Homogeneous Femtomolar Detection of P-tau181 via Proximity Extension and CRISPR/Cas Technique

Résumé fourni par la source

Accurate quantification of site-specific tau phosphorylation in plasma holds great promise for the noninvasive early diagnosis of Alzheimer’s disease (AD). Here, we integrated the proximity extension assay (PEA) with nucleic acid amplification techniques-polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA)-and coupled them with CRISPR/Cas12a-mediated fluorescence detection to enable quantitative and homogeneous measurement of threonine-181-phosphorylated tau (p-tau181), a key biomarker of AD. Binding of two PEA probes to a single p-tau181 molecule induces proximity-mediated probe hybridization and extension, thereby converting the protein signal into an amplifiable nucleic acid signal. The resulting double-stranded DNA is subsequently amplified by PCR or RPA and detected through Cas12a trans -cleavage activity. The limits of detection (LODs) for the PEA–PCR–CRISPR/Cas and PEA–RPA–CRISPR/Cas assays were 149.0 fM (6.8 pg·mL –1 ) and 45.4 fM (2.1 pg·mL –1 ), respectively. In fetal bovine serum, LODs of 231.4 fM (10.6 pg·mL –1 ) and 139.2 fM (6.3 pg·mL –1 ) were achieved, demonstrating excellent antimatrix performance. The accuracy of the PEA–RPA–CRISPR/Cas assay in human serum was further validated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. This homogeneous, wash-free approach combines operational simplicity with ultrahigh sensitivity, showing great potential for routine clinical detection and early stage monitoring of AD biomarkers.

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