2.2.1. Antibody Adsorption

The electrodes were cleaned by plasma treatment. A 0.05 μL drop of an anti-troponin I solution, diluted with phosphate buffered saline (PBS) to 5 μg/mL, was deposited on the working electrode. After 4 h of incubation at 6 ◦C and rinsing with PBS, the chip was assembled with the PDMS microfluidic channel (see Section 2.1.2).

#### 2.2.2. Application of Thiol-SAMs with Hydrocarbon Spacer, Antibody Immobilization

To test chemicals for use as SAM, the electrodes were cleaned by plasma treatment, and 10 μL of an ethanolic solution containing 50 mM 4-mercaptobenzoic acid, 1,4-benzenedithiol, or 6-mercapto-1-hexanol was deposited on both working and counter electrodes. After incubation overnight at ambient temperature, the electrodes were rinsed with ethanol and the chip assembled with the PDMS channel (see Section 2.1.2).

A schematic representation of the antibody immobilization via SAM with hydrocarbon spacer and subsequent assay is given in Figure S1 in Supplementary Material. The electrodes were cleaned by plasma treatment, and a 0.05 μL drop containing 20 mM 4- mercaptobenzoic acid dissolved in ethanol was deposited on the working electrode. After 24 h of incubation at 6 ◦C, the chip was rinsed with ethanol. Another plasma cleaning step of the counter electrode was performed, during which the working electrode was covered with a piece of polystyrene. A 10 μL drop containing 20 mM 1,4-benzenedithiol dissolved in ethanol was deposited on the counter electrode and incubated overnight at 6 ◦C. After rinsing with ethanol and drying, 10 μL of a freshly prepared aqueous solution containing 0.05 M N-hydroxysuccinimide (NHS) and 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was deposited on the electrodes and incubated for 30 min. The mixture was supposed to react only with the carboxyl groups of the 4-mercaptobenzoic acid SAM on the working electrode resulting in an active ester [24]. After rinsing with PBS, 0.05 μL of an anti-troponin I solution, diluted with PBS to 5 μg/mL, was deposited on the working electrode and incubated for 30 min. After rinsing with PBS, 0.05 μL of an aqueous solution of ethanolamine hydrochloride, 1 M, pH = 8.5, was deposited on the working electrode and incubated for 30 min to deactivate potentially available still reactive active ester groups [24]. Finally, the impedance biosensor chip was thoroughly rinsed with PBS, dried, and assembled with the PDMS microfluidic channel (see Section 2.1.2).

#### 2.2.3. Application of Thiol-SAMs with DNA Spacer, Antibody Immobilization

Figure S2 shows the sequences of the ssDNA used below, including the schematic arrangemen<sup>t</sup> in the sensing layer. A schematic representation of the antibody immobilization via SAM with DNA spacer is given in Figure S3. Electrodes were cleaned by plasma treatment. SH-ssDNA (5−thiol-C6-TTT TTT TTTTCC TGC GTC GTT TAA GGA AGT AC-3, purchased from Metabion, Planegg, Germany) was coimmobilized with thiol compounds with hydrocarbon spacer for stabilization of the DNA-based SAM. The immobilization mixture contained either 0.025 mM SH-ssDNA and 15 mM 6-mercapto-1-hexanol or 0.033 mM SH-ssDNA and 13.3 mM 1,4-benzenedithiol dissolved in ethanol. A 0.05 μL drop of the immobilization mixture was deposited on the working electrode and incubated overnight at 6 ◦C. After rinsing with ethanol, the counter electrode was again cleaned by plasma treatment, while the working electrode was covered with impermeable polystyrene. A 10 μL drop containing 20 mM 1,4-benzenedithiol dissolved in ethanol was deposited on the counter electrode and incubated overnight at 6 ◦C. After that, 0.05 μL amino-ssDNA (5- amino-C6-GTA CTT CCT TAA ACG ACG CAG G-3, purchased from Metabion, Planegg, Germany), which was diluted with phosphate buffer to a concentration of 0.1 mM, was deposited onto the working electrode and incubated overnight at 6 ◦C. To convert the amino groups to carboxyl groups for antibody coupling, glutaric anhydride was dissolved in 8 M sodium hydroxide solution at a concentration of 0.5 mg/μ<sup>L</sup> [25]; 10 μL of this solution was applied on the electrodes. The glutaric anhydride was supposed to react only with the amino groups of the functionalized working electrode. After 48 h of incubation at 6 ◦C, the chip was rinsed with bidistilled water. The following protocol of antibody coupling via EDC/NHS mixture and subsequent bonding of the PDMS channel was the same as described in the section before (Section 2.2.2).

#### *2.3. Measurements with the Microfluidic Impedance Biosensor Chip*
