Dual aptamer-immobilized surfaces for improved affinity through multiple target binding in potentiometric thrombin biosensing

• Published in: Biosensors & bioelectronics 2015-11, Vol.73, p.174-180
• Main Authors: Goda, Tatsuro, Higashi, Daiki, Matsumoto, Akira, Hoshi, Toru, Sawaguchi, Takashi, Miyahara, Yuji
• Format: Article
• Language: English
• Subjects: Aptamers, Nucleotide; Binding Sites; Biosensing Techniques - methods; Biosensing Techniques - statistics & numerical data; Chronocoulometry; Cyclic voltammetry; DNA aptamer; Field-effect transistors; Humans; Immobilized Nucleic Acids; Ligands; Limit of Detection; Models, Molecular; Potentiometry; Self-assembled monolayers; Surface Properties; Thrombin; Thrombin - analysis; Thrombin - chemistry
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2015.05.067

We developed a label-free and reagent-less potentiometric biosensor with improved affinity for thrombin. Two different oligomeric DNA aptamers that can recognize different epitopes in thrombin were introduced in parallel or serial manners on the sensing surface to capture the target via multiple contacts as found in many biological systems. The spacer and linker in the aptamer probes were optimized for exerting the best performance in molecular recognition. To gain the specificity of the sensor to the target, an antifouling molecule, sulfobeaine-3-undecanethiol (SB), was introduced on the sensor to form a self-assembled monolayer (SAM). Surface characterization revealed that the aptamer probe density was comparable to the distance of the two epitopes in thrombin, while the backfilling SB SAM was tightly aligned on the surface to resist nonspecific adsorption. The apparent binding parameters were obtained by thrombin sensing in potentiometry using the 1:1 Langmuir adsorption model, showing the improved dissociation constants (Kd) with the limit of detection of 5.5nM on the dual aptamer-immobilized surfaces compared with single aptamer-immobilized ones. A fine control of spacer and linker length in the aptamer ligand was essential to realize the multivalent binding of thrombin on the sensor surface. The findings reported herein are effective for improving the sensitivity of potentiometric biosensor in an affordable way towards detection of tiny amount of biomolecules. •Label-free reagent-less potentiometry was employed using FET biosensors.•Electrodes were modified with two DNA aptamers to induce multiple binding.•Binding affinity was improved by the cooperative binding with thrombin.•Spacer and linker lengths were critical for the best sensing performance.