Guided Electrokinetic Assembly of Functionalized Latex Beads for Fluorescence Signal Enhancement Using AC Electro-Osmosis

Avidin-Functionalized Gold Nanoparticles Enable Cationic Isotachophoresis for Enhanced Lateral Flow Assays.

Lateral flow assays (LFAs) are widely used for rapid, point-of-care diagnostics but struggle with sensitivity in part due to passive migration and interference from high-abundance serum proteins. Here, we introduce the lateral isotachophoresis-guided migration analysis (LIGMA) assay, a novel diagnostic platform that leverages cationic isotachophoresis (+ITP) to enhance biomolecular separation and detection in LFAs. Using avidin-functionalized gold nanoparticles (AV-S-GNPs) as positively charged nanocarriers, this system enables the direct electrophoretic migration of negatively charged analytes, overcoming a major limitation of conventional cationic electrokinetic focusing methods. The functionalized nanoparticles exhibited superior stability across physiological pH ranges and maintained consistent electrophoretic mobility under + ITP conditions. LIGMA achieved a limit of detection of 1.97 nM and a limit of quantification of 4.12 nM for human immunoglobulin G (IgG), requiring only 1 μL of sample volume and under 2 min of assay time. Compared to standard LFAs, ITP-enhanced migration produced sharper capture line resolution, improved analyte focusing, and minimized background interference from abundant serum proteins. This work establishes the LIGMA assay 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.

Ultrafast Self-Assembly of Zeolitic Imidazolate Framework-8 Enables Antibody Orientation for Ultrasensitive Lateral Flow Immunoassays.

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.

Nanoconfinement-Driven Solid-State Ratiometric Fluorescent Aptasensor for 17β-Estradiol Detection in Complex Matrices

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.

Ru(bpy)32+-Conjugated Magnetic Nanoparticle as Fluorescent-Electrochemiluminescent Biological Label.

Electrochemiluminescence (ECL) has emerged as an ultrasensitive detection technique with zero background signal, and coupling of ECL with a commonly used fluorescence imaging technique can greatly advance the bioimaging/detection applications. However, molecular ECL luminophores have poor cellular uptake and cell-targeting properties. Here, we show 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. In particular, we have designed a colloidal magnetic nanoprobe that is composed of a 6-7 nm iron oxide core and surface terminated with a ruthenium complex that acts as both a fluorescent and ECL probe. The nanoparticles are functionalized with targeting ligands that enable the receptor-mediated uptake by specific cells. The designed nanoprobe has been used for imaging/detection of biotin/galactose overexpressed cells via both fluorescence and ECL, along with magnetic extraction of labeled cells for signal enhancement. Results demonstrate that molecular ECL luminophore can be transformed into a fluorescent-ECL nanoprobe with more advanced bioimaging and detection applications.

Homogeneous fluorescence polarisation immunoassay for the quantification of extracellular vesicles from whole blood.

Extracellular vesicles (EVs) are important biomarkers in disease diagnosis and monitoring. However, their quantification typically relies on laboratory instruments, limiting their application in point-of-care testing. In this study, we report a homogeneous fluorescence polarisation immunoassay for the quantification of EVs from whole blood using an antibody tracer. The assay uses a portable fluorescence polarisation analyser with microfluidic devices to simplify EV quantification after pretreatment. To reduce nonspecific binding of the tracer to the microfluidic channel walls, we employed polyethylene glycol-modified PDMS, which eliminates the need for blocking agents. Consequently, this study enabled homogeneous quantification using a portable fluorescence polarisation analyser, achieving a limit of detection of 5.0 × 107 particles/mL and a limit of quantification of 6.6 × 107 particles/mL. Overall, this approach provides a simple, and reagent-efficient method for EV quantification from complex biological matrices, highlighting the potential of fluorescence polarisation immunoassay as a practical tool for future EV-based clinical studies.

Cascade-driven dual-signal attenuation antifouling immunosensor for femtogram-level sensing for SCCA.