Immobilization of nanoparticles (NPs) is a technique suitable for the preparation of large-scale substrates for surface-enhanced vibrational spectroscopy including micro- and nano-spectroscopic applications. The developed immobilization method provides the enhancing properties of the roughened substrate surface to be maintained for techniques like surface-enhanced Raman scattering (SERS) spectroscopy, however, at the same time the morphology is not limiting for related near-field (scanning probe) techniques. The study is focused on the comparison of different immobilization procedures of Ag nanoparticles and finding the relationship between preparation procedures leading to convenient surface morphology and the quality of the observed signal of the model analyte (riboflavin) using SERS. Amino-linker (3-aminopropyl)trimethoxysilane (APTMS) and four thio-linkers (cysteine, 3-mercaptopropanoic acid, 2-mercaptoethanol, and 2,2'-oxydiethanthiol) using five immobilization procedures at three different temperatures (23 °C, 40 °C, and 70 °C) were compared. Surface morphology was monitored by scanning electron microscopy and atomic force microscopy. The SERS spectra of riboflavin were evaluated in terms of the intensity and the resolution of individual bands. The spectral dataset was inspected by multivariate statistical methods - principal component analysis and discriminant analysis. The evaluation of spectra and statistical models show the influence of the used linker and AgNPs immobilization procedure on the spectral output. APTMS linker is less suitable; much more appropriate are thio-linkers deposited on an evaporated Au layer on a glass slide. The best spectral parameters were obtained for 2,2'-oxydiethanthiol and 23 °C.

Department of Analytical Chemistry Faculty of Chemical Engineering University of Chemistry and Technology Prague Technická 5 Praha 6 166 28 Czech Republic

Department of Low Dimensional Systems J Heyrovský Institute of Physical Chemistry Czech Academy of Sciences v v i Dolejškova 3 Prague 8 18223 Czech Republic; Department of Physical Chemistry Faculty of Chemical Engineering University of Chemistry and Technology Prague Technická 5 Praha 6 166 28 Czech Republic

Department of Physical Chemistry Faculty of Chemical Engineering University of Chemistry and Technology Prague Technická 5 Praha 6 166 28 Czech Republic

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