3.2. Fabrication

Milling and Optical Transparency
The milling increased the surface roughness of the COC plates also increases the surface area. Inhibition of the amplification can be caused by large surface areas as the used chemicals have more surface to have interaction with [44]. The created surface roughness is visualized using a FEI Sirion high resolution scanning electron microscope (HR-SEM) (FEI Company, Hillsboro, OR, USA) and measured using a Bruker Icon Dimension AFM in tapping mode with Bruker Tespa-V2 cantilevers (Bruker Nano Surfaces, Santa Barbara, CA, USA) and Gwyddion 2.52 open source freeware [62]. The results are shown in Figure 8. The surface roughness of pristine COC had a RRMS of 3.5 nm. This increased two orders of magnitude after milling (RRMS of 310.1 nm). With the reported surface treatment [44,59] we were capable of decreasing the surface roughness to a value even lower than that of pristine COC and the lowest reported in literature (RRMS of 0.9 nm). For this grade of COC (TOPAS 6017) it worked the best to do four short exposures of 5 s, with N2 blow drying after each exposure, instead of one longer exposure, as is more common in other grades of COC [44,59]. The difference in duration for the cyclohexane vapor post-treatment can be explained by the different ratios of the copolymers present in each grade. As the grade number increase, the ratio changes towards more norbornene monomers and less linear ethene monomers. The norbornene is more apolar due to the bridged cyclic hydrocarbon present in its molecular structure and therefore, will dissolve faster in non-polar solvents, like cyclohexane (vapor).
Lowering the surface roughness also increased the optical transmittance fivefold. Transmittance measurements in the visible range are done using a Woollam M-2000UI ellipsometer (J.A. Woollam Co., Lincoln, NE, USA). The results can be seen in Figure 9 and Figure 10. Having a high optical transparency in the visible range can be desired when in situ fluorescence detection will be implemented (e.g., EvaGreen fluorescence dye has an excitation wavelength of 500 nm and emission wavelength of 525 nm [63]). However, as in situ fluorescence detection is not used yet in this system and can also be done through the transparent PCR plate sealing foil, no further effort is put into optimizing this procedure to get even better optical transmittance.