The present invention relates to nontoxic, water soluble and functionalized transition metal ion doped semiconductor nanocrystals and a process for the preparation thereof.

More particularly the present invention relates to nontoxic, water soluble and functionalized transitional metal ion doped ZnS/Se semiconductor nanocrystals having high quantum yield, stable emission and enhanced shelf life.

The present invention further relates to a process for the preparation of the high quality nontoxic, water soluble and functionalized transition metal ion doped semiconductor nanocrystals having high water solubility and emission stability.

These doped nanocrystals are useful for their application as bio-conjugates for fluorescent biological labeling and other biomedical applications. The said nanocrystals are further useful for preparation of light emitting diodes and solar cells, and energy converters.

BACKGROUND AND PRIORART OF THE INVENTION;

Fluorescing semiconductor nanomaterials e.g. CdSe, which are commonly known as quantum dots, are found by the researchers as better and also stable bio-label agents for biological imaging as compared to traditionally or conventionally used organic dyes. These quantum dots when shelled with higher band-gap materials e.g. ZnS to make CdSe-ZnS core shell, they achieve much higher emission intensities. Further, said quantum dots do not photo- bleach easily when used for bio-imaging. In spite of their improved properties, these materials are not readily and widely accepted by the biological community for wide-spread, real-life applications because of the adverse toxicity of cadmium.

Quantum Dot (QD) based cellular imaging is of considerable current interest because they have bright, sharp, stable and tunable fluorescence which can be excited by a wide range of light from UV to visible region. Current research efforts include search for alternative non cadmium based i.e. non-toxic QD, understanding the surface chemistry to develop suitable surface coatings that can help to derive various QD-bioconjugates, minimization of non-specific binding, exploring various bio-affinity molecules to improve labeling specificity and cellular trafficking mechanism of QD-bioconjugates. These QD based bio- probes appear as powerful tool in studying the protein dynamics and function at single molecule sensitivity in the sub-cellular length scale which can provide the opportunity for molecular level understanding of disease mechanism.

However, most widely used QD is composed of cadmium based materials for example CdSe, CdTe or CdS. Further extensive research shows that these materials have severe cytotoxicity effect due to heavy metal cadmium which limits their in-vitro and in-vivo applications. In addition, environmental pollution issue of cadmium based material is a serious deterrent to any widespread use of such materials in biomedical and other applications. Hence, even these Quantum Dots which already proved as better conjugate for bio-imaging were considered to be unfit for future applications in the community and in day to day life. Thus, researchers are continuously searching for alternative, nontoxic, water-soluble nanocrystals with high and stable emissions for bio- labeling applications.

A reference may be made to the US Patent publication No. 20070269382, published on November 22, 2007 disclosing nanoparticles and their use for multifunctional bioimaging, comprising a CdS: Mn/ZnS core/shell quantum dot, wherein said quantum dot is fluorescent, radio-opaque, and paramagnetic. The said nanoparticles comprise a further outer shell coating surrounding said ZnS shell which is amine fαnctionalized. These quantum dots however contains cadmium and therefore not suitable for biological applications.

There are several common approaches of converting hydrophobic nanoparticles into water soluble nanoparticles. Reference may be made to Guo, W.; Li, J. J.; Wang, Y. A.; Peng, X. J. Am. Chem. Soc. 2003, 125, 3901 and Yu. W.; Liu, H..; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Paele, F.; Bruchez, M. P. Nat. Biotech. 2003, 21, 41, which disclose ligand exchange based replacement of original hydrophobic surfactant by thiolated small molecule or by silica coating and polymer coating. The water soluble particles produced by those approaches have either poor shelf life or larger size that limits their application.

Another reference may be made to Pradhan, N.; Peng, X. J. Am. Chem. Soc; (Article); 2007; 129(11); 3339-3347 disclosing that the transition metal ion doped nanocrystals are capable of acting as an alternative to cadmium semiconductor nanocrystals. These doped semiconductor nanocrystals do emit different colors in the visible window but the reproducible synthesis of such materials in high quality still remains a real challenge. This document further reports that Mn doped ZnSe with 50% Quantum Yield (QY) have been developed and commercialized, but this material still remains toxic due to the presence of selenium. Further the emission stability and water solubility remains serious challenges for any possible bio labeling applications of these doped nanocrystals.

Another reference may be made to US Patent No. 6,262,129, that discloses a method for producing nanoparticles of transition metals where the method comprising the steps of forming a metal precursor solution from a transition metal; introducing said metal precursor solution to a surfactant solution, wherein said forming and introducing performed in an inert atmosphere with the exclusion of oxygen, said surfactant solution comprises a combination of an organic stabilizer and a phosphine to protect said nanoparticles, and said organic stabilizer being a long chain organic compound of the form R--X, where R is a member selected from the group consisting of l) a hydrocarbon chain in straight or branched formation, said hydrocarbon chain comprising 6 to 22 carbon atoms, and 2) a fluorocarbon chain in straight or branched formation, said fluorocarbon chain comprising 6 to 22 carbon atoms, and where X is selected from the group consisting of carboxylic acid, phosphoric acid, phosphinic acid, sulfonic acid, sulfinic acids, and thiol adding a flocculent to cause nanoparticles to precipitate out of solution without permanent agglomeration; and adding a hydrocarbon solvent for one of re-dispersing and re-peptizing said nanoparticles. This document further reports that the particles were well-dispersed in a typical polymer linkage pattern, indicating that particles prepared in accordance with the present invention have great potential in biological labeling and imaging. However, this work uses metal nanoparticles which are non-fluorescent. Further, the coating is hydrophobic and they are not water soluble.

Further another reference may be made to US Patent No. 7405002, which discloses coated water-soluble nanoparticles comprising semiconductor core and silica coating. The patent further reports that the silica shell can be functionalized or derivatized to include compounds, atoms, or materials that can alter or improve properties such as water solubility, water stability, photo- stability and biocompatibility. For example, a silica shell can include moieties such as polyethylene glycol (PEG) and other glycols. These nanoparticles, with and without PEG, have been shown to be non-toxic to living cells for extended periods, and it is believed that the nanoparticles are also non-toxic in vivo due, at least in part, to the isolation of the toxic core within the polymerized silica shell. The nanoparticles are soluble in an aqueous solution having a pH of less than or equal to 7. The document also reports the silica shell to be stable for 24 hours. However, the main drawback of the invention is the low quantum yield (10-20%) of the nanoparticles. Further this method used Cd based quantum dot and made them non-toxic using polymer coating. So coating makes them non- toxic for some application (such as bio-labeling) but material is still toxic for in- vivo imaging and environmental pollution issue is still there.

Yet another reference may be made to the article from Nanomaterials and Nanofabrication Laboratories, LLC (NN-Labs); Xiaogang Peng, Department of Chemistry and Biochemistry, University of Arkansas, Presidential Green Chemistry Challenge Award Program 2008, Environmental Protection Agency, United States, that reports doped semiconductor nanocrystals as heavy-metal- free Quantum Dots. This article discloses that these heavy-metal-free Quantum Dots are nontoxic with pure dopant emission (over 99 percent) at fluorescence quantum yields greater than 80 percent. These doped quantum dots do not suffer the re-absorption self-quenching inherent in intrinsically emitting quantum dots, due to the large Stokes shift between the host absorption and dopant emission. In addition, these high-quality, doped quantum dots have far greater thermal and photo stabilities than do conventional quantum dots. The superior quality of these new, heavy-metal-free, doped quantum dots will enable quantum dot applications to reach the commercial level without introducing toxins into the environment, which can be effectively used for biomedical labeling. However, the main drawback of this work produces Mn doped ZnSe, where Se is toxic. The process transfers the particles with convention thiol based ligand exchange. This method produces water soluble particles which have poor shelf life and limited functionalization scope.

Our pending Indian Patent Application No 1473/KOL/2OO8 discloses synthesis of high quality Mn doped ZnS that overcomes the problems mentioned in connection with previously synthesized nanoparticles. The said application further discloses efficiently doped transitional metal ions such as Mn, Ni, Cu, Co and Fe in a variety of nanocrystals such as ZnS, ZnSe and ZnTe. However these nanoparticles are not soluble in water and functionalized and not suitable for fluorescent biological labeling with high quantum yield. In spite of the above mentioned references, the inventors of the present invention surprisingly found nontoxic, water soluble and functionalized transition metal ion doped ZnS semiconductor nanocrystals, useful for fluorescent biological labeling that obviates the drawbacks of the above mentioned citations.

OBJECTIVES OF THE INVENTION:

The main objective of the present invention is to provide nontoxic, water soluble and functionalized transitional metal ion doped ZnS/Se semiconductor nanocrystals having high quantum yield and stable emission and enhanced shelf life.

Another objective of the present invention is to provide nontoxic, water soluble and functionalized transition metal ion doped ZnS/Se semiconductor nanocrystals useful for fluorescent biological labeling in various bio-imaging applications.

Still another objective of the present invention is to provide nontoxic, water soluble polymer coated nanoparticles with 20-40% Quantum Yield.

Yet another objective is to provide nontoxic, water soluble and functionalized Mn doped ZnS/Se semiconductor nanocrystals having shelf life in pH range of 4 to 10, which is stable for at least up to 200 days without any loss of fluorescence.

Yet another objective of the present invention is to provide polymer coated nontoxic, water soluble and functionalized transition metal ion doped semiconductor nanocrystals useful as bio-conjugates for both in-vitro and in- vivo applications . SUMMARY OF THE INVENTION:

The present invention provides nontoxic, water soluble and fαnctionalized transition metal ion doped semiconductor nanocrystals having high quantum yield and stable emission and enhanced shelf life.

Accordingly, the present invention provides nontoxic, water soluble and functionalized transitional metal ion doped semiconductor nanocrystals having high quantum yield and stable emission and enhanced shelf life.

In an embodiment of the present invention, the nontoxic, water soluble and functionalized Mn doped ZnS/Se semiconductor nanocrystals have high and stable emission for bio labeling applications.

In another embodiment of the present invention, the nontoxic, water soluble and functionalized transition metal ion doped semiconductor ZnS/ Se nanocrystals have long term stability in aqueous buffer solutions having enhanced shelf life and suitable for commercialization.

In yet another embodiment of the present invention, the said nontoxic, water soluble and functionalized transition metal ion doped semiconductor nanocrystals have the following characteristics properties:

i. Quantum Yield in the range of 20 -40%; ii. Size in the range of 2.5 nm to 100 nm; iii. Emission Stability at temperature range of 0 ⁰ C to 100 ⁰ C; iv. Water Solubility in the range of 1-100 mg/ml; v. pH in the range of 4 to 10; vi. Shelf Life for at least up to 200 days in various aqueous buffer solutions. In still another embodiment of the present invention, the transitional metal ion dopant is selected from the group comprising Mn, Cu and Ni.

In still another embodiment of the present invention, the host nanocrystal is selected from the group comprising ZnS and ZnSe.

In yet another embodiment of the present invention, the nanocrystals have long term stability in aqueous solutions.

In still another embodiment of the present invention, the nontoxic, water soluble and functionalized transition metal ion doped ZnS semiconductor nanocrystals are having 20-40% of Quantum Yield.

Further in another embodiment of the present invention, the nontoxic, water soluble and functionalized transition metal ion doped ZnS semiconductor nanocrystals are capable of being used for fluorescence labeling, where the fluorescence is stable for at least up to 24 hours.

In still another embodiment of the present invention, the preparation of the nontoxic, water soluble and functionalized transition metal ion doped semiconductor nanocrystals comprises the following steps:

(a) synthesizing transitional metal doped nanocrystals according to the process of our co-pending patent application number

(b) coating the doped nanocrystals as obtained from step (a) with suitable polymer in reverse micelle;

(c) dissolving polymer forming precursor monomer and cross-linker in reverse micelle followed by mixing them with hydrophobic particles as obtained from step (b);

(d) adding an initiator to the nanocrystals solution as obtained from step (c) at inert or nitrogen atmosphere to initiate polymerization; (e) separating the coated particles from the mixture as obtained from step (d) by precipitating said particles from reverse micelle by adding alcohol into the system;

(f) purifying the nanocrystals as obtained from step (e) in a known manner and dissolving in water or buffer solution;

(g) functionalizing the water soluble particles as obtained from step (f) with different chemical and bio-chemicals via different bio- conjugation chemistry;

In yet another embodiment of the present invention, the functionalized transitional metal doped semiconductor nanocrystals obtained are subjected to fluorescent biological labeling of the cell.

In still another embodiment of the present invention, the nontoxic, water soluble transition metal ion doped semiconductor nanocrystals are coated with suitable polymers selected from the group comprising polyacrylate, polyethylene glycol, primary amine, carboxylic acid derivatives and mixture of different functional groups on the surface of the said nanocrystals, thereby making them useful as bio-conjugates.

Further in another embodiment of the present invention, the micelle used is Igepeal-cyclohexane reverse micelle.

In yet another embodiment of the present invention, the initiator used is persulphate for initializing the polymerization.

In still another embodiment of the present invention, the alcohol used is ethanol.

In yet another embodiment of the present invention, the pH of the buffer solution at which the functionalized nanocrystals are synthesized is in the range of 4 to 10. In still another embodiment of the present invention, the pH of the buffer solution at which the functionalized nanocrystals are used for labeling is in a range of 5 to 9.

In yet another embodiment of the present invention, different colored emissions can be achieved by doping different transition metal ions Mn, Ni and Cu into the ZnS/Se nanocrystals and making them water-soluble without any loss in their emission efficiencies.

In yet another embodiment of the present invention, the nanocrystals are in the form of powder or dispersed in aqueous solution.

Further in another embodiment of the present invention, the lower cytotoxicity of these probes made using the nontoxic, water soluble and functionalized transition metal ion doped semiconductor nanocrystals provides advantage over cadmium based Quantum Dots for long term imaging in biological system.

BRIEF DESCRIPTION OF THE DRAWINGS:

The present invention will be described herein below in detail with reference to the drawings accompanying this specification wherein -

Figure 1 illustrates a schematic representation of polymer coating processes producing amine functionalized particle followed by the fuctionalization of Mn doped ZnS nanocrystals.

Figure 2 illustrates stable colloidal solution of polyacrylate coated Mn doped ZnS in a range of different pH. Nanoparticles have mixture of amine and polyethylene glycol functional group on their surface.

Figure 3 illustrates labeling of human muscle cell using oleyl functionalized Mn doped ZnS. Cells were incubated with particle for one hour, followed by washing. Imaging was performed in blue excitation using normal fluorescence microscope.

DETAILED DESCRIPTION OF THE INVENTION:

According to the present invention there is provided nontoxic, water soluble and functionalized transitional metal ion doped ZnS/Se semiconductor nanocrystals having high quantum yield and stable emission and enhanced shelf life.

It has been the endeavor of the applicant to produce doped semiconductor nanocrystals which can emit different colors in the visible window and at the same time are biologically acceptable, thus making them safe labeling reagents in different biological applications.

The present invention further includes within its ambit water soluble transition metal ion doped semiconductor nanocrystals which has long term stability in aqueous buffer solutions having enhanced shelf life and suitable for commercialization. These nanocrystals are having high and stable emission for bio-labeling applications. These transition metal ion doped semiconductor nanocrystals can be effectively used as non-toxic conjugate for in-vitro and in- vivo applications due to absence of cadmium as compared to cadmium based materials. These nanocrystals are environmental pollution free, non-toxic, biologically acceptable and non-cytotoxic in nature.

The nontoxic, water soluble transition metal ion doped semiconductor nanocrystals are coated with suitable polymers selected from the group comprising polyacrylate, polyethylene glycol, primary amine, carboxylic acid derivatives and mixture of different functional groups on the surface of the said nanocrystals, thereby making them useful for bio-conjugation chemistry. The polymer coating provides the protection of nanoparticles from adverse experimental condition and also gives the opportunity to functionalize the particles with various chemicals and bio-chemicals.

According to the present invention there is further provided a process for the preparation of nontoxic, water soluble transition metal ion doped semiconductor nanocrystals, which comprises the following steps of:

(a) synthesizing doped nanocrystals according to the process of Patent application number 1473/KOL/2008;

(b) performing polyacrylate coating of said doped nanocrystals in Igepeal-cyclohexane reverse micelle;

(c) dissolving polymer forming precursor monomer and cross-linker in reverse micelle followed by mixing them with hydrophobic particles as obtained from step (b);

(d) adding persulphate initiator to the nanocrystals solution as obtained from step (c) at inert or nitrogen atmosphere to initiate polymerization;

(e) separating the coated particles from the mixture as obtained from step (d) by precipitating said particles from reverse micelle by adding alcohol into the system;

(f) purifying said particles in a known manner and dissolving in water or buffer solution; and

(g) functionalizing the water soluble particle with different chemical and bio-chemicals via different bio-conjugation chemistry.

The functionalized nanocrystals obtained are subjected to fluorescent biological labeling of the cell.

In the above mentioned polymer coating process, the polymer quoted nanoparticles may have polyethylene glycol, primary amine, carboxylic acid or mixture of different functional group on their surface. The coating method is found to be generic in the sense that different colored emissions have been achieved by doping different transition metal ions like Mn and Cu in to ZnS/Se nanocrystals and making them water-soluble without any loss in their emission efficiencies. This has paved the way for generating a range of different colors for bio-labeling applications based on non-toxic materials.

The polyethylene glycol and oleyl functionalized water soluble Mn doped ZnS nanoparticles are found to be useful to label cells. The fluorescence is stable for a long time, i.e., at least for 24 hrs in the labeled cell, suggesting that these doped nanoparticles based label can be used not only for short-term but also for various long term cell imaging applications.

For polyacrylate coating reverse micelle was used as the reaction medium and room temperature, normal atmospheric pressure was used.

The following examples are represented by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1

Preparation of the transition metal ion doped ZnS/Se semiconductor nanocrystals:

The Mn doped ZnS/Se nanocrystals were initially synthesized by the process of our co-pending application no. 1473/KOL/2OO8 followed by coating the said nanocrystals with polymers such as polyacrylate, polyethylene glycol, primary amine, carboxylic acid derivatives on the Igepeal-cyclohexane reverse micelle. The polymer was then dissolved forming a precursor monomer and cross-linker in reverse micelle, which was further mixed with hydrophobic nanoparticles. Persulphate initiator was added to the nanocrystals solution at inert or nitrogen atmosphere to initiate polymerization. The coated particles were separated from the mixture by precipitating said particles from reverse micelle by adding alcohol into the system. The nanocrystals were then purified and dissolved in water or aqueous buffer solution. Functionalizing the water soluble particles was performed with different chemical and bio-chemicals via different bio- conjugation chemical reactions.

Table i demonstrates the different emission properties of the transition metal ion doped semiconductor ZnS/Se nanocrystals.

Table l

Example 2

Oleyl fuctionalization of the nanoparticles:

Oleyl functionalized particles are prepared using polymer coated particle that have both carboxylate and polyethylene glycol on their surface. The carboxylate group of this particle was covalently conjugated with oleyl amine via N-Ethyl- N'-(3-dimethylaminopropyl) carbodiimide (EDC) coupling chemistry. EDC coupling was performed in room temperature using buffer solution of pH 6.0 using excess EDC (10 times molar excess compared to oleyl amine) and reacting for 6 hours followed by removal of excess EDC by overnight dialysis. Example 3

Non-toxicity of the transition metal ion doped ZnS semiconductor nanocrystals :

Human muscle cells (obtained from Biological Chemistry department, IACS) were sub cultured in well plates using 5θθμL of media, followed by overnight incubation at 37 ⁰ C for cell attachment on the well plate surface. Next, 10-iooμL of particle solution were added and mixed with the cell culture medium. Cells were observed under microscope after different incubation periods from 1 hour to 24 hours. Cells remain healthy, grow and multiply even in presence of particle.

Example 4

Labeling the human muscle cells with functionalized nanoparticles:

Human muscle cells (obtained from Biological Chemistry Department, IACS) were sub cultured in well plates using sooμL of media, followed by overnight incubation at 37 ⁰ C for cell attachment on the well plate surface. Next, loμL of particle solution were added and mixed with the cell culture medium. After 2 hours of incubation at 37 ⁰ C, cells were washed with buffer solution, and the fresh cell culture medium was added. Cells were then imaged under fluorescence microscope. (As shown in Figure 3)

Example 5

Labeling the HepG2 cells with functionalized nanoparticles:

HepG2 cells (obtained from Biological Chemistry department, IACS) were sub cultured in well plates using 2θθθμL of media, followed by overnight incubation at 37 ⁰ C for cell attachment on the well plate surface. Next, lOoμL of particle solution were added and mixed with the cell culture medium. After 2 hours of incubation at 37 ⁰ C, cells were washed with buffer solution, and the fresh cell culture medium was added. Cells were then imaged under fluorescence microscope.

Example 6

Stability of the transition metal ion doped nanocrystals at various pH ranges:

Water solubility was performed for polymer coated particles of different functional groups. Presence of carboxylate, amine and polyethylene groups is required for water solubility. Carboxylate functionalized particles are soluble in the pH range 4-10, but slowly precipitate in pH less than 4. Amine functionalized particles are soluble in pH range of 4-8 but precipitates at pH higher than 8. Presence of a mixture of amine-polyethylene and carboxylate- polyethylene group on the particle surface can stabilized the particle in the pH range of 4-10. (As shown in Figure 2)

Example 7

Long term stability of the functionalized nanocrystals:

Aqueous solution of polymer coated particles was mixed with different buffer solution of varying pH and then solubility was checked by observing precipitate. The colloidal stability (dispersion stability of particles/ shelf life) of coated particles in different aqueous buffer was checked for more than 2oodays and they become stable without any sign of precipitation. Fluorescence study of particle solution shows that there was no significant change in fluorescence intensity even after 200 days. The stability was further tested by boiling the colloidal solution and they remain stable in boiling water.

Table 2 demonstrates different stability properties of the said transition metal ion doped ZnS/Se semiconductor nanocrystals.

Table 2

Example 8

Transitional metal ion doped semiconductor nanocrystals as bioconjugates :

The polymer coated particle was functionalized glucose via a bioconjugation method. Glucosamine and gluteraldehyde was mixed in 1:1 ratio in carbonate buffer solution of pH 9.5. After one hour this solution was mixed with amine- polyethylene glycol functionalized particle, followed by borohydride reduction of imine bond (formed during aldehyde and amine group). Dialysis was then performed to separate free glucosamine. The resultant particle is water soluble and collidally stable. Their biochemical activity was tested via their interaction with glycoprotein Concanavalin A. Example Q

Water solubility of the transition metal ion doped semiconductor nanocrystals :

Water soluble doped nanocrystals are prepared by the following methods - (i) Polymer coating. After the purification the doped nanocrystals were dissolved in 6 ml cyclohexane. This solution was taken in a 50 ml 3-necked flask. In a 2 ml centrifuge tube, O.O2mmol MBA was taken with looμl water. To this mixture Igepal (0.5ml) and cyclohexane (1.5 ml) were added. The mixture was shaken to get a clear solution. In another 2 ml centrifuge tube, o.2θmmol N-(3-Aminopropyl) methacrylamide hydrochloride was taken and dissolved in looμl water. Igepal (0.5ml) and cyclohexane (1.5ml) were added to it and this mixture was shaken to get a clear solution. The last two solutions were added to the above reaction flask. The mixture was stirred and degassed for 2θmins under Ar atmosphere. After that, a solution of o.oi3mmol ammonium persulphate dissolved in iooul water was injected into the reaction flask and degassing was continued for όomins. Persulphate will start polymerization process which will continue for όomins. When the reaction will be completed ethanol was added to precipitate the polymer coated doped nanocrystals and the precipitate was washed with chloroform and ethanol. Then water was added to the precipitate and stirring was continued for hours to get a dispersed solution.

(ii) Di-thiol ligand exchange. In a 2 ml centrifuge tube cyclohexane (o. 5 ml) and Igepal (0.5 ml) were taken to make 1 ml solution. The mixture was shaken to get a clear solution. To this mixture purified nanocrystals dissolved in cyclohexane was added. The mixture was stirred to get a homogeneous solution. To this mixture an aqueous solution (0.1 ml) of dimercaptosuccinic acid (0.1M) was added followed by 0.1 M NaOH (0.1 ml) aqueous solution. During this process nanocrystals will start to precipitate. The ligand exchange reaction was continued for lomins. When all nanocrystals will be precipitated, cyclohexane was added to wash excess Igepal. Then the precipitate was dissolved in water and stirred for hours to get dispersed solution.

APVAJSfTAGES OF THE INVENTION:

The non-toxic, water-soluble transition metal ion doped semiconductor nanocrystals of the present invention are useful for wide varieties of applications and advantages, some of which are stated herein below -

1. As the functionalized transitional metal doped semiconductor nanocrystals are nontoxic (free from Cd), it can be used as a safe labeling reagent in different biological applications.

2. Stable fluorescence and lower cytotoxicity of these probes gives advantage over Cd based QD for long term imaging in biological system.

3. High quantum yield (about 40%) for Mn doped ZnS nanocrystals in water makes these nanocrystals as superior than all existing doped nanocrystals designed for these purposes.

4. The successful polymer coating gives the opportunity to derive various doped nanoparticles based bioconjugates (peptide, antibody, oligonucleotide, vitamin etc) useful for different biomedical applications.

5. The stability of polymer coated and functionalized nanoparticles against a wide range of pH and long term stability in aqueous buffer solutions enhances their shelf-life, essential for commercialization.