Heterofunctional Solid-Binding Peptides for Nucleic Acid Sensing Towards the Development of Cancer Biosensors

Abstract

A key challenge in designing biosensors is achieving high detection sensitivity without compromising target specificity. Field-effect transistors based on 2D solids offer enhanced sensitivity due to their atomically thin characteristics. Still, such biosensors must possess several critical attributes to detect ultra-low target concentrations within a bodily fluid: 1) Probes must be securely immobilized onto the 2D-layer substrate; 2) Optimal molecular packing of the probe must be controlled for efficient target capture; 3) Non-specific adsorption of off-target molecules must be prevented; and 4) Non-covalent functionalization of the sensing surface is favored to avoid creating surface defects that could affect sensing properties. Achieving these attributes presents several major obstacles. In the present research, these challenges are addressed to set the foundation for further development towards a versatile cancer diagnostic device. An anti-fouling graphene-binding peptide (GrBP) was used to confer passivating properties onto the sensing surface which mitigates non-specific protein adsorption, while a heterofunctional GrBP chimera bearing a peptide nucleic acid linker was used (GrBP-PNA) to immobilize DNA probes onto the sensing surface. Sensor surface functionalization, followed by subsequent target capture, was confirmed using surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) analysis. Raman spectroscopy of the graphene FET device revealed a pristine surface ready functionalization in order to detect nucleic acid biomarker targets. The results provide a viable biosensing strategy for versatile nucleic acid sensing using a modular probe design. This modular approach and anti-fouling method can allow for the eventual goal of collecting miRNA expression profiles from complex biological liquids for clinical diagnosis and prognosis of various diseases.