(J-370) Piezoelectric Plate Sensor For SARS-CoV-2 Detection In 30 Minutes Without Gene Isolation and Amplification

Introduction:: SARS-CoV-2 is responsible for the current Covid-19 pandemic. As of April 2023, so far it has more than 687M cases and 6.8M deaths worldwide. Although several effective vaccines were developed, new variants of the virus continue to emerge, and each new variant proves more transmittable than the one it has replaced. Even people who are fully vaccinated could still get infected. The only way to contain the spread of the virus is by rapid and accurate testing on the onset of infection. Current diagnostic tests to detect the presence of the virus with high sensitivity is PCR. However, PCR is a laboratory test requiring expensive equipment and highly trained personnel. Moreover, it requires several steps such as gene isolation, reverse-transcription and amplification and it typically takes 1-2 days to get the results. Although at home rapid antigen tests can detect SARS-coV-2 in 15 min they cannot detect the virus in samples with a viral load 10,000 viruses/ml. Piezoelectric plate sensor (PEPS) as shown in figure 1a is a highly sensitive biosensor developed in our lab. It has remarkable sensitivity that it can detect DNA in-situ with PCR-like sensitivity and specificity without the need for DNA isolation or amplification. What is unique about PEPS is that its detection Δf is inherently enhanced more than 1000 times by the unique property of the patented piezoelectric layer due to the stress induced upon binding. Previously PEPS exhibited an unprecedented 150 CFU/ml sensitivity for in situ DNA detection of bacteria directly from patient stool.


Materials and Methods:: The goal of this study is to develop a SARS-CoV-2 genetic test assay directly from patient samples in 30 min without gene isolation and amplification. To detect the viral RNA a 28-nt amine-functionalized probe DNA 5’-GTTCCATCTCTAATTGAGGTTGAACCTC-3’ to target a highly conserved orf1ab gene such that it can specifically detect all variants. Furthermore, this probe was blasted against several respiratory pathogens and human genes using NCBI BLAST to ensure the probe only perfectly matched the targeted SARS-CoV-2 gene. The detection temperature was set at 58˚C to ensure only SARS-CoV-2 RNA can bond to the probe on the PEPS. Specific detect of the viral RNA directly from samples was carried out in a temperature controlled flow system where samples with 15 vol% sodium dodecyl sulfate and 3 vol% bovine serum albumin were loaded in a reservoir kept at 95°C to lyse the virus and denature the viral RNA for 10 min, and flow to the detection cell kept at 58 ˚C where a PEPS with probes immobilized on its surface is situated at the center of the flow to catch and detect the viral RNA in the flow.


Results, Conclusions, and Discussions:: Figure 1a shows the comparison of the total frequency shift after 30 min detection between synthetic RNA and viral particles at different concentrations. There was a downshift of around 700Hz & 1.8kHz for 10² and 104 cp/ml concentration. Figure 1b shows relative frequency downshift vs time during detection for patient sample with a concentration ranging from 10²-10⁶ cp/ml. There was no frequency downshift when the sample concentration was 0 cp/ml.

In this study, PEPS was used to detect SARS-CoV-2 viral RNA directly from patient sample with gene isolation, amplification, and RNA reverse transcription. Effective viral particle lysing was confirmed by com-parable detection Δf values of the same concentrations. Testing of 15 covid-19 patient samples and comparison with PCR indicates PEPS had a 100 cp/ml sensitivity in direct detection of viral RNA from patient swaps in 30 min.


Acknowledgements (Optional): : This work was supported by NIH Grant #. 1R43AI152716-01 and No. R03EB030215

References (Optional): : 1. https://www.cdc.gov/.
2. Han, S. Biosensors and Bioelectronics, 130, 73-80.