A pneumatic valve controlled microdevice for bioanalysis

Abstract

This paper describes a pneumatic valve controlled microdevice for performing mixing and reaction. This microdevice combined the degassed polydimethylsiloxane (PDMS) pumping method with a syringe-actuated valve system to control the dispensing and mixing of nanoliter solutions. The syringe was used to manually generate vacuum and to open the valves. Upon the opening of the valve, the microchamber was filled with the solution, which was driven by the external atmosphere through the degassed PDMS microchannel. With this microdevice, the enzymatic kinetics of alkaline phosphatase converting the fluorescein diphosphate was studied, and the Michaelis-Menten kinetics was analyzed. The microdevice has the advantages of simplicity and low cost in fabrication and operation.

Figure 1

(a) The microphotograph of three parallel dispensing and mixing units in the pneumatic-valve controlled microdevice. On the microwell layer, there are three types of microwells for enzyme, substrate, and buffer solutions, respectively. On the valve layer, there are one group of valves for mixing and two groups of dispensing valves: Valve group #1 for buffer dispensing and Valve group #2 for substrate solution dispensing; (b) The dispensing strategy by degassed PDMS and the valve. Upon opening the dispensing valves, the external atmosphere will drive the liquid reagents into the microchannel due to the internal vacuum in the microchamber generated by the degassed PDMS. The red dots represent gas molecules in air. (c) The valve-controlled mixing: (Top view) Upon opening the mixing valves, the external atmosphere will drive the liquid reagents in the enzyme microwells to mix with other liquid reagents over the boundaries between the microwells.

Figure 2

The microdispensing system with pneumatic valves is a multiplayer PDMS microdevice: (a) The configuration of the multiplayer PDMS microdevice. The microwell layer shows only the reaction area in the scheme. (b) The photograph of the syringe-controlled valve and the dispensing system.

Figure 3

Demonstration of the pneumatic valves controlled solutions dispensing. The device contains six reaction units, each of which has three microwells of different volume ratios for serial analysis. (a) and (b) Enzyme solution was aspirated to the microwells directly through the branch channels. At the same time, the dispensing valves for buffer were activated to dispense the buffer solution. (c) and (d) After the buffer solution was dispensed completely, the buffer solution in channel was removed (e) and (f) the third solution of substrate was dispensed by opening the substrate dispensing valves. For better illustration, dye molecules were added into the three solutions: [Fe(phen)₃]²⁺ for enzyme, [Fe(SCN)_x]^3−x for buffer and KMnO₄ for substrate solutions.

Figure 4

Demonstration of the pneumatic valves controlled mixing. After all the microwells were filled with the proper solutions, and there was no solution in the channels, the large mixing valves were opened to initiate the reaction. The solution turned dark red when Fe(NO₃)₃ mixed with KSCN.

Figure 5

Results of the FDP calibration experiment. The straight line is the linear fitting of the data points. The error in the data points ranged from 3% to ∼10% RSD. The inset shows the fluorescent images of the reaction units containing different FDP concentrations.

Figure 6

(a) Plot of the fluorescence signals against time in the 96-well microplate after alkaline phosphatase (0.1 U/ml) and FDP (1 μM) was mixed. (b) Lineweaver-Burk plot of the initial reaction velocity versus [FDP]⁻¹ with microdevice. The values of V_max, K_m, and k_cat were obtained by analyzing the plots, V_max = 28 nM s⁻¹, K_m = 2.9 μM, and k_cat = 156 s⁻¹.