Existing nuclear acid test with quantitative reverse transcription polymerase chain reaction (qRT-PCR) is regarded as the golden rule in detection of the novel coronavirus (SAR-CoV-2). However, the limitation of qRT-PCR in detection of the viral genome is also obvious.
- At first, the SAR-CoV-2 genome consists of 30,000 nucleotides and almost every part of it mutates continuously ever since. In contrary, the length qRT-PCR probe is only about 20 nucleotides, so it is much likely to miss the complementary target which is subjected to mutation.
- Secondly, qRT-PCR requires the polymerase enzyme to build and amplify the target sequence by forward and reverse primers. If these viral fragments also mutate, the primers are also possible to miss their targets thus detection fails.
- Thirdly, the build-degenerate-amplify procedure by the polymerase enzyme are not error-free. Thus, it introduces replication error to the amplified product and generates erroneous result.
- Finally, the number of viral fragments can be detected by qRT-PCR in a single experiment is rather limited due to fluorescent color crosstalk. So alternative multiplexing scheme is urgently needed for high throughput multi viral fragment detection.
Unfortunately, existing multiplexing biosensing systems such as Next generation Sequencing (NGS) or Single Molecule Array (SIMOA) are very time consuming for clinical applications. Yet, these systems are far from fulfilling the four important characteristics i.e. multiprocessing, quick, precise, and cost-effective (MQPC).
In contrary, genetic editing tool are becoming popular in the area of biosensing. Researchers from MIT and Harvard establish a nucleic acid detection technology with sensitivity reaching the level of angstrom (single copy) and specificity reaching single base, namely a nucleic acid detection platform SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) [1, 2] based on CRISPR-Cas13a which is known as genetic scissor for high-sensitivity detection of trace nucleic acid of SARS-CoV-2 virus. Herein, we employ the CRISPR-Cas13 genetic editing technology with corresponding combinations of crRNA to split the SARS-CoV-2 viral spike genome into fragments. Thereafter, these fragments are detected by direct hybridization with complementary RNA probes by thermoplasmonic heating and quantum optical measurements. The most disruptive technological breakthroughs of our QPLoC biosensor for SARS-CoV-2 detection fulfill the MQPC aspects which include,
- Multiplex detection of over 100 viral genome fragments at once in label-free manner,
- Quick label-free detection with thermoplasmonic effect in 15 minutes,
- Precise SARS-CoV-2 viral genome measurement with attomole sensitivity i.e. 500 copies per mL,
- Cost-effective biochip made of Titanium Nitride Nanocubes by 3D printing to replace gold material.
Therefore, we applied for the Innovative Technological Fund (ITF) of the Hong Kong SAR Government with City University of Hong Kong (CityU). The project title is “A thermoplasmonic lab-on-chip microarray for in-vitro diagnostics of SARS-CoV-2 RNAs, CRP/039/22”. To verify our performance, we did benchmark tests with SARS-CoV-2 qRT-PCR toolkits from a well-known international manufacturer, the test was conducted by trained personnel from CityU and performed in the Hong Kong Science Park (HKSTP) Biomedical Technology Support Centre. Without further due, let’s take a look at the biosensing scheme, corresponding raw data and comparison with qRT-PCR. The results show that our QPLoC biosensor in combination with CRISPR-Cas13a outperforms the qRT-PCR in terms of detection limit and system linearity, i.e. particularly in extremely low concentrations.
Figure 1. Schemeatic of label-free biosensing for SARS-CoV-2 spike RNA with TiN nanocubes and CRISPR-Cas13a where (a) shows the splitting of the viral spike RNA to fragments specified by two clipping crRNA and CRISPR-Cas13a nuclease. (b) shows the combination of crRNAs and CRISPR-Cas13a nuclease which split the long viral spike RNA to multiple fragments. (c) indicates the fragments detected by thermoplasmonic effect on hybridization with corresponding RNA probes functionalized on the TiN nanocubes.
Figure 2. Corresponding functionalization of different complimentary RNA probes on the biochip.
Figure 3. Raw data of thermoplasmonic hybridization with and without functionalization of the microwells. The pixel in non-functionalized microwell shows no response whereas the counterpart in complementary functionalized microwell shows significant oscillations on target-probe hybridization.
Figure 4. Raw data from selected pixels in corresponding to positive control SARS-CoV-2 sample solutions of different Ct values.
Figure 5. Benchmark comparison between qRT-PCR and our QPLoC biosensing to different SARS-CoV-2 concentrations. Linearity down to Ct 40 is observed on the QPLoC biosensor whereas signal saturation is observed on qRT-PCR.
Reference
[1] SHERLOCK: nucleic acid detection with CRISPR nucleases
[2] Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA