Development of an Electrochemical Paper-Based Analytical Device for Trace Detection of Virus Particles

Abstract

Viral pathogens are a serious health threat around the world, particularly in resource limited settings, where current sensing approaches are often insufficient and slow, compounding the spread and burden of these pathogens. Here, we describe a label-free, point-of-care approach toward detection of virus particles, based on a microfluidic paper-based analytical device with integrated microwire Au electrodes. The device is initially characterized through capturing of streptavidin modified nanoparticles by biotin-modified microwires. An order of magnitude improvement in detection limits is achieved through use of a microfluidic device over a classical static paper-based device, due to enhanced mass transport and capturing of particles on the modified electrodes. Electrochemical impedance spectroscopy detection of West Nile virus particles was carried out using antibody functionalized Au microwires, achieving a detection limit of 10.2 particles in 50 μL of cell culture media. No increase in signal is found on addition of an excess of a nonspecific target (Sindbis). This detection motif is significantly cheaper (∼$1 per test) and faster (∼30 min) than current methods, while achieving the desired selectivity and sensitivity. This sensing motif represents a general platform for trace detection of a wide range of biological pathogens.

Figure 1.

a) Reaction scheme with b) representative corresponding cyclic voltammograms and c) EIS for Au microwire electrodes at different stages of modification with biotin, for capture of SA particles on a static-ePAD.

Figure 2.

Comparison of monothiol (11-mercaptoundecanoic acid) vs dithiol (lipoic acid) modification of Au microwires, for detection of 100 nm SA particles in static-ePADs, n = 4, sensitivity = 11.7 and 17.5 particles⁻¹ mL, and R² = 0.9062 and 0.9963 for the mono- and dithiol, respectively.

Figure 3.

Effect of SA particle size on %ΔR_ct for 1 × 10⁶ SA particles mL⁻¹ captured on biotin-modified microwires in a static-ePAD, R² = 0.9866, n = 4.

Figure 4.

Calibration curve for flow-ePADs (blue triangles, a inset, R² = 0.9932) and static-ePADs (red circles, b inset, R² = 0.9963) for detection of different concentrations of 100 nm diameter SA particles, 50 μL aliquot in phosphate buffer, n = 4.

Figure 5.

SEM images a) before and b) after modification with 1 × 10⁶ SA particles mL⁻¹ (100 nm diameter) in a static-ePAD.

Figure 6.

Optimization of 4G2 antibody concentration for Au microwire modification. %ΔR_ct measured for [Fe(CN)₆]^3/4– impedance before and after addition of 1 × 10⁶ WNV particles mL⁻¹ (blue circles) or viral media (red squares) to flow-ePADs (n = 4).

Figure 7.

Calibration curve (blue triangles) for different concentrations of WNV particles in a 50 μL aliquot of cell culture media (R² = 0.9650, n = 4) and a nonspecific test with Sindbis virus particles (red circle, n = 2) added to antibody-modified microwires in a flow-ePAD.