Rapid quantitation of SARS-CoV-2 antibodies in clinical samples with an electrochemical sensor.

The current coronavirus disease 2019 (COVID-19) pandemic is caused by several variants of severe acute respiratory syndrome coronavirus-2 virus (SARS-CoV-2). With the roll-out of vaccines and development of new therapeutics that may be targeted to distinct viral molecules, there is a need to screen populations for viral antigen-specific SARS-CoV-2 antibodies. Here, we report a rapid, multiplexed, electrochemical (EC) device with on-chip control that enables detection of SARS-CoV-2 antibodies in less than 10 min using 1.5 µL of a patient sample. The EC biosensor demonstrated 100% sensitivity and specificity, and an area under the receiver operating characteristic curve of 1, when evaluated using 93 clinical samples, including plasma and dried blood spot samples from 54 SARS-CoV-2 positive and 39 negative patients. This EC biosensor platform enables simple, cost-effective, sensitive, and rapid detection of anti-SARS-CoV-2 antibodies in complex clinical samples, which is convenient for evaluating humoral-responses to vaccination or infection in population-wide testing, including applications in point-of-care settings. We also demonstrate the feasibility of using dried blood spot samples that can be collected locally and transported to distant clinical laboratories at ambient temperature for detection of anti-SARS-CoV-2 antibodies which may be utilized for serological surveillance and demonstrate the utility of remote sampling.

Biofabrication of Multiplexed Electrochemical Immunosensors for Simultaneous Detection of Clinical Biomarkers in Complex Fluids.

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.

Ultrarapid Method for Coating Electrochemical Sensors with Antifouling Conductive Nanomaterials Enables Highly Sensitive Multiplexed Detection in Whole Blood.

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.

Enabling Multiplexed Electrochemical Detection of Biomarkers with High Sensitivity in Complex Biological Samples.

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) with assay times of 37 and 15 min when integrated with a microfluidic system. These biosensors developed demonstrate the vast potential of solving the biofouling problem, and how it can enable potential clinically important diagnostic applications. This Account reviews our antifouling surface chemistry and the multiplexed EC sensor-based biodetection method we developed and places it in context of the various innovative contributions that have been made by other researchers in this field. We are optimistic that future iterations of these systems will change the way diagnostic testing is done, and where it can be carried out, in the future.

Recent innovations in cost-effective polymer and paper hybrid microfluidic devices.

Hybrid microfluidic systems that are composed of multiple different types of substrates have been recognized as a versatile and superior platform, which can draw benefits from different substrates while avoiding their limitations. This review article introduces the recent innovations of different types of low-cost hybrid microfluidic devices, particularly focusing on cost-effective polymer- and paper-based hybrid microfluidic devices. In this article, the fabrication of these hybrid microfluidic devices is briefly described and summarized. We then highlight various hybrid microfluidic systems, including polydimethylsiloxane (PDMS)-based, thermoplastic-based, paper/polymer hybrid systems, as well as other emerging hybrid systems (such as thread-based). The special benefits of using these hybrid systems have been summarized accordingly. A broad range of biological and biomedical applications using these hybrid microfluidic devices are discussed in detail, including nucleic acid analysis, protein analysis, cellular analysis, 3D cell culture, organ-on-a-chip, and tissue engineering. The perspective trends of hybrid microfluidic systems involving the improvement of fabrication techniques and broader applications are also discussed at the end of the review.