Green fluorescent silver nanodots by use of naringenin and a process of preparation thereof

Abstract

Present invention discloses a novel green fluorescence silver nanodots prepared with naringenin, a flavanone, by a near green synthesis without adding harsh reducing agent such as sodium borohydride and others. Particularly, the present invention provides silver nanodots (quantum dot like nanostructure) of average size 3.5 nm. The preparation method is a unique kind of single pot green synthesis method wherein no common and strong reagent like sodium borohydride, ascorbic acid surfactant, thiol containing reagent or protein solutions are used. Alternatively, a simple flavanone, aromatic organic molecule that contains three OH groups is used both as a mild reducing and stabilizing agent for the preparation of the particles at room temperature (25°C). The aqueous suspension of the invented nanodots product is quite stable in room temperature. It produces strong green fluorescence with average fluorescence lifetime ~2.37 ns and produces a large stocks shift.

Description

[0001] GREEN FLUORESCENT SILVER NANODOTS BY USE OF NARINGENIN AND A PROCESS OF PREPARATION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to silver nanodots / quantum dots by the use of naringenin. Particularly, the present invention relates to a method of preparation of the silver nanodots using a simpler green synthesis method. More particularly, the present invention relates to the preparation method and basic characterization that confirm silver nanodots / quantum dots sizes, nature, and unique green fluorescence properties in aqueous suspension.

[0004] BACKGROUND OF THE INVENTION

[0005] Synthesis and utilization of nanoparticles (nanodots / quantumdots / nanocluster) of different sizes with multi-variant surface features are big thrusts of recent years. Thus, the Nobel Prize in Chemistry in 2023 was given in chemistry for the discovery and development of quantum dots, a kind of nanoparticles where quantum confinement of light is possible and depending further on the shape and sizes optoelectronic behaviour of the materials also varies. The quantum dots, sometimes called nanodots, semiconductor nanocrystals, artificial atoms etc and, the sizes vary between 2-8 nm. Theoptical and electronic properties of these nano- size particles differ from those of bulk material due to different types of quantum mechanical effects. However, quantum dots (QD) / nanodots show properties intermediate between bulk semiconductors and simple atoms or molecules. Their optoelectronic behaviour also critically depends on the size and shape of the particle. Generally bigger size (5-6 nm) QDs emit light of longer wavelengths and produce colours such as orange, or red. However, particles of sizes of 2-5 nm give emission of shorter wavelengths such as blue and green light. Invented particle is of average size of ~3.5 nm and produced green fluorescence in a significant high level. Familiar silver QDs are usually composed of metal sulphide and / with other inorganic composite; oxidation state of silver of these quantum dots are often with non-zero oxidation state. However, invented particles are of silver dots and prepared with pi-electron rich flavanone, naringenin; and preferred to call them as silver nanodots.

[0006] Several earlier studies were carried out in the preparation of silver ‘nanoclusters’ (NC), sizes of which were smaller than the quantum dots / nanodots; they were nanometre to sub- nanometrescale. Thus, for ‘nanoclusters’ sizes approaching the Fermi-wavelength of an electron ~1 nm. In the bigger side of nanoscale, particles often named as ‘nanoparticles’, the sizes are bigger than 10 nm. Sizes of quantumdots / nanodots, however, are often in between, i.e. ~ 2-10 nm. For the preparation of some of these particles, nanoclusters (sizes close or 1 nm) chemical reduction and the photo -reduction are the most commonly used methods. The commonly using reducing agents are sodium borohydride, ascorbic acid or ultraviolet light irradiation reduction. Amino acids, enzymes, DNA or polysaccharides have also been used as stabilizing ligands for the preparation of Ag nanocluster. Similarly, silver ‘nanoparticles’ (sizes are generally bigger than 10 nm) are also usually prepared by the chemical reduction of silver salts precursors with strong reducing agents like sodium borohydride, formaldehyde, ammonia, or, similar other reducing agents. The toxicity of these reagents often limit the use of resulting nanoparticles in biological applications. To alleviate this, different methods based on the treatment of aqueous solutions of silver salt. Salts with laser light, ultrasonic waves, gamma rays, ionizing or ultraviolet (UV) light radiations are used as alternative preparation methods.

[0007] An important issue with synthesized nanoparticle of different sizes and nature are their stabilization in aqueous suspension. Without stabilization, these particles strongly interact with each other and aggregate irreversibly to reduce their surface energy. A proper stabilizing scaffold is thus indispensable. Surface covered molecules (scaffolds), not only provide the stability from coagulation / agglomeration, but it also often module opto -electronic and biochemical behaviours of the particles. Scaffolds that allowed stabilization of few-atom metal / silver nanoclusters was earlier mentioned already in the 1970s, it was the cryogenic noble gas matrix.

[0008] References may be made to Journals “Science 1996, 274 (5291), 1353-1355” and “Inorg. Chem.1978, 17 (1), 155-163”, which pioneered the production of matrix-isolated metal nanoclusters. Also zeolites as scaffolds for nanoclusters were greatly investigated. Silver nanoclusters have unique optical properties: produce significantly high luminescence (fluorescence), and some of the nanocluster has strong ability to convert light energy into heat with high efficiency. Fluorescence and other optoelectronic properties of nanoclusters however, depend on the sizes of the particles and the scaffold that stabilizes it. Stabilizing agents, for instance, carboxylic acid groups containing molecules usually produce fluorescence in the red region. The amine containing groups as a stabilizing partner often found to produces emission in the yellow range. Antimicrobial behaviour of silver nanocluster also depends on the surface morphology (size, shape, surface charge), surface coating, and the chemical nature of the stabilizing ligand.

[0009] For several reasons current thrust is to find organic scaffolds for fluorescent silver nanoclusters / nanoparticles. These are important as the interaction between the ligands and nanoclusters can be adjusted to finely tune the spectroscopic properties. For example, different form DNA sequences were used to synthesize silver nanoclusters that emit from the blue to near infrared region. Earlier investigations noted that there are usually have been used thiol (- SH), amine (-NH2), protein or etc. for synthesizing these fluorescence silver nanoparticles.

[0010] References may be made to Journal “J. Am. Chem. Soc.2009, 131 (39), 13972-13980” which used cyclohexylamine for stabilizing the fluorescence silver nanoparticle.

[0011] References may be made to Journal “J. Chem. Educ.2020, 97 (1), 239-243”, which synthesized silver nanoclusters with the help of thiol (-SH) containing dihydrolipoic acid (DHLA).

[0012] Thus, the formation and stabilization of silver nanoclusters / nanoparticles in solution have been accomplished in various ways. However, the method and the product, a silver nanodots prepared with flavanone, naringenin aromatic ring is novel and not reported; both the product (silver nanodots by using naringenin) and the method of its preparation are new and novel. The invented material is small enough where quantum confinement of light produces green fluorescence upon interaction with UV / deep violet (340-370 nm) light. The sizes (average size 3.5nm) are much below the ordinary silver nanoparticles, however, bigger than nanoclusters which are often of sub-nanometer scale. The scaffolds used for the nanodots particles are aromatic small flavanone molecule, naringenin that contains three hydroxyl groups and aromatic rings. Naringenin is easily available and not used earlier for the preparation of nanodots. The invented material was prepared by milled reduction of Ag+ with OH groups those are attached to aromatic rings of naringenin. The method is one pot synthesis at room temperature and involved no major hazardous chemicals (except dilute aqueous sodium hydroxide solution).

[0013] OBJECTS OF THE INVENTION Main object of the present invention is to provide silver nanodots / quantum dots by the use of naringenin.

[0014] Another object of the present invention is to provide a method of preparation of the silver nanodotswhich is similar in sizes of quantum dots but produce high green fluorescence.

[0015] Yet another object of the present invention is to establish a preparation method of the silver nanodots using naringenin, a flavanone as a mild reducing and stabilizing agent.

[0016] Yet another object of the present invention is to analyse the final productivity of these silver nanodots by using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES).

[0017] Yet another object of the present invention is to carried out size and shape analysis of the silver nanodots by transmission electron microscopy (TEM).

[0018] Yet another object of the present invention is to measure the spectroscopic signature (excitation and emission) of nanodots by UV-Vis spectroscopy and fluorescence spectrophotometer.

[0019] Yet another object of the present invention is to determine the relative fluorescence quantum yield of the produced nanodots.

[0020] Yet another objectof the present invention is to measure the fluorescence lifetime of the produced nanodots.

[0021] SUMMARY OF THE INVENTION

[0022] Accordingly, present invention provides green fluorescent silver nanodots, wherein the size of the silver nanodots with average size of 3.5 nm.

[0023] In an embodiment of the present invention, the silver nanodots exhibit significant fluorescence with a peak maximum at -532 nm with excitation at 365 nm.

[0024] In another embodiment of the present invention, the relative fluorescence quantum yield in aqueous solution is - 2.6 times higher than the fluorescence of l-Anilino-8-naphthalene sulfonate (ANS). In yet another embodiment of the present invention, the fluorescence lifetime of the silver nanodots is -2.37 ns±().l ns.

[0025] In yet another embodiment, present invention provides a process for preparation of silver nanodots by use of naringenin comprising the steps of: i. dissolving solid naringenin in ethanol to obtain stock naringenin solution; ii. adding the stock naringenin solution as obtained in step (i) to water followed by stirring in the range of 3000 rpm to 4000 rpm on hot plate at room temperature in the range of 25°C to 30°C for a period in the range of 10-15 min to obtain aqueous naringenin solution; iii. dissolving silver nitrate in water to obtain stock silver nitrate solution; iv. mixing stock silver nitrate solution as obtained in step (iii) with the aqueous naringenin solution as obtained in step (ii) for a period in the range of 5 - 10 min followed by stirring to obtain a mixture; v. adjusting the pH of the mixture as obtained in step (iv) to 8.0-8.5 followed by stirring for a period of 12 to 15 h to obtain a reaction mixture having pH in the range of 7.4- 7.6; vi. centrifuging the reaction mixture as obtained in step (v) for a period in the range of 10 -30 min at 15000 - 18000 rpm to obtain a supernatant; vii. ultra-centrifuging the supernatant as obtained in step (vi) at 60000-65000 rpm for a period in the range of 2 -3 h to obtain an upper portion (solution phase) as final supernatant; viii. filtering and dialyzing the final supernatant solution as obtained in step (vii) in water for a period in the range of 48-60 h at a temperature in the range of 4°C-10°C to obtain highly suspended purified silver nanodots suspension.

[0026] In yet another embodiment of the present invention, mixture in step (iv) is prepared in the volume parts of silver nitrate solution (100 mM) and naringenin (20 mM) aqueous solution with the ratio in the range of 6: 1.

[0027] In yet another embodiment of the present invention, the molar ratio of silver nitrate and naringenin in the reaction mixture is in the ratio is 30:1 (error in concentration less than ±5%). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 represents the left Panel (A) that shows the clear suspension of silver nanodots under visible (white LED) light and right side Panel (B) that shows the green fluorescence upon excitation at 365 nm light using UV lamp.

[0029] FIG. 2 represents the spectroscopic signature and properties of silver nanodot product. A. UV- Vis absorbance spectra of the nanodot suspension in spectroscopic grade HPLC water. Two absorption bands were observed at 276 nm and 322 nm in the prepared solution. B. Excitation spectra (dash-dot) where Xmax of excitation at - 368 nm (associated with band gap energy) and emission spectra (solid black) Z,max of emission at - 532 nm obtained by exciting at 365 nm wavelength. C. Fluorescence spectra of the nanodot in aqueous suspension, obtained upon excitation at different wavelengths: 365 nm (a), 360 nm (b), 355 nm (c), 350 nm (d), 345 (e) and 340 nm (f) . It always shows A,max of emission at -532 nm.

[0030] FIG. 3 represents (A)Field Emission Gun-Transmission Electron Microscope image of the synthesized silver nanodots. (B). Size distribution graph based on the TEM analysis.

[0031] FIG. 4 represents the fluorescence lifetime of silver fluorescence nanodots in water (circle) with fit (black line) excitation at -365 nm. Instrument response also included (square).

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Present invention provides silver nanodots / quantum dots by the use of naringenin where silver atoms clumped and produce quantum dot like nanostructure of average diameter of -3.5 nm.

[0034] The aqueous suspension of the nanodots is fairly stable in room temperature. It produces strong green fluorescence with much longer fluorescence lifetime -2.37 ns. Shows large Stokes shift in its luminescence / fluorescence behavior. The product produces unique green fluorescence with high quantum yield (-2.6 times than ANS in water).

[0035] A simple flavanone, naringenin, an aromatic organic molecule, that contains three OH groups is used both as a mild reducing and stabilizing agent for the preparation of the quantum dots. The method is very simple, cost effective and the production can be made at room temperature (25°C). The preparation method was close to a green synthesis method; used no common and strong / harsh reagent like sodium borohydride, ascorbic acid surfactant, thiol containing reagent, protein solutions or ultraviolet light irradiation reductions which are earlier reported for other kind of nanoparticles. It does not require any protein, amine or thiol containing compounds. Simply the alkaline solution of silver nitrate was treated with naringenin, incubated at 25°C for 12h under 3000 rpm using magnetic hotplate stirrer and it produced the quantum dot size (~3.5 nm) nanodots.

[0036] Silver nanodots are prepared with aromatic compound that contain two benzene like 7t-electron systems. Polyhydroxy groups of naringenin in alkaline condition reduce silver ion and accumulate few silver atoms to produce unique nanodots that gives green fluorescence.

[0037] The excitation peak appeared at 368 nm and the fluorescence peak maximum at 532 nm, a large stock shift (difference in excitation energy and emission energy) are the unique features of the silver nanodots.

[0038] EXAMPLES

[0039] Following examples are given as a way of illustration only and should not be construed to limit the scope of the present invention.

[0040] EXAMPLE 1

[0041] A stock of 20 mM naringenin was made by dissolving solid naringenin (Sigma-Aldrich, Catalogue No- N5893-5G) in minimum amount of absolute alcohol (obtained from Merck Millipore, CAS No: 64-17-5) followed by addition of HPLC grade water. 100 pL of 20 mM stock naringenin solution is added to 19 mL spectroscopic grade HPLC water in a round bottom flask (size 100 mL). The solution was stirring by magnetic bead at 3000 rpm on hot plate at room temperature (25 °C). After 10 minutes 600 pL of 100 mM silver nitrate solution is mixed with the naringenin solution. The stock 100 mM solution of AgNOs (purchased from ACROS Organics) was prepared by dissolving required amount of AgNOs in HPLC water. The naringenin and AgNOssolutions were mixed for five minutes and the above stirring condition. After 5 minutes 25 uL 1(M) NaOH solution was added to above mixture. The reaction was continued for 12 h. The initial pH was 8.3 and the final pH of the solution was ~ 7.4. The reaction mixture was subsequently centrifuged for 10 min at 15000 rpm and the supernatant was collected. The supernatant was again ultra-centrifuged at 60000 rpm for 2 hours by using Sorvall Ultracentrifuge, T890 rotor (Sorvall WX ULTRA90); the upper portion (solution phase) was collected. The final supernatant solution was passed through 0.22 Millipore syringe filter and dialyzed in water using 3.5 kDa ‘D tube dialyzer’ (Merck, Novagen, Cat no-71742- 4) for 48 hours at 4 C temperature and obtained the highly suspended purified silver nanodots suspension. The spectroscopic signature (given below) was recorded with the product after ultracentrifugation, however the nature of the spectra of the product obtained after dialysis was very similar.

[0042] The reaction mixture was prepared in the volume parts of silver nitrate solution (100 mM) and naringenin (20 mM) aqueous solution with the ratio of 6: 1. Final concentration, the ratio of silver nitrate and naringenin in the reaction mixture was 30: 1.

[0043] The method for preparing the silver nanodots, is characterized in that the silver nitrate solution and naringenin was under agitation (3000 rpm) under magnetic stirrer for 12 h agitation under room temperature (25°C). Synthesized nanodot was obtained and purified by centrifugation, ultracentrifugation and followed by dialysis.

[0044] The synthesized silver nanodots exhibit significant fluorescence with a peak maximum at -532 nm with excitation at 365 nm. The calculated fluorescence quantum yield in aqueous solution was - 2.6 times higher than the fluorescence of l-Anilino-8-naphthalene sulfonate (ANS).

[0045] The average fluorescence lifetime of the produced in aqueous suspension is 2.37 ns.

[0046] According to Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES) it was established that the silver nanodot in the aqueous suspension ~lmg / L.

[0047] At low temperature i.e at 4°Cthe yield was significantly less compared to yield at room temperature (25°C). The fluorescence intensity (signature of the product) was very low compared to the product obtained at 25°C. At higher temperature, such as at 40 °C, the reaction was very fast and the products are unstable and quickly coagulated. The composition is chosen for good yield at room temperature (25°C-30°C). The productivity of nanodots by using different molar concentration ration of starting materials i.e varying the naringcnin: AgNOs ratio has been measured. All the reactions have been done at room temperature (25°C). At naringenin to AgNOsmolarconccntration ratio of 1:3, 1:5 no significant amount of product could be obtained; from fluorescence intensity it is found negligible. With relative increases of concentration of AgNOs. fluorescence intensity due to the formation of the product found to increases implying the gradual increase of the yield. The productivity at 1:30 concentration ratio was maximum. Increasing the concentration ratio further caused quick aggregation (unwanted products).

[0048] In the starting condition, just after mixing of the solutions measured pH is (8.0-8.5) and final pH of 7.4-7.6(pH noted after incubation for 60 minutesjshow maximum fluorescence with maximum productivity. Productivity of the green nanodot in other pH conditions was less.

[0049] So naringenin mediated fluorescent particle synthesis is temperature dependent, concentration dependent and pH dependent.

[0050] 1. Characterization of synthesized silver nanodots

[0051] A. UV Vis spectroscopy and fluorescence spectroscopy analysis

[0052] The absorption bands were found in 276 nm, 322.With excitation by 365 nm wavelength light high green emission was found in 532 nm. At a constant emission wavelength of 532 nm, an excitation spectra were recorded where kma is ~ 368 nm (FIG. 2B). These quantum dots showed green fluorescence at 532 nm with the change of different excitation wavelengths from 340 nm to 365 nm (FIG. 2C). It produces strong fluorescence with average fluorescence lifetime of 2.37 ns.

[0053] B. Size measurement by transmission electron microscope (TEM)

[0054] Size and morphological analysis of these nanodots are analyzed by JEOL JEM-2100F ultra high-resolution Field Emission Gun Transmission Electron Microscope (UHR-FEG TEM). Carbon coated copper 300 mesh grid has been used for sample deposition and recorded by Gatanorius CCD camera. From TEM images it was observed that fluorescent quantum dots are usually very small and uniform in size and spherical shape visible in FIG. 3A. Size of these nanodots around ~ 3.5 nm in average diameter obtained from FIG. 3B. These particles are quite stable and easily synthesized. They show strong green fluorescence upon excitation at 365 nm and produce a large stocks shift.

[0055] C. Fluorescence life time measurement

[0056] Fluorescence lifetime has been done by using time -correlated single photon counting set up from Horiba Jobin- Yvon where an excitation wavelength of 365 nm was applied by Nano-led light source. The luminescence decay data were collected on a Hamamatsu MCP photomultiplier (R3809) and were analyzed by using Origin 8 software. The lifetime decay curve was fitted by double exponential functions. In this case, the average fluorescence lifetime of synthesized nanodots in water was -2.37 ns (FIG. 4).

[0057] D. Fluorescence quantum yield analysis

[0058] Determination of quantum yield is calculated by using l-aniIino-8-naphthalene sulfonate (ANS) as an emission standard. Here selection of the reference is very important for spectral overlapping observation. Absorbance and emission spectra were recorded of ANS and nanodots. We calculated quantum yield by using integrated fluorescence vs absorbance curve. We observed that synthesized silver nanodots 2.6 times greater quantum yield to ANS by using the following equation

[0059] Where QA^VD is quantum yield of silver nanodots (AgND) and QAVS is quantum yield of reference sample (here ANS carry out for standard). lAgND is the integrated fluorescence intensity of AgND and IANS is the integrated fluorescence intensity of ANS. AANS and AAgND is the absorbance of ANS and AgND, respectively.

[0060] ADVANTAGES OF THE INVENTION

[0061] • The flavanone compound is easily available. The quantum confinement of light in this size silver particle produces high green fluorescence which could have several uses. It also shows large stokes shift. • For the preparation of metal nano-clusters (sizes close or <1 nm) chemical reduction and the photo-reduction are the most commonly used methods. The commonly using reducing agents are sodium borohydride, ascorbic acid or ultraviolet light irradiation reduction, amino acids, enzymes, DNA or polysaccharides have also been used as stabilizing ligands for the preparation of Ag nanocluster. It is also noted that earlier investigations used thiol (-SH), amine (-NH2), protein or etc. for synthesizing this fluorescence silver nanoparticles (not nanodots). No such hazardous chemical in the synthesis method has been used in the present invention.

[0062] • The method is very mild and kind of one-pot synthesis at room temperature. It does not require any protein, amine or thiol containing compounds. Polyhydroxy groups of naringenin in alkaline condition reduce silver ion and accumulate few silver atoms as nanodots.

[0063] • OH groups attached to the aromatic molecule act as reducing agent and molecule armored the nanodot sphere and prohibits its coagulation. In addition, aromatic pi electronic system may contribute to optoelectronic behavior of the nanodots.

[0064] The aqueous suspension was quite stable at room temperature (25°C).

Claims

WE CLAIM1. A green fluorescent silver nanodots, wherein the size of the silver nanodots with average size 3.5 nm.

2. The silver nanodots as claimed in claim 1, wherein the silver nanodots exhibit fluorescence with a peak maximum at 532 nm with excitation at 365 nm.

3. The silver nanodots as claimed in claim 1, wherein a relative fluorescence quantum yield in aqueous solution is 2.6 times higher than the fluorescence of l-Anilino-8-naphthalene sulfonate (ANS).

4. The silver nanodots as claimed in claim 1, wherein a fluorescence lifetime of the silver nanodots is 2.37 ns±0.1 ns.

5. A process for preparation of silver nanodots by use of naringenin, comprising the steps of: i. dissolving solid naringenin in ethanol to obtain a stock naringenin solution; ii. adding the stock naringenin solution as obtained in step (i) to water followed by stirring in the range of 3000 rpm to 4000 rpm on a hot plate at room temperature in the range of 25°C to 30°C for a time period in the range of 10-15 minutes to obtain an aqueous naringenin solution; iii. dissolving silver nitrate in water to obtain a stock silver nitrate solution; iv. mixing the stock silver nitrate solution as obtained in step (iii) with the aqueous naringenin solution as obtained in step (ii) for a time period in the range of 5-10 minutes followed by stirring to obtain a mixture; v. adjusting pH of the mixture as obtained in step (iv) to 8.0-8.5 followed by stirring for a time period of 12 to 15 hours to obtain a reaction mixture having pH in the range of 7.4-7.6; vi. centrifuging the reaction mixture as obtained in step (v) for a time period in the range of 10-30 minutes at 15000 - 18000 rpm to obtain a supernatant; vii. ultra-centrifuging the supernatant as obtained in step (vi) at 60000-65000 rpm for a period in the range of 2 -3 hours to obtain an upper portion or solution phase as final supernatant;viii. filtering and dialyzing the final supernatant solution as obtained in step (vii) in water for a time period in the range of 48-60 hours at a temperature in the range of 4°C- 10°C to obtain highly suspended purified silver nanodots suspension.

6. The process as claimed in claim 5, wherein the mixture in step (iv) is prepared in the volume parts of silver nitrate solution (100 mM) and naringenin (20 mM) aqueous solution with the ratio in the range of 6: 1.

7. The process as claimed in claim 5, wherein the molar ratio of silver nitrate and naringenin in the reaction mixture is in the ratio is 30: 1.

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