**Microfluidic Impedance Biosensor Chips Using Sensing Layers Based on DNA-Based Self-Assembled Monolayers for Label-Free Detection of Proteins**

**Khaled Alsabbagh 1, Tim Hornung 1, Achim Voigt 1, Sahba Sadir 2, Taleieh Rajabi 1 and Kerstin Länge 1,\***


**Abstract:** A microfluidic chip for electrochemical impedance spectroscopy (EIS) is presented as bio-sensor for label-free detection of proteins by using the example of cardiac troponin I. Troponin I is one of the most specific diagnostic serum biomarkers for myocardial infarction. The microfluidic impedance biosensor chip presented here consists of a microscope glass slide serving as base plate, sputtered electrodes, and a polydimethylsiloxane (PDMS) microchannel. Electrode functionalization protocols were developed considering a possible charge transfer through the sensing layer, in addition to analyte-specific binding by corresponding antibodies and reduction of nonspecific protein adsorption to prevent false-positive signals. Reagents tested for self-assembled monolayers (SAMs) on gold electrodes included thiolated hydrocarbons and thiolated oligonucleotides, where SAMs based on the latter showed a better performance. The corresponding antibody was covalently coupled on the SAM using carbodiimide chemistry. Sampling and measurement took only a few minutes. Application of a human serum albumin (HSA) sample, 1000 ng/mL, led to negligible impedance changes, while application of a troponin I sample, 1 ng/mL, led to a significant shift in the Nyquist plot. The results are promising regarding specific detection of clinically relevant concentrations of biomarkers, such as cardiac markers, with the newly developed microfluidic impedance biosensor chip.

**Keywords:** biosensor; immunosensor; cardiac troponin I; single-strand DNA; electrochemical impedance spectroscopy; label-free; proteins; microfluidic chip; self-assembled monolayers
