CRISPR-based ultrasensitive multiplexed array for the detection of multiple plasma biomarkers for early Alzheimer’s disease diagnosis

Mild cognitive impairment (MCI) is an intermediate stage between normal cognition and dementia, with a high risk of progression to Alzheimer’s disease (AD). As an important threshold for the intervention and prevention of AD, accurate diagnosis of AD-related MCI based on plasma biomarkers is crucial. However, early detection of AD-related MCI still poses a huge challenge. Herein, we propose an ultrasensitive CRISPR-based multi-protein detection array (UCMDA) that integrates the array device with CRISPR/Cas12a and antibody pair-based multi-recombinase polymerase amplification to facilitate inexpensive, sensitive, and specific detection of multiple markers in plasma. With only one fluorescence probe, UCMDA can simultaneously detect Aβ40, Aβ42, p-tau181, p-tau217, p-tau231, and p-tau396,404 within 1 h with a detection limit of 1 fg/mL. The applicability of UCMDA was demonstrated in 155 clinical plasma samples from normal senile controls (NCs), individuals with AD-related MCI, and patients with AD. We found that the UCMDA platform combined with machine learning algorithms achieved accurate early diagnosis of AD-related MCI with a detection accuracy, sensitivity, and specificity exceeding 90.38%. We anticipate that this inexpensive, ultrasensitive, and multi-detection platform can also be widely used to detect other protein-related biomarkers besides AD.