FULLY AUTOMATED BEAD-BASED AFFINITY ASSAYS WITH PHOTOTHERMALLY ACTUATED WAX VALVES

Abstract

This paper reports on a compact fully automated microfluidic platform for the implementation of affinity assays. Many wax valves can be easily integrated, which allows the control of the different reagents involved in the assay with a single plunger actuator. This is a clear advantage in comparison with previous systems requiring one plunger per reagent blister [1]. The concept of photothermal control of multiple-actuation wax valves was presented in [2]. Here we demonstrate the integration of these valves in a microfluidic chip fabricated with rapid prototyping techniques (laser cutting of pressure sensitive adhesive layers). We also demonstrate that the present wax valves can be used to retain functionalized beads while allowing the flow of reagents, to carry out bead-based affinity assays. The structure and actuation of these wax valves are detailed schematically in Figure 1. A photothermal heater is used to transform the energy of the light emitted by the LED into heat. The wax that is in close contact with the heater melts and, owing to a negative pressure applied on the outlet of the plug, this melted wax is ejected creating a tunnel. The procedure for closing is the same as for opening, except that no pressure is applied. In this case, the melted wax is not ejected, but it refills the tunnel. The chip and the instrument composing the microfluidic platform are presented in Figure 2. The microfluidic chip has a pumping chamber which is actuated by a stepper-motor linear actuator on the instrument. Elastomeric posts inside the chamber enable volume squeezing and expansion so that both positive and negative pressure can be generated to control the fluid flow inside the chip. The chip aligned to an array of LEDs in the instrument. An Arduino board is used to controls the pumping and valve opening/closing sequence that performs the complete ELISA protocol on a 5-valve chip. A red LED and a photodetector are used for absorbance measurements on the fluid displaced from the beads area. A photograph of the closed and opened states of a valve with dyed solutions is shown in Figure 3. Figure 4 demonstrates that beads are retained in the valves. A TNF¿ immunoassay was implemented on the chip using four concentrations. The resulting absorbance values shows a good correlation with those obtained with the same assay performed in a 96 well-plate (Figure 5). The presented platform main advantages are the absence of electrical and pneumatic connections and the easy scale-up, which will allow multiplexing in future designs. The current version of the instrument can control up to 64 valves with only 16 digital outputs (row-column addressing of the LED array). The characteristics of the presented platform are suitable for a wide range of applications requiring complex analytical protocols.