Rapid, accurate and frequent detection of the RNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and of serological host antibodies to the virus would facilitate the determination of the immune status of individuals who have Coronavirus disease 2019 (COVID-19), were previously infected by the virus, or were vaccinated against the disease. Here we describe the development and application of a 3D-printed lab-on-a-chip that concurrently detects, via multiplexed electrochemical outputs and within 2 h, SARS-CoV-2 RNA in saliva as well as anti-SARS-CoV-2 immunoglobulins in saliva spiked with blood plasma. The device automatedly extracts, concentrates and amplifies SARS-CoV-2 RNA from unprocessed saliva, and integrates the Cas12a-based enzymatic detection of SARS-CoV-2 RNA via isothermal nucleic acid amplification with a sandwich-based enzyme-linked immunosorbent assay on electrodes functionalized with the Spike S1, nucleocapsid and receptor-binding-domain antigens of SARS-CoV-2. Inexpensive microfluidic electrochemical sensors for performing multiplexed diagnostics at the point of care may facilitate the widespread monitoring of COVID-19 infection and immunity.
Conspectus The ability to perform multiplexed detection of various biomarkers within complex biological fluids in a robust, rapid, sensitive, and cost-effective manner could transform clinical diagnostics and enable personalized healthcare. Electrochemical (EC) sensor technology has been explored as a way to address this challenge because it does not require optical instrumentation and it is readily compatible with both integrated circuit and microfluidic technologies; yet this approach has had little impact as a viable commercial bioanalytical tool to date. The most critical limitation hindering their clinical application is the fact that EC sensors undergo rapid biofouling when exposed to complex biological samples (e.g., blood, plasma, saliva, urine), leading to the loss of sensitivity and selectivity. Thus, to break through this barrier, we must solve this biofouling problem. In response to this challenge, our group has developed a rapid, robust, and low-cost nanocomposite-based antifouling coating for multiplexed EC sensors that enables unprecedented performance in terms of biomarker signal detection compared to reported literature. The bioinspired antifouling coating that we developed is a nanoporous composite that contains various conductive nanomaterials, including gold nanowires (AuNWs), carbon nanotubes (CNTs), or reduced graphene oxide nanoflakes (rGOx). Each study has progressively evolved this technology to provide increasing performance while simplifying process flow, reducing time, and decreasing cost. For example, after successfully developing a semipermeable nanocomposite coating containing AuNWs cross-linked to bovine serum albumin (BSA) using glutaraldehyde, we replaced the nanomaterials with reduced graphene oxide, reducing the cost by 100-fold while maintaining similar signal transduction and antifouling properties. We, subsequently, developed a localized heat-induced coating method that significantly improved the efficiency of the drop-casting coating process and occurs within the unprecedented time of <1 min (at least 3 orders of magnitude faster than state-of-the-art). Moreover, the resulting coated electrodes can be stored at room temperature for at least 5 months and still maintain full sensitivity and specificity. Importantly, this improved coating showed excellent antifouling activity against various biological fluids, including plasma, serum, whole blood, urine, and saliva. To enable affinity-based sensing of multiple biomarkers simultaneously, we have developed multiplexed EC sensors coated with the improved nanocomposite coating and then employed a sandwich enzyme-linked immunosorbent assay (ELISA) format for signal detection in which the substrate for the enzyme bound to the secondary antibody precipitates locally at the molecular binding site above the electrode surface. Using this improved EC sensor platform, we demonstrated ultrasensitive detection of a wide range of biomarkers from biological fluids, including clinical biomarkers, in both single and multiplex formats (N = 4) wi...
The commercialization of electrochemical (EC)-sensors for medical diagnostics is currently limited by their rapid fouling in biological fluids, and use of potential antifouling coatings is hindered by the complexity and cost of application methods. Here, a simple ultrafast (< 1 min) method is described for coating EC-sensors with cross-linked bovine serum albumin infused with conductive, pentaamine-functionalized, graphene particles that can be stored at room temperature for at least 20-weeks, which provides unprecedented sensitivity and selectivity for diagnostic applications. The antifouling coating is applied directly on-chip using rapid heating via simple dip-coating, which provides unprecedented high levels of electrode conductivity for up to 9-weeks in unprocessed biological samples. This method is leveraged to develop a multiplexed platform for detecting clinically relevant biomarkers including myocardial infarction and traumatic brain injury using only 15 μL of blood. Single-digit pg mL −1 sensitivity is obtained within minutes in unprocessed human plasma and whole blood, which is faster and at least 50 times more sensitive than traditional enzyme-linked immunosorbent assays, and the signal generated is stable enough to be measured after 1 week of storage. The multiplexed EC-sensor platform is validated by analyzing 22 patient samples and demonstrating excellent correlation with reported clinical values.
Here we describe an ultra-fast (< 1 min) method for coating electrochemical (EC) sensors with an anti-fouling nanocomposite layer that can be stored at room temperature for months, which provides unprecedented sensitivity and selectivity for diagnostic applications. We leveraged this method to develop a multiplexed diagnostic platform for detection of biomarkers that could potentially be used to triage patients with myocardial infarction and traumatic brain injury using only 15 microliters of blood. Single-digit pg/mL sensitivity was obtained within minutes for all the biomarkers tested in unprocessed human plasma samples and whole blood, which is much faster and at least 50 times more sensitive than traditional ELISA methods, and the signal was stable enough to be measured after one week of storage. The multiplexed EC sensor platform was validated by analyzing 22 patient samples, which demonstrated excellent correlation with reported clinical values.
Simultaneous detection of multiple disease biomarkers in unprocessed whole blood is considered the gold standard for accurate clinical diagnosis. Here, this study reports the development of a 4-plex electrochemical (EC) immunosensor with on-chip negative control capable of detecting a range of biomarkers in small volumes (15 μL) of complex biological fluids, including serum, plasma, and whole blood. A framework for fabricating and optimizing multiplexed sandwich immunoassays is presented that is enabled by use of EC sensor chips coated with an ultra-selective, antifouling, and nanocomposite coating. Cyclic voltammetry evaluation of sensor performance is carried out by monitoring the local precipitation of an electroactive product generated by horseradish peroxidase linked to a secondary antibody. EC immunosensors demonstrate high sensitivity and specificity without background signal with a limit of detection in single-digit picogram per milliliter in multiple complex biological fluids. These multiplexed immunosensors enable the simultaneous detection of four different biomarkers in plasma and whole blood with excellent sensitivity and selectivity. This rapid and cost-effective biosensor platform can be further adapted for use with different high affinity probes for any biomarker, and thereby create for a new class of highly sensitive and specific multiplexed diagnostics.
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