A new, 3D-printed, lab-on-chip system integrates eRapid and SHERLOCK technologies into a unique, postcard-sized system that can simultaneously detect the presence of both SARS-CoV-2 RNA and antibodies against the virus in a patient saliva. The detection, which takes under two hours, occurs through multiplexed electrochemical outputs.
In Nature Biomedical Engineering, the prototype prototype is described as a lab-on-a-chip for the concurrent electrochemical detection of SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies in saliva and plasma.
Everyone was working on diagnostics that could detect either the SARS-CoV-2 virus or antibodies against it, but not both. We understood that we could successfully detect the presence of DNA and RNA molecules electrochemically, thanks to our research on Lyme disease. Helena de Puig, PhD, said: We decided to develop a one-on-one drug to help track infections and address the pandemic.
Because viral particles and antibodies can be found there, the team chose saliva as their sample material. For the SHERLOCK portion of the diagnostic, which detects the presence of SARS-CoV-2 RNA, the device must be able to extract, concentrate, and amplify viral RNA from a saliva sample, then mix it with CRISPR reagents, and deliver the resulting solution to the eRapid chip portion for detection.
A microfluidic system of several reservoirs, channels, and heating elements was developed in order to automatically mix and transfer substances within the prototype device. In the first chamber, saliva is combined with an enzyme that breaks open any viruses outer envelopes to expose their RNA. Then the sample is then pumped into a reaction chamber, where it is heated and mixed with loop-mediated reagents that enhance the viral RNA. After 30 minutes of amplification, a mixture of SHERLOCK reagents is added
The mechanism for the detection of SARS-CoV-2 genetic material is that single-stranded (ssDNA) molecules which have been attached to them bind to peptide nucleic acid (PNA) on the electrodes surface. The biotin then dissolves into a poly-HRP-streptavidin, which causes the solid TMB to precipitate out of the liquid solution. This change is identified as a difference in the amount of electrical current flowing through the electrode, indicating that the
The Cas12a within the SHERLOCK mixture cuts the biotin molecule as well as the ssDNA. This cutting action does not cause the TMB to precipitate onto the electrode, indicating a positive test result.
The combination of the PNA-based assay and the poly-HRP-streptavidin-TMB reaction chemistry that we used for this product allowed us to detect the presence of SARS-CoV-2 with four times greater sensitivity than our original fluorescence-based SHERLOCK technology in the same amount of time, according to Joshua Rainbow, a former visiting graduate student at the Wyss Institute. It was also able to detect the presence of viral RNA with 100% accuracy.
The two remaining eRapid electrodes were combined with two COVID-related antibodies. The S1 subunit of the Spike protein (S1), the ribosomal binding domain within that subunit (S1-RBD) and the N protein, which is present in most coronaviruses (N). If a patient saliva sample contains one or more of these antibodies, they bind to their partners antibodies on the electrodes. A secondary antibody that is attached to biotin will then bind
Cue samples of human plasma from patients who previously tested positive for SARS-CoV-2, were tested by scientists. The method was able to distinguish between antibodies against S1, S1, S1, and N with greater than 95 percent accuracy.
Sanjay Sharma Timilsina, a former postdoctoral fellow at the Wyss Institute, believes that being able to easily distinguish between different types of antibodies is extremely beneficial for determining whether patients are immune to vaccines or infection, and improving the effectiveness of those different immunity levels over the years.
Using saliva from SARS-CoV-2 patients, the team analyzed the combined viral RNA and antibody electrodes. Despite the fact that the multiplexed chips correctly identified positive and negative RNA and antibody samples with complete accuracy, at the same time.
The low-cost and compact design is user-friendly and reduces the number of steps a patient requires to perform, reducing the possibility of user error. Customized cartridges may be easily developed to detect antigens and antibodies from different diseases, and may be placed in a reusable housing and readout device that a user might keep in his house.