Aug 2026· Italian National Conference on Sensors· Vol 26· 0 citations· 39 references
Medicine
Abstract
Fluorescence-based immunoassays are widely used for sensitive and specific biomarker detection; however, further improvements in sensitivity remain desirable for detecting low-abundance analytes without increasing assay complexity. In this work, we present a proof-of-concept demonstration of guided electrokinetic assembly of functionalized latex beads as a post-assay signal enhancement strategy using alternating-current electro-osmosis (ACEO). Carboxyl-modified 1 μm polystyrene beads conjugated with Alexa Fluor 647-labeled anti-IgG were localized within lithographically defined windows on carbon interdigitated electrode arrays, producing localized fluorescence enhancement through physical bead localization without enzymatic amplification or additional labeling chemistries. Compatibility of the approach with fluorescence-based immunoassays was demonstrated through adaptation of a TNF-α ELISA workflow. Electro-osmotic localization of functionalized bead conjugates was achieved within 120 s, producing an approximately 12-fold increase in corrected total fluorescence relative to the corrected signal of the pre-electro-osmosis condition while demonstrating negligible enrichment of unbound fluorescent protein. Application of the platform to a TNF-α sandwich immunoassay yielded an approximately 5.5-fold enhancement in fluorescence signal, and robust bead localization was maintained across anti-IgG concentrations ranging from 1 to 4 μg/mL. These findings demonstrate that guided electrokinetic bead localization provides an effective signal enhancement strategy for fluorescence-based immunoassays and represents a promising approach for improving the detection of low-abundance analytes.
The LIGMA assay is established as a viable electrokinetic strategy for improving paper-based biosensors, providing a foundation for expanded applications in clinical diagnostics, multiplexed detection, and portable field-deployable biosensing platforms.
D. McCornack, C. Ivory, Zhihong Zhang et al.· Analytical Chemistry· 0 citations
Gold-nanoparticles-based lateral flow immunoassays (AuNPs-LFIAs) are widely used for point-of-care testing, yet their sensitivity remains fundamentally limited, likely because random antibody orientation on AuNP surfaces restricts Fab accessibility and antigen recognition efficiency. Precise control of antibody orientation is thus critical for maximizing probe activity, yet existing directional conjugation strategies are often multistep, technically complex, and poorly scalable. Here, we report an ultrafast biomimetic mineralization strategy that enables one-step, room-temperature self-assembly of zeolitic imidazolate framework-8 (ZIF-8) with antibodies and dual-ligand AuNPs, producing uniform hybrid nanoprobes within 5 min. ZIF-8 shell preserves the plasmonic properties of AuNPs while simultaneously enhancing colloidal stability and optical signal intensity. Molecular dynamics simulations suggest a two-step orientation model: (i) rapid electrostatic attraction between negatively charged Fc regions and Zn2+ ions initiates nucleation, followed by (ii) Fc-associated interfacial interactions that promote preferential Fc-oriented binding and anisotropic Fc/Fab distribution, thereby enhancing Fab exposure for efficient antigen recognition. This controlled assembly increases Fab accessibility more than 3-fold relative to conventional adsorption or co-precipitation strategies, minimizes nonspecific adsorption, and amplifies signal output. When applied to competitive LFIAs, the oriented nanoprobes enabled ultrasensitive detection of chloramphenicol down to 13 pg/mL, corresponding to approximately 1 order of magnitude higher sensitivity than AuNPs-based LFIA gold standard. By integrating mechanistic insight with operational simplicity, this work establishes a generalizable platform for ultrafast, scalable, and antibody-orientation-controlled nanoprobe fabrication for next-generation LFIAs in food safety, clinical diagnostics, and environmental monitoring.
Precise quantitative monitoring of 17β-estradiol (E2) is important for reproductive management in precision livestock farming. However, E2 determination in complex biological matrices remains challenging because of matrix-derived background and signal variability. Here, we developed a nanoconfinement-assisted solid-state ratiometric fluorescent aptasensor integrating target-induced strand displacement (TISD), magnetic separation, and anodic aluminum oxide (AAO) nanochannel confinement. The sensing probe consisted of streptavidin-coated magnetic nanoparticles (MNPs) carrying a FAM-labeled cDNA internal reference and a Texas Red-labeled E2 aptamer reporter. E2 binding promoted dissociation of the Texas Red-labeled aptamer from the magnetic probe. Magnetic separation and washing reduced soluble matrix-derived interference, while subsequent deposition of the sensing complexes onto an AAO membrane mitigated coffee-ring-associated nonuniformity and produced a more spatially uniform dual-color fluorescence distribution for ratiometric analysis. Under matrix-matched calibration conditions, linear ranges of 5.0–50.0 pM were obtained in tap water and sow saliva, 5.0–40.0 pM in whole milk, and 5.0–15.0 pM in post-estrus sow urine. The LOD determined in tap water was 3.62 pM. The different calibration slopes obtained among the four matrices indicated that residual matrix-dependent effects remained and that matrix-specific calibration was required for quantitative analysis. Matrix-matched spike recoveries ranged from 86.92% to 119.54% across the investigated matrices. The aptasensor exhibited the strongest response toward 17β-E2 among the tested compounds; however, cross-reactivities of 77.3% for E3 and 47.3% for 17α-E2 indicated preferential rather than exclusive recognition. Molecular docking suggested a putative binding pose but did not experimentally establish the molecular recognition mechanism. Overall, the platform demonstrated laboratory-scale analytical feasibility in pretreated tap water, sow saliva, whole milk, and post-estrus sow urine. Further development of sample preparation, magnetic handling, membrane loading, probe selectivity, and portable fluorescence readout will be required before in situ or on-site application.
It is shown that transforming a molecular ECL luminophore into a colloidal nanoprobe can increase its cellular uptake as well as enhance its application potential via both fluorescence and ECL.
Abu Raihan Sarkar, Puja Garai, Rajkumar Sahoo et al.· ACS Applied Materials and In...· 0 citations
A homogeneous fluorescence polarisation immunoassay for the quantification of EVs from whole blood using an antibody tracer using a portable fluorescence polarisation analyser with microfluidic devices to simplify EV quantification after pretreatment.
Hao Liu, M. Fukuyama, Sheng Yuan Leong et al.· Talanta: The International J...· 0 citations
A cascade-driven dual-signal attenuation strategy that holds great promise for high‑performance electrochemical biosensing in complex biological samples.
Ge Song, Jiaqing Wang, Xianrui Jiang et al.· Analytical and Bioanalytical...· 0 citations
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