CRISPR/Cas12a-Derived electrochemical aptasensor for ultrasensitive detection of COVID-19 nucleocapsid protein

• Published in: Biosensors & bioelectronics 2022-03, Vol.200, p.113922-113922, Article 113922
• Main Authors: Han, Cong, Li, Wenjin, Li, Qian, Xing, Wenping, Luo, Hang, Ji, Haishuo, Fang, Xiaona, Luo, Zhaofeng, Zhang, Liyun
• Format: Article
• Language: English
• Subjects: Aptamer; Aptamers, Nucleotide; Biosensing Techniques; COVID-19; CRISPR-Cas Systems; CRISPR/Cas12a system; Electrochemical aptasensor; Electrochemical Techniques; Electrodes; Gold; Humans; Limit of Detection; Nucleocapsid protein; Nucleocapsid Proteins; SARS-CoV-2
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2021.113922

Fast, affordable, portable, and sensitive technology to detect COVID-19 is critical to address the current outbreak. Here, we present a CRISPR/Cas12a-derived electrochemical aptasensor for cost-effective, fast, and ultrasensitive COVID-19 nucleocapsid protein (Np) detection. First, an electrochemical sensing interface was fabricated by immobilizing methylene blue labeled poly adenines DNA sequence (polyA-MB electrochemical reporter) on a gold electrode surface. Second, an arched probe was prepared via hybridization of Np aptamer and an activator strand. In the presence of COVID-19 Np, the activator strand could be released from the arched probe due to the specific interaction between the target and the aptamer, which then activated the trans-cleavage activity of the CRISPR/Cas12a system. Subsequently, the polyA-MB reporters were cleaved from the electrode surface, decreasing the current of differential pulse voltammetry (DPV) at a potential of −0.27 V(vs. Ag/AgCl). The CRISPR/Cas12a-derived electrochemical aptasensor shows a highly efficient performance for COVID-19 Np detection in 50 pg mL-1 to 100 ng mL-1 with a limit of detection (LOD) low to 16.5 pg mL-1. Notably, the whole process of one test can be completed within 30 min. Simultaneously, the aptasensor displays a high selectivity to other proteins. The further measurements demonstrate that the aptasensor is robust in a natural system for point-of-care testing, such as in tap water, milk, or serum. The aptasensor is universal and expandable and holds great potential in the COVID-19 early diagnosis, environmental surveillance, food security, and other aspects. •Integrating CRISPR/Cas12a with the electrochemical assay has achieved ultrasensitive detection of COVID-19 Np.•The aptasensor provides an effective platform for practical, economic, and deployable detection of COVID-19.•The aptasensor is independent of large instruments and proprietary laboratories, making analysis flexible and convenient.•The aptasensor holds great potential as point-of-care testing (POCT) for COVID-19 in tap water, milk, or serum.