UVA mediated synthesis of gold nanoparticles in

UVA mediated synthesis of gold nanoparticles in pharmaceuticalgrade heparin sodium solutions.

M. del P. Rodríguez Torres1 ,L.A. Diaz-Torres1 ,M. Olmos López2 ,P Salas3,Clara Gutiérrez3. 1 Grupo de Espectroscopia de Materiales Avanzados y Nanoestructurados (EMANA), Centro de Investigaciones en Óptica A. C., León, Gto. 37150 México. 2 Centro de Investigaciones en Óptica A. C., León, Gto. 37150 México. 3 Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, A.P. 1-1010, Querétaro, Qro. 76000, México.

ABSTRACT A photochemical-based method in which UVA light (λ=366 nm) is used for synthesizing gold nanoparticles is presented by irradiating gold (III) chloride hydrate (HAuCl4) in the presence of pharmaceutical-grade heparin sodium (PGHEP) as a reducing and stabilizing agent in aqueous solution. Different HAuCl4 to PGHEP concentration ratios were exposed to UVA for up to seven hours. The as-synthesized nanoparticles were characterized by UV-VIS and Raman spectroscopy, transmission electron microscopy (TEM), and pH measurements. The synthesized AuNPs present spherical as well as anisotropic shapes, such as oval, triangular, hexagonal sheets, rods, and some other faceted forms, with dimensions ranging from 20 nm to 300 nm. All obtained products show good temporal stability in solution. Surface plasmons differ when varying HAuCl4 to PGHEP concentration ratio. The obtained samples exhibit two absorption peaks, one in the region between 500-600 nm, and another one in the near-IR between 900-1200 nm; both peaks shift to longer wavelengths and increase their absorption intensity as the HAuCl4 to PGHEP concentration ratio increase. TEM images show the change in nanoparticles yield as well as the shape and sizes change depending on HAuCl4 to PGHEP concentration ratio variation. Ph measurements suggest that acidic media promote anisotropic nanoparticle formation. Raman spectroscopy was used to find out which heparin sodium main groups attached to the nanoparticles surface, and in what amount. In summary, it is found that when modifying the reactants concentrations and keeping the UV exposition time as the only fixed parameter, different nanoparticles with distinctive characteristics can be attained. Keywords: Gold nanoparticles, colloidal dispersions, biomolecules, polymers, photochemistry, UV light,TEM,UV-Vis spectroscopy.

1. INTRODUCTION Interest in nanomaterials has been growing in the last years in any of their forms, for example as carbon-based (fullerenes and nanotubes), dendrimers (providing interior cavities for other molecules), composites and metal-based materials such as quantum dots, metal oxides and nanocolloids, these last ones being synthesized using different techniques: solvothermal reaction, sol-gel, micellar, structured media, metal vapor techniques ,seeding growth, sonochemistry, radiolysis and photochemistry1. This last mentioned technique is a very useful and novel one having advantages such as the spatial and temporal control. Photochemical techniques also depend on the generation of reducing species in the photodecomposition of molecules; some of them do not contain a reducing moiety before they are excited by light2,so in comparison with thermal methods this results in a new path for nanoparticle synthesis. Photochemical methods are top-down and bottom-up as shown in figure 1.

Plasmonics: Metallic Nanostructures and Their Optical Properties XI, edited by Mark I. Stockman, Proc. of SPIE Vol. 8809, 88092R · © 2013 SPIE · CCC code: 0277-786X/13/$18 · doi: 10.1117/12.2024441

Proc. of SPIE Vol. 8809 88092R-1

Figure 1.Photochemical synthesis paths for nanoparticle synthesis.

In this work, a photochemical method based on the reduction of a gold precursor, HAuCl4, using pharmaceutical-grade heparin (PGHEP), distilled water as a solvent and a home-made UVA (λ=366 nm) reactor to trigger the synthesis reaction is presented as a new, inexpensive technique way on the photochemistry trends to prepare gold nanocolloids with quasispherical and anisotropic shapes.Heparin-based nanoparticles have already been synthesized by thermal methods and getting spherical or quaispherical products mainly and modifying heparins structure4,5,6 which we did not. Heparin is a member of the glycosaminoglycans family, which are basically polysaccharides consisting of a repeating disaccharide unit, which in this case would be one consisting of 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine as shown in figure 2. Heparin is a highly sulfated material being that the reason why it has the highest negative charge of any known material. Commercial preparations such as the one we used are commonly between 7,000 and 25,000 Da3. Heparin is used as an anticoagulant and to treat pulmonary embolisms in Medicine.

Figure 2. Heparin disaccharide unit.


1.Experimental. Reagents and materials. A pharmaceutical-grade sodium heparin solution with a 5000 UI/mL concentration was obtained from Pisa Laboratories (Mexico), hydrogen tetrachloroaurate (HAuCl4) was obtained from Sigma-Aldrich. As the solvent, distilled water was used. All reagents were used without further purification. All volumes were measured exactly using a micropipette. Three different samples solutions were prepared in the following reagent order varying their concentrations: Distilled water was poured in a vial, then the metal precursor,HAuCl4 and last, heparin .The solution was stirred afterwards to homogeneize the components and finally it was poured in a 10 mm quartz cell. To trigger the reaction, a home-made UV reactor was used to irradiate the syntheses solutions, it is made of aluminium with three vertically-oriented UVA lamps, and on top of it there is a fan to avoid overheating inside. The quartz cell was placed inside it for seven hours.The arrangement used is shown in figure 3.

Figure 3. Heparin-based nanoparticles photochemical reaction procedure.

The synthesized samples were characterized by UV-Vis spectra were measured in a Perkin Elmer Lambda 900 UV/VIS/NIR spectrophotometer with using a 10-mm quartz cuvette, the measurements were done in air at ambient temperature.For ATR spectra measurement a Perkin Elmer Spectrum BX FTIR system was used. The pH changes were monitored by using a Conductronic pH120 pH meter. Transmission Electron Microscopy (TEM) was carried out in a JEOL model JEM-1010 operated at 80 kV using a carbon grid. TEM samples were prepared in the traditional way; an aliquot of the solution was dropped on a 3 mm diameter lacey carbon copper grid and then left to dry at room temperature for the solvent to evaporate.Finally, Raman measurements were taken with a Renishaw InVia Raman


microscope using the 785 nm laser-line, the samples were measured in solution. Heparin was analyzed directly as contained in its vial (5000 IU/mL) and the gold colloids were measured directly as well. Results. Absorbance spectra show different traits for the syntheses. Firstly, Sample 1 shows one peak at 542 nm which is big in optical density and a shoulder in the IR region at approximately 900 nm with weaker intensity indicating the presence of anisotropical nanoparticles of different sizes, mostly because of its width. In sample 2, the peak in the IR region shifts to shorter wavelengths (737 nm) and there is one at 532 nm indicating there might be either spherical or quasispherical products but of different sizes and in less amount than the nanoparticles in Sample 1 because of the lowering in absorbance. Finally, for Sample 3,the absorbance spectra is almost weak and undetectable, there is an almost flat bump at approximately 534 nm and the peak in the IR does as well at 741 nm, it has to be pointed out that for the first two samples, the plasmon peaks blue-shift, while in the case of the third one they red-shift, which might indicate that what might have happened was that the reagents concentrations were so low that firstly, there was not enough metal precursor nor heparin for the reaction as it was taking place, being that the reason for the spectrum weak absorbance signal because not many were produced and that the particles generated just reached a limiting size, that is not getting to have smaller sizes because of the lack of heparin.

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Wavelength (nm) Figure 4.Absorbance spectra of the synthesized nanoparticles. TEM images show in figures 5a to 5f are in agreement with UV-Vis spectra, offering besides some extra information. For Sample 1 TEM images, it is shown that anisotropic particles, for example, plates ranging from to , as well as some spherical and quasispherical products of in diameter. It can be seen that there are some polygons of in length and some trapezoidal nanoparticles, too, whereas in Sample 2, the products do not show the big plates in Sample 1 ,but it does have some triangles and polygons but in much less amount than sample 1 and another aspect is that very few rod-like products are present as well , which may be responsible for the IR peak decrement in the UV-Vis spectrum. Some spherical and quasispherical nanoparticles are aggregated, this could explain the broadness of the peak at 532 nm. Sample 3 shows nanoparticles which are quasispherical and in very little amount in accordance with their UV-Vis spectrum.


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Figure 5.TEM images for the three varying concentrations syntheses of nanoparticles.


pH measurements in Figure 6 show a decrease in value, from 6.99 to 5.79 ,indicating that heparin effectively acts as a reducing agent because it is injecting electrons during the reaction and since the initial solution normally shows a very light yellow tone and as the reaction is taking place the color changes until it reaches colors in purple shades, these ones go from very light for the lowest concentration up to a very intense one when the highest concentration is attained. All this indicating there has been a change in the oxidation state of gold from ionic to the zero-valent state in which nanoparticles are formed.It has been proved that acid media promotes nanoplate formation7.

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Irradiation time. Figure 6. pH measurements of pharmaceutical-grade heparin in solution. The pharmaceutical grade heparin has distinctive peaks in the following positions as shown in figure 7: 806,842,866 cm-1 (C-O-C stretching vibrations); 1000 cm-1 (C-N stretching in the N-sulfated-glucosamine monosaccharide),1030 cm-1 (NSO3 vibration),1043 cm-1 (6-O-SO3 vibration),1060 cm-1 (3-O-SO3 vibration),1263 cm-1 (S=O bonds) and 1320 (-OH)8,9. Considering that in Raman spectroscopy the fact that the concentration of a species influences the signal strength, the heparin-based nanoparticles respect the rule quite well. Also in sample 1 there is a sharp peak at 731 cm-1 which corresponds to a C-Cl bond considering that the metal precursor is HAuCl4, this peaks diminishes its intensity as the reagents participating in the nanoparticle synthesis reaction decrease and that does not appear in the heparin spectrum, meaning that a complex between gold and heparin has been formed. As for the other peaks. The C-N peak stretching does not appear anymore in the nanoparticles solutions spectra indicating its removal, possibly to the fact that it participates in the metal precursor reduction process as the reaction is taking place. The other characteristic peaks belonging to heparin only suffer very slight shifts in their positions, but the fact they are still there indicates they are attached to the nanoparticles surface especially sulfates, these ones are responsible for heparin´s negative charge and also make the synthesized products negative in their charge as well. As for their intensity, the fact that it decreases has to do with the concentration used but also to the degradation suffered by the interaction with UV light. The -OH group region which has only one peak in the heparin solution spectrum shows the appearance of two shoulders are due to the protonation and deprotonation in the heparin compound.


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Raman shift (cm-1) Figure 7 .Raman spectra of heparin and the samples obtained in the photochemical-based synthesis.

Conclusion. A new, inexpensive, room temperature and facile photochemical method to synthesize nanoparticles based on the usage of UV light is presented. The shapes and nanoparticle amount that can be obtained are concentration-dependent in the time exposition time used (seven hours) taking into account that since the medium is acid anisotropic shapes are favored. It was also shown that the glucosamine monosaccharide heparin plays a reducing role in the synthesis and that the sulfate groups are attached to the nanoparticles surface. Although not demonstrated in this paper, it can be said that heparin.based nanoparticles can be used for plasma and biomedical applications. Acknowledgments. This work was supported by CONACYT under PhD grant No. 166107 for M. del P. Rodríguez-Torres, and the Nanophotonics and Nanomaterials and Materials laboratories at Centro de Investigaciones en Óptica,A.C as well as CFATA for the TEM measurements aid. References. [1] Jingfang Zhou, John Ralston , Rossen Sedev and David A. Beattie," Functionalized gold nanoparticles: Synthesis, structure and colloid stability,"Journal of Colloid and Interface Science 331, 251–262 (2009) [2] Juan C. Scaiano, Paul Billone, Carlos M. Gonzalez, Luca Maretti, M. Luisa Marin, Katherine L. McGilvray, and Nathan Yuan, "Photochemical routes to silver and gold nanoparticles," Pure Appl. Chem. Vol. 81, 635-647 (2009) [3] Rebecca Lever, Barbara Mulloy and Clive P. Page, [Heparin - A Century of Progress] , Springer, 473 (2012). 4] Yanli Guo,Hongtao Yan, "Preparation and Characterization of Heparin-Stabilized Gold Nanoparticles," Journal of Carbohydrate Chemistry 27, 309-319 (2008)


[5] Melissa M. Kemp, Ashavani Kumar, Shaymaa Mousa, Tae-Joon Park, Pulickel Ajayan, Natsuki Kubotera, Shaker Mousa, Robert J. Linhardt, "Synthesis of Gold and Silver Nanoparticles Stabilized with Glycosaminoglycans having Distinctive Biological Activities," Biomacromolecules 10,589-595 (2009). [6] Youmie Park, A-Rang Im, Yoo Na Hong, Chong-Kook Kim and Yeong Shik Kim,"Green Synthesis and Nanotopography of Heparin-Reduced Gold Nanoparticles with Enhanced Anticoagulant Activity," Journal of Nanoscience and Nanotechnology 11, 7570–7578 (2011) [7] Xiong Y, McLellan JM, Chen J, Yin Y, Li ZY and Xia Y, "Kinetically controlled synthesis of triangular and hexagonal nanoplates of palladium and their SPR/SERS properties," J Am Chem Soc.127,17118-17127 (2005) [8] Donald H. Athax, Adolfas K. Gaigalas, and Vytas Reipa, "Structural Analysis of Heparin by Raman Spectroscopy," Journal of Pharmaceutical Sciences Vol. 85,53-56 January (1996) [9] N. I. Sushko, S. P. Firsov, R. G. Zhbankov, M. Tsarenkov, M. Marchewka, and Ch. Ratajczak , "Vibrational spectra of heparins," Journal of Applied Spectroscopy Vol. 61, 5-6(1994)
