1 PREPARATION OF BIOTIN-GLUTATHIONE COATED QUANTUM DOTS Libor Janů Supervisor: doc. Ing. René Kizek, PhD. Department of Chemistry and Biochemistry, Faculty of agronomy, Mendel University

2 Introduction Semiconductor nanocrystals (2-10 nm)Semiconductor nanocrystals (2-10 nm) Fluorescent labeles in a variety of biological investigations (biological active compounds, Hg 2+,…)Fluorescent labeles in a variety of biological investigations (biological active compounds, Hg 2+,…) Better spectroscopic properties and longer lifetime in comparison to classical molecular dyesBetter spectroscopic properties and longer lifetime in comparison to classical molecular dyes

3 Introduction Solution phase synthesis of quantum dotsSolution phase synthesis of quantum dots Usually CdTe or CdSe QDUsually CdTe or CdSe QD Low quantum yield and toxic properties of naked QDLow quantum yield and toxic properties of naked QD Modifications: TOPO (trioctylphosphine oxide) coating – biocompatible, better spectroscopic properties but hydrophobicModifications: TOPO (trioctylphosphine oxide) coating – biocompatible, better spectroscopic properties but hydrophobic Solubility improving: ligand exchange (mercaptoacetic acid or DHLA), silanization, amphiphilic copolymers, …Solubility improving: ligand exchange (mercaptoacetic acid or DHLA), silanization, amphiphilic copolymers, … Long reaction times, multistep process, high temperatures, inert atmosphere.Long reaction times, multistep process, high temperatures, inert atmosphere.

4 Introduction Fig.1: Quantum dots modifications. Source: Min Zhou, Indraneel Ghosh, Current Trends in Peptide Science, Quantum Dots and Peptides: A Bright Future Together, PeptideScience Volume 88 / Number 3, 327 Thiol chelation Silanization Ligand exchange Hydrophobic interaction Copolymer Phospholipids

5 Material and methods The aim of the work: 1)Biotinylated quantum dots 2)Glutathione as the coating agent providing solubility in water 3)One step synthesis in water Synthesis of biotin-glutathione Solid phase peptide synthesis on Pioneer Peptide SynthesiserSolid phase peptide synthesis on Pioneer Peptide Synthesiser Biotin-glutathione was synthesized using peptide bonding of the biotin carboxy group and amino group of the gamma-glutamic acidBiotin-glutathione was synthesized using peptide bonding of the biotin carboxy group and amino group of the gamma-glutamic acid Biotin + avidin-biomolecule QD GSH

6 Material and methods Synthesis of biotin-glutathione quantum dots Reaction CdCl 2 + Na 2 TeO 3 in the presence of the biotin-glutathioneReaction CdCl 2 + Na 2 TeO 3 in the presence of the biotin-glutathione Mercapto group of glutathione thiolates the quantum dot surfaceMercapto group of glutathione thiolates the quantum dot surface Carboxilic acid provides solubility in waterCarboxilic acid provides solubility in water Na 2 TeO 3 is air stable avoiding the need of inert atmosphere during synthesisNa 2 TeO 3 is air stable avoiding the need of inert atmosphere during synthesis Reaction conditions: 95°C, synthesis time 2,5 hourReaction conditions: 95°C, synthesis time 2,5 hourReaction: 4TeO BH 4 - → 4Te 2- +3BO H 2 O CdCl 2 + Te 2- + MPA → Cd - (MPA) x Te y +2Cl - Cd - MPA x Te y → CdTe Junling Duan et.al.: One-Pot Synthesis of Highly Luminescent CdTe Quantum Dots by Microwave Irradiation Reduction and Their Hg2+-Sensitive Properties. Nano Res (2009) 2: 61 68

7 Results Fig. 2: MALDI-TOF MS of biotin-glutathione. Peaks with the diferent mass are HCCA matrix and Na + adducts. Purity of crude product ≥ 80%, purity of pure peptide ≥ 98%. (Ryvolova et. al.:Biotin-modified glutathione as a functionalized coating for bioconjugation of CdTe-based quantum dots. Electrophoresis 2011, 32, 1619–1622) B-GSH B800 B-GSH m/z Intensity (a.u.) HCCA matrix [M-2H+Na]-

8 Results Fig.3: Electropherogram of crude quantum dot-biotin-glutathione. UV detection at 214 nm. (Ryvolova et. al.:Biotin-modified glutathione as a functionalized coating for bioconjugation of CdTe-based quantum dots. Electrophoresis 2011, 32, 1619– 1622) B-GSH GSH B-GSH – QD Absorbance 214 nm (AU) Migration Time (min)

9 Results Fig.4: Electropherogram of crude quantum dot-biotin-glutathione. LIF detection (488 nm/530 nm); inset: B-GHS-QDs under ambient light (left), quantum dot-biotin- glutathione under UV light (right). (Ryvolova et. al.:Biotin-modified glutathione as a functionalized coating for bioconjugation of CdTe-based quantum dots. Electrophoresis 2011, 32, 1619–1622) B-GSH – QD Fluorescence (a.u.) Migration Time (min)

10 Results Fig.5: Electropherogram of the mixture of the B-GSH-QDs and avidin solution; LIF detection (488 nm/530 nm). (Ryvolova et. al.:Biotin-modified glutathione as a functionalized coating for bioconjugation of CdTe-based quantum dots. Electrophoresis 2011, 32, 1619–1622) B-GSH – QD Avidin-B-GSH – QD

11 Conclusion Biotinylated quantum dots as a probe with specific afinity to avidin and streptavidinBiotinylated quantum dots as a probe with specific afinity to avidin and streptavidin No need the multistep and time consuming synthesis in organic solvents.No need the multistep and time consuming synthesis in organic solvents. Next synthesis efficiency enhancement possibilities: microvawe synthesis – homogenous heating of the sample, smaller surface defects, shorter reaction times.Next synthesis efficiency enhancement possibilities: microvawe synthesis – homogenous heating of the sample, smaller surface defects, shorter reaction times.

12 Acknowledgements doc. Ing. René Kizek, PhD. Ing. Markéta Ryvolová, PhD. Ing. Jana Chomoucká, PhD. Mgr. Natalia Cernei Ing. Pavlína Šobrová Mgr. Ondřej Zítka This study was supported from IGA AF Mendelu,IP 13/2011.

13 Thank you for your attention!