Aptamers dimerization inspired biomimetic clamp assay towards impedimetric SARS-CoV-2 antigen detection.

Antigen-detecting rapid diagnostic testing (Ag-RDT) has contributed to containing the spread of SARS-CoV-2 variants of concern (VOCs). In this study, we proposed a biomimetic clamp assay for impedimetric SARS-CoV-2 nucleocapsid protein (Np) detection. The DNA biomimetic clamp (DNA-BC) is formed by a pair of Np aptamers connected via a T20 spacer. The 5'- terminal of the DNA-BC is phosphate-modified and then anchored on the surface of the screen-printed gold electrode, which has been pre-coated with Au@UiO-66-NH2. The integrated DNA-material sensing biochip is fabricated through the strong Zr-O-P bonds to form a clamp-type impedimetric aptasensor. It is demonstrated that the aptasensor could achieve Np detection in one step within 11 min and shows pronounced sensitivity with a detection limit of 0.31 pg mL-1. Above all, the aptasensor displays great specificity and stability under physiological conditions as well as various water environments. It is a potentially promising strategy to exploit reliable Ag-RDT products to confront the ongoing epidemic.

Customization of aptamer to develop CRISPR/Cas12a-derived ultrasensitive biosensor.

The CRISPR/Cas systems have provided wide biosensing applications. Particularly, the aptamer-involved CRISPR/Cas sensor system powerfully expanded to non-nucleic-acid targets. However, tailoring the sequence of the aptamer to explore the relationship between affinity and the activation of CRISPR/Cas12a trans-cleavage activity has not been reported yet. Herein, we developed a series of new aptamers toward the spike protein 1(S1) of SARS-CoV-2. Surface plasmon resonance measurements showed that the affinity of these aptamers to S1 was at the nM level. Subsequently, a "SET" effect (Sequence Essential Trans-cleavage activity) is discovered for the activation of CRISPR/Cas12a trans-cleavage activity. That is, an aptamer, as the activator, sequence needs to be tailored to activate CRISPR/Cas12a efficiently. A balance should be reached between affinity and activation ability. On the one hand, high affinity ensures target recognition performance, and on the other hand, activation can achieve adequate amplification and output of recognition signals. The optimized sequence (with 27 nucleotides, for short 27-nt) not only recognizes the target with a high affinity and specificity but also can trigger the CRISPR/Cas12a trans-cleavage activity efficiently, showing an excellent detection performance in electrochemical biosensors. The detection limit for SARS-CoV-2 S1 can be low at 1.5 pg mL-1. The new CRISPR/Cas12a-derived aptasensor also displays a remarkable ability to detect Beta, Delta, and Omicron variants but is selective toward other kinds of proteins. Above all, it is robust for point-of-care testing (POCT) in complex biological fluids, such as saliva, urine, and serum, and provides a universal and scalable detecting platform. Our results provide new insights into aptamer development and a different strategy for COVID-19 antigen detection and biosensor development.

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

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.

Aptamer-Functionalized Nanochannels for One-Step Detection of SARS-CoV-2 in Samples from COVID-19 Patients.

With the outbreak of COVID-19, which is fast transmitting and highly contagious, the development of rapid, highly specific, and sensitive detection kits has become a research hotspot. The existing assay methods for SARS-CoV-2 are mainly based on enzymatic reactions, which require expensive reagents, hindering popular use, especially in resource-constrained areas. Herein, we propose an aptamer-based method for the assay of SARS-CoV-2 via binding of the spike protein using functionalized biomimetic nanochannels. To get the analogous effect of human ACE2, a receptor for the spike protein, the aptamer to bind to the spike S1 protein has been first screened by a SELEX technique and then immobilized on the previously prepared nanochannels. In the presence of SARS-CoV-2, the changes in steric hindrance and charge density on the surface of the nanochannels will affect the ion transport, along with a rapid electrochemical response. Our method has been successfully applied to detect the viral particles in clinical pharyngeal swab specimens in one step without sample treatment. We expect this rapid, reagent-free, and sensitive assay method to be developed as a useful tool for diagnosing COVID-19.

Discovery of sandwich type COVID-19 nucleocapsid protein DNA aptamers.

Here, we report for the first time DNA aptamers targeted toward the COVID-19 nucleocapsid protein (Np). Np is one of the most abundant structural proteins and it serves as a diagnostic marker for the accurate and sensitive detection of COVID-19. After five rounds of selection, we obtained four DNA sequences with an affinity below 5 nM. The best one displayed a superb binding performance toward Np with a Kd value of 0.49 nM. Interestingly, we found that the four pairs of aptamers could bind to Np successively, suggesting a sandwich-type interaction. Using these sandwiched aptamers in ELISA and colloidal gold immunochromatographic strips, we were able to detect Np at the tens of pM level. The results demonstrate that aptamers are powerful molecular tools for virus detection, diagnosis, and antiviral therapy.