Over the last few years, the dramatic proliferation of emerging viruses such as Zika has resulted in a pressing need to develop inexpensive point-of-care diagnostic devices for rapid, sensitive, and accurate screening of patients. The ability to diagnose active infections on-site can improve patient compliance, allow physicians to tailor treatment options, and help prevent transmission to uninfected people. While commercially available point-of-care tests exist for bacterial infections, there are currently no approved point-of-care diagnostic assays for use in health clinics or in the field that detect viral infections. We are developing a point-of-care diagnostic device for viral detection that is inexpensive and can carry out all assay steps in an automated format. Our earlier work characterized electrochemical paper-based analytical devices (ePADs) coupled with metallic microwire electrodes as a disposable sensing system suitable for this application.^1,2 Toward development of virus particle sensors, we have functionalized the electrodes with bioaffinity reagents to drive specific analyte binding, optimized electrode-integrated paper-based microfluidic platforms, and employed electrochemical impedance spectroscopy (EIS) to detect low levels of virus-sized particles. As shown in the figure, the functionalized electrode enables the binding of analyte to its surface and forms an insulating layer, increasing the impedance at the surface. In a proof of concept study, biotin-modified electrodes were incubated with streptavidin conjugated beads of size similar to viruses and investigated as a representative specific binding system. Two different electrode systems have been investigated. In the first, freestanding Au microwires were used because they can be easily modified with specific recognition elements using well-established coupling techniques prior to incorporation into the ePAD. As an alternative, Au nanoparticles were electrodeposited on screen-printed carbon electrodes, functionalized via the same chemistry, and compared for detection limits, sensitivities, and linear ranges. Preliminary optimization tests were achieved in a paper-based wax-confined static well fabricated as previously described.¹ After demonstrating detection in static solutions, we tested the system using a flow-through ePAD system² for improved sensitivity. To create steady-state flow, a fan-shaped passive pump was connected to the channel inlet and the microwire electrodes were embedded between two paper layers for electrochemical analysis. The two layers of paper create a channel and generate relatively high flow rates and more effective mass transport to the electrode surfaces. Key elements of flow rate, paper/electrode geometry, and number of electrodes were optimized for the method. Furthermore, multiple wires modified with the same affinity group were added in an overlapping, parallel configuration to increase surface area and enhance the signal. Sensor response was characterized with EIS using ferri/ferrocyanide as the mediator before and after analyte incubation. EIS provides significantly lower detection limits compared to traditional colorimetric lateral flow devices, while being inexpensive to manufacture and readily multiplexed for multiple analyte detection. The optimized electrochemical affinity assay makes ePAD particularly promising to rapidly detect virus-size particles in a multiplexed sample with high sensitivity, selectivity, and stability.

(1) Adkins, J. A.; Henry, C. S. Analytica Chimica Acta 2015, 891, 247-254.

(2) Adkins, J. A.; Noviana, E.; Henry, C. S. Anal Chem 2016, 88, 10639-10647.