Palacký University in Olomouc Interaction of nanoparticles with amino acids and a physiologically important model protein studied by spectroscopic techniques Karolína M. Šišková, Dept. of Biophysics, CRH Faculty of Science Palacký University in Olomouc Czech Republic Advanced Materials & Processing, September 07-08, 2017, Edinburgh Scotland

Graphical abstract of this talk Ag NP W C H R E T P UV-Vis and SERS spectroscopies K F D M S Y Au NP Advanced Materials & Processing, September 07-08, 2017, Edinburgh Scotland

Ag and Au NPs Unique optical properties Ag NPs = Antimicrobial agents Scholl J.A. et al. Quantum plasmon resonances of individual metallic nanoparticles. Nature, 2012, 483, 421-427 Kahraman M. et al. Fundamentals and applications of SERS-based bioanalytical sensing. Nanophotonics, 2017, 6 (5), 831-852 Rai M. et al. Silver nanoparticles: The powerful nanoweapon against multidrug-resistant bacteria. J. Appl. Microbiol. 2012, 112, 841−852 Commercially exploited their toxicity and ecotoxicity need to be carefully evaluated Siskova K.M. et al. Revisiting spontaneous silver nanoparticles formation: a factor influencing the determination of minimum inhibitory concentration values? AIMS Environ. Sci. 2015, 2 (3), 607-622 Their main features: Localized surface plasmon - dependent on composition, size, shape, aggregation state Electric double-layer - changed by surface functionalization

Ag and Au NPs in deionized water 9.8 + 2.7 nm 9.5 + 2.9 nm UV-Vis spectra: surface plasmon extinction Here example of isolated spherical NPs prepared by borohydride reduction TEM images: size and shape of NPs

Ag NPs Electric bilayer (EB) enveloping each NP in aqueous solution; negative zeta potential values of NPs If EB destroyed (e.g. adsorption of AA molecules), aggregation proceeds – observable by naked eye:

Interaction of Ag nanoparticles with amino acids based on UV-Vis absorption measurements Type of NPs-AA system 0.01 mM AA 0.1 mM AA 1 mM AA Agbh-C - Agbh-M + Agbh-E Agbh-D Agbh-R Agbh-K Agbh-F Agbh-Y Agbh-H Agbh-W Agbh-P Agbh-S Agbh-T 9 from 13 AAs interact with Ag NPs! Functional groups of AAs play a key role; AA concentration is also important. Notes: final concentrations of AA are mentioned; + means aggregation observed, - no aggregation

Au NPs Aggregation observable by naked eye > Recorded by UV-Vis extinction

Interaction of Au nanoparticles with amino acids based on UV-Vis absorption measurements Type of NPs-AA system 0.01 mM AA 0.1 mM AA 1 mM AA Aubh-C - Aubh-M Aubh-E Aubh-D + Aubh-R Aubh-K Aubh-F Aubh-Y Aubh-H Aubh-W Aubh-P Aubh-S Aubh-T 2 from 13 AAs interact with Au NPs! Functional groups of AAs play a key role; AA concentration is also important. Notes: final concentrations of AA are mentioned; + means aggregation observed, - no aggregation

Surface-Enhanced Raman Scattering - principle Not in real scale = only scheme: l hn - light Adsorbate molecule Ag NP and/or Au NP Oscillating dipole Enhancement (G) of initial and Raman scattered light G ~ E2laser* E2Raman ~ E4

Example of phenylalanine (at pH 7): Raman scattering vs Example of phenylalanine (at pH 7): Raman scattering vs. SERS (using Agbh NPs) Region of Ag-O, Ag-S, Ag-N vibrations Fingerprint region Raman shift (cm-1)

Interaction of Agbh NPs with AAs (1mM) based on SERS type of NPs-AA system exc. 532 nm signal; AA final charge in solution at pH 7 at pH 9 at pH 10 at pH 11 Agbh-C no; 0 no; -I yes; -I yes; -II Agbh-M yes; 0 yes, but decrease; -I x Agbh-E yes (decrease after 3h); -I yes, but decrease; -II Agbh-D yes ; -I Agbh-R yes; +I yes, but decrease; 0 Agbh-K Agbh-F yes (increase after 3h); 0 Agbh-Y Agbh-H yes, but changes; -I Agbh-W Agbh-P yes (after 4h); -I no; -II Agbh-S yes (after 3h); 0 (yes); -I (yes), decrease; -I Agbh-T yes (after 2,5h); 0 Notes: "x" means not measured; "no"/"yes" if signal not observed and/or observed (immediately when no time mentioned or after several hours as indicated); "yes, but decrease" means the signal decreased at the particular pH in comparison to less basic pH; "(yes)" means that the signal was very weak and only three or less characteristic peaks were observed. C interacted with AgNPs after pH increase due to HS/-S transition Positively charged AAs (K, R) = more attractive for negatively charged AgNPs Neutral AAs with AgNPs = good SERS signals (except C) Negatively charged AAs (E, D, P) - the time of interaction matters.

Interaction of AgCitr NPs with AAs (1mM) based on SERS Type of NPs-AA system excitation laser line 532 nm AgCitr-C no AgCitr-M AgCitr-E AgCitr-D yes AgCitr-R AgCitr-K AgCitr-F AgCitr-Y AgCitr-H AgCitr-W AgCitr-P AgCitr-s Agcitr-T Citrates take more space than borates + bound through carboxylic groups => NPs less reactive Positively charged AAs = more attractive for negatively charged AgNPs (citrates on surface) Neutral AAs with AgCitr = no SERS signals Notes: yes/no indicates if SERS signal observed or not Length of AA carbon chain matters: D vs. E

Conclusions Outlook Evidenced that AuNPs are less reactive than AgNPs. Species on the surface of NPs play a crutial role (citrates vs. borates). Zeta potential value of NPs vs. charge of amino acids in solution of the particular pH is an important factor. Ionic vs. covalent interaction between amino acids and NPs can be distinguished in SERS. Outlook SERS signal from different AuNPs at 633 nm excitation Influence of pH changes on SERS spectra of AuNPs SERS signal + Raman optical activity measurements from a physiologically important model protein, Na+/K+-ATPase Fluorescence spectra of NPs + amino acids (namely Y, F, W)

Thank you for your attention! Acknowledgement Dr. Eva Kocisova and Assoc. Prof. Peter Mojzes Dept. of Physics of Biomolecules, Institute of Mathematics and Physics, Charles University, Prague, Czech Republic Assoc. Prof. Martin Kubala, Dept. of Biophysics, CRH, Dr. Josef Kapitan, Dept. of Optics, Faculty of Science, Palacky University in Olomouc, Czech Republic Thank you for your attention!