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Sensors 2020, 20, 1333 4 of 15 on the COC substrate for investigating the reachable effective surfaces and the influence on the performance of the 2D IPE array. The electrodes of the 2.5D array featured 6 Au layers. 2.3. Characterization of the Printing Process The measurement of the resistances of the printed conductive paths was carried out by four-wire measurement using a point probe station. The determination of cracks and the measurement of the structure dimensions were done using a microscope. The layer thickness and the cross-sectional area for the determination of the specific resistance were measured using a white light interferometer. The adhesion of the printed structures on the COC substrate was tested by a cross-cut test according to DIN EN ISO 2409:2013. Depending on the amount of the peeled off surface area, a cross cut value can be determined. A cross cut value of 0 means that no surface area at all was peeled off. If more than 65% of the tested surface area is peeled off, the cross cut value will be 5. With this test, the influences of the pretreatment by low-pressure oxygen plasma and the sintering temperature on the adhesion were investigated. Electrochemical measurements were performed with Interface 1010B potentiostats (Gamry Instruments, Warminster, PA, USA) in noise-reject mode and potentials were recorded vs. an external Ag/AgCl/KCl (sat.) electrode, if not stated otherwise. Cyclic voltammetry (CV) and square wave voltammetry (SWV) were recorded with 2 mV step size at scan-rates and frequencies as indicated in the text. SWV pulse size was set to 50 mV, which corresponds to the peak-to-peak amplitude of the square-wave for this software version (Gamry Framework v7.06). 2D arrays were contacted via a 30 pin card-edge connector delivered with the 8X220AT electrode arrays from DropSens. A custom-made PCB with spring contacts was used to contact the 2.5D array. The effective surface of the 2D IPE and SPE arrays was determined electrochemically in two ways: first, CVs were performed in deaerated 2 mM Ru(NH3)6Cl3 in 0.1 M KCl between 0.05 V and −0.45 V. The active surface area (ARuHex) was derived from the reduction peak of Ru(NH3)6Cl3 using Randles-Sevcik equation [23] assuming a diffusion coefficient of 8.43 × 10−6 cm2 s−1 [24]. Second, CVs were conducted with 200 mV/s in deaerated 0.5 M sulfuric acid between −0.2 V and 1.5 V. The reduction peak (between 1.15 V and 0.7 V) of chemisorbed oxygen was evaluated to determine the surface AOxide related to the number of gold atoms exposed to the solution assuming a charge density of 390 μC cm−2 for an oxygen monolayer [25]. Since the reaction of Ru(NH3)6Cl3 is a fast, diffusion-controlled process, the contribution of possible pores is negligible. ARuHex can thus be a good indicator for the footprint of the electrode, while Aoxide includes all the surface in contact with H2SO4 [26]. 2.4. Immobilization of DNA Capture Probes Before functionalization, the electrode arrays were rinsed with isopropanol and DI water. 2D electrode arrays were then cycled in 0.5 M sulfuric acid as described above, afterwards extensively rinsed with DI water and finally dried by flushing with nitrogen. The immobilization solution consisted of 20 μM thiolated capture probes, 1X Nexterion Spot buffer in DNase/RNase-free DI water. We included 20 μM MCH in the immobilization solution according to other reports [27,28]. The immobilization solution was spotted on the WE (3.0 μL and 0.5 μL for electrode diameters of 2.56 mm and 1 mm, respectively) and allowed to react for 2 h in a humid environment. Afterwards, the electrode arrays were rinsed with 1 mM MCH and then incubated in 1 mM MCH for 1 h. Finally, the arrays were washed for 15 min in 1 X SSC and 0.1% SDS at 45 ◦C, 5 min in 0.1 X SSC and 0.1% SDS at 45 ◦C, and 5 min in ddDI water at room-temperature. The sensors were dried by flushing with nitrogen and stored in nitrogen atmosphere until use. Capture probe surface coverage was quantified for 2D electrode arrays comparably to the method established by Steel et al. [29]: Chronocoulometry (stepped from 0.05 V to −0.45 V) was performed in deaerated Tris buffer (adapted to pH 7.4 with HCl) first without and then with 0.15 mM Ru(NH3)6Cl3. To remove the Ru(NH3)6Cl3 after the measurement, the electrode array was immersed for 5 min in 0.1 M sodium phosphate buffer and 1 min in ddDI water.PDF Image | Inkjet-Printing Nanoparticle Gold Silver Ink Cyclic Olefin
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