3.1. Design and operational principle of the centrifugal microfluidic system
The centrifugal microfluidic disc designed in this work is composed of six identical sections to allow six simultaneous dilution processes. Each section mainly consists of two diluent chambers, a single dilution chamber, seven product chambers, and connecting channels between them, as shown in Figure 2(a). The diluent needs to be loaded to the two identical diluent chambers prior to the operation, and the user needs to apply only the target analyte solution to the dilution chamber to start the serial dilution process. After the diluent and analyte solutions are loaded through the green-colored inlet holes above the respective chambers, the inlet and outlet holes are all sealed using a pressure-sensitive adhesive tape. Each diluent chamber has three outlet channels at different levels, such that the diluent chamber is radially occupied by three equivalent volumes of the diluent. The six outlet channels were initially all closed by the ferrowax microvalves (DV1 ~ DV6, diluent valve) to isolate the dilution chamber from the diluent chambers. The main channel to elute the diluted sample from the dilution chamber to the product chamber has seven initially open ferrowax microvalves (EV1 ~ EV7, elution valve), which is disconnected to the seven final product chambers by the closed ferrowax microvalves (FV1 ~ FV7, final valve) located at the entry of the seven respective branch channels.
The process begins by eluting the original 1 X analyte solution by rotating the disc at 2,000 rpm after opening FV1. Then, predetermined volume of 1 X solution was eluted to the first product chamber, while the rest of the 1 X solution remained in the dilution chamber. For the first dilution, EV1 was closed to isolate the 1 X product chamber from the main channel, and DV1 was opened, followed by centrifugation at 2,000 rpm. Then, one third the initial volume of diluent was eluted to the dilution chamber. For the complete mixing of the analyte solution and the diluent, the disc was shaken by repeatedly rotating at 60 degrees clockwise and counterclockwise. After shaking, the first diluted solution was eluted to the second product chamber by opening the FV2 and rotating the disc at 2,000 rpm. The second and more consecutive dilutions can be accomplished by repeating the same dilution procedure but appropriately changing the microvalves to be opened and closed (Figure 2(b)).
As illustrated in Figure S4, the ferrowax was loaded in the groove of the top layer, which was then integrated with the bottom layer having discontinuous deep channel and the middle layer having the channel part peeled off. The ferrowax microvalves within the fabricated channels on the disc were initially all in an open state, but the DVs and FVs were made to be closed prior to the dilution process. The valve-closing process was automatically executed by irradiating the laser to the groove part to melt the ferrowax. Due to the capillary force, the melted ferrowax would be guided to the nearby shallow channel, where the ferrowax was solidified, consequently blocking the channel. Whenever needed, the closed channels were reopened by irradiating the laser to the shallow channel blocked by the ferrowax. The melted ferrowax was removed away by flowing in the same direction with the solution.