It is well known that the engrailed-2 (EN2) protein, a biomarker for prostate cancer, strongly binds to a specific DNA sequence (5'-TAATTA-3') to regulate transcription. Based on this intrinsic property, DNA probes with additional flanked sequences were designed and optimized. Various measurements, such as electrophoresis mobility shift assay, surface plasmon resonance, and quantitative fluorescence assay were performed to investigate the feasibility of the DNA probes. Then, the affinities of the DNA probes to the target protein were quantitatively determined using FAM-modified DNA probes and magnetic beads, resulting in dissociation constants ranging from 61.03 to 98.84nM. To develop an early diagnosis platform for prostate cancer, an ultrasensitive electrochemical biosensor based on the electrodeposition of gold nanoparticles was designed. The EN2 protein was quantitatively detected using the electrochemical biosensor, and the calculated detection limit was found to be 5.62fM. Finally, the specificity and applicability of the biosensor were verified using several proteins and an artificial urine medium. The impedance signals increased in the cases of EN2, suggesting that the system exhibited high selectivity to only EN2.
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