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PREPARATION OF BIOTIN-GLUTATHIONE PREPARATION OF BIOTIN-GLUTATHIONE COATED QUANTUM DOTSCOATED QUANTUM DOTS

Libor JanůLibor Janů

Supervisor: doc. Ing. René Kizek, PhD.Supervisor: doc. Ing. René Kizek, PhD.

Department of Chemistry and Biochemistry,Department of Chemistry and Biochemistry,Faculty of agronomy, Faculty of agronomy,

Mendel University Mendel University

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IntroductionIntroduction• Semiconductor nanocrystals (2-10 nm) Semiconductor nanocrystals (2-10 nm)

• FFluorescent labeles in a variety of biological investigationsluorescent labeles in a variety of biological investigations (biological active compounds, Hg(biological active compounds, Hg2+2+,…),…)

• Better spectroscopic properties and longer lifetime in Better spectroscopic properties and longer lifetime in comparison to classical molecular dyescomparison to classical molecular dyes

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IntroductionIntroduction• 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) Modifications: TOPO (trioctylphosphine oxide) coating – biocompatible, better spectroscopic coating – biocompatible, better spectroscopic properties but hydrophobicproperties but hydrophobic

• Solubility improving: ligand exchange (mercaptoacetic acid or DHLA), Solubility improving: ligand exchange (mercaptoacetic acid or DHLA), silanization, amphiphilic copolymers, … silanization, amphiphilic copolymers, …

• Long reaction times, multistep process, high temperatures, inert Long reaction times, multistep process, high temperatures, inert atmosphere.atmosphere.

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IntroductionIntroduction

Fig.1: Quantum dots modifications. Source: Min Zhou, Indraneel Ghosh, Current Trends in Peptide Science, 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, 327Quantum Dots and Peptides: A Bright Future Together, PeptideScience Volume 88 / Number 3, 327

Thiol chelation

Silanization

Ligand exchange

Hydrophobic interaction

Copolymer

Phospholipids

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Material and methodsMaterial and methodsThe aim of the work:The aim of the work:1)1) Biotinylated quantum dots Biotinylated quantum dots 2)2) Glutathione as the coating agent providing solubility in waterGlutathione as the coating agent providing solubility in water3)3) One step synthesis in waterOne step synthesis in water

Synthesis of biotin-glutathioneSynthesis of biotin-glutathione • Solid phase peptide synthesis on Pioneer Peptide Synthesiser Solid phase peptide synthesis on Pioneer Peptide Synthesiser • BBiotin-glutathione was synthesized using peptide bonding ofiotin-glutathione was synthesized using peptide bonding of the biotin the biotin

carboxy group and amino group of the carboxy group and amino group of the gammagamma-glutamic acid-glutamic acid

Biotin + avidin-biomoleculeQD

GSH

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Material and methodsMaterial and methodsSynthesis of biotin-glutathione quantum dotsSynthesis of biotin-glutathione quantum dots

• Reaction CdClReaction CdCl2 2 + Na+ Na22TeOTeO3 3 in the presence of the biotin-glutathionein 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• NaNa22TeOTeO3 3 is air stable avoiding the need of inert atmosphere during synthesisis 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 hour

Reaction:Reaction:

4TeO4TeO332-2- +3BH +3BH44

-- → 4Te → 4Te2-2- +3BO +3BO22-- +6H +6H22OO

CdClCdCl22 + Te + Te2-2- + MPA → Cd + MPA → Cd-- (MPA) (MPA)xxTeTeyy+2Cl+2Cl--

CdCd-- MPA MPAxxTeTeyy → CdTe → CdTe

Junling Duan et.al.: One-Pot Synthesis of Highly Junling Duan et.al.: One-Pot Synthesis of Highly Luminescent CdTe Quantum Dots by Microwave Luminescent CdTe Quantum Dots by Microwave Irradiation Reduction and Their Hg2+-Sensitive Irradiation Reduction and Their Hg2+-Sensitive Properties. Nano Res (2009) 2: 61 68Properties. Nano Res (2009) 2: 61 68

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Results

Fig. 2: MALDI-TOF MS of biotin-glutathione. Peaks with the diferent mass are HCCA matrix and Fig. 2: MALDI-TOF MS of biotin-glutathione. Peaks with the diferent mass are HCCA matrix and NaNa+ + adducts. Purity of crude product ≥ 80%, purity of pure peptide ≥ 98%. (Ryvolova et. 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 al.:Biotin-modified glutathione as a functionalized coating for bioconjugation of CdTe-based quantum dots. Electrophoresis 2011, 32, 1619–1622)quantum dots. Electrophoresis 2011, 32, 1619–1622)

532.185

0

200

400

600

Inte

ns. [a

.u.]

500 600 700 800 900 1000m/z

N

N

S

O

H

H

ONH

OH

O O

NH

SH

O

NH

OH

O

B-GSH

B800

B-GSH

m/z

Inte

nsi

ty (

a.u

.)

HCCA matrix

HCCA matrixHCCA matrix

[M-2H+Na]-

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Results

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

B-GSH

GSH

B-GSH – QDAb

sorb

ance

214

nm (

AU

)

Migration Time (min)

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Results

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

B-GSH – QDF

luor

esce

nce

(a.

u.)

Migration Time (min)

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Results

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

B-GSH – QD

Avidin-B-GSH – QD

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ConclusionConclusion• Biotinylated quantum dots as a probe with specific afinity Biotinylated quantum dots as a probe with specific afinity

to avidin and streptavidinto avidin and streptavidin

• No need the multistep and time consuming synthesis in No need the multistep and time consuming synthesis in organic solvents. organic solvents.

• Next synthesis efficiencyNext synthesis efficiencyenhancement possibilities: enhancement possibilities: microvawe synthesis – microvawe synthesis – homogenous heating of the sample,homogenous heating of the sample,smaller surface defects, smaller surface defects, shorter reaction timesshorter reaction times..

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AcknowledgementsAcknowledgements

• 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 fromIGA AF Mendelu,IP 13/2011.

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Thank you for your attention!Thank you for your attention!