Chitosan Clad Manganese Doped Zinc Suiphide Nanoparticles as Biological Labels

Proceedings of the 2nd IEEE International Conference on Nano/Micro Engineered and Molecular Systems January 16 - 19, 2007, Bangkok, Thailand

Chitosan Clad Manganese Doped Zinc Suiphide Nanoparticles as Biological Labels Hemant C. Warad 1, Chanchana Thanachayanont 2, Gamolwan Tumcharern 3, and Joydeep Dutta '^ lCentre of Excellence in Nanotechnology, School of Engineering and Technology, Asian Institute of Technology, Thailand. 2 National Metal and Materials Technology Center, Thailand. 3 National Nanotechnology Center, Thailand

(2 acetamido-2-deoxy b-1, 4-D-glucan) commonly known as chitin, which is found in a wide range of natural sources (crustaceans, fungi, insects, annelids, molluscs etc.). It is insoluble in alkaline, strong acids and in neutral pH solutions due to its rigid crystalline structure and intra and inter molecular hydrogen bonding network [10], but is highly soluble in dilute acids. The dissolution of chitosan in dilute acidic solution, results in the protonation of amine groups (-NH2) with the formation of positively charged NH3, imparting a polycationic nature to the polymer, that has been utilized for polyelectrolyte applications [11]. The amine groups of chitosan are the major effective binding sites for metal ions forming stable complexes by co-ordination, which is of interest when used as capping agent for nanoparticles [12, 13]. This property makes chitosan to be used as a flocculent, gasselective membrane, plant disease resistance promoter, anticancer agent, wound healing promoting agent, and antimicrobial agent [14 - 20]. Long chain chitosan with its large number of cationic amine groups form multiple binding sites with metal particle surfaces. This effectively encapsulates the particle, providing steric hindrance from agglomeration of the particles in a colloidal system. This inherent property of chitosan makes it an interesting polymer to be used for capping nanoparticles to avoid agglomeration. The unattached amines can allow the possibility of being functionalized for different targeting purposes. Cytotoxicity of chitosan coated ZnS nanoparticles has also been studied and will be reported. We will discuss the synthesis of ZnS:Mn2+ nanophosphors and discuss about the photoluminescence properties of the QDs. We will also present the preliminary studies on the attachment of ZnS nanoparticles to fibroblast cells and to anionic cell walls of Bacillus cereous.

Abstract- Here we report the synthesis in aqueous media of redispersible zinc sulphide quantum dots doped with manganese, capped by biocompatible 'chitosan', molecules. The nanoparticles show highly efficient luminescence with a peak at around 590 nm that has been correlated to the manganese dopants. The synthesis involves very simple precipitation techniques that may lead to the development of a cost effective alternative of using nanocrystals to replace fluorophores for biological staining. The biocompatible capping agent, chitosan, renders the particles suitable for bio-labels (markers). The 40 nm sized spherical nanoparticles consists of multiple crystallites of about 4 - 5 nm as observed in High Resolution Transmission Electron Microscopy studies.

Keywords-Colloid, Fluorescence, Marker, Quantum Dot, Zinc Compound

I.

INTRODUCTION

Biomolecules labeled with luminescent colloidal semiconductor quantum dots have potential applications in fluoro-immunoassays and biological imaging [1, 2]. Quantum dots (QDs) have several advantages over conventional organic fluorophores as they are more efficient in luminescence compared to organic dyes and their emission spectra are narrow, symmetric and tunable according to the particle sizes and material composition which show excellent photostability. Due to their broad absorption spectra they can be excited to all colors of the QDs by a single excitation light source [3]. Also the excitation and emission peaks are easily distinguishable as excitation can be done in any wavelength in the broad absorption band [4]. Due to inherent advantages of QDs over organic dyes it could allow the integration of nanotechnology and biotechnology leading to major advances in medical diagnostics, molecular biology and cell biology [5, 6]. The utility of QDs to sense biological surfaces depend critically on QDs surface properties and surface functionalization. For most biological applications, biocompatible polymers like Polyvinvy Alcohol or proteins like lysine, streptavidin, biotin etc. have been used [7, 8]. Proteins are expensive but offer attractive possibilities for biological applications. In this work we have used chitosan for steric stabilization of the synthesized colloids that ineffect also form the biocompatible coating around ZnS nanoparticles. Chitosan is an inexpensive, biocompatible, biodegradable biopolymer that has free amine groups which serve as functional elements for biological applications [9]. Chitosan is a partially deacetylated polymer of acetyl glucosamine

II. EXPERIMENTAL SECTION The inorganic wet chemical synthesis used to prepare ZnS:Mn2+ nanocrystals passivated with chitosan is performed at room temperature and under ambient conditions. Medium molecular weight chitosan with a 75 - 85 % degree deacetylation were obtained from Sigma-Aldrich. Zinc acetate, manganese acetate and sodium sulphide were purchased from Univar, Fluka Chemika and Panreac respectively. Dilute acetic acid used to dissolve chitosan polymer was prepared from glacial acetic acid obtained from Sigma-Aldrich. Stock solutions of 0.25 M zinc acetate and 0.25 M sodium sulphide were prepared by dissolving 2.74 g and 3.00 g of respective salts in 50 ml of de-ionized water. 5 mM stock

*Contact author phone: +66-2-524-5680; fax: +66-2-524-5697; e-mail:

joygait.ac.th

1-4244-0610-2/07/$20.00 C)2007 IEEE

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concentration of Zn2+ ions), replace some of the Zn2+ in the ZnS lattice thereby doping the ZnS which gives ZnS its characteristic Orange red luminescence (590 nm). Nanoparticles have a strong tendency to agglomerate and settle down due to their Van der Waal's interactions. To avoid agglomeration of nanoparticles a repulsive force must be added between the particles to balance the attraction process. Chitosan exhibits polycationic structure, inducing steric hindrances to show net positive charge due to the presence of the amine groups. The key idea behind this work is, capping the surface of the ZnS nanoparticles with chitosan so that the net unattached positive amine groups will decorate the nanoparticles surface, serving to functionalize them.

solution of manganese acetate was prepared by dissolving 61.27 mg of the salt in 50 ml of de-ionized water. Stock solution of 0.1 0% polymer was prepared by dissolving 100 mg of chitosan in 100 ml of dilute acetic acid (1 % concentration). To synthesize nanoparticles of ZnS:Mn2+ , 40 ml de-ionized water was heated to boil. To this 1 ml of the above prepared zinc acetate and manganese acetate was added and stirred with continuous heating. Upon boiling, 7 ml of 0.1 % chitosan solution was then added with continuous stirring and heating following which 1 ml of sodium sulphide was added to the solution. Upon the addition of sodium sulphide a white precipitate of ZnS nanoparticles is formed instantaneously and the solution is rapidly cooled in ice. A pH of around 5.4 results during the synthesis of ZnS:Mn2+ nanoparticles. The supernatant was then centrifuged at 4000 rpm for 20 minutes to sediment agglomerated particles. To the colloid, dilute acetic acid was added to remove excess chitosan hanging from the particles. The supernatant was then dialyzed to remove other un-reacted ions. In some of the experiments (e.g. for X-Ray diffraction studies), after washing the colloid several times with de-ionized water, the sample was freeze dried to obtain fine powder of ZnS:Mn2+ nanoparticles. The morphology of the nanoparticle were studied in a JEOL, JSM-6301F scanning electron microscope (SEM) operating with primary electron energy of 20 KeV. High resolution electron microscopy (HRTEM) was carried out in a JEOL, JEM-210OF transmission electron microscope (TEM) operating at a source voltage of 200 kV. The TEM sample was prepared by dispersing the sample in dilute acetic acid (diluted thrice) and dropping it onto a lacy holey carbon coated copper grid. X-ray diffraction (XRD) patterns were recorded on a JEOL, JDX-3530 diffractometer using Cu-radiation (Cu Ka, wavelength = 1.540600A). UV-Visible absorption spectra were obtained with an Elico spectrophotometer SL164 and the photoluminescence and excitation spectra with a Perkin-Elmer LS 50B spectrometer. Photo correlation spectra measurements were done in a Zetasizer Nano ZS Zen-3600 from Malvern Instruments.

Figure 1. SEM micrograph of ZnS:Mn21 nanoparticles showing average agglomerate sizes of around 30-40 nm

A typical Scanning Electron Micrograph of ZnS:Mn2+ nanoparticles are shown in figure 1. SEM study shows that the particles have smooth surfaces due to the surface passivation of the luminescent nanocrystals by chitosan polymer. We observe that the agglomerate sizes are around 30 - 40 nm. The stabilizing agent passivates the surface of the particle as has been further confirmed from infrared spectroscopy (FTIR). 60 50 Ox00

40

III. RESULTS AND DISCUSSIONS The general sequence of the formation of Zinc Sulphide nanoparticles in co-precipitation techniques proceed by nucleation, growth and Ostwald ripening followed by agglomeration or aggregation that are dominated by the surface energy of the nanoparticles [21]. Here, manganese doped zinc sulphide (ZnS:Mn ) nanoparticles were synthesized from a homogenous solution of zinc acetate that was reacted with sodium sulphide in the presence of manganese acetate in aqueous media. Zinc acetate in solution (de-ionized water in our case) dissociates into zinc (Zn2+) and Acetate (Ac-) ions. When sodium sulphide (which itself dissociates into sodium [Na+] and sulphide [S2-] ions) is added to the solution containing the Zn2+ ions, the S2- reacts with the Zn2+ ions forming zinc sulphide (ZnS). The Na+ ions present in the solution plays a very important role in deciding the size of the ZnS nanocrystals. The manganese (Mn2 ) ions which are present in small quantities compared to the Zn2+ ions (1/100th the

30 20 10 Co Cl Co

CW

Co

OC

Wavenumber (cm'1) Figure 2. FTIR spectra of vigerously washed chitosan capped ZnS:Mn2. Peaks at 1560 cm-1 and 659 cm-1 are representative of amine group present sterically capping the nanoparticles.

It is clear from the spectra (figure 2) that the particles are capped with chitosan that are indicated by the presence of strong peak at 1560 cm-' and weak one at 659 cm-', that are indicative of the presence of amine groups. The presence of amine group even after rigorous washing of the colloidal nanoparticles suggests further that chitosan enrobes the

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nanoparticles preventing agglomeration of particles by steric hindrance [22].

ZnS has a direct band gap of 3.6 eV with an effective energy band gap of 339 nm at room temperature. Upon doping zinc sulphide with manganese, the Mn2+ ions substitute the Zn2+ ions in the ZnS crystal acting as trap sites for the electrons and holes. An electron can undergo photo-excitation process in the host ZnS lattice of nanoparticles and subsequently decay via a non-radiative transition to the 4T, level to the 6Al level [24]. The strong emission could be attributed to the radiative decay between these localized states of manganese inside the ZnS bandgap [21]. I

0.8 4 0.6

Figure 3. HRTEM reveals the individual grain boundaries. Approximate crystallite sizes are 4-5 nm

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High Resolution Transmission Electron Microscopy analysis reveals that the 40 nm agglomerates are polycrystalline having an average crystallite of approximately 4 - 5 nm as shown in figure 3. This is reasonably consistent with the estimation of the crystallite sizes obtained from the X-ray powder diffractogram (XRD) analysis [23]. Figure 4 shows the XRD of nanocrystalline ZnS:Mn2+ that reveal the quantum dots of ZnS:Mn2+ have zinc blende structure with planes at { 111 }, {220} and {3 11 } respectively. The nanocrystals have lesser lattice planes compared to bulk which contributes to the broadening of the peaks in the diffraction pattern. This broadening of the peak could also arise due to the micro-straining of the crystal structure arising from defects like dislocation and twinning etc. These defects are believed to be associated with the chemically synthesized nanocrystals as they grow spontaneously during chemical reaction since chemical species get very little time to diffuse to an energetically favorable site. It could also arise due to lack of sufficient energy needed by an atom to move to a proper site in forming the crystallite. The crystallite size determined from the Debye-Scherrer formula from the major peak (from { 111 } plane) centered at 20 = 28.520 was established to be about 1.97 nm.

0.2

O 275

{2 2 O}

{3 1 1}

V 115 40 20

30

40

Angle (20)

475 575 Wavelength (nm)

675

The colloid containing ZnS:Mn2+ nanoparticles have an optical absorption edge at around 265 nm and under ultraviolet (UV) exposure (figure 5), it glows with an orange-red color. The strong emission peaking at 590 nm is attributed to radiative decay between localized states of manganese within ZnS bandgap. The emission peaking around 425 nm is typical luminescence of undoped ZnS resulting from the transition of electrons from shallow states near the conduction band to sulphur vacancies present near the valence band. of detecting microbial Conventional methods contamination have rely on time-consuming steps, followed by biochemical identification, having a total assay time of up to 1 week in certain cases [25]. A great deal of research has been focused on the development of biological sensors for the detection of microorganisms, allowing rapid and "real-time" identification [26]. Most bacteria range from 0.2 - 2.0 ptm in breadth and about 2 - 8 pm in length. The basic shapes of bacteria vary from spherical coccus, rod shapes bacillus and spiral. Since the cell walls of bacilli are anionic in nature, any functionalized material having a net positive charge (for example luminescent nanocrystals capped with chitosan at slightly acidic pH) will attach and organize on the cell walls leading to biolabeling. In a simple experiment to demonstrate that the luminescent nanocrystals attach to the walls of the bacillus, some cultured bacilli were fixed on to a glass slide. Freeze dried powder of ZnS: Mn2+ was sprinkled on the surface of the microorganisms and the slide was rinsed with deionized water to wash away unattached nanoparticles. Upon observation under a fluorescent microscope, distinct orange red glow was observed which took

t 265

v, 190

375

Figure 5. Normalized UV-Vis absorption and fluorescence emission spectra of ZnS:Mn2+ These nanoparticles have an optical edge at 285 nm and with a strong emission at 590 nm. Emission peak at 424 nm is from ZnS nanocrystallites.

{111}

340

0.4

50

Figure 4. XRD scan of ZnS:Mn21 nanoparticle showing broadening of the peaks at { 111 }, {220} and {3 11 } due to nanocrystalline nature of the crystal. These quantum dots have zinc blende structure. The crystallite size as calculated by Debye-Scherrer formula is 1.97 nm.

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the shape of the bacilli, (figure 6). As a control experiment, some of the colloidal suspension of the nanocrystals were smeared on to the glass slide (without the bacilli) and heated. After vigorous washing, no luminescence was observed, conclusively proving that the luminescent nanocrystals indeed attach strongly to the cell wall of the bacilli.

ZnS nanoparticles attaches to bacillus and upon irradiation with UV light glows with an orange color. [1] [2] [3] [4]

[5] [6]

Figure 6. Chitosan capped ZnS: Mn2+ with bacillus as seen under a Fluorescence Microscope. The orange glow is from bio-functionalized ZnS:Mn2+attached to bacillus.

[7]

Though no specific experiments were conducted to prove selective attachment of the chitosan capped ZnS:Mn2+ nanocrystals to specific species of bacteria, initial results show that these nanocrystals preferentially attach to gram-positive bacteria. Current focus of our research is to modify the capping agent of the nanoparticles to enhance specificity. Cytocompatibility of the nanoparticles, were studied by adding colloidal ZnS:Mn2+ nanoparticles to a culture dish containing human 3T3 fibroblast cells. The cells were observed under a microscope over intervals of time. It was observed that the cells did not perish even after 8 days. It was also noticed that the growth of new cells were inhibited by the presence of chitosan, which is a known bactericidal agent [27]. The biopolymer chitosan, as capping agent makes these nanoparticles cytocompatible, allowing them to be used as biolabels.

[8] [9] [10]

[11] [12] [13]

[14]

CONCLUSION Synthesis of colloidal suspensions of zinc sulphide doped with manganese was achieved from a simple precipitation reaction leading to particle sizes of 30 - 40 nm consisting of polycrystalline consisting of crystallites of 4 - 5 nm. The particles were stabilized in the colloidal suspension with a biocompatible polymer 'chitosan'. It was found that chitosan provides an effective passivation of the unsaturated bonds on the particle surfaces, eliminating the non-radiative pathways for the excited electrons, thus leading to a high luminescence efficiency of the ZnS:Mn2+ nanoparticles. ZnS:Mn2+ has zinc blende structure, as observed by X-ray diffraction, and has a crystallite size of 1.97 nm as calculated by Debye-Scherrer formula. Under UV exposure, the colloidal suspension or powder of ZnS:Mn2+ glows with an orange-red luminescence peaking at around 590 nm. The biopolymer chitosan, as capping agent makes these nanoparticles biocompatible, allowing them to be used as bio-labels. We have demonstrated that the chitosan capped

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