gene therapy in GM1 gangliosidosis mice. The probe exhibited a gradual increase in MR relaxation rate upon incubation with β-gal in the presence of 4.5% HSA. Following intravenous administration, AAV-treated GM1 mice demonstrated distinct MR signal enhancement and kinetic profiles compared to untreated β-gal-deficient controls. This study establishes an enzyme-activated, protein-binding MR imaging strategy for real-time, noninvasive monitoring of AAV gene therapy.

Paper-based analytical devices (PADs) are promising platforms to achieve Point-of-Care Testing (POCT) due to their low cost, ease of operation, disposability, and simplicity of assaying. Inspired by the bioanalytical potential of marine luciferins as excellent optical indicators, we evaluated a panel of 30 coelenterazine (CTZ) analogs for the specific imaging of important serum proteins from various species. This panel included 20 novel CTZ analogs, recently developed by the authors and designated as the "G-series". Remarkably, several G-series CTZ analogs are exclusively bright with specific serum albumins and collectively create unique intensity patterns for these proteins. These G-series CTZ indicators produced highly quantitative luminescence signals with distinct spectral profiles across both trace and physiological albumin concentrations. This luminescence was utilized to determine kinetic constants, identify reaction sites, and establish dose-response curves for the various albumins. Finally, the optimized imaging system was transitioned to paper microplates for a rapid, on-site assay of serum albumins. HSA and BSA deposits dried on paper microplates were successfully imaged with CTZ indicators, achieving high signal-to-background ratios. This platform offers a cost-effective and portable approach to the rapid assay of serum albumins from various animal sources.

Several autoantigens relevant to the immune system, especially those targeted by autoantibodies induced by antitumor responses, tend to be rich in disordered regions and are prone to aggregation. This inherent instability presents significant challenges for the production, purification, and analysis of autoantigens in laboratory settings. Cysteine-specific cationization can effectively solubilize and purify these challenging proteins, allowing the isolation of full-length water-soluble antigens in their denatured state. The purified antigens enable accurate multiplex autoantibody assays using a suspension Luminex bead array platform. However, well-validated positive control antibodies are essential to ensuring precise clinical diagnosis. In this study, we prepared and characterized a panel of control antibodies by immunizing rabbits with cysteine-specific S-cationized antigens. The resulting antibodies predominantly recognized linear epitopes and were highly effective as quality control reagents in autoantibody array assays. Additionally, these antibodies maintained their ability to bind to their native, unmodified intracellular counterparts, highlighting the usefulness of this approach for producing antibodies against intrinsically disordered proteins. Although a modest immune response against the S-cationized modification site was observed, it remained minimal and did not affect the usefulness of the antibodies for assay validation. We propose this versatile cysteine-specific cationization platform for managing unstable proteins rich in disordered regions, supporting antigen production for diagnostics, and antibody development for research and validation purposes.

Photoinduced affinity labeling for cross-linking biomolecules in close spatial proximity has become a powerful strategy in life science studies to identify interaction partners in fundamental research as well as biomarkers in applied studies. Next-generation photo-cross-linkers additionally provide inducible fluorogenic properties to enable a visual read-out. Azido-substituted coumarin is nonfluorescent, but UV irradiation initiates the formation of a highly reactive nitrene radical that can act as a cross-linker while restoring the fluorescence activity of the coumarin chromophore. In this study, we present a 7-azidocoumarin derivative that is used as a suitable building block for solid-phase synthesis and demonstrates easy access to a variety of glycan-based photo affinity probes. Applications of photo-cross-linkers for glycans and their respective binding proteins are still rare. We show several azidocoumarin glycan-presenting probes and their selective targeting and covalent linking to lectins, accompanied by a turn-on fluorescence activity of the coumarin fluorophore. Selective recognition of specific target lectins from the presented glycan photo affinity probes is further demonstrated in complex biological environments, which now open opportunities for identifying and localizing both known and previously unidentified glycan receptors in cells, tissues, or patient samples.

Chronic neuroinflammation and dysfunction of the neuro-glial-vascular unit (NGVU) are central mechanisms driving the progression of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Neuropeptides, as key regulatory signaling molecules in the central nervous system (CNS), bind to specific G protein-coupled receptors (GPCRs) on the surfaces of microglia, astrocytes, oligodendrocytes, and cerebrovascular elements. Through cell type-specific biased signaling, they precisely regulate the threshold for inflammatory activation, coordinate phagocytosis and autophagy, maintain metabolic homeostasis, and support the function of the blood-brain barrier. This review systematically analyzes the immune-regulatory roles of key neuropeptides, including neuropeptide Y (NPY), vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide (VIP/PACAP), substance P (SP), and calcitonin gene-related peptide (CGRP). We focus on how these systems contribute to CNS homeostasis and disease-relevant processes, including myelin repair and neuroinflammatory regulation. Integrating evidence from preclinical models and human samples, it clarifies the pathological mechanisms linking these neuropeptides to disease progression. The review also outlines a translational research pathway focused on ligand structure engineering, targeted delivery, and biomarker-guided patient stratification, emphasizing receptor subtype selectivity and CNS permeability for precise therapy. By integrating the neuropeptide-mediated neuro-immune network, this work offers new insights into immune pathology in neurodegenerative diseases and provides a foundation for next-generation immune regulation.

CD38 is an established biomarker of multiple myeloma (MM), and peptide-based radiopharmaceuticals targeted to this receptor offer a route to molecularly specific imaging. In this work, we identified a novel CD38-targeted peptide sequence (HAPWFRGGGGS) through phage display and synthesized it using automated solid-phase peptide synthesis. The peptide was modified by introducing a PEG₄ spacer and on-resin conjugation of the DIAMSAR chelator, which forms a stable complex with Copper-64 (Cu-64), yielding DIAMSAR-PEG₄-HAPWFRGGGGS (Monomer_L). [⁶⁴Cu]Cu-Monomer_L was radiolabeled with high molar activity (>98% yield, ∼65 MBq/nmol) but showed suboptimal serum stability (∼45% intact at 2 h). To improve in vivo stability, the l-amino acid sequence was replaced with d-amino acid (Monomer_D), resulting in >90% serum stability and enhanced binding affinity toward CD38, as demonstrated by molecular docking and cell-binding assays in CD38-expressing MOLP2 human MM cells. To further increase avidity, a dimeric analog (Dimer_D) was designed by linking two Monomer_D units via a PEG₄ linker. In viable MOLP2 MM cells, tracer uptake ranked as [⁶⁴Cu]Cu-Dimer_D > [⁶⁴Cu]Cu-Monomer_D > [⁶⁴Cu]Cu-Monomer_L, and was markedly reduced by excess unlabeled peptide, confirming CD38-specific binding. Binding specificity and functional engagement of CD38 were further supported by antibody-blocking, enzymatic activity inhibition, and cellular internalization studies. Replacement of L-with d-amino acids improved binding affinity, lowering the K_d from 1043 nM ([⁶⁴Cu]Cu-Monomer_L) to ∼740 nM ([⁶⁴Cu]Cu-Monomer_D). The dimerization further lowered the K_d (∼730 nM) with markedly higher B_max (6993 fmol/mg vs 3024 fmol/mg), consistent with avidity-driven enhancement in receptor engagement. In vivo small animal dynamic PET/CT and ex vivo biodistribution were performed in disseminated and subcutaneous MOLP2-CBR-GFP MM models with naïve controls. Uptake increased with peptide valency, showing maximum femoral uptake of 1.52 ± 0.35 and 2.93 ± 0.68% ID/mL for [⁶⁴Cu]Cu-Monomer_D and [⁶⁴Cu]Cu-Dimer_D, respectively, whereas naïve mice exhibited <1% ID/mL over 0-2 h post injection (3-4 MBq; 45-50 pmol). In the subcutaneous model, [⁶⁴Cu]Cu-Dimer_D enabled clear tumor visualization at 2 h post injection with 4.66 ± 0.20% ID/mL uptake and a T/M ratio of 10.6 ± 3.1. Ex vivo tissue biodistribution confirmed higher femoral uptake (2.26 ± 0.42% ID/g) and femur-to-muscle ratio (18.17 ± 3.26). Autoradiography of excised tissues corroborated tracer localization to tumor-rich regions. Overall, [⁶⁴Cu]Cu-Dimer_D demonstrates high stability, avidity, and translational promise as a CD38-targeted PET tracer for MM.

Targeting intracellular signaling molecules that translocate to the nucleus is a promising approach for peptide-based therapeutics. Here, we focused on Smad2/3, key mediators of TGF-β signaling that act as transcription factors upon nuclear entry. To inhibit their nuclear localization, we delivered an Smad2/3-binding SARA peptide into the cytoplasm using our previously developed cell-penetrating carrier, cpPG. A conjugate, SARA-cpPG, was synthesized by linking the SARA peptide to Mal-DKDKC₁₂-K₅, a cpPG derivative with an N-terminal maleimide. In A549 cells, SARA-cpPG showed uptake over 20-fold higher than that of the SARA peptide alone and retained cytoplasmic localization. Functionally, SARA-cpPG suppressed TGF-β1-induced actin polymerization and cell migration, comparable to the effects of receptor kinase inhibitor LY2157299. These effects were not observed with cpPG alone or a control conjugate (SARAm-cpPG) containing a nonbinding mutant peptide. Mechanistic studies revealed that SARA-cpPG did not inhibit Smad2/3 phosphorylation but reduced TGF-β target gene expression, suggesting a blockade of nuclear translocation. This suppression was absent in treatments with SARAm-cpPG, cpPG alone, or a non-cytoplasm-specific carrier conjugate (SARA-R8). These findings demonstrate that cpPG enables efficient cytosolic delivery of functional peptides and supports a strategy for intracellular peptide therapeutics targeting nuclear signaling pathways.