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.
Abstract
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.
Lateral flow immunoassay (LFIA) has been widely used for rapid on-site analysis because of its simplicity, portability, and low cost. However, its performance in competitive assays, particularly for small molecule toxins, remains limited, as the properties of antibodies and the reliance on single-mode signal often lead to insufficient sensitivity and reliability. To address this challenge, we developed an enhanced colorimetric-photothermal LFIA by integrating linker-optimized bivalent nanobodies (BvNbs) and gold-liposome nanocomposites (Au-LNCs). Using tetrodotoxin (TTX) as a representative analyte, a series of BvNbs with different linker lengths were constructed. Molecular docking and molecular dynamics simulations further demonstrated that linker length modulates BvNbs detection performance by regulating conformational stability, local flexibility, and persistent interactions with free target molecules. In parallel, photothermal Au-LNCs were prepared, which exhibited broadband near-infrared absorption and a photothermal conversion efficiency as high as 78.06%. The resulting LFIA achieved a photothermal LOD of 1.89 ng/mL, corresponding to an approximately 10.7-fold improvement in sensitivity compared with conventional colloidal gold-based LFIA, while maintaining good reliability in real samples. Overall, this study provides mechanistic insight into the rational design of BvNbs and a promising strategy for improving LFIA performance.
Jinzhi Han, Hongzhi Liang, Lin Luo et al.· Biosensors & bioelectronics· 0 citations
Electrochemical Lateral Flow Immunoassays (eLFIA) are emerging as powerful possibility for in vitro diagnostic tools due to their sensitivity, small size and affordable price. However, the development of non-toxic and biodegradable nanolabels that enable sensitive quantification remains a challenge. In this study, three nanocarriers (liposomes, niosomes, and lipid-polymer micelles) were synthesized and characterized, encapsulating the starch-polyiodide complex as electrochemical probe for eLFIA. After evaluating their size, morphology, molecular structure, encapsulation efficiency, and colloidal stability, micelles were selected as the ideal nanolabel for this eLFIA. A proof of concept was performed using the biotin-neutravidin model system, thus evaluating its sensitivity and detection limit, reaching a detectable concentration of 17 nM by performing a fixed-potential coulometry for three minutes, hence, employing just a little over 20 min for the total assay. This system constitutes, to the authors' knowledge, the first in vitro diagnostic device based on sustainable bio-derived nanoparticles, paving the way for future environmentally friendly tests.
C. Saweres-Argüelles, A. Sánchez-Calvo, Gemma Gutiérrez et al.· Colloids and Surfaces B: Bio...· 0 citations
A dual-mode colorimetric–photothermal LFIA platform was successfully constructed for the detection of thrombosis-related biomarkers, including thrombomodulin and α2-plasmin inhibitor–plasmin complex, in clinical serum samples, enhancing analytical sensitivity and reliability.
Jun Yin, Yu Qiu, Yao Nie et al.· RSC Advances· 0 citations
Small extracellular vesicles (sEVs) have emerged as promising biomarkers for liquid biopsy; however, the limited specificity of single-marker detection and the intrinsic heterogeneity of circulating sEVs hinder their reliable clinical application. Herein, we develop a programmable dual-aptamer amplified luminescent proximity homogeneous assay (AlphaLISA) for rapid and wash-free detection of tumor-derived sEVs. In this strategy, EpCAM and PD-L1 aptamers simultaneously recognize two membrane proteins on the same vesicle, enabling proximity-induced interaction between donor and acceptor beads to generate a chemiluminescent signal via singlet oxygen-mediated energy transfer. This dual-recognition design effectively improves analytical specificity by implementing a molecular "AND" logic requirement for signal generation. The proposed assay exhibits a broad linear range of 5.3 × 105 to 5.3 × 109 particles mL-1 with a limit of detection of 1.63 × 104 particles mL-1. Good analytical performance was demonstrated by recovery rates of 93.9-107.5% with relative standard deviations below 5%, indicating high accuracy and reproducibility. Notably, the assay enables signal readout within 1 min under homogeneous conditions, highlighting its operational simplicity and rapid response. Clinical applicability was evaluated using serum samples from 20 lung cancer patients and 20 healthy controls, achieving an area under the ROC curve of 0.87 (95% CI: 0.75-0.97), with a sensitivity of 95% and a specificity of 75%. Overall, this programmable dual-aptamer AlphaLISA platform provides a rapid, homogeneous, and highly adaptable strategy for tumor-derived sEVs analysis, offering promising potential for noninvasive cancer diagnostics.
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.
T. Zhou, Alfonso Shin, L. Kulinsky· Italian National Conference...· 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