In a recent study published in Nature Biomedical Engineering, researchers described the development and applications of a three-dimensional (3D)-printed lab-on-a-chip (LOC) device that quickly detected the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patient's saliva.
The coronavirus disease 2019 (COVID-19) pandemic highlighted the need for multi-functional and cost-effective diagnostics methods that could seamlessly diagnose acute and convalescent SARS-CoV-2 infections and a patients’ immunization status following vaccination. Despite the advances in SARS-CoV-2 diagnosis, such a LOC platform for point-of-care (POC) use is yet unavailable.
The clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have garnered the attention of the scientific community recently. They capitalize on endonucleases, such as CRISPR-associated protein 12a (Cas12a), and yield highly specific results under physiological conditions. When coupled with amplification techniques, such as loop-mediated isothermal amplification (LAMP), these CRISPR-based assays become truly worthwhile. Then, they can perform SARS-CoV-2 ribonucleic acid (RNA) detection in clinical samples, typically in the attomolar (10−18) range.
Previously published POC CRISPR-based electrochemical detection platforms have not been sensitive enough to detect clinically relevant quantities of SARS-CoV-2 RNA. Although the graphene field effect transistor-based EC biosensors had similar sensitivity to SARS-CoV-2 RNA, those assays lacked multiplexing capabilities and were validated using viral transport media-based nasopharyngeal swab samples.
About the study
In the present study, researchers described a LOC multiplexed diagnostic device capable of simultaneously detecting SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies from unprocessed saliva samples. They ensured that saliva samples were exposed to a proteinase K solution and then heated to 55°C for 15 minutes. Another round of heating at 95 °C for five minutes in high-power resistors allowed SARS-CoV-2 RNA lysis and nuclease inactivation. The saliva samples were pumped over the polyethersulfone (PES) membrane within the reaction chamber to ensure the proper binding of viral RNA. Conversely, saliva for antibody detection was pumped over the EC sensor chip directly, which the researchers read using a potentiostat. This device had an integrated microfluidic chip to enable automated liquid handling for saliva sample preparation while ensuring proper RNA amplification for CRISPR-based RNA detection. Additionally, it had a sensitive readout for SARS-CoV-2 RNA and host antibodies on the same electrochemical (EC) biosensor chip.
The researchers designed a biotinylated single-stranded deoxyribonucleic acid (ssDNA) replication protein A (RP), which partially hybridized to peptide nucleic acid (PNA) capture probes and integrated the CRISPR-based molecular assay onto the EC sensor platform. Further, they varied the concentration and incubation time of the RP to obtain a rapid, high signal-to-noise ratio. Next, they incubated functionalized EC biosensors with saliva samples containing a mixture of the LAMP/Cas12a, which collaterally cleaved the biotinylated ssDNA RP. This led to a reduction in the binding of polystreptavidin-horseradish peroxidase (HRP); thus, a reduction in the precipitation of tetramethylbenzidine (TMB) deposited locally on the electrode surface. In other words, the LAMP amplification increased the signal sensitivity over and above the sensor’s limit of detection (LOD).
The use of polystreptavidin-HRP/TMB-based reaction chemistry for readout enabled further amplification of the EC signal for both the serological and CRISPR-based RNA sensors. The team measured peak current or reduced precipitation of TMB using cyclic voltammetry (CV); varying the voltage between −0.5 and 0.5 volts. Lastly, the researchers validated the CRISPR-EC sensor platform used in the study to determine its clinical value.
The study describes a novel device that used polystreptavidin-HRP/TMB-based reaction chemistry for readout to amplify the EC signal for both the serological and CRISPR-based RNA sensors. Further, this device used a customized molecular assay which, through optimization of RNA-dependent Cas12a cleavage of a biotinylated ssDNA RP, detected target nucleic acid with more sensitivity than fluorescence-based CRISPR-Cas12a assays but in a similar timeframe.
Of all the RP concentrations tested, one nanomolar (nM) RP and five minutes of incubation produced the highest signal with no background noise. The CRISPR-EC sensor platform yielded a LOD of 0.8 copies per µl, displaying a sensitivity of nearly four times compared to the fluorescence-based assays used for validating the primers. The current from the EC electrodes clearly distinguished the SARS-CoV-2-negative and positive saliva samples. Additionally, receiver operating characteristic (ROC) curve analysis demonstrated a correlation between reverse transcriptase-quantitative polymerase chain reaction (RT–qPCR) and CRISPR-Cas12a-based detection assay. Furthermore, its EC sensor detected IgG at an accuracy of 100% in clinical samples unlike the enzyme-linked immunosorbent assay (ELISA).
Additionally, the researchers noted that the simultaneous multiplexed detection of different viral antigens, including spike (S)1-receptor-binding domain (RBD), S1, and nucleocapsid (N), led to increased diagnostic sensitivity. Furthermore, this device used saliva, a better alternative to nasopharyngeal swabs for SARS-CoV-2 diagnosis. Saliva is easier to collect than nasopharyngeal swabs. Additionally, it provides nucleic acid and serological data during and after infection or vaccination.
The compact and sealed design of this diagnostic device, reduced user steps to avoid contamination or human error, makes it an attractive POC testing system even for untrained end-users. Most importantly, this microfluidic LOC platform clearly distinguished four types of SARS-CoV-2 clinical saliva samples within two hours, which further highlights its utility as a POC testing system.
To summarize, this user-friendly, inexpensive, 3D-printed LOC device, with streamlined workflow and multiplexing capabilities, could empower clinicians and the public alike, simultaneously making it easier to quickly and easily monitor the infection and immune status of COVID-19 patients. This device could provide insights into SARS-CoV-2 infection stages to help curb COVID-19 spread; likewise, it could provide data on anti-SARS-CoV-2 antibody titers to help understand how novel SARS-CoV-2 variants might affect individuals with immunity acquired through infection, vaccination, or a combination of both.