Coupling Capillary-Driven Microfluidics with Lateral Flow Immunoassay for Signal Enhancement

Abstract

Microfluidics has emerged as a versatile technology that is applied to enhance the performance of analytical techniques, among others. Pursuing this, we present a capillary-driven microfluidic device that improves the sensitivity of lateral flow immunoassay rapid tests thanks to offering an automated washing step. A novel multilevel microfluidic chip was 3D-printed with a photocurable black resin, sealed by an optically clear pressure-sensitive adhesive, and linked to the lateral flow strip. To depict the efficacy of microfluidics and the washing step, cortisol was measured quantitatively within the proposed device. Measuring cortisol levels is a way to capture physiological stress responses. Among biofluids, saliva is less infectious and easier to sample than others. However, higher sensitivity is demanded because the salivary cortisol concentrations are much lower than in blood. We carried out a competitive lateral flow immunoassay protocol with the difference that the microfluidic device applies an automated washing step after the sample is drained downstream. It washes the trapped quantum-dot-labeled antibodies out from nitrocellulose, diminishing background noise as these are bonded to cortisols and not to the immobilized receptors. Fluorescence spectroscopy, as a high-precision analysis, was successfully applied to determine clinically relevant salivary cortisol concentrations within a buffer quantitatively. The microfluidic design relied on a 3D valve that avoids reagent cross-contamination. This cross-contamination could make the washing buffer impure and undesirably dilute the sample. The proposed device is cost-effective, self-powered, robust, and ideal for non-expert users.

Keywords: 3D-printing; capillary valve; capillary-driven microfluidics; cortisol; fluorescence spectroscopy; lateral flow assay.

Figure 1

The chip schematic encompasses a microfluidic device and lateral flow strip. The microfluidic comprises a main microchannel that is connected to the washing buffer by way of a valve.

Figure 2

The effect of appending an automated washing step by the microfluidic device on the lateral flow immunoassay of cortisol. For the blank sample, the light intensity difference (ΔI) between the targeted detection circle and the rest of the nitrocellulose is increased by 47%. Also, LOD (by SD₀ from 3 replicates) is enhanced by 1.87 times.

Figure 3

Coupling the microfluidic device with the competitive lateral flow immunoassay for quantitative cortisol measurement. All the top rows (a–e) represent the schematics of the immunoassay steps, and the bottom rows show the capillary-driven flow experiment at the corresponding immunoassay stage. To have a clear vision of the fluid dynamic steps, the colored aqueous phases were used in a transparent microfluidic chip. While fluorescent spectrometry was done within the 3D-printed black chips. (a) Introducing the sample into the capillary-driven microfluidic device. (b) Capillary action drives the flow from the inlet into the downstream lateral flow, which contains immobilized cortisol-BSA on nitrocellulose. Free antibodies bond to the cortisol-BSAs. (c) The sample (blue) flows downstream, and the suction of the absorption pad empties the main microchannel until the shallow resistor before the valve junction. It opens a venting channel, upstream of the washing reservoir. Then, the downstream suction displaces air from the void into the deep branch. It moves the washing buffer (red) into the void and connects it to the blue liquid through the second branch. (d) The valve is opened, and the washing buffer goes through the lateral flow part. (e) The washing step is finished, which removes the free QD-Ab from the nitrocellulose. It increases the fluorescent light intensity difference between the detection point and the nitrocellulose background. (f) The obtained calibration curve for cortisol. The 3D-printed chip is black to decrease surrounding noise. By increasing the cortisol concentration in the sample, fewer free QD-Abs are available to bond to the receptors. So, cortisol concentration shows a reverse relation with the fluorescent light intensity (y = −11.44 ln⁡(x) + 92.556). The maximum and minimum reference lights are for blank and 1000 ng/mL samples, respectively (SD from 3 replicates).