Substances were dissolved in deionized water (DIW) (18 M cm at 298 K), was employed to prepare sample solutions that mimic biological environments. To prepare the RM containing Fc, ferrocenyl methanol (FcMeOH) was employed. FeMeOH was very first dissolved in ethanol, then, the option was mixed into the RM. On the other hand, the mixture of potassium hexacyanoferrate(III) [K4 Fe(CN)six ] and potassium hexacyanoferrate(II) [K3 Fe(CN)six ] was also utilised to examine the redox response of the sensors. HRP, GluOx, and FcMeOH (95 ) had been purchased from Sigma ldrich Inc. Sodium HEPES (99 ) was purchased from Dojindo Laboratries. NaCl, KCl, MgCl2 , H2 O2 (30.0 ), ethanol (99 ), K4 Fe(CN)six H2 O (99.five ), and K3 Fe(CN)six (99.0 ) were purchased from Wako Pure Chemical Industries, Ltd.Sensors 2021, 21,4 of2.4. Measurement Procedure Firstly, the output of your sensor devoid of the PIC membrane was measured applying the remedy of K3 Fe(CN)six and K4 Fe(CN)six with many mixture ratios to examine the redox response. Then, the response to H2 O2 was measured in the sensor with the enzymes immobilized and Fc as a AVE5688 supplier mediator. The schematic illustration of your measurement setup is shown in Figure 2. A 90 of your RM containing 500 FcMeOH was very first put on the array, then, 10 of sample solution containing H2 O2 and 500 FcMeOH was added dropwise. The measurements have been carried out for the H2 O2 concentration range of 10-8 0-4 M. Right after the measurements for each of the concentrations, the sample option was removed in the surface each time as well as the sensor surface was washed by the RM various times. The output distribution amongst the pixels and the concentration dependence with the output have been analyzed employing the output values at 300 s right after the addition of H2 O2 , at which we assumed the enzymatic reactions were sufficiently progressed. The sensing characteristics of Glu have been also measured with a comparable process to that for H2 O2 sensing. A reference electrode of Ag/AgCl with 3 M NaCl was utilised. Despite the fact that KCl inner resolution for Ag/AgCl reference electrode is advantageous with regards to ion mobility, the leakage of potassium ions from a high-density option might be dangerous taking into account the application to cell measurements. For that reason, three M NaCl was employed as inner solution within this study.Ref. electrode Ag/AgCl (3 M NaCl)Pipette Analyte solutionFeFeFe Charge E E E E E E E + + ++ + + + + +++Au/Ti Sensor chipFigure 2. Schematic illustration of setup for H2 O2 and Glu measurements.three. Benefits and Discussion three.1. Fabricated Device Optical microscopy pictures from the sensor chip (a) prior to and (b) right after Au deposition are shown in Figure 3. The bluish region shown in Figure 3a corresponds to the sensing area. In Figure 3b, it was observed that the Au layer was deposited in each with the sensing regions. As schematically depicted in Figure 1, the surface of your sensing area is lowered than the surrounding locations as a result of the passivation layer. The flux of Au vapor incident into the sensor surface was practically perpendicular to the sensing location, and hence, the thickness of an Au layer deposited on the sidewalls was negligible. As a result, the Au layer deposited around the pixels was separated from that on the neighboring pixels. The incident from the vapor might be regarded as practically perpendicular to the sensor surface when it comes to the deposition on the sidewall but not absolutely perpendicular, which brought on the so-called shadowing impact, as are going to be discussed later.Sensors 2021, 21,5 of(a) Before Au d.