Peptide–nucleic acid aptamer pair biosensor for disease biomarker detection in clinical samples

Abstract

Precise monitoring of biomarkers is of great significance for early warning, diagnosis and prognosis of diseases. Nevertheless, restricted by limited surface area and single kinetic responsiveness, sensitive and specific detection of targets using an interface-based biosensing method remains challenging. Here, a biolayer interferometry-based peptide–nucleic acid aptamer pair (BLI-PNAP) biosensing strategy is introduced for highly specific monitoring of a disease biomarker at a picomolar concentration. Based on the synergistic effect of PNAP, the biosensor achieved good responsiveness by increasing the molecular assembly density on the surface, and ultra-high specificity by molecular dynamics selectivity of paired receptors in target recognition, and significantly enhanced sensitivity by introducing gold nanoparticles to amplify the response signal. Using the BLI-PNAP biosensor, a wide linear window in the 0.01–1 nM range and high sensitivity for VEGF165 was demonstrated, with a detection limit of 6 pM. Molecular dynamics simulations were utilized to further explore the molecular mechanism and key driving force underlying the paired recognition of VEGF165 by the two aptamers. Moreover, the biosensor had excellent performance when applied to clinical serum samples. The developed biosensor enables rapid, automated, real-time online and high-throughput analysis of biomarkers and has great potential for point-of-care diagnosis of diseases.