Diffusion-free valve for preprogrammed immunoassay with capillary microfluidics

Abstract

By manipulating the geometry and surface chemistry of microfluidic channels, capillary-driven microfluidics can move and stop fluids spontaneously without external instrumentation. Furthermore, complex microfluidic circuits can be preprogrammed by synchronizing the capillary pressures and encoding the surface tensions of microfluidic chips. A key component of these systems is the capillary valve. However, the main concern for these valves is the presence of unwanted diffusion during the valve loading and activation steps that can cause cross-contamination. In this study, we design and validate a novel diffusion-free capillary valve: the π-valve. This valve consists of a 3D structure and a void area. The void acts as a spacer between two fluids to avoid direct contact. When the valve is triggered, the air trapped within the void is displaced by pneumatic suction induced from the capillary flow downstream without introducing a gas bubble into the circuit. The proposed design eliminates diffusive mixing before valve activation. Numerical simulation is used to study the function and optimize the dimensions of the π-valve, and 3D printing is used to fabricate either the mould or the microfluidic chip. A comparison with a conventional valve (based on a constriction-expansion valve) demonstrates that the π-valve eliminates possible backflow into the valve and reduces the mixing and diffusion during the loading and trigger steps. As a proof-of-concept, this valve is successfully implemented in a capillary-driven circuit for the determination of benzodiazepine, achieving the successive release of 3 solutions in a 3D-printed microfluidic chip without external instrumentation. The results show a 40% increase in the fluorescence intensity using the π-valve relative to the conventional value. Overall, the π-valve prevents cross-contamination, minimizes sample use, and facilitates a sophisticated preprogrammed release of fluids, offering a promising tool for conducting automated immunoassays applicable at point-of-care testing.

Keywords: Chemistry; Electrical and electronic engineering.

Fig. 1. Sketch of the diffusion-free valve (π-valve).

$\pi$-valve encompasses: (1) inlet channel, (2) stop junction, (3) void (air chamber), (4) reagent inlet, (5) main channel, (6) shallow branch, (7) deep branch, and (8) activation resistance component. The valve avoids diffusive mixing of the reagents by using the isolating air void before their activation. The detailed step-by-step process is described in Table 2

Fig. 2. Conceptualization of the π-valve and its operations in the form of its nearest electronic equivalent (FET).

a Electronic analogy of the π-valve. The meniscus is a negative potential source. The liquid flows toward the potential with the lowest magnitude. ${\mathrm{P}}_1$ and ${\mathrm{P}}_2$ are the capillary retention pressures upstream of the main channel and branch, respectively; P_c is the capillary pressure downstream; ${\mathrm{R}}_1$, ${\mathrm{r}}_1$, and ${\mathrm{r}}_2$ are the activation resistance, deep branch, and shallow branch, respectively; G, S and D are the typical terminals of a transistor, the gate, the source and the drain, respectively; ${\mathrm{V}}_{\mathrm{G}}$ is the gate voltage; and ${\mathrm{V}}_{\mathrm{c}}$ is the channel voltage. b Transistor circuit analogy. The void operates like the channel of a FET. Conditional equations from the figure must be considered for correct operation. When the gate potential is sufficiently large to stop the current downstream in the main channel, its field effect on the void pressure opens the transistor channel. Consequently, the valve is activated

Fig. 3. The valve diffusion analysis using the colour intensity evolution.

a Color intensity during π-valve loading and activation. Solid red and blue lines are the color intensities of the red and blue liquids at position $P_2$, respectively. The dotted blue line is the color intensity of the blue liquid at position $P_1$. b Picture of the valve during activation. The scale bar is 1 mm. c Color intensity during activation at position 2 ($P_2$). The solid lines are the average intensity at 20 Hz, and the dashed lines are their respective standard deviations. Background noise is subtracted by selecting a second ROI close to the detection site. Intensities are taken from Video 3 (SI), which shows the activation and release of the 3D printed $\pi$-valve

Fig. 4. Comparison of the diffusion of liquids between π-valve and conventional valve (based on constriction-expansion).

Functional time steps are defined as (1) loading; (2) air trapping/interface breaking; (3) air equilibration/pressure equilibration; (4) activation/trigger; and (5) release. a Percentage of blue intensity at P₁. b Percentage of red intensity at P₂. P₁ is located 1 mm upstream from the stop junction of the void, and P₂ is located 1 mm downstream from the connection between the shallow branch and the main channel. Backflow into the reservoir is observed for the conventional valve (I). Furthermore, the reagent diffuses into the main channel before valve activation (II), and mixing occurs when the valve is released (III). No mixing is observed for the $\pi$-valve before release. The scale bars are 1 mm

Fig. 5. Sketch of the preprogrammed π-valve array within a capillary circuit.

The components are the (1) inlet of the main channel, (2) deep branch, (3) activation resistance, (4) reagent reservoir, (5) retention burst, (6) shallow branch, (7) reagent inlet, (8) stop junction, (9) void area, (10) detection site or reaction chamber, (11) capillary pump, and (12) air vent

Fig. 6. The microfluidic sequence for benzodiazepine detection.

Competitive immunoassay for benzodiazepine detection using capillary-driven microfluidic devices with a $\pi$-valve array (a) and without a $\pi$-valve array (b). The determination consists of an activation step (step 0) and 3 subsequent flow events. The trigger step (0) starts with the introduction of 30 µL of sample with the anti-drug in the main channel (5 µg/mL anti-benzodiazepine in 5% BSA without benzodiazepine). In step (1), the solution of reservoir 1, which is filled with 10 µL of 5% BSA for blocking, was eluted and passed through the detection zone. Subsequently, in step (2), reservoir 2, which is filled with 10 µL of the secondary fluorescent QD-Abs (1:100 QD-Abs in 5% BSA), was captured by the drug-BSA present in the nitrocellulose membrane. Finally, in step (3), reservoir 3, which is filled with 10 µL of PBS, was used to wash the remaining fluorescence reagents in the detection zone. Note that a higher intensity than that in (b) is apparent in (a)

Fig. 7. Relative fluorescence intensity versus drug presence.

Red: the preprogrammed assay with a π-valve array. Blue: the preprogrammed assay without π-valves. Gray: outside microfluidics by manually pipetting into the nitrocellulose strip the same volume and chronological sequence. The nitrocellulose background light is the reference (0%). The standard deviation is determined from the measurement of three replicates

Fig. 8. Quantitative competitive immunoassay for benzodiazepine using microfluidics with -valve.

a Light evolution at the detection line by labelling and washing the blank sample. b Relative fluorescence intensity changes with the drug concentration within the sample. The maximum and minimum reference lights are considered for blank (0 ng/mL) to 1000 ng/mL (SD from three replicates) samples