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

Abstract

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.

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

“…Cleaned gold sensors were placed on a hot plate set to 85 °C for 2 min. After the sensors were equilibrated to 85 °C, 70 μL of nanomaterial coating was drop cast over each sensor for 45s ( Timilsina et al 2021 , 2022b ). Thereafter, sensors were immediately rinsed in PBS followed by drying for further use.…”

Rapid quantitative electrochemical detection of SARS-CoV-2 antibodies in plasma and dried blood spot samples

“…70 µL of anti-fouling nanocomposite was then drop cast to each chip and incubated for 45s. [15] The sensors were immediately washed by dipping in PBS, rinsed at 400 rpm for 10 min, and dried with a slide spinner (Millipore Sigma, no. 674664).…”

Portable and Label-Free Sensor Array for Discriminating Multiple Analytes via a Handheld Gas Pressure Meter

“…1,2 In the past few years, a series of strategies have been developed for simultaneous detection of multiple targets, including microbead-based assays, 3 mass spectrometry, 4 and multichannel paper-based and electrochemical chip-based assays. 5,6 Although these methods provide high accuracy and sensitivity, it is only applicable to biosensors with specific interactions. After all, not all analytes have "lockand-key"-specific recognition, especially the complex analytes.…”

“…Simultaneous detection of multiple analytes in a complex sample has received extensive consideration because of the increasing need for complex sample analysis. Compared with detection of a single target, the simultaneous detection of multiple analytes can shorten the analysis time, reduce the cost, and decrease false positives. , In the past few years, a series of strategies have been developed for simultaneous detection of multiple targets, including microbead-based assays, mass spectrometry, and multichannel paper-based and electrochemical chip-based assays. , Although these methods provide high accuracy and sensitivity, it is only applicable to biosensors with specific interactions. After all, not all analytes have “lock-and-key”-specific recognition, especially the complex analytes.…”