FIELD OF INVENTION:
The present invention in particular relates to the field of preparation of sulfur doped graphene quantum dots (S-GQDs) and more specifically relates to a synthesis method involving hydrothermal reaction on cane molasses to synthesize graphene quantum dots under moderate temperature and autogenous pressure.
DESCRIPTION OF THE RELATED ART:
The overall GDP growth in India is majorly standing on the agriculture industries and agro products. Most of these products are not profitable goods but eatables and raw material. Sugar Industry by-products have the most utility in the energy, organic chemicals and recombinant drug sector and one of its major by-product, molasses, is being underutilized inspite of its high potential. The world is now moving to carbon nanomaterial and quantum dots to find effective bioimaging solution from conventional dyes and dangerous gamma radiations. There are many loopholes seen in the conventional materials such as their poor optical properties, toxicity and fast average photoluminiscent decay making it difficult to be detected.
Several methods are known from prior art for the synthesis of graphene quantum dots for bioimaging application, one of which includes graphene quantum dots from a carbon source (e.g., coal, coke, and combinations thereof) by exposing the carbon source to an oxidant disclosed in patent application WO2014179708 Aland separating the formed graphene quantum dots from the oxidant or reducing the higher carbon source such a graphene oxide, fullerenes etc to graphene quantum dots.
Publication No. CN105621407 (A) relates to a method for preparing a sulfur having excellent optical properties of doped graphene quantum dots. The surface of pyren'e is nitrated at low temperature, the nitro group on the surface of pyrene is removed through a hydrothermal reaction at high temperature and high pressure, the hexagonal annular structure of pyrene is cut. The obtained graphene quantum dots are synthesized with sublimed sulfur at a temperature of 150-200°

C through a hydrothermal reaction in one step to prepare the sulfur-doped graphene quantum dots with average granularity of grains is 4-6 nm.
Publication No. CN103833029 (B) relates to a multicolor fluorescence efficiency of water-soluble sulfur, oxygen co-doped graphene quantum dot preparation. An inexpensive sugar, fructose, sucrose, glucose and sulfuric acid as raw materials, a one-pot synthesis (One-Pot Synthesis), will co-doped sulfur, oxygen, and synthesis of graphene quantum dots combine two one. Under hydrothermal reaction conditions, the sugar provides a source of carbon and oxygen, whereas sulfuric acid was added as raw materials.
Publication No. WO2014176519 (Al) relates to a method for sensing a graphene quantum dot which includes heating an organic starting material to a temperature within 20°C of the organic starting material's boiling point for a time no longer than ten minutes to form a graphene quantum dot, exciting the graphene quantum dot with light having a first wavelength, and measuring light emitted by the excited graphene quantum dot at a second wavelength different from the first wavelength.
Publication No. US2016256403 (Al) relates to a process for the preparation of composition comprising fluorescent graphene quantum dots (GQDs) which has been embedded in a polymer matrix of polyethylene glycol (PEG) (PEG-GQDs). The biocompatible composition with reduced cytotoxicity comprising graphene quantum dots (GQDs) with a particle size ranging from 5-10 nm embedded in polyethylene glycol (PEG) matrix with a particle size ranging from 80-100 nm, for drug delivery and biomedical applications.
The article entitled "Sulfur-doped graphene quantum dots as a novel fluorescent
probe for highly selective and sensitive detection of Fe talks about the S-GQDs
which is used as an efficient fluorescent probe for highly selective detection of
Fe3+. Upon increasing of Fe3+ concentration ranging from 0.01 to 0.70 |aM, the
fluorescence intensity of S-GQDs gradually decreased and reached a plateau at 0Q0 MM

This fluorescent probe has been successfully applied to the direct analysis of Fe in human serum, which presents potential applications in clinical diagnosis and may open a new way to the design of effective fluorescence probes for other biologically related targets [Li SI, Li Y, Cao J, Zhu J, Fan L, Li X.; NCBI, 2014 Oct 21].
The article entitled "Time-efficient syntheses of nitrogen and sulfur co-doped graphene quantum dots with tunable luminescence and their sensing applications" talks about the heteroatom doped graphene quantum dots (GQDs) which are particularly promising in bioimaging and fluorescent sensing. The two nitrogen and sulfur co-doped GQDs (N,S-GQDs) with varied fluorescence emission wavelength has been synthesized via HNO3, vapour cutting route [Hongbo Xu, ShenghaiZhou,a Lili Xiao, Qunhui Yuan and Wei Gan; Pubs.rsc, 06 Apr 2016].
The article entitled "Sulphur doping: a facile approach to tune the electronic structure and optical properties of graphene quantum dots." talks about the S-GQDs has been prepared by a hydrothermal method using fructose and sulphuric acid as source materials. Absorption and photoluminescence investigations show that inter-band crossings are responsible for the observed multiple emission peaks [Li X, Lau SP, Tang L, Ji R, Yang P.; NCBI, 2014 May 21].
Top-down and bottom-up approaches are being employed to synthesize GQDs was displayed in the review article of Mitchell Bacon et al (2014), Graphene Quantum Dots, Part. & Part. Syst. Charact, 31, 415-428. In top-down methods, GQDs are obtained from bulk carbonaceous materials, whereas, carbon containing small molecular precursors are being converted to GQDs in bottom-up techniques. Significant arrays of polysaccharides, such as cellulose, chitosan, starch, have already been reported to be utilized for development of CNDs were mention in the Y. Liu, C. Y. Liu and Z. Y. Zhang et al. Colloid Interface Sci., 2011, 356,416, S. Chandra, S. H. Pathan, S. Mitra, B. H. Modha, A. Goswami

and P. Pramanik, RSC Adv., 2012, 2, 3602 and B. Zhang, C. Y. Liu and Y. Liu et al Eur. J. Inorg. Chem., 2010, 28, 4411 published articles.
Recently it has been reported that complex polysaccharides containing products such as egg shells ash, orange juice, soya- milk, willow bark, pomelo peel, rice husk etc. were used for the synthesis of these carbonaceous materials.
The disadvantages of the above-mentioned methods involve a pre-treatment step that uses toxic substances that needs to be removed after reaction and requires following a tedious and expensive process. It is a well-established fact that the use of chemical irradiation compromises the biocompatibility of graphene quantum dots making them unfit for the use in human body. The Industrial up scaling of some of these reactions is a difficult task. Major drawbacks like poor yield, weak optical properties and not well studied biocompatibility issues have been reported in the published literature.
Therefore, while existing carbon material for bioimaging such as quantum dots, carbon nanodots (CND), graphene oxide nanodots, graphene quantum dots, etc. synthesized via multistep reactions, there is still an urgent need for a method to prepare carbon material using an eco-friendly, sustainable, economical, single-step reaction that can be easily scaled-up for mass production and does not involve the use of any toxic chemical irradiation. Simultaneously, there is also a need of a biocompatible material that possess superior optical properties (i.e. high quantum yield and large decay curve) and is smaller in size (< 5nm) that can serve as an efficient bioimaging tool providing new dimension in the field of diagnosis.
Developing a method for preparation of material with high biocompatibility for bioimaging is a big concern and a top priority for researchers working in the field of biomedical imaging and/or diagnostics. If the precursor of the developed material is affordable, easily accessible and is from an agro-industrial bio waste then this could be an 'icing on the cake'.

Thus, in view of the above prior arts, the present invention aims to provide a method to harness true potential of sugar industries by-products by developing Sulphur doped graphene quantum dots that could be used as an important bioimaging and diagnostic solution for the whole world.
OBJECTS OF THE INVENTION:
The principal object of the present invention is to provide a method for facile and sustainable synthesis of highly fluorescent S-doped graphene quantum dots from cane molasses through hydrothermal reaction under moderate temperature (180°C) and pressure, in relatively less time, thereby, avoiding the harsh reaction conditions.
Another object of the present invention is to provide a method for mass production of S-doped graphene quantum dots from cane molasses.
Yet another object of the present invention is to provide highly biocompatible, cytocompatible, hemocompatible, histocompatible quantum dots.
Still another objective of the present invention is to provide a method that does not involve harmful chemicals and harsh reaction conditions for the synthesis of graphene quantum dots.
Yet another objective of the present invention is to provide starting material, a heterogeneous mixture, which is a by-product of sugar industry, obtained after industrial process of developing sugar from sugarcane.
Still another objective of the present invention is to provide a competent candidate to be used as a bioimaging tool and for diagnostic purposes.
SUMMARY OF THE INVENTION:
In accordance with the objects of the disclosure the present invention provides a facile and sustainable synthesis method involving hydrothermal reaction on cane molasses to synthesize graphene quantum dots under moderate temperature and jautQsenous nressurjs. This method is, of considerable interest due to sustainable,

affordable, clean, biocompatible and cost effective fabrication. Graphene quantum dots produced by this method are found to be highly biocompatible with high quantum yield and large decay time that can be used for various bioimaging applications.
The S-GQDs synthesized according to the present invention are water soluble having lateral size and height in the range of-2- 5 nm and -3-5 nm, respectively. This makes the material more autopermeable to the cells making it a competent candidate to be used as a bioimaging tool. The resultant graphene quantum dots from this process can serve as replacement of the other carbon dots developed from the mild reaction like microwave and organic dyes due to their superior optical properties and high biocompatibility. In order to check the biocompatibility the material is subjected to three systems. Firstly, cytocompatibility is evaluated on the primary (UMNSAH/DF-1) cell line, second hemocompatibility is checked on. the mouse blood cells and finally histocompatablity is studied using histopathology of vital organs of mouse after giving the material for 14 consecutive days through intraperetonial and oral routes. This method results into graphene quantum dots that had autopermeablizing property in HEPG2 cancer cell line, even at very low concentration of 50 mg/ml.
BRIEF DESCRIPTION OF THE DRAWINGS:
Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the accompanying drawings and wherein:
Scheme 1 shows hydrothermal synthesis of GQDs;
Fig. 1 FTIR of S-GQD (a)FTIR spectra of S-GQDs and (b) crude molasses (CM) Fig. 2 shows (a) Particle size distribution with modal frequency (<10 nm) (b) HRTEM- showing GQDs at (lnm and lnm) respectively with SEAD pattern (c) UV-Vis spectra of GQDs (d) PL emission spectra (with progressively longer excitation wavelengths from 340 nm in 10 nm increment) QY=~47% ; (e) Excited state intensity decay data of GQDs in water at ambient conditions. Excitation was carried out using a 340nm NanoLED, and emission was collected at 425nm and 450nm. The bottom curve (blue) denote the instrumental response function (IRF)

measured using a dilute glycogen suspension and the lower panels show weighted residuals for the corresponding fits.
Table 1: Recovered excited state intensity decay parameters along with the goodness-of-the-fit (x) for S-GQDs dissolved in water. Excitation at 340 nm using NanoLED. Error in recovered decay times is < 5%. x's are the recovered decaytimes and a's are the corresponding pre-exponential factors and tavgindicates average life time. Table 1 :
Xem/Xcx Tt/hs(di) TafflS (d^) Ta/hS (da) Tt»g/hS X*

425/340 1.51 (di = 0.77) 7.65(02=0.23) 2.9 1.00
450/340 1.49 (di = 0.76) 755(02=054) 2.9 0.98
425/340 0.42 (di = 0.63) 2.71 (02=058) 8.95(03=0.09) 1.86 1.00
450/340 0.28 (Qi= 0.70) 2.47(02=052) 8.57(03=0.08) 1.42 1.06
Fig. 3 shows (a) Differential peaks generated in XRD of S-GQDs with proper
characterization (b) Raman of Molasses cake (before work-up) (c) XRD of the S-
GQDs (Molasses filtrate), cane molasses (vacuum dried) and molasses cake
(before work-up) (d) XPS survey peaks of the GQDs;
Fig. 4 shows (a) Shows tabular representation of concentration dependent cell
cytotoxicity & (b) Hemolysis assay of RBCs; where /Control' represent sample
contain saline only,cS-GQDs5 represent sample contain test drug, 'Triton X'
represent sample contain Triton X. (c) RBC aggregation assay; fic' represents the
sample contains test sample, (d) represent sample contain saline only;
Fig. 5 shows Fluorescent micrograph (a) DIC image of GQDs+DFl (chicken
fibroblast cells) (b) Fluorescent excited image of GQDs +DF1 (chicken fibroblast
cells) (c) Overlay Image a) and b) micrograph;
Fig. 6 shows comparison of Intensity in lab scale and after pilot scale-up to be
relatively similar.
DESCRIPTION OF THE PREFERRED EMBODIMENTS:
Discussed below are some representative embodiments of the present invention. The invention in its broader aspects is not limited to the specific details and representative methods. The illustrative examples are described in this section in connection with the embodiments and methods provided. The invention according

to its various aspects is particularly pointed out and distinctly claimed in the attached claims read in view of this specification.
Through the whole document of present invention, the term "moderate" is used to define the temperature and pressure conditions used for the synthesis of the graphene quantum dots where a temperature of only 180°C and autogenous pressure gives a high yield.
Through the whole document of present invention, the term 'Bioimaging' refers to the technique of producing high-resolution contrast images of biological materials or living systems due to photoluminescence properties. They have been successfully employed for imaging of various dynamics inside the cell.
Through the whole document of present invention, the term 'Sustainable' means to be remaining productive throughout without compromising its optical properties and biocompatibility.
Through the whole document of the present disclosure, the term "graphene" refers to "a conductive material in which carbon atoms are arranged in a two-dimensional Sp hybridised honeycomb form and which has a thickness of few atomic layers, typically 1-8".
Through the whole document of the present disclosure, the term "graphene quantum dot" refers to "a zero- dimensional material which has a size of 2-20 nm or less and a height of about 10 nm or less."
Through the whole document of the present disclosure, the term 'Hydrothermal synthesis' include the various techniques of crystallizing substances from high-temperature aqueous solutions at high vapour pressure.
The invention is described in detail with reference to the examples given below. The examples are provided just to illustrate the physico-mechanical properties of the invention and therefore, should not be construed to limit the scope of the invention.
EXAMPLES Example 1

Briefly, 5ml of cane molasses was stirred continuously for 2 h using glass beads and The mixture was poured into a Polytetrafluoroethylene (PTFE) unit (3/4 volume), sealed and assembled within a hydrothermal bomb unit accordingly for hydrothermal reaction at 180°C for 4 h kept in an oven. After allowing sufficient time for the bombs to cool down, the PTFE units were extracted and the contents were filtered using a Gooch crucible (grade 3) funnel and vacuum pump attached to the filtration unit. Water is used to wash the material until little or no color leaching is observed in the wash. The filtrate obtained was dried in rotaevaporator for further application. Optimization of the parameters for the synthesis was achieved by carrying out the synthesis at varying temperature and time. The synthesized material was characterized by UV-Vis spectroscopy (Shimadzu), Fluorescence
Spectroscopy, Transmission Electron Microscopy, X-ray Diffraction, Fourier Transform Infrared spectroscopy.
Example 2
FTIR measurement
The FTIR measurements were done on an FTIR-ATR model Alpha-P, Bruker. Oven dry molasses and above obtained GQDs were subjected to FTIR scan. The IR spectrum of both materials was obtained by scanning at a resolution of 4cm"1 and by averaging 24 scans.
To get an insight into the hyperfine chemical structure, the FTIR spectra of S-GQDs and crude molasses (CM) were measured as shown Fig (1). The surface chemistry and changes in the functional groups in the product (S-GQDs) from the starting material (CM) can be envisage/correlated from the characteristic FTIR peaks. A broad peak at 3400 cm'1 was seen in the starting (CM) material which was attributed to the hydroxyl (-OH) stretching vibration. Crude Molasses contains high percentage of invert sugar (aldoses & ketoses) which are linked with the help of glycosidic linkages. During the reaction some of these linkages are broken giving rise to freely exposed (-OH) groups, which are involved in intermolecular hydrogen bonding in aqueous medium resulting in high solubility of S-GQDs.

The vibration peak at around 2790 cm"1 and 1720 cm"1 corresponding to aldehyde. and keto group, respectively, in the spectra of starting material (CM), becomes almost invisible in case of S-GQDs. Asymmetric and symmetric stretching vibration of graphite like -CH2- linkages around 2925 cm"1 and 2851 cm"1 arising due to the reduction of keto and aldehyde groups of the CM were also seen in the spectra of S-GQDs. The characteristic conjugate peaks -1378 cm"1 and 1158 cm'1 arises from asymmetric and symmetric SO2 stretching, respectively. Moreover, peaks -1037 cm"1 and 780 cm"1 in the spectra of S-GQDs were attributed to thiocarbonyl bending and C=S stretching vibrations, respectively. These newly emerging peaks in S-GQDs predict effective doping of sulfur into the carbon matrix. Furthermore, the presence of sulfur was also confirmed through ED AX of CM.
A sharp peak at around 1636 cm'lin S-GQDs also confirms the presence of C=C stretching mode of polycyclic aromatic hydrocarbons indicating that the S-GQDs possesses the structure of graphene.
Example 3
Raman spectra, XRD, UV-Vis Spectra, fluorescence spectra The synthesized material was characterized by UV-Vis spectroscopy (Shimadzu), Fluorescence spectroscopy (Varian Cary), Transmission Electron Microscopy (TECHNAI G2), X-ray Diffraction (Bruker India). Obtained results were analyzed and plotted as shown in Fig. 1 and 2.
Example 4
Biocompatibility
(a) Cell cytotoxicity assay onprimary (UMNSAH/DF-1) cell line
Concentration dependent cell cytotoxicity was performed to check primary
cytocompatibility of material and to find out dose response curve. Cells were
trypsinized with IX Trypsin/EDTA solution, washed in PBS and 106 cells were
taken in each well of a 12 well plate. 3-[4,5-Dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium (MTT) dye assay was conducted as per J. Mosmann method

with modification. Dye and media absorptions were taken to minimize background error. Control and test samples (GQDs) at different concentration were incubated at 37°C in 10% C02for 4 h with MTT salt solution (Hi-Media RM1131, 3 mg/ml). Finally 500jil of (0.04N HC1) Acid isopropanol was added in the each well. The plates were gently stirred to enhance dissolution of the crystals and kept for 5 min incubation at 37°C. Percentage of viability was calculated as per formula given in supplementary. All experiments were carried out in biological triplicate. Cells along with media with or without S-GQDs in different concentrations were added in plates and incubated for 24hrs followed by cytotoxicity assay. Concentration was expressed in log and graph was plotted between log concentrations vs. % viability. The percentage survival was calculated as:
absorbance of test- absorbance of blank
X 100
absorbance of control — absorbance of blank
T-test was used to perform statistical analysis. A P-value of < 0.05 was considered significant.
Cells treated with S-GQDs in concentrations from 200(ig/ml to 10 mg/ml were found to be viable when compared to untreated controls (Figure 4). LD-50 value of the S-GQDs was found to be at 9.311mg/ml. Significant cell viability (88.7%) was observed at treatment of 2mg/ml of S-GQDs and further higher concentrations showed considerable cell death. The toxicity effect reduces to normal in 72 hrs.
(b) Hemolysis
Normal saline equilibrated sample was prepared with freshly isolated un-coagulated blood for the estimation of hemolysis. The specific amount from prepared sample (100|aL) was incubated with 2mg/ml S-GQDs at 37°C for 2 hour in 5% CO2 incubator at 95% humidity [6]. The absorbance was observed at 540 nm which account for the total hemoglobins content in blood plasma, hence indicating the total concentration of lysis. Normal saline or 1% triton X combination with blood sample was considered as positive and negative controls respectively. All the experiments were done in biological triplicate.

RBC hemolysis with addition of S-GQDs after 1 h incubation was evaluated by employing fresh blood against saline or triton X treated group (Fig 3). No significant change in hemolysis was observed in test groups when compared to saline treated control, whereas triton X treated groups showed complete hemolysis as revealed from absorption data. The HR ratio of the test sample obtained was 1.03%. Moreover, hemocompatibility of test sample was also assessed by blood cell aggregation method and the end results showed no significant aggregates in GQD treated sample (Fig 3). Compared to control sample (Fig 3).
(c) Assessment of toxicity in vital organs
Healthy rats were treated with 1ml of normal saline (both oral and /./?.) or S-GQDs (lOmg/kg, p.o/Lp).
Liver being the most vital organ for enzymatic activities and metabolism, study of liver function, including SGOT/SGPT level (65,35) in blood serum was carried out to find any detrimental effect of S-GQDs on liver function. In brief, the assessment of SGOT/SGPT catalyzes has done using the commercially available kit for the study of liver function with respect to test drug (GQD). Absorbance of test was assessed versus blank spectrophotometrically (LABINDIA 3000, India) at 340 nm. Both SGPT/SGOT value has expressed as IU/L. Further organ toxicity was assessed on heart, liver, kidney, and brain against structural changes by getting histology of respective organs. Cryo-sections of tissues were stained with HE (Haematoxylin and Eosin) and mounted slides were observed on 400X magnification.
Numerous modifications and adaptations of the system of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the true spirit and scope of this invention.

WE CLAIM:
1. A process for mass production of sulfur doped graphene quantum dots from cane molasses in relatively less time, comprising hydrothermal reaction of cane molasses at moderate temperature and autogenous pressure.
2. The process as claimed in claim 1 wherein the process comprises the steps of - continuous stirring of cane molasses later subjecting to hydrothermal reaction in a Polytetrafluoroethylene (PTFE) sealed in a hydrothermal bomb under 180°C temperature and autogenous pressure for 4 h in a hydrothermal unit (hot air oven); after cooling, extracting and filtering the obtained material using a grade 3 Gooch crucible filter while washing with distilled water; and finally, collecting, dialyzing, rotaevaporating the obtained filtrate and storing in a dessicator for further applications.
3. The process as claimed in claim 1 wherein cane molasses used as a starting material, is a heterogeneous mixture of many biomolecules including sulfur which results into sulfur doped graphene quantum dots, introducing an extra transition band increasing the decay time for better detection and sustained fluorescence intensity.
4. The process as claimed in claim 1 wherein the process excludes use of any external doping agent to dope sulfur in graphene quantum dots.
5. The process as claimed in claim 1 wherein the graphene quantum dots prepared are water soluble having lateral size and height in the range of ~2-5 nm and -3-5 nm, respectively.
6. The process as claimed in claim 1 wherein the graphene quantum dots possess high photo stability and sustained fluorescence giving an averaged lifetime (zav) of 2.9 ns.
7. The process as claimed in claim 1 wherein graphene quantum dots have tunable fluorescence and slow decay time.
8. The process as claimed in claim 1 wherein sulfur doped graphene quantum dots have high degree of crystallinity as per the in plane and basal lattice parameters in XRD indicating peaks corresponding 004,100,00 and exhibit a graphitic structure.

9. The process as claimed in claim 1 wherein the sulfur doped graphene quantum dots exhibit high crystal quality with less crystal defects as exhibited by the Raman spectra Rowing the ordered G Band is much stronger than the disorder D band.