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

Abstract

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...

Novel Immunoprobe Based on MOF-818 Synergizing with an Antifouling Sensing Interface to Improve Immunosensors

“…12−15 Owing to the porosity, huge specific surface area, and bioprotective properties, [16][17][18][19][20]56,57 metal−organic framework (MOF) materials were commonly employed in immunoprobes to enhance the sensitivity of sensors. MOFbased immunoprobes were usually composed of three modules: (1) The large specific surface area of a carrier unit was taken advantage to adsorb signal molecules, 21,22 but some MOFs caused signal leakage due to instability to other material precursors. For instance, metal ions or signal molecules with an aromatic heterocycle caused the competitive etching of MOFs.…”

“…Electrochemical immunosensors are widely manipulated to detect trace markers in biological fluids due to their high sensitivity, simple operation, miniaturization of devices, and intuitive readout of results. − Because of the tiny concentration alterations of biomarkers in disease early stages and the complexity of biological fluids, , greater performance requirements are necessary. In addition, insufficient precision can result in false positive results, leading to unnecessary testing and treatment. − To circumvent the aforementioned stumbling block, it is an urgent priority to formulate new immunosensors.…”

Biofabrication of multiplexed electrochemical immunosensors for simultaneous detection of clinical biomarkers in complex fluids

“…But while significant basic research has been conducted on developing multiplexed EC POC devices for disease diagnosis, very few products have been translated to the clinical setting. [5] There are various reasons for this, including biofouling of electrodes by components with complex biological fluids (sweat, saliva, blood, plasma), the complexity of building bioassays on the electrodes, challenges related to development of multiplexing capabilities, sensitivity to changes in environmental cues, and the need to develop sophisticated readouts and analytical tools for analysis of complex data generated with these types of devices. [6] We have recently developed an engineering solution to address these challenges and create a clinically translatable EC platform for rapid detection and quantification of clinically relevant disease biomarkers in small volumes of complex biological fluids, including whole human blood.…”

“…Key to this advance was the development of a novel antifouling electroconductive nanocomposite coating to combat EC biosensors, which enabled engineering of an EC sensor platform that provides effective and accurate detection of several biomarkers that may be used for diagnosis of various diseases with clinical significance. [5,7] The coating method involves brief (1 minute), localized, heat-induced coating of EC sensors with a nanocomposite composed of denatured bovine serum albumin cross-linked with pentaamine functionalized graphene oxide cross-linked with glutaraldehyde (BSA/prGOx/GA). [8] Multiplexing is a critical requirement for POC devices because disease and pathophysiology often involves interplay among many complex biological processes, and hence accurate diagnosis requires detection of several molecules rather than a single biomarker entity.…”

Programmable DNA-Fueled Electrochemical Analysis of Lung Cancer Exosomes

“…Herein, we integrate programmable DNA synthesis with the CRISPR-Cas12a trans-cleavage and develop a new method to determine PD-L1-expressing exosomes in lung cancer by using an electrochemical technique that is promising for high sensitivity, good portability, and ease of use. , Scheme depicts the main concept of our method. PD-L1-expressing exosomes are first enriched at PD-L1 antibody-functionalized immune-magnetic beads (anti-PD-L1@IMBs).…”