Aug 2026· Analytical Chemistry· Vol 98 32, pp.
23806-23818
· 0 citations· 34 references
Medicine
TL;DR
This programmable dual-aptamer amplified luminescent proximity homogeneous assay (AlphaLISA) provides a rapid, homogeneous, and highly adaptable strategy for tumor-derived sEVs analysis, offering promising potential for noninvasive cancer diagnostics.
Abstract
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.
Extracellular vesicles (EVs) have emerged as a prominent and rapidly evolving class of biomarkers, garnering an increasing amount of attention in recent years. The analysis of diverse EV-derived biomolecules, including proteins, mRNA, and miRNA, has driven the development of numerous innovative analytical technologies for EV detection. Nevertheless, despite significant advancements, most current EV analysis methods still require substantial improvements to address critical limitations, such as operational complexity, insufficient sensitivity, and the persistent issue of false-positive signals. In this study, we present a targeted fusion and nonenzymatic amplification strategy for ultrasensitive detection of tumor-derived EV miRNAs. The proposed approach integrates an aptamer-anchored liposome system that facilitates specific recognition and fusion with tumor EVs, coupled with a spatial confinement nonenzymatic signal amplification system to significantly enhance detection sensitivity. In conjunction with flow cytometry, the platform demonstrates a detection limit of 4 ng/mL for EV- associated proteins (0.2 fM for EV miR-21) and effectively discriminates cancer patients from healthy controls. This innovative strategy shows substantial promise for diverse biomedical applications, potentially revolutionizing early cancer diagnosis, metastasis monitoring, and prognostic evaluation while also offering opportunities for the detection and management of various other diseases.
Small extracellular vesicles (sEV) are increasingly reported as biomarkers for the early diagnosis of pancreatic cancer (PC), but the current techniques for isolation and detection of sEV rely on expensive instruments and tedious protocols. In this work, a facile and rapid sEV isolation and detection method (LAPT-sEViso) was developed, which is based on the specific aggregation of GPC-1-positive PC-derived sEV and an aptamer-functionalized DNA long chain produced by rolling circle amplification (RCA-APT). The LAPT-sEViso can efficiently isolate sEV from cell culture medium and serum, showing 45 times higher yield (5.5 × 106 particles mL-1), 1.1 times higher purity (1.66 × 1010 particles mg-1) and 4.9 times higher recovery (80.9%) comparing to the traditional ultracentrifugation method, with only $1000 common instruments and $2.88 reagents/materials in 1 h. Moreover, after simple filtration and on-membrane ELISA, sEV concentration can be instrument-free detected with a limit of detection of 5.62 × 103 particles μL-1 (linear range from 5.0 × 103 to 5.0 × 107 μL-1). The LAPT-sEViso provides an efficient and practical approach for the rapid isolation and detection of sEV, providing a novel approach for the sEV-based liquid biopsy.
A novel, enzyme-free surface-enhanced Raman scattering (SERS) platform based on a "dual-hotspot amplification" strategy that enhances detection sensitivity but also provides a useful design concept for the ultrasensitive analysis of carbohydrate-derived biomarkers.
Meiqi Bao, Xiaoyan Ma, Hongcai Liu et al.· Biosensors & bioelectronics· 0 citations
The rapid diagnosis of high-risk viral pathogens is critical for public health, yet current nucleic acid-based methods often necessitate pre-amplification steps. These processes can introduce amplification bias and hinder accurate quantification. To address these limitations, we report a Cas12a-mediated Field-Effect Transistor (FET) biosensor designed for the amplification-free detection of viral nucleic acids, including SARS-CoV-2, Respiratory Syncytial Virus (RSV), and Influenza A.
Our system utilizes an extended gate (EG) electrode functionalized with single-stranded DNA (ssDNA). By leveraging the synergistic combination of the FET’s high-sensitivity transduction and the signal amplification provided by Cas12a’s collateral cleavage activity, we achieved a detection sensitivity 10,000-fold higher than traditional fluorescence-based assays. Furthermore, the platform demonstrates high practical utility through the simultaneous detection of multiple viral sequences on a single 8-well EG chip, facilitating the identification of co-infections. The use of a universal EG design allows for target-independent mass production and improved cost-efficiency. Finally, we implemented a sensitivity-based EG selection strategy to mitigate measurement errors stemming from device-to-device variation, ensuring robust and reproducible performance for point-of-care applications.
Part of this presentation is based on our previously reported study on Cas12a-mediated FET biosensing systems (Sens Act B: Chemical, 443 (2025) 138212).
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
Simultaneous and accurate detection of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) is critical for the early diagnosis and monitoring of inflammatory diseases. However, conventional analytical methods are often cumbersome, time-consuming, and limited in the capacity for simultaneous multiplex analysis and rapid biomarker discrimination. Herein, an integrated multiplex PEC sensing platform is developed for the synchronous, highly sensitive detection and intelligent discrimination of IL-6 and TNF-α. The sensor is constructed by integrating a copper phthalocyanine-based covalent organic framework as the photoactive material with a catalytic hairpin assembly (CHA) cycling amplification strategy. Upon target recognition by specific aptamers, DNA signal strands are released and trigger a CHA reaction on the electrode surface, forming Y-shaped DNA structures and enabling detection in a "signal-off" mode. The sensing system functions as an OR logic gate capable of responding to the presence of either target and distinguishing between individual and coexisting target states through combinatorial logic operations. Different from conventional multiplex sensing strategies that rely on complex instrumentation or multichannel configurations, the approach uses a single electrode for signal readout, offering simple fabrication, rapid response, and low cost. This work provides an effective strategy for multiplex detection of inflammatory markers and highlights the potential of intelligent diagnostic platforms for point-of-care applications.
Yuhang Su, Qinwei Zhao, Ying Tang et al.· Analytical Chemistry· 0 citations