Toward Continuous Molecular Testing Using Gold-Coated Threads as Multi-Target Electrochemical Biosensors

Abstract

Analytical systems based on isothermal nucleic acid amplification tests (NAATs) paired with electroanalytical detection enable cost-effective, sensitive, and specific digital pathogen detection for various in situ applications such as point-of-care medical diagnostics, food safety monitoring, and environmental surveillance. Self-assembled monolayers (SAMs) on gold surfaces are reliable platforms for electroanalytical DNA biosensors. However, the lack of automation and scalability often limits traditional chip-based systems. To address these challenges, we propose a continuous thread-based device that enables multiple electrochemical readings on a functionalized working electrode Au thread with a single connection point. We demonstrate the possibility of rolling the thread on a spool, which enables easy manipulation in a roll-to-roll architecture for high-throughput applications. As a proof of concept, we have demonstrated the detection of recombinase polymerase amplification (RPA) isothermally amplified DNA from the two toxic microalgae species Ostreopsis cf. ovata and Ostreopsis cf. siamensis by performing a sandwich hybridization assay (SHA) with electrochemical readout.

Keywords: chronoamperometry; isothermal DNA amplification; metal-coated threads; roll-to-roll; sandwich hybridization assay; self-assembled monolayers.

Figure 1

Schematic of functionalized threads and device: (a) A photograph of patterned Au and Ag threads. (b) Schematic of patterned, SAM functionalized Au thread used as the working electrode. (c) Schematic of the glass slide device and its components. The right side shows a photograph of the completed device for comparison. WE = functionalized Au thread working electrode, CE = two Au threads acting as a counter electrode, RE = Ag thread pseudo-reference electrode.

Figure 2

Results for the electrochemical biosensor: (a) Schematic overview of the sandwich hybridization assay. The amplified DNA product with 5′ and 3′ single-stranded tails hybridizes with the capture probe and subsequently the HRP-tagged oligonucleotide reporter probe. HRP catalyzes a reaction where the TMB substrate is oxidized with hydrogen peroxide, which is reduced back during chronoamperometric measurements. (b) Results of chronoamperometry with two targets. The upper half shows a photograph of the finished glass slide device with mounted electrode threads, clips connected to the electrode threads, and 100 µL liquid (water as a demonstration) added to the fourth region from the connection region. The box plot at the bottom shows the average current of chronoamperometry for positive and negative samples of both O. cf. ovata (OO) and O. cf. siamensis (OS). There was a significant difference in current between negative (mean M = −1.037 µA, standard deviation SD = 0.4143 µA) and positive (M = −2.063 µA, SD = 1.061 µA) OO samples (n = 7, p = 0.032), as well as between negative (M = −1.067 µA, SD = 0.6069 µA) and positive (M = −3.716 µA, SD = µA) OS samples (n = 7, p = 0.0028). Legend: ns means no significant difference, the asterisks (*) mean significant difference (one: p ≤ 0.05, two: p ≤ 0.01) (c) Photograph of a patterned, functionalized working electrode Au thread rolled onto a spool with a 4-pin cogwheel-shaped cross-section with functionalized regions aligned with the grooves. The thread is longer than those typically used in experiments to be able to roll it more than two revolutions around the spool for demonstration purposes. (d) Chronoamperometric detection on threads that have been rolled onto a spool after functionalization. The photograph on the top shows a functionalized Au thread that has been rolled out from a spool onto a glass slide device. The thread is longer than those typically used in the experiments for demonstration purposes. The box plots at the bottom show the average current for positive and negative samples of O. cf. ovata (OO). There was a significant difference in current between negative (M = −0.7537 µA, SD = 0.1765 µA) and positive (M = −4.460 µA, SD = 0.8264 µA) samples (n = 6, p < 0.0001). Legend: ns means no significant difference, the asterisks (*) mean significant difference (four: p ≤ 0.0001).

Figure 3

Device characterization: (a) Schematic of the alternative approach used to compare signal strength unaffected by variation in amplification yield. The capture probes used bind directly to the reporter probe. (b) Test for signal drop over a distance of 50.0 cm. Box plots show average chronoamperometric current after 1 s for reaction regions positioned 41 mm and 541 mm from the connection points. There was no significant difference (n = 5, p = 0.3105) in current between the measured region closest to the connector (M = −2.257 µA, SD = 0.5945 µA) and the one 50.0 cm down the thread (M = −1.979 µA, SD = 0.2276 µA). Legend: ns means no significant difference.