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Label-free detection of biospecific interactions in peptide arrays using core-shell nanoparticle films

Güvenc, Haci Osman

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A novel nanostructured material with mutually coupled optical and biological functionalities was developed to facilitate the label-free read-out of biospecific binding events in high-density peptide arrays. The nanostructured material consists of a monolayer of dielectric nanoparticle cores deposited on a planar substrate and coated with a metal shell. Upon reflection of white light, these core-shell nanoparticle films exhibit pronounced plasmonic extinction peaks in a wide wavelength regime. Upon molecule adsorption the peaks shift to longer wavelengths due to the change in the refractive index close to the surface, thus, providing a label-free detection mechanism. The optical properties of the biosensor surfaces were analyzed with three different instrumental set-ups; (i) a standard UV-Vis reflection set-up, (ii) a LSPR imaging set-up based on a scanning unit and (iii) a homemade CCD-based fast read-out system for simultaneous analysis of extended surface areas. The UV-Vis reflection set-up was used to evaluate the performance and sensitivity of the proposed and prepared biosensor surfaces by nonspecific adsorption of proteins whereas the others were used to detect biomolecular reactions in an array format. In particular, biospecific interactions in high density peptide arrays were investigated. To optimize the wavelength shift induced by protein adsorption, various features were changed in the biosensor configuration, and the impact of these parameters on biosensor performance was tested. Metal shell thickness and roughness, the layer structure of the underlying substrate and the metal shell material (Au or Ag) were found to have an impact on biosensor performance. The most significant improvement, however, was obtained when operating biosensors with rough metal shells, prepared by seeding and consecutive electroless plating, at long wavelength plasmonic resonances. Here, an approximately five-fold increase in sensitivity towards protein adsorption could be achieved with respect to state-of-the art core-shell nanoparticle sensors. Also, the use of densely-packed monolayer films prepared by a so-called floating technique proved to be advantageous in the analysis of high-density arrays compared to films generated by statistical adsorption of nanoparticles. The optical homogeneity of the core-shell nanoparticle film was found to be another crucial parameter in label-free detection of specific interactions in high density peptide arrays. Core-shell nanoparticle films with improved optical homogeneity were obtained by changing the shell preparation technique from seeding and consecutive electroless metal plating to sputter coating. In collaboration with the Cancer Research Center (DKFZ) Heidelberg high density peptide arrays were transferred to the core-shell nanoparticle film by cleavage from a synthesis slide preserving spot size and lateral distances. Both the CCD-based fast read-out system and the scanning unit were used to detect protein/peptide interactions in these arrays and yielded consistent results in terms of wavelength shift. The antibody-stained peptide arrays were estimated to contain slightly more than 1 ng/mm2 of protein which resulted in 3.6 nm wavelength shift. In future experiments, the use of biosensors with seeded and plated metal shells, operated at long wavelength plasmonic resonances, should provide even higher sensitivity in array analysis. Core-shell nanoparticle films were also used to enhance the intensity of weak Raman signals of molecules, in this case methylene blue (MB) and fibrinogen via electromagnetic and chemical amplification mechanisms due to their strong surface plasmon resonance (SPR) response in Surface Enhanced Raman Scattering (SERS).

Item Type: Dissertation
Supervisor: Dahint, Prof. (apl.) Dr. Reiner
Place of Publication: Heildeberg
Date of thesis defense: 26 March 2013
Date Deposited: 03 Apr 2013 12:54
Date: 2013
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Controlled Keywords: Label-free Detection, Core-shell nanoparticles, peptide arrays
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