An aptamer/CRISPR electrochemical (ACE) biosensor for Plasmodium falciparum histidine-rich protein II and SARS-CoV-2 nucleocapsid protein

The trans-cleavage activity of CRISPR Cas12 and Cas13 on single-stranded DNA has been widely applied for biosensing and diagnostic applications. Typically, such approaches have traditionally been limited to the sensing of nucleic acids. Here, we have combined CRISPR Cas12 with nucleic acid aptamers to enable protein recognition by an electrochemical approach. To demonstrate the versatility of this approach, we have successfully detected two important protein disease biomarkers: Plasmodium falciparum histidine-rich protein II (PfHRP2) as a biomarker of malaria, and SARS-CoV-2 nucleocapsid (N) protein as a biomarker of COVID. We designed activable aptasensors by annealing aptamers to a complementary locking strand. CRISPR Cas12 trans-cleavage is initiated by strand displacement upon protein binding, thereby cleaving a redox reporter conjugated to DNA on an electrode, transducing into an electrochemical signal. The limits of detection for PfHRP2 and COVID N protein are 45.5 nM and 8.18 nM respectively with high specificity towards their targets. Protein detection by such CRISPR-assisted ACE biosensors can be potentially expanded and multiplexed across several critical biomarkers in parallel.