Nanogravimetric and Optical Characterizations of Thrombin Interaction with a Self-Assembled Thiolated Aptamer

Jane Politi,Principia Dardano,Fabrizia Nici,Ilaria Rea,Luca De Stefano,Monica Terracciano,Giorgia Oliviero,Nicola Borbone,Gennaro Piccialli

doi:10.1155/2016/3561863

Abstract

Efficient biorecognition of thrombin (TB), a serine protease with crucial role in physiological and pathological blood coagulation, is a hot topic in medical diagnostics. In this work, we investigate the ability of synthetic thrombin aptamer (TBA), immobilized on a gold substrate, to bind thrombin by two different label-free techniques: the quartz crystal microbalance (QCM) and the spectroscopic ellipsometry (SE). By QCM characterization in the range from 20 to 110 nM, we demonstrate high specificity of TBA-TB interaction and determine affinity constant (Kd) of17.7±0.3 nM, system sensitivity of0.42±0.03 Hz nM−1, and limit of detection (LOD) of240±20 pM. The interaction between TBA and TB is also investigated by SE, an all-optical method, by quantifying the thickness increase of the TBA film assembled on gold substrate. AFM characterization of TBA and TB molecules deposited on flat silicon surface is also supplied.

Highlights

Thrombin (TB) is a strong vasoconstrictor involved as main agent in vasospasm [1]
We investigate the ability of synthetic thrombin aptamer (TBA), immobilized on a gold substrate, to bind thrombin by two different label-free techniques: the quartz crystal microbalance (QCM) and the spectroscopic ellipsometry (SE)
We studied the interaction between TBA, once immobilized on a gold substrate, and TB by two different label-free methods: quartz crystal microbalance (QCM) and spectroscopic ellipsometry (SE)

Summary

Introduction

Thrombin (TB) is a strong vasoconstrictor involved as main agent in vasospasm [1]. Beyond its key role in the dynamic process of thrombus formation, TB has a pronounced proinflammatory character, which may influence the development and progression of atherosclerosis [2]. The SELEX process, a multisteps procedure, selects the oligonucleotide sequences, which will form the aptamer, on the basis of their specific binding affinity to a target analyte, chosen from a library of sequences. By this kind of approach, a large number of aptamers have been discovered for diagnostics, therapeutics, and other biotechnical applications [9]. The results of gravimetric analysis were completed by spectroscopic ellipsometry, a powerful optical technique to investigate optical properties (complex refractive index) and thickness of thin films Both analytical techniques demonstrated the ability of TBA to selectively bind TB, even if it is covalently bound on a modified gold surface

Experimental Section

Results and Discussion

Conclusions