Panasonic Viera Channel List Editor Download Chip
Idara Viengxay
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The COVID-19 pandemic has highlighted the need for cost-effective diagnostics for SARS-CoV-2 RNA as well as for the detection of antibodies generated by the host in response to infection. This type of multifunctional detection method will be particularly useful for diagnosis of both acute and convalescent infections, as well as for assessing patient immunization status following vaccination. The clinical timeline of SARS-CoV-2 infection consists of an acute phase, when viral RNA is detectable in clinical samples, such as saliva or nasopharyngeal swabs, followed by a convalescent phase when serology biomarkers, such as IgG antibodies, are present in saliva and serum1. Therefore, simultaneous analysis of these different biomarkers in clinical samples as the disease progresses could provide more accurate results for disease monitoring and management.
Molecular diagnostics, on the other hand, commonly involve methods such as quantitative polymerase chain reaction (qPCR), which require rigorous sample preparation and temperature control, cold storage of reagents, expensive instrumentation (requiring routine maintenance) and trained personnel to run the tests. While there are home diagnostic tests approved for use by the US Food and Drug Administration (FDA), many of these tests either involve self-collection and mailing to a central laboratory or are based on rapid antigen tests, which have shown to be less accurate than nucleic acid tests, such as qPCR14. During the last few years, powerful diagnostic techniques that capitalize on CRISPR (clustered regularly interspaced short palindromic repeats) technology and associated programmable endonucleases have gained interest due in part to their high specificity, programmability and capacity to work at physiological conditions15,16,17,18,19. CRISPR-based diagnostics capitalize on endonucleases, such as Cas12a, which has a specific cleavage activity towards double-stranded DNA (dsDNA) fragments matching its guide RNA (gRNA) sequence. Once the Cas12a-gRNA complex binds to its dsDNA target, it activates and subsequently engages in indiscriminate collateral hydrolysis of nearby single-stranded DNA (ssDNA)20,21.
EC biosensors offer a particularly promising solution to achieve ultrasensitive, selective, multiplexed, quantitative and cost-effective LOC detection of both nucleic acids and proteins; they also offer the potential to interface with electronic medical records, integrated cloud systems and telemedicine. Despite their potential, EC diagnostics have only been used to detect either nucleic acids or proteins individually26,29 and have been limited to methods that require multiple liquid handling steps and specialized equipment. Here we describe a low-cost, three-dimensional (3D)-printed, self-contained LOC diagnostic platform that is capable of concurrent detection of SARS-CoV-2 nucleic acids and host antibodies directed against the virus from unprocessed saliva samples. This device integrates microfluidics that enable automated liquid handling for sample preparation with a simple and sensitive readout for both viral RNA as well as host antibodies. The simplicity of this device makes it user-friendly and should enable its use for POC testing within hospitals and in COVID-19 testing clinics.
a, Overview of the microfluidic chip designed for an LOC sample-to-answer saliva detection of SARS-CoV-2 RNA and antibodies. (1) The user inputs saliva onto the antibody detection reservoir and a saliva and proteinase K mixture into the sample preparation reservoir, where it incubates. (2) The saliva is pumped over the PES membrane inside the reaction chamber for RNA capture and heated to denature potential reaction inhibitors. (3) The LAMP solution is then pumped from the reservoir into the reaction chamber and incubated. (4) The CRISPR mixture is pumped into the reaction chamber, incubated and then pumped over the EC sensor chip. (5) The saliva for antibody detection is pumped over the EC sensor chip. (6) After the addition of polystreptavidin-HRP and TMB, results from the EC sensor chip are read with a potentiostat. b, An exploded view of the multiplexed system, which includes a heater system, a sealed microfluidic chip and a multiplexed EC sensor chip. c, Photograph of the microfluidic system with a quarter dollar for scale.
We next worked to combine both EC sensors to create a multiplexed assay for simultaneous viral RNA and serological biomarker detection on-chip to facilitate increased sensitivity and specificity of SARS-CoV-2 detection44. Saliva is an excellent source of both viral RNA as well as host antibodies (IgG, IgM and IgA) in SARS-CoV-2 patients51, and hence is an ideal sample for a multiplexed assay for viral RNA and serology. Unfortunately, the patient saliva samples used for this study had to be heat-inactivated before use as saliva from SARS-CoV-2 infected patients are of a highly contagious nature. Because high heat denatures the antibodies1, we spiked a National Institute for Biological Standards and Control (NIBSC) SARS-CoV-2 IgG calibrant into heat-inactivated saliva samples at 1:20 dilution to simulate the ratio of IgG present in saliva. After confirming that the signal outputs from the spiked saliva samples were consistent with the signals generated from the plasma samples (Supplementary Fig. 24), we modified the EC sensor chip so that the four electrodes could be used individually to detect the three antigens (S1, N and S1-RBD) and PNA (Fig. 4a).
In the present study, we described a sample-in-answer-out diagnostic platform that integrates ultrasensitive and highly specific multiplexed EC sensors within an LOC microfluidic chip to rapidly and simultaneously detect both clinically relevant quantities of SARS-CoV-2 viral RNA and antibodies within patient saliva samples. This platform incorporates multiple innovations including: (1) engineering of a high-specificity and high-sensitivity multiplexed EC sensor chip that enables detection of both proteins and nucleic acids with clinically relevant samples of biological fluids perfused through a single microfluidic channel; (2) development of a CRISPR-based detection assay optimized to function in a multiplexed EC assay in parallel with antibody-based detection assays, the optimized assay being amenable to POC applications; (3) development of methods for automated microfluidic extraction and amplification of RNA from raw patient saliva samples; and (4) fluidic integration of the sample preparation process with the EC sensor chip.
We validated the EC sensor for serology and obtained an accuracy of 100% (100% sensitivity and 100% specificity) for IgG as compared with traditional ELISA, with S1-RBD showing the highest accuracy in detecting IgG in clinical samples. This increased specificity for S1-RBD may be explained by the RBD domain being a highly immunogenic epitope for development of neutralizing antibodies to SARS-CoV-2 during the humoral response47. In addition, we characterized a multiplexed EC sensor that detects antibodies against relevant viral structural proteins (S1-RBD, S1 and N) for a variety of antibody isotypes (IgG, IgM and IgA), and enables a more robust understanding of the humoral response in patients. Importantly, we also found that simultaneous multiplexed detection of different viral antigens led to increased diagnostic sensitivity.
Finally, we designed and tested a microfluidic LOC platform that integrates with the multiplexed EC sensor for simultaneous detection of both RNA and IgG in clinical saliva samples. Saliva is an excellent alternative to nasopharyngeal swabs and nasal swabs for SARS-CoV-2 diagnosis, as it is simple to collect, does not require extensive collection equipment, and has been shown to provide both nucleic acid and serological data during and post infection or vaccination1,30,65. The microfluidic chip eliminates the need for RNA extraction kits by automating raw saliva sample preparation, RNA amplification and CRISPR-based RNA detection steps. With its compact and sealed design, our LOC platform limits user steps to avoid possible sources of contamination or human-introduced error to allow for device use by untrained end-users, further increasing its potential as a POC testing system. With our integrated device, we are able to perform both serological and RNA detection from a saliva sample without requiring specialized collection reagents or equipment, and with results reported concurrently. To our knowledge, there has so far been no report of an EC diagnostic device that is multiplexed, highly sensitive and capable of processing raw biological samples such as saliva.
While our LOC platform is promising, there are bottlenecks that need to be addressed before this technology can be readily adopted for clinical POC settings. Currently, our system uses peristaltic pumping for fluid movement and a potentiostat for the readout of the EC sensor chips. Further integration of the electronics, peristaltic pumping and potentiostat-based readout would allow for a robust reusable system that could make this multiplexed diagnostic tool useful for healthcare and clinical POC settings. Similarly, the 3D-printed microfluidic chips can be made at scale by transitioning to injection moulding techniques, with the LAMP and the CRISPR detection reagents pre-measured, lyophilised and sealed within the cartridge for streamlined assay use.
As the COVID-19 pandemic has shown, there is a critical need to rapidly adapt current testing strategies to more quickly and easily monitor both the infection and immune status of patients. Knowledge of infection stages can help curb disease spread, while insights on antibody titre levels can help with understanding how novel variants may affect individuals with immune protection through infection, vaccination or a combination of both. The streamlined workflow and multiplexing capabilities of our EC sensor represent important steps towards building the infrastructure necessary to provide this information to clinicians and members of the public alike.
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