Introduction: the pain and swelling of rheumatoid arthritis, and

Introduction:Gold is a chemical element with symbol Au (from Latin: aurum) and atomic number 79. In its purest form, it is a bright,slightly reddish yellow, dense, soft, malleable and ductile metal.

The pure metal melts at 1063°C and boils at 2966°C.It has an atomic weight of196.967 with a density of 19.32 g cm?3at 20°C. Its beauty and rarity has led to the use in jewelry and in coinage. Chemically, gold is a transition metal and a group 11 element.

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Common oxidation state ofgold is +III and +I, although it can show oxidation states from –I to + V. Itis one of the least reactive chemical elements, and is solid under standard conditions. The metal therefore occursoften in free elemental (native) form.

Gold resists attacks by individual acids, but it can be dissolved by aqua regia (1:3 mixture of nitric acid andhydrochloric acid). The acid mixture causes the formation of a soluble gold tetrachloride anion. Gold metal also dissolvesin alkaline solutions of cyanide, which are used in mining and electroplating. It is insoluble in nitric acid, which dissolves silver and base metals.Its insolubility in nitric acid is used to refine it from silver or other basemetals. Gold because of its malleability andductility is used in various purposes such as infra-red shields, heat resistantsuits.Gold as we know is probably the most ancientadministered medicine.

 In the 19th century gold was used as a “nervine,” therapy fornervous disorders. Depression, epilepsy, migraine, and glandular problems such as amenorrhea and impotence were treated with the help of gold. 1 Some gold salts do have anti-inflammatory properties and at present two are stillused as pharmaceuticals in the treatment of arthritis and other similarconditions in the US (sodium aurothiomalate and auranofin). These drugs have been explored as ameans to help to reduce the pain and swelling of rheumatoid arthritis, and also (historically) against tuberculosis and some parasites 2 Gold Nanoparticles because of their uniqueoptical, electronical and molecular recognition properties are used widely inbiological fields for various purposes.  Nanoparticles can be produced through various methods.

Chemical processare the most popular methods for nanoparticle production but some of thechemical processes use toxic chemicals and hence can not be used for biologicalpurposes. Synthesis of Au nanoparticles using microbial, plant, plant extractsand enzymes are the suggested eco-friendly ways. There are many microbes whichare known to produce nanostructured mineral crystals and metallic nanoparticleswith properties similar to chemically synthesised materials, while exercisingstrict control over size, shape and composition of the particles.  A fungusVerticillium sp. when exposed to aqueous AuCl4? ions results in reduction of the metalions and formation of gold nanoparticles of around 20 nm diameter.3 The gold nanoparticles formed are reported to be on both the surface andwithin the fungal cells (on the cytoplasmic membrane) with negligible reductionof the metal ions in solution.4 Manybacteria such as Thermomonospora sp, 5  Rhodococcus sp.

, Rhodopseudomonascapsulate ,6Pseudomonas aeruginosa, 7Delftia acidovorans 8 produce gold nanoparticles by the process ofreduction of gold salt. Rhodococcus sp. is reported to produce goldnanoparticle intracellularly 9 while others produce extracellularly. Plantextract have also been used for the preparation of gold nanoparticle. Forexample, extract of Cymbopogon flexuosus formnanoparticles extracellularly 10 while live alfalfa plant produce intracellularly 11.  One of the advantage of using plant fornanoparticle production instead of using bacteria and fungi is the lack ofpathogenicity of plant nanoparticles. These organisms probably formnanoparticles as a surviving mechanism adapted by the organism to cope with thehigh levels of metal in the environment. The mechanisms may involve alterationof the chemical nature of the organisms so that the metal no longer causestoxicity.

In this process the metal can be reduced and nanoparticle can beproduced. Thus, it can be said that the production of nanoparticle isby-product of resistance mechanism evolved by the organism against the specificmetal, and therefore this process is exploited for the green synthesis ofnanoparticle. Todate, there are numerous techniques for synthesizing nanoparticles. However,these techniques fall into two broad approaches and can be defined as either atop down approach or a bottom up approach 12.

The top down approachstarts with a material of interest, which then undergoes size reduction viaphysical and chemical processes to produce nanoparticles. Importantly,nanoparticles are highly dependent on their size, shape, and surface structureand processing tends to introduce surface imperfections. These surfaceimperfections can significantly impact on the overall nanoparticle surfacephysicochemical properties 13. In the bottom up approach, nanoparticlesare built from atoms, molecules and smallerparticles/monomers 1415 16. In either approach, theresulting nanoparticles are characterized using varioustechniques to determine properties such asparticle size, size distribution, shape, and surface area. This is ofparticular importance if the properties of nanoparticlesneed to be homogeneous for a particular application.

17Duringnanoparticle synthesis the gold salt is reduced by any reducing agent whetherchemical or biological which results into change in colour. This is the firstqualitative indication that the nanoparticle is formed. For further study ofnanoparticles various spectroscopy and microscopy techniques are being usedsuch as UV-Visible spectroscopy, dynamic light scattering (DLS), atomic forcemicroscopy (AFM), transmission electron microscopy (TEM),scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS),powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy(FT-IR), and Raman spectroscopy. Microscopy based techniquessuch as AFM, SEM and TEM obtain data from images taken of the nanoparticles.

Inparticular, both SEM and TEM have been extensively used to determine size andmorphological features of nanoparticles. While spectroscopy based techniquessuch as UV-vis, DLS, XRD, EDS, FT-IR, determine data related to composition,structure, crystal phase and properties of nanoparticles. In case UV-Visible spectroscopywavelengths between 300 and 800 nm are generally used for characterizingmetallic nanoparticles ranging in size from 2 nm up to around 100 nm 18. Forexample, gold (Au) nanoparticles are generally detected by the presence ofpeaks between 500 and 550 nm.19  DLS spectroscopy is used to determine sizedistribution and quantify the surface charge of nanoparticles suspended in aliquid.19 20 Researchershave shown that different pH can also affect the formation of nanoparticles.Importance of pH in the biosynthesis of colloidal gold using alfalfa biomasshave been shown and it is being concluded that the size of nanoparticleschanges with the change in pH. 21 Large nanoparticles are produced at lowerpH(2-4) 22.

 In case of Avenasativa, it has been shown that formation of gold nanoparticle is highlydependent on the pH value. At pH 2, large-sized nanoparticles (25?85 nm) arereported, although in low quantity. At pH 3 and 4, smaller-sized nanoparticlesin a large quantity are reported. They speculated that at low pH (pH 2), thegold nanoparticles prefer to aggregate to form larger nanoparticles rather thanto nucleate and form new nanoparticles. In contrast, at pH 3 and 4, morefunctional groups (carbonyl and hydroxyl) are available for gold binding; thus,a higher number of new Au (III) complexes would bind to the biomass at the sametime that will nucleate separately and form nanoparticles of relatively smallsize.

23 Temperaturecan also affect the synthesis of nanoparticle. It is reported that at highertemperatures rate of formation of nanoparticle increases. 24 Theyhave also reported synthesis of nanorod and platelet-shaped gold nanoparticlesat higher temperatures and formation of spherical-shaped nanoparticles at lowertemperatures. Therefore, it can be concluded that temperature is also one ofthe crucial factors which determine the size and shape of nanoparticles. Theexact mechanism how the nanoparticles are formed is not understood still.

It isrequired to understand so that controlled and definite size and shapednanoparticle can be synthesized.    Objectives :1.      To screen small biomolecules which can form AuNp.2.      To understand how biomolecules are effecting the formation of AuNp.

3.      To understand how the AuNp formed are degraded when given tobiological system.Material and Method:1.      HAuCl4 HAuCl4 and all the nucleotides and nucleosides – Adenosine,AMP, ADP,ATP, Cytosine, CMP, CDP, CTP,Guanosine, GMP, GDP , GTP, Uridine,UMP,UDP and UTP were bought from Sigma Aldrich. Synthesis of AuNP:AuNp was synthesized by mixing HAuCl4 with the respective nucleotide/nucleoside and then mix it for 5 mins. Characterization of Gold NanoparticleUV-visspectrophotometerThe reduction of pure HAuCl4 wasmonitored using UV-vis spectroscopy. The colloid Au solution was scannedusing a UV spectrophotometer (SpectrumX-5).Transmissionelectron microscopyMicroscopic imageof Au-NPs was studied using Transmissionelectron microscopy (TEM).

Analysis of the sample was done using a Technai TM G2 spirit (FEI, The Netherlands) with Gatan CCDdigital camera operated at an accelerating voltage of 80 kV. A drop of the solution was placed on carbon-coatedcopper grid. The grid was then dried in desiccators by keepingovernight at room temperature.

Scanning electron microscopySEM analysis of the sample was done using Quanta450 (FEG, FEI, The Neetherlands).Zetapotential and Size measurementZeta potential (?)and surface conductivity (?sc) were measured using a Malvern Nano ZSinstrument using phase analysis light scattering technique. Diluted sampleswere sonicated for 5 min prior to measurements.

Results:      1. AuNp prepared withNucleotides and nucleosidesHAuCL4 with Adenosine, AMP, ADP,ATP, Guanosine, GMP, GDP and GTPchanges colour within 5 mins indicating the formation of AuNp. (Fig. 1 a)Au-GDP and GTP shows peak at 460 nm (Fig. 1 b) Fig.1 (a) Au Nanoparticle prepared with different nucleotides andnucleosides( Adenosine, Cytidine, Guanosine, Uridine, AMP, CMP, GMP,UMP,ADP,CDP,GDP,UDP,ATP,CTP,GTP,UTP). (b) UV spectra of Au nanoparticles produced with different nucleotidesand nucleosidesDLS measurement: Sample name Z-Average d.nm.

Pdi A 872.8 0.568 C 3641 0.973 G 2065 0.

744 U 1.31E+04 0.709 AMP 5401 0.883 CMP 6005 0.968 GMP 1898 0.985 UMP 6389 1 ADP 1863 0.

783 CDP 7810 0.947 GDP 1699 0.869 UDP 1906 1 ATP 649 0.532 CTP 1721 0.

858 GTP 908.7 0.59 UTP 2748 0.

835           GDP-AuNP: functioningas a UV- Sensor GDP-AuNP   Au (C) Au (C) Au (C) GDP-AuNP   GDP-AuNP   UV                                                            Fig-2.aGDP-AuNpdecolourises when subjected to heat ( Table 2). The decolourised solution isstable at 4? but it changes to pink colour when exposed to UV light.

(Fig-2.a)UVSpectra  shows that the GDP-AuNp prepared(immediately)gives peak at 490 nm showing the presence of nanocluster. but after treatmentof UV, a distinct peak at 550 is observed which indicates the presence of AuNp.Fig.-2.

b.Zetapotential of GDP_AuNp (Table-1) shows that the nanoparticle after UV exposureare stable. Their average size ranges from 185nm to 73 nm to 59 nmrespectively. Their polydispersity index is 0.

2 to 0.4 and 0.4 respectively.

                                                              Fig. 2.bEstimation of hydrodynamic radius and charge by DLS. SAMPLE GDP-AuNp (freshly prepared) GDP-AuNP (decolourised) GDP-AuNp  (UV treated) DLS Z-Average (d.nm) 185.

1 73.51 59.64 PDI 0.

230 0.404 0.44 ZETA POTENTIAL (mV) -16.8 -29.1 -37.

5 Table-1Time required by GDP-AuNp to decolourise after their treatment withtemperature. TEMPERATURE ?C TIME REQUIRED FOR DECOLOURISATION 30 33 hours 34.8 15 hours 39.

3 10 hours 45.3 4 hours 49.9 3 hours 53 2 hours 30 mins 55 1 hour 50 mins 59.9 50 mins 64.3 45 mins 70.

3 28 mins 75 20 mins 78.1 14 mins 80 11 mins Table-2TEM, SEM of GDP-AuNPTEM shows that the size of GDP-AuNP (preparedimmediately) ranges from 1.52 nm to 4.0 nm while that of GDP-AuNp afterdecolourisation is 3-4nm and after UV exposure it is                   Fig.-31-Initial                                1.      After decolourisation             3-After treating with UV             Fig.

-3        Effect of different pH of HAuCl4 on GDP-AuNpformation:With increase of pH colour intensifies till ph 5 andthen starts declining. Fig.4.

a. and UV spectra shows increasing curve till ph 7and then it also starts declining.Zeta potential shows that except for GDP-AuNp at pH 1all are stable. Table -3.  Control pH 1    2       3         4        5         6       7             8        9     10   Fig.

4 a Fig.4.b.Estimation of hydrodynamic radius and surface charge with the help ofDLS    Sample Name     Z-Average (d.

nm)      Pdi     Zeta potential HAuCL4  pH1 315.4 0.445 -20.

3 HAuCL4  pH2 401.3 0.385 -37.

9 HAuCL4  pH3 157 0.219 -44.9 HAuCL4  pH4 88 0.243 -36.

1 HAuCL4  pH5 76.73 0.24 -46.6 HAuCL4  pH6 251.

5 0.329 -52.3 HAuCL4  pH7 161.6 0.227 -48 HAuCL4  pH8 128.5 0.

207 -49.5 HAuCL4  pH9 222.4 0.287 -20.

6 HAuCL4  pH10 238.9 0.381 -53.9 Table-3 Effect of different conc. of HAuCL4 on GDP- AuNpformation:By increasing anddecreasing the concentration of HAuCl4, there is no effect on colour but UVSpectra shows distinct peak at 440 nm (10 mM conc.).

Fig. 5.a,b.  Zeta potential shows that GDP-AuNp preparedwith effective conc. of HAuCl4 -10mM is the most stable. Table -4  GDP (C) HAuCl4 (mM)                        25          10           5 Fig.

5.a. Fig.5.b.Estimation of hydrodynamic radius and surface charge with the help ofDLS   Sample Name      Z-Average         (d.

nm)  Pdi          Zeta potential HAuCL4 25 mM 115.7 0.076 -38.

8 HAuCl4 10 mM 856.6 0.77 -51 HAuCl4 5 mM 63.37 0.144 -23.7 Table-4.

DISCUSSION:HAuCL4 when treated with Adenosine, AMP, ADP, ATP,Guanosine, GMP, GDP and GTP changes colour within 3-5 mins. indicatingformation of gold nanoparticle. But GDP nanoparticle when subjected to heat fora specific period of time becomes colourless and when this colourless solutionis exposed to UV, it changes to pink solution. Zeta potential shows that thenanoparticle after UV exposure becomes stable.TEM shows that the size of nanoparticle in all the threecases is less than the 10 nm. Increase in pH of HAuCl4 till pH 5 stabilizes thethe nanoparticle but above that stability starts declining. Nanoparticle prepared by HAuCl4 with effective concentrationof 10 mM was most stable, increasing or decreasing the concentration led toinstability.