25. 11.2009 ® Drno PREPARATION, PHYSICO-CHEMICAL PROPERTIES AND APLLICATION OF QUANTUM DOTS IN BIOANALYSES Ivona Svobodová Institute of Analytical Chemistry of the ASCR, v.v.i., Veveří 97, 602 00 Brno, Czech Republic svob odova@i ach. cz Semiconductor nanocrystals - quantum dots - semiconductor nano-scale inorganic crystals (1-10 nm) -core from elements of II. and VI. or III. and V. group -sufrace modified by polar organic molecules to increase hydrophilicity mm* ■ti ■'■ ■' fvn Quantum dots - modification - different materials (ZnS; ZnSe; PbS; CdS; CdSe CdTe) - surface coated (CdSe/ZnS, polymery) - silanization - ligands (TOP/TOPO, oleic acid, dithiotreitol, thioglycol, 3-mercaptopropionic acid, 2-mercaptoethylamine) Quantum dots - preparation lststep: preparation of hydogentelluiide -4 NaBH4 + 2 Te + 7 H20 -► 2 NaHTe + Na2B407 + 14 H2 a) 6hoursO°C b) 30 minute 80 °C c) over night 2 - 8 °C 2nd step: quantum dots formation CdCI2 + NaHTe + MPA or MA + heating MPA 3-mercaptopropionic acid: HS-CH2-CH2-COOH MA 2-mercaptoethylamin: HS-CH2-CH2-NH2 Quantum dots - coating CdTe + CdCI2 + Na2S + MPA or MA + heating CdTe/CdS/ZnS CdTe/CdS + ZnCI2 + Na2S + MPA or MA + heating Quantum dots - preparation Dependence of QDs size on refluction time - different refluction times in second reaction step - increase of emmision wavelength and particle diameter with refluction time Refluxing 10 min lh 2.5 h 13.5 h 16.5 h 27 h 41h 44 h SZ. 498 517 536 652 667 701 737 750 Particle 1.99 2.67 2.86 4.02 4.17 4.80 4.87 5 25. 11.2009 Quantum dots - preparation Eychmuller, A.; Rogach, A. L. Chemistry and photophysics of thiol-stabilized ll-VI semiconductor r\ar\ocrysta\sPure and Applied Chemistry 2000, 72, 179-188. Quantum dots - structure : HRTEM image Symmetry space group F -43m a = 6.48 Á , : ■■■.■■-' ' 5 Í Quantum dots - fluorescence spectrum "*» «*, - wide excitation spectra ^ with maximum at 469 nm Bff**"*^ - narrow emission spectra with maximum at 600 nm - bandwidth 58 nm at half height Quantum dots - optical properties Excitation and emmision spectra of fluorescence Quantum dots 600 nm - excitation maximum 469 nm -emmision maximum 600 nm 1 ■ 1 A ■ 0,4 Á A AV V / \ S 0,5. f> v/ I 1 0,2 300 400 500 600 700 Fluorescein - excitation maximum 494 nm - emmision maximum 521 nm Quantum dots - optical properties fs 3 of U U Emission spectrum of QDs wfthMAonthesurface K 25. 11.2009 Quantum dots - optical properties Fluorescence lifetimes Quantum dots - optical properties High chemical- and photostability ri p*M w íl^ 5: ÍV, -í im; mc i- rtif.í ~ý" «U ťífWťrŕniľs—iŤlFi Quantum dots - conjugation A/, Aľ-caibonyldiimidazole (CDI): carboxylic group of MPA \ on the QDs surface catalyzers: CDI amino group from ovalbumin TOkranse formation of peptide bond V lírjgřSlílf ŤinWufl Irwin* *iou -V:r:í ■lFř-_:^i Quanti im dots - conjugation Sulfosuccinimidyl -4-(N-maleinimidom ethyl) -cyklohexane-1-carboxylate (Sulfo-SMCC): GíMranřn en řt -. T--'.. amino group of MA on the QDs surface catalyzers: Sulfo-SMCC thiol group from anti-ovalbu I | min 1 formation of thioether bond TÝM •J CAflfLÍJpífr ff 28 Oxidation of antibody glycan:' Quantum dots - conjugation carboxylic -^ group of MPA on the QDs surface modified by adipic acid dihydrazide m^X^** oxidation of antibody glycan by NalQ4 formation of etheric bond ' I** H« F Quantum dots - conjugation ^T~4 *^ "M @*^Y» Avidin-biotin interaction: carboxylic group of MPA on the QDs surface catalyzers: EDC (i-ethyi-3-(3- dimethylaminopropylbodiimide hydrochloride) amino group from avidin formation of peptide bond afinity interaction avidin-biotinylated antibody 25. 11.2009 Quantum dots - conjugates separation Checking of EDC/NHS conjugating process I aisled antibody addition ofnor-conjugated GDs Characterization of 3.5 nm CdTe QDs and their conjugates by CZE-LIF, uncoated capillary 12/20 cm, voltage 6 kV, injection 1 s, 100 mM TRIS/TAPS buffer pH = 8.3 Quantum dots - conjugates separation £? 250- löteiSÖ CdTe-WPI ^a ■e Quantum dots - conjugates separation Conjugation of antigen via N, AT-carbonyldiimidazole (CDI) labeled antigen ^^wrtWr»M..-.-^ Characterization of 3.5 nm CdTe QDs and their conjugates by CZE-LIF, uncoated capillary 15/20 cm, voltage 6 kV, injection 5 s, 100 mM TRIS/TAPS buffer pH = 8.3 Quantum dots - conjugates separation CE Competitive imunoassay ... UMail^i ^iai-uUuflJ * r" I imunoosifMc • A I-3 A -"■■■ y \j _"..■ j v > 1 i i Characterization of 3.5 nm CdTe QDs and their conjugates by CZE-LIF, uncoated capillary 15/20 cm, voltage 6kV, injection 5 s, 100 mM TRIS/TAPS buffer pH = 8.3 Quantum dots - conjugates separation - Magnetic particles conjugated with antibody via oxidation of antibody glycan - QDs or FITC conjugated with antigen via EDC/Sulfo-NHS QDs conjugate FITC conjugate ■^■v ■■ ^j ■ ■ Quantum dots - selective cell labeling B-lymphocyte Quantum dots were conjugated with antibody against CD3 protein, a membrane protein specific for T-lymphocytes while B-lymphocytes do not contain it. Antibody was also conjugated with FITC for control. T-lymphocyte 25. 11.2009 Quantum dots - application Vizualization of organelles in cells - analyte migration monitoring in cells, pathogenes detection nucleus-655 nm Golgi apparatus - 585 nm microtubuls -525 nm Hep-2 cells Mouse small intestine Quantum dots - application Cancer tissues labeling - imaging and localization of cancer tissues -■ - using conjugated QDs with specific cancer antibodies 5 * Quantum dots - application Solar cells Ecsá - Solar cells from CdSe nanorods (Prof. A.Paul Alivisatos, Lawrence Berkeley National Laboratory) - QDs absorbed broader spectrum of sun light - QDs allows deposit photosensitive layer on wide range of materials Acknowledgment Foret František, Klepárník Karel, Hezinová Věra, Lišková Marcela, Přikryl Jan - Institute of Analytical Chemistry AS CR HRTEM Dr. Mariana Klementová-Institute of Inorganic Chemistry AS CR Fluorescence and fluorescence lifetimes measurement Mgr. Petru Táborskému, Ph.D., doc. Janu Preislerovi, Ph.D. and doc. Přemysl Lubal, Ph.D. -Department of Chemistry, Faculty of Science, Masaryk University Financial support GAAV - KAN400310651 and KJB400310709 GACR-GA203/08/1680 MSMT - LC06023 AV0Z40310501