In a recent study posted to the Analytica Chimica Acta*, researchers reported the efficacy of their self-developed CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection) biosensor in naked-eye detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) using gold nanoparticles (AuNPs).
Background
The quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is the gold standard for viral detection. However, it is expensive and technique-sensitive and thus unsuitable for rapid point of care (PoC) diagnosis. Commonly used PoC tests such as antigen tests and serological assays have low reliability and longer duration, respectively. Thus, the need arises for diagnostically accurate, rapid, and economic PoC techniques for SARS-CoV-2 diagnosis. Although the AuNPs -CRISPR technique of viral detection is promising, it has not been investigated extensively.
The CASCADE sensor comprises Cas CSIPR nuclease protein. AuNPs are coated with single-stranded RNA (ssRNA) oligonucleotide sequences. The Cas nuclease forms a complex with ribonucleoprotein (RNP) and guide RNA (crRNA) which is complementary to nucleic acid targets. The crRNA-target interaction activates the Cas13 enzyme. On target recognition, ssRNA degradation by the activated Cas nuclease enzyme and subsequent AuNP aggregation takes place. This leads to a reduced absorption intensity and a color shift to higher wavelengths, visible to the eyes. The combination of these changes facilitates SARS-CoV-2 RNA detection.
About the study
In the present study, researchers designed the CASCADE biosensor for naked-eye detection of SARS-CoV-2 RNA extracted from SARS-CoV-2-positive nasopharyngeal swabs using AuNPs.
The Cas protein is of two types – Cas12 and Cas13. The trans-cleavage activity of Cas13 is multiple-turnover, and thus, the signals can be amplified using many unspecific nucleic acid oligonucleotides. Further, Cas13 does not require a protospacer adjacent motif (PAM) sequence. The incorporation of the Cas13 nuclease increased the versatility of the sensor.
The team synthesized AuNPs of three diameters-34, 22, and 12 nm, and coated them with RNA3 oligonucleotides in 1.5 pmol µl-1 for detecting SARS-CoV-2-ORF1ab and spike (S) gene targets. The 12 nm AuNP enabled the most rapid target detection with intense color changes within 15 minutes and thus was selected for the sensor.
The length and amount of the ssRNA coating on the AuNPs were determined to evaluate AuNP stability. The longer the ssRNA length, the more stable the AuNP would be with more efficient Cas13 enzymatic degradation. Therefore, three RNA3 sequence lengths were assessed: 33, 23, and 13 nt. Of these, the 33nt RNA3 provided the strongest target detection and greatest absorbance reduction. To assess the coating amount, three loads were tested (2.3, 1.5, and 0.7 pmol µL-1), of which the 2.3 pmol µL-1 was most efficient and chosen for the sensor.
The long-term stability was also assessed by storing the ssRNA3-AuNPs at 4°C for five months before use. Post storage, their performance was similar to that of freshly prepared AuNPs. This indicates that the ssRNA3-AuNPs had high long-term stability, which also increased the portability of the sensor.
The most appropriate AuNP concentration was determined by evaluating three concentrations: 12, 23, and 47 nM, of which, the 23.4 nM concentration demonstrated the greatest absorption decrease and color change. Thus, this AuNP concentration was selected for the sensor.
The sensor’s sensitivity was analyzed based on the crRNA design. Two crRNAs were used for the recognition of specific regions, R1 and R2 of the Orf1ab gene target, and another two crRNAs for the S gene targets, S1 and S2. As low as 0.5 nM and 0.1 nM concentrations of R2, S1, and R1 were efficiently detected in 30 minutes and one hour, respectively. This indicates the sensor was highly sensitive. Further, the isothermal amplification of the target RNAs by coupling recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) also increased the sensor’s sensitivity. The sensor could detect concentrations as low as three femtomolar and 40 attomolar within two hours.
The detection speed and specificity of the sensor were also evaluated using the Orf1ab target, which led to AuNP aggregation with visually detectable spectral changes within two minutes. This indicates that the sensor detected viral RNA rapidly. Of note, if the SARS-CoV-2 target sequence was absent or target sequences of other human coronaviruses were present, no color change occurred. This is indicative of the high specificity of the sensor.
Conclusion
The study findings showed that the CASCADE biosensor is a rapid, versatile, and portable device that can be used for effective naked-eye SARS-CoV-2 RNA detection in clinical samples with high, specificity, reliability, and sensitivity.
