SYNTHESIS AND MODIFICATION OF QUANTUM DOTS FOR MEDICAL APPLICATIONS

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1 SYTESIS AD MDIFICATI F QUATUM DTS FR MEDICAL APPLICATIS Jana CMUCKA a, Marketa RYVLVA b, Jana DRBLAVVA a, Libor JAU b, Vojtech ADAM b, Jan PRASEK a, Rene KIZEK b and Jaromír UBALEK a a Brno University of Technology, Department of Microelectronics Technicka 3058/10, Brno, Czech Republic, chomoucka@feec.vutbr.cz b Mendel University in Brno, Department of Chemistry and Biochemistry, Zemedelska 1, Brno, Czech Republic Abstract There is a growing interest in using semiconductor quantum dots (QDs) as optical labels for biosensing events. The size-controlled fluorescence properties of QDs, the high fluorescence quantum yields of QDs and their stability against photobleaching makes QDs superior optical labels for multiplex analysis. In this paper, biotin-conjugated glutathione (B-GS) was synthesized using peptide bonding of the biotin carboxy group and amino group of the γ-glutamic acid to create an alternative coating for CdTe QDs. This type of coating combines the functionality of biotin with the fluorescent properties of quantum dots to create a specific, high affinity fluorescent probe able to react with avidin, streptavidin and/or neutravidin. Biotinfuncionalized glutathione-coated QDs were prepared by a facile one step reduction route using a 2 Te 3 as the Te source in an aqueous environment. The synthesis parameters of this simple and rapid approach, including the reaction temperature and time, the p of the reaction solution and the molar ratio of the GS stabilizer to Cd 2+, have considerable influence on particle sizes and photoluminescence quantum yield of the CdTe QDs. btained QDs were separated from the excess of the B-GS by capillary electrophoresis employing 300 mm borate buffer with p 7.8 as a background electrolyte. The detection of sample components was performed by the photometric detection at 214 nm and laser-induced fluorescence employing Ar + ion laser (488 nm). Keywords: quantum dots, 1. ITRDUCTI Quantum dots (QDs) are nanometer-sized crystals made of metallic or mostly of semiconductor materials. QDs nano-particles generally fall within 2 10 nm size range [1, 2] and possess tunable optical and electrical properties [3]. QDs were found as an alternative to organic dyes and fluorescent proteins and thus they can be used for various biosensing purposes. The photo-physical properties which make QDs interesting compared to classic organic dyes are: broad absorption spectra, very narrow emission spectra, long fluorescence lifetime and high stability against photobleaching [4, 5]. QDs have also high quantum yield, high molar extinction coefficients [6] and large effective Stokes shift [7]. QDs always emit the same wavelength of light no matter what excitation wavelength is used [8]. Therefore, multiple QDs with different emission spectra can be simultaneously visualized using a single excitation light source. Since the emission spectrum of each QDs is narrow, the fluorescence signal of each QD can be readily separated and individually analyzed based on the emission spectrum in order to achieve multiplexed imaging [7]. The dimension of the core determines the bandgap and hence the colour of emission. It is known, that an increase in particle sizes produces a redshift in the emission spectrum [9] (see Fig. 1.). In principle, the emission of QDs can be coarse-tuned by the choice of the material and later fine-tuned by playing with the size of the core.

2 Increasing QDs size Fig. 1 An example of QDs sorted by size emitting light of different colors excited simultaneously by a single excitation wavelength. 2. QUATUM DTS PREPARATI Up to now, there are a number of methods for the preparation of QDs through colloidal chemistry (such as sonochemical method, microwave irradiation method or organometallic precursor method) [10] and/or nano nanoscale patterning [11], i.e. employing lithography-based technology. Chemical synthesis of QDs represents a typical approach, which is generally divided into water phase and organic phase approaches. Compared with organic approach, aqueous synthesis is effective, less toxic and more reproducible method [12]. Furthermore, the products often show improved water stability and biological compatibility. 2.1 The organic phase method The organometallic way produces QDs, which are generally capped with hydrophobic ligands (e.g. trioctylphosphine oxide TP or trioctylphosphine - TP) and hence cannot be directly employed in bioapplications. To be used in biological applications, QDs need to be soluble in aqueous solutions and require surface modifications to achieve biocompatibility and stability [13]. Coordinating solvents stabilize the bulk semiconductors and prevent aggregation as the QDs grow. The semiconductor core material must be protected from degradation and oxidation to optimize QDs performance. Shell growth and surface modification enhance stability and increase photoluminescence of the core. The inorganic core-shell semiconductor nanoparticles are soluble in nonpolar solvents only. owever, a further process is needed for the QDs used in biological system. Therefore numerous effective methods have been developed for creating of hydrophilic QDs, which can be divided into two main categories. The first route is commonly designated as cap exchange. The hydrophobic layer of organic solvent can be replaced with bifunctional molecules containing a soft acidic group (usually a thiol, e.g. sodium thiolycolate) and hydrophilic groups (e.g. carboxylic or amino groups) which point outwards from the QDs surfaces to bulk water molecules [14, 15]. In fact, substitution of monothiols by polythiols or phosphines usually improves stability. The second route is native surface modification, for example adding a silica shell to the nanoparticles using a silica precursor during the polycondensation [16]. Amorphous silica shells can be further functionalized with other molecules or polymers. The method of QDs encapsulation into solid lipid nanoparticles, which are composed of high biocompatible lipids with physical and chemical long-term stability, was also successfully tested. These lipid nanoparticles are more convenient than small molecules (e.g. mercaptopropionic acid) traditionally used for QDs surface modification, which are rather unstable since they can be easily degraded by hydrolysis or oxidation of the capping ligand. 2.2 The water phase method The second way is the aqueous synthesis route, producing QDs with excellent water solubility, biological compatibility, and stability. ne of the most widespread approaches to creating water-soluble QDs is ligand

3 exchange with thioalkyl acids such as mercaptoacetic acid, mercaptopropionic acid, mercaptoundecanoic acid or reduced glutathione (GS). These QDs have lower quantum yields than the above mentioned ones (up to 10%) without any following treatment. From these ligands, GS seems to be very perspective molecule, since provides an additional functionality to the QDs due to its key function in detoxification of heavy metals in organisms. The fluorescence is considerably quenched at the presence of heavy metals and therefore GS-QDs were successfully employed for determination of heavy metals. In addition, GS-QDs exhibit high sensitivity to 2 2 produced from the glucose oxidase catalyzing oxidation of glucose and therefore glucose can be sensitively detected by the quenching of the GS-QDs florescence [17]. 3. QUATUM DTS APPLICATIS Concerning the QDs biological applications, two main groups may be cited: biosensors [18] and labels in biological imaging [19]. A few examples of each group can be seen on the schema below (Fig. 2.). Fig. 2 An example of QDs bioanalytical and biomedical application. For in vivo biological imaging applications of QDs, the fluorescent emission wavelength ideally should be in a region of the spectrum where blood and tissue absorb minimally but still detectable by the instruments. Thus the QDs should emit at approximately nm in the IR region to minimize the problems of indigenous fluorescence of tissues. Moreover, the spectroscopic properties of IR QDs would allow imaging deeper penetration than conventional near-infrared dyes [20]. Beside the application as simple sensors, QDs have much higher impact as unique fluorescent labels. Various specific labeling strategies are known and most of these approaches are based on bioconjugation with other biomolecule exhibiting some specific affinity to the target compound. Summary of these approaches was recently presented in a review article published by Algar et al. [21]. ne of these strategies utilizes the biotin-avidin (respectively streptavidin and neutravidin) interaction exhibiting very high specificity. Modification of QDs by the streptavidin proved to be a very successful method evaluated in various publications [22-24] and due to this success streptavidin-qds are nowadays also commercially available. Also biotin-functionalized QDs were developed to exploit the same interaction [25]. owever, so called multicolor QDs, which means particles modified by several different molecules, are now of great interest. Therefore the aim of this study was to prepare QDs based on the good properties due to presence of GS and also with the possibility to be employed in modern biotechnological biotin-avidin (or its homologues) applications [26]. 4. EXPERIMETAL 4.1 Preparation of biotin- modified glutathione (B-GS) Biotin and GS were conjugated via standard peptide bond using carboxy group of the biotin and amino

4 Intensity (a.u.) Intens. [a.u.] , Brno, Czech Republic, EU group of the γ-glutamic acid. The biotinylation at the -end of the tripeptide was the last step of the peptide synthesis. Final product was analyzed by mass spectrometry. Matrix-Assisted Laser Desorption/Ionization- Time of Flight Mass Spectrometry (MALDI-TF MS) was carried out using an Ultraflex III instrument (Bruker Daltonik, Germany). 4.2 Synthesis og glutathione coated CdTe QDs The synthesis of CdTe QDs and their subsequent coating was adapted from the work of Duan et. al. [27]. 1mL of the CdCl 2 solution (c = 0.04 mol/l) was diluted with 10.5 ml of water. During constant stirring, 25 mg of sodium citrate, 1mL of a 2 Te 3 solution (c = 0.01 mol/l), 70 mg of B-GS and 20 mg of ab 4 were added into water-cadmium(ii) solution. The mixture was kept at 95 C under the reflux cooling for 2.5 hours. 4.3 Capillary electrophoresis Synthesized B-GS-QDs were analyzed by capillary electrophoresis (Beckman Coulter, PACE 5500) with absorbance detection at 214 nm and with the laser-induced fluorescence detection (Ar +, ex nm/ em nm). Separation of the excess of B-GS and GS was carried out using uncoated fused silica capillary with 50 m internal diameter and 375 m b outer diameter. Total length was 47 cm and the effective length was 40 cm. Borate buffer (300 mmol/l, p 7.8) was used as a background electrolyte. 5. RESULT AD DISCUSSI Biotin and GS were conjugated via standard peptide bond using carboxy group of the biotin and amino group of the γ-glutamic acid [28]. The biotinylation at the -end of the tripeptide was the last step of the peptide synthesis. Purification of the product was carried out using high performance liquid chromatography and the purity of 99 % was reached. Final product (for structure see Fig. 3.) was analyzed by mass spectrometry. The major peak in the spectrum has the molecular mass of , which is in good agreement with the theoretical molecular mass of Da calculated for the B-GS. B B-GS S B-GS 400 S 200 CCA matrix [M-2+a]- CCA matrix CCA matrix m/z m/z Fig. 3 MALDI-TF MS spectrum obtained for B-GS and structure of the B-GS. Then, B-GS was used as an alternative coating for CdTe based QDs. Sodium telluride was used as the Te source. Due to the fact that sodium telluride is air stable, all of the operations were performed in the air avoiding the need for inert atmosphere. The synthesis pathway is thus free of complicated vacuum manipulations and environmentally friendly. The prepared GS-CdTe QDs emit at 504 nm and their emission spectrum showed quite symmetric and narrow shape.

5 fluorescence (a.u.) absorbance 214 (a.u.) , Brno, Czech Republic, EU The typical electropherogram of the GS CdTe QDs solution is shown in the Fig. 4. The identification GS signal was done by the standard addition method and identification of the GS-QDs signal was done by CE- LIF. 0,4 0,4 B-GS LIF (488 nm/530 nm) 214 nm 0,040 0,035 0,3 B-GS - QDs 0,030 0,3 0,2 0,025 0,020 0,2 0,1 0,1 GS 0,015 0,010 0,005 0,0-0,1 0, migration time, min -0,005 Fig. 4 Electropherogram of the mixture of the B-GS-QDs and excess B-GS and GS, UV detection at 214 nm, LIF detection (488 nm/530 nm). 6. CCLUSI It follows from the results obtained that biotinylated glutathione is suitable coating for the single step synthesis of thiol stabilized CdTe QDs. btained QDs are of good properties for the fluorimetric detection with the excitation by Ar + laser at the wavelength of 488 nm and emission of 530 nm. Moreover, we show that capillary electrophoresis is an efficient method for separation of the GS and B-GS excess from the B- GS-QDs and for stability control. ACKWLEDGEMETS The financial support from the grant anobiotecell GA CR P102/11/1068 and ASEMED GA AV GA KA is highly acknowledged. LITERATURE [1.] FERACVA, A. and LABUDA, J. (2008), DA Biosensors Based on anostrucutred Materials, Wiley-VC, Weinheim, Germany. [2.] KRAL, V., et al., anomedicine - Current status and perspectives: A big potential or just a catchword?, Chemicke Listy, 2006, 100, 1, 4-9. [3.] WALLIG, M.A., et al., Quantum Dots for Live Cell and In Vivo Imaging, Int. J. Mol. Sci., 2009, 10, 2, [4.] GALIA, R.E. and DE LA GUARDIA, M., The use of quantum dots in organic chemistry, Trac-Trends in Analytical Chemistry, 2009, 28, 3, [5.] LI,.C., et al., Progress in the toxicological researches for quantum dots, Science in China Series B-Chemistry, 2008, 51, 5, [6.] XIG, Y., et al., Semiconductor Quantum Dots for Biosensing and In Vivo Imaging, Ieee Transactions on anobioscience, 2009, 8, 1, [7.] CAI, W.B., et al., Are quantum dots ready for in vivo imaging in human subjects?, anoscale Research Letters, 2007, 2, 6,

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