Immunoassays are essential tools in clinical diagnostics, food safety surveillance, and environmental monitoring; however, a persistent discrepancy exists between the theoretical performance of bioreceptors and their practical efficacy in sensor devices. This performance gap is largely rooted in the stochastic nature of antibody anchoring at sensing interfaces, which frequently causes surface-induced denaturation, orientational heterogeneity, and steric occlusion. This review provides a microscopic framework to elucidate these deleterious interfacial behaviors and advocates a shift from empirical trial-and-error practices toward rational, controllable interface design. We systematically categorize and evaluate strategies for controlled antibody anchoring along a passive-to-active regulation spectrum. Passive positioning strategies, including affinity-mediated capture and site-defined chemical anchoring, use external binding mediators or localized antibody modifications to guide antibodies into functional postures. By contrast, active programming approaches, including genetically engineered antibodies and anchoring guided by intrinsic antibody properties, elevate antibodies into autonomous structural components that direct their own spatial integration at interfaces. Furthermore, exploratory concepts from adjacent disciplines and possible long-range conformational effects of antibody anchoring are also briefly considered as complementary perspectives for future interface design. To resolve the field's fragmented evaluation metrics, we propose a reporting framework that integrates key measurements needed to support claims of improved antibody anchoring. Finally, challenges and future directions are outlined, emphasizing the integration of computational interface engineering, AI-guided protein design, and standardized evaluation protocols. Together, these advances chart a clear path toward predictable, robust, and optimized immunosensing platforms.
The reliable electrochemical detection of immunoglobulin G (IgG) in complex biological fluids is severely hampered by nonspecific protein fouling and enzymatic degradation. Inspired by natural lasso peptides, we engineer a multifunctional lasso-like peptide (LaP) composed entirely of D-amino acids that uniquely integrates a terminal cysteine anchoring domain, a zwitterionic antifouling segment, and a specific IgG recognition segment into a single topology-constrained architecture. The covalently closed lasso topology sterically restricts protease access to cleavage-sensitive peptide bonds, while the all-D-amino acid backbone is inherently invisible to naturally occurring proteases. Together, these features provide dual physical and stereochemical protection while preserving the conformational freedom of the recognition domain for efficient target binding. Compared with a branched peptide (BrP) of identical sequence, the LaP-modified interface exhibits markedly superior resistance to nonspecific protein adsorption in undiluted serum. Molecular dynamics simulations reveal that the lasso constraint induces a superficial, low-affinity binding pose toward carboxypeptidase Y (CPY), whereas BrP penetrates deeply into the enzyme active pocket. Consequently, the LaP-based biosensor shows negligible signal change after CPY treatment, whereas BrP loses most of its activity. The LaP/AuNPs/PANI electrochemical biosensor achieves excellent selectivity and maintains its analytical performance even in complex serum matrices. This work establishes a paradigm for designing topology-constrained, multifunctional peptide interfaces that overcome the longstanding trade-off between biofouling and enzymatic degradation, opening new avenues for high-fidelity electrochemical biosensing in complex biological matrices.
We developed a site-directed immobilization strategy exploiting the competitive inhibitor Remazol Brilliant Blue R (RB) to orient horseradish peroxidase (HRP) on functionalized glass surfaces. RB was covalently bound to poly(ethylene-alt-maleic anhydride) (PEMA)-coated substrates, enabling HRP binding via its active site. Subsequent contact with a second PEMA-functionalized surface facilitated covalent immobilization of HRP with controlled orientation. Interfacial layer assembly and the comparative random control were characterized by AFM and XPS analyses, while successful RB acetylation was confirmed by NMR and ATR-IR spectroscopy. Directed contact approaches using stepwise RB and PEMA+RB functionalization yielded HRP loadings with specific activities approximately twofold higher than non-directed immobilization. Our inhibitor-mediated method preserves the native enzyme structure without genetic modification, offering a straightforward platform for enhancing enzyme performance. This strategy is promising for applications in biosensors, biocatalysis, and enzyme-based devices requiring efficient enzyme immobilization.
Anne Muschter, S. Rosencrantz, T. Chouki et al.· Molecules· 0 citations
Bispecific aptamers (BsApts) enable dual-target recognition, which is essential for rapid antibiotic detection. However, the practical application of BsApts is often hindered by structural instability, which compromises their binding affinity. To address this, we employed a "sequence-structure-dynamic simulation" framework to design BsApts targeting sulfadiazine and sulfamethoxazole. We introduced a novel C-type configuration utilizing poly-T linkers with complementary terminal poly-A sequences, facilitating self-assembly via A-T base-pairing. Molecular dynamics simulations revealed that this C-type design possesses superior stability, with an RMSD of ∼0.41 nm, significantly lower than traditional L-type constructs (>0.83 nm). Fluorescence assays confirmed that the optimized variant (C10_SME-SDZ) achieved nanomolar affinity (Kd = 52.81 nM for SDZ and 63.18 nM for SME), representing a 23-fold and 19-fold enhancement for SDZ and SME, respectively, compared to parental aptamers. Conversely, L-type aptamers exhibited weak or lost recognition. This study provides a robust computational strategy for engineering high-affinity, stable bispecific aptamers for enhanced food safety monitoring.
Shuang Jiang, Yue-Xiang Ren, Xue Wang et al.· Analytica Chimica Acta· 0 citations
Detecting target molecules in complex samples with both high sensitivity and selectivity remains a central challenge in biosensing, owing to low analyte abundance and interference from coexisting substances. Here, we present a membrane scaffold based on a ZZ-tag-displaying hepatitis B virus-derived L protein (ZZ-L membrane) that enables the oriented immobilization of sensing molecules. On quartz crystal microbalance sensor chips, the ZZ-L membrane enables controlled orientation of immunoglobulin G (IgG), leading to up to ~200-fold improvements in detection sensitivity and markedly enhanced binding capacity for purified food allergens, including ovomucoid, lectin, and tropomyosin, compared with direct immobilization. Whereas conventional methods failed to detect targets in complex matrices, the ZZ-L membrane enabled sensitive and selective detection of gliadin in wheat gluten and tropomyosin in heated shrimp extracts. Oriented immobilization of anti- hemagglutinin IgG further reduced the detectable amount of UV-inactivated influenza A virus by approximately 12-fold. The approach also extends beyond antibodies: Fc-fused leptin receptors were similarly immobilized, resulting in an approximately 15-fold increase in detection sensitivity. Across all targets, the number of bound analyte molecules per sensing molecule was consistently increased, indicating an increased active fraction at the molecular recognition interface. Together, these results demonstrate that membrane-mediated orientation control improves molecular accessibility and increases the active fraction, establishing the ZZ-L membrane as a robust platform for biosensing in complex samples with broad applicability in food safety, viral diagnostics, and biomarker detection.
Antibody-drug conjugates (ADCs) face challenges in accurate total antibody quantification due to their complex composition, low in vivo concentrations, and interference from the serum matrix. The dynamic decrease in the drug-antibody ratio (DAR) during metabolism, varying affinities among different DAR species, and discrepancies between standards and actual samples further compromise the analytical accuracy. Herein, inspired by an aptamer targeting the non-complementarity determining region (nCDR), we developed a novel spatially driven DNA-based probe proximity assay for rapid, sensitive, and high-throughput total antibody quantification, using two oligonucleotides separately labeled with fluorescent or quenching groups. The high affinity and selectivity of the probes for trastuzumab and ADCs were confirmed by microscale thermophoresis, native PAGE, and molecular docking. Systematic spatial screening was used to further identify the optimal stem, spacer, and orientation between the two probes for collaborative recognition. Moreover, the proposed homogeneous detection method achieved superior recovery for trastuzumab and successfully quantified total antibodies in trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). In contrast to the conventional indirect enzyme-linked immunosorbent assay (ELISA), our method reduces the recovery loss caused by decreased affinity from payload conjugation and delivers comparable signals for trastuzumab, T-DM1, and T-DXd, which provides a novel approach to address accuracy issues arising from affinity changes due to ADC dynamics in vivo. Finally, the proposed assay was applied to a series of spiked serum samples and clinical samples, demonstrating its feasibility and proof of concept. This strategy may facilitate the development of bioanalytical techniques for ADC characterization and monitoring.
