2.1. Design and fabrication of the centrifugal microfluidic disc
The serial dilution disc was designed using computer-aided design software (AutoCAD, Autodesk, Inc., Sausalito, CA, USA). The disc was fabricated by assembling three separate layers: a 5 mm polycarbonate bottom plate (IDEL Corp., Chilgok, Korea), a 1 mm polycarbonate top plate (IDEL Corp., Chilgok, Korea), and a 0.1 mm middle layer of double-sided polyimide tape (930H, Coretech, Gimpo, Korea). Figure 1(a) shows an expanded view of this assembly, while its assembled form is shown in Figure 1(b). The top and bottom layers were milled by a computer numerical control machining center (HI-M1300, Hwacheon, Seoul, Korea). The bottom layer was further treated with a diluted chloroform solution to obtain a smooth and transparent milled-surface, and both the top and bottom layers were cleaned with 70% ethanol and distilled water (DW). The layers were additionally autoclaved when they were used to dilute the Escherichia Coli O157:H7 (ShigatoxigenicEscherichia coli (STEC)) samples. The bottom layer was mainly composed of six identical sections containing two diluent chambers, one dilution chamber, seven product chambers, and associated deep channels, while the top layer was designed to consist of the appropriately positioned ferrowax grooves, venting holes, inlet holes, and outlet holes. A double-sided polyimide tape for the middle layer was fabricated using a cutting plotter (Silhouette Cameo 3, Silhouette America® Inc., Orem, UT, USA). The middle layer was used to bind the top and bottom layers after the areas corresponding to the chambers and channels are peel off.
To facilitate the convenient and accurate assembly of the three layers, we employed 3D printed jig fabricated by 3D printing (3DWOX 2X, Sindoh Co., Ltd., Seoul, Korea) of a polylactic acid filament (Figure S1), which enabled the accurate alignment of the three layers having a perforated structure of the central axis and six alignment holes. Prior to the assembly, the ferrowax prepared by mixing the same volumes of paraffin wax (Tm = 50–52°C, Fluka Chemie GmbH, Buchs, Switzerland) and ferrofluid (APG 314, Ferrotec Inc., Santa Clara, CA, USA) was loaded onto the ferrowax grooves of the top layer. The assembled disc was finally laminated using a steel roller to eliminate any gaps or air bubbles between the tape and polycarbonate plates. A pressure-sensitive adhesive tape (HJ-Bioanalytik GmbH, Mönchengladbach, Germany) was used to seal the inlet and outlet holes during the dilution operation.