Danyu Li,Siyi Zou,Ziyang Huang,Congcong Sun,Guozhen Liu
Extracellular vesicles (EVs) carry disease-specific molecular profiles, demonstrating massive potential in biomarker discovery. In this study, we developed an integrated biochip platform, termed EVID-biochip (EVs identification and detection biochip), which integrates in situ electrochemical protein detection with on-chip antifouling-immunomagnetic beads modified with CD81 antibodies and zwitterion molecules, enabling efficient isolation and detection of neuronal EVs. The capability of the EVID-biochip to isolate common EVs and detect neuronal EVs associated with Parkinson's disease in human serum is successfully demonstrated, using the transmembrane protein L1-cell adhesion molecule (L1CAM) as a target biomarker. The EVID-biochip exhibited high efficiency and specificity for the detection of L1CAM with a sensitivity of 1 pg/mL. Based on the validation of 76 human serum samples, for the first time, this study discovered that the level of L1CAM/neuronal EV particles in serum could serve as a reliable indicator to distinguish Parkinson's disease from control groups with AUC = 0.973. EVID-biochip represents a reliable and rapid liquid biopsy platform for the analysis of complex biofluids offering EVs isolation and detection in a single chip, requiring a small sample volume (300 µL) and an assay time of 1.5 h. This approach has the potential to advance the diagnosis and biomarker discovery of various neurological disorders and other diseases.
The early, non-invasive diagnosis of Alzheimer's disease (AD) remains a significant clinical challenge. MicroRNAs carried by L1CAM-immunocaptured small extracellular vesicles (sEVs) in blood, particularly miR-106b-5p, have emerged as promising candidate biomarkers for AD. However, their detection requires methods that are sensitive, specific, stable, and suitable for point-of-care (POC) applications. Catalytic hairpin assembly (CHA) combined with lateral flow immunoassay (LFIA) offers a potential POC solution, but its practical application is limited by high background leakage caused by “DNA breathing” and poor probe stability. Herein, we propose an integrated POC platform that combines immunomagnetic enrichment of L1CAM-positive sEVs with a novel “Fold-Adsorb-Block-Immobilize-Lyophilize” (FABIL) process for CHA probe stabilization. The FABIL process utilizes magnetic graphene oxide (MGO) to adsorb and physically separate hairpin probes (H1 and H2), thereby suppressing nonspecific hybridization and generating lyophilized CHA microspheres with room-temperature stability. When integrated into a lateral flow test strip, the FABIL-NE-miR platform achieves a visual detection limit of 10 fM and a liquid-phase limit of detection of 1.14 fM, overcoming the traditional trade-off between low background and high sensitivity. Clinical validation using 100 serum samples, including 32 AD cases and 68 non-AD controls, demonstrated that FABIL-NE-miR outperformed traditional low-background CHA systems, with an area under the ROC curve (AUC) of 0.9563, a sensitivity of 90.63%, and a specificity of 94.12%. Overall, the FABIL-NE-miR platform enables rapid visual detection of low-abundance AD-associated miRNAs from L1CAM-immunocaptured sEV fractions. By addressing key challenges such as background leakage and probe instability, this study provides a practical tool for AD screening and supports the feasibility of the FABIL strategy as a universal platform for next-generation nucleic acid diagnostics.
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