A new aptamer immobilization strategy for protein recognition

• Published in: Sensors and actuators. B, Chemical Chemical, 2017-11, Vol.252, p.222-231
• Main Authors: Guarisco, M., Gandolfi, D., Guider, R., Vanzetti, L., Bartali, R., Ghulinyan, M., Cretich, M., Chiari, M., Bettotti, P., Pavesi, L., Pederzolli, C., Pasquardini, L.
• Format: Article
• Language: English
• Subjects: Atomic force microscopy; Bioassays; Biomarkers; Biomarkers detection; Biomolecules; Biosensors; Buffer solutions; Buffers (chemistry); Coating; Contact angle; Copolymers; Crosslinking; Deoxyribonucleic acid; DNA; DNA-aptamers; Fluorescence; Gene sequencing; Immobilization; Molecular biology; Organic chemistry; Proteins; Recognition; Self-assembly; Silicon nitride; Substrates; Surface functionalization; Thrombin
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2017.05.133

[Display omitted] •An aptamer-based surface for specific protein capture was set up.•An easy and efficient surface coating allowed a higher aptamer mobility.•A high specificity even in complete human serum was obtained. The interest towards aptamer-based surfaces in the field of bioaffinity assays is constantly growing. A crucial aspect is related to the ability of maintaining the high specificity of these molecules once immobilized on the surface. In this article we compare the immobilization of aptamers on a silanized silicon nitride surface as well as on a soft polymeric layer. An innovative immobilization approach, based on a copolymer, N-dimethylacrylamide-N-acryloyloxysuccinimide-3-(trimethoxysilyl)propylmethacrylate (DMA-NAS-MAPS) coating, able to crosslink itself to the substrate and to bind amino-modified biomolecules is proposed. Comparing this coating with a more classical functionalization process based on silane chemistry, we propose that the better results obtained on the polymeric layer are due to an increased binding efficiency of the aptamers bound to a soft material. The high specificity of immobilized DNA-aptamers is demonstrated using two sequences specific for thrombin detection and a non-sense sequence as negative control. The coating provides higher sensitivity compared to classical self-assembled silane coatings, probably due to a better mobility of bound aptamers. The aptamer immobilization on both surfaces was characterized and optimized using atomic force microscopy, X-ray photoelectron spectroscopy, contact angle and fluorescence microscopy. The comparison between the two different functionalizations highlights the better performances of the copolymer coating in terms of protein recognition, demonstrating thrombin detection down to 0.011nM in buffer solution and 4.9nM in complete human serum. Moreover, the localized immobilization of the aptameric sequences, utilized in this work, suggests the possibility of employing this platform also for multianalyte detection.