Summer Research Fellowship Programme of India's Science Academies


A S Preedhi

Integrated MSc. Chemistry, Amrita Vishwa Vidyapeetham, Coimbatore

Guided by:

Prof. Puspendu K Das

IPC Department, Indian Institute of Science, Bangalore 560 012


Gold nanoparticles are gaining wide applications in biomedical fields. By controlling the size and manipulating the surface one can increase the biocompatibility by several folds. A major drawback of using these nanoparticles is its relative instability in biological media. The present work is focused on the synthesis and characterization of stabilized gold nanoparticles so that they can be used as an effective drug delivery and gene delivery agent. Here, we use polyethylene glycol having a molecular weight 10000 (PEG 10000) as the stabilizing agent. PEG is one of the commonly used stabilizing agents and is a principal component of many therapeutic drugs. The work involves two steps. Initially, citrate capped gold nanoparticles of 15nm are synthesized and then the citrate ligands are exchanged with PEG 10000. The synthesis requires chloroauric acid, citric acid, sodium citrate and PEG 10000. The process involves the reduction of Au from +3 to 0 oxidation state, which is indicated by the formation of wine red colour. Another widely used one step synthetic strategy of pegylated gold nanoparticles involves using NaOH as the reducing agent and PEG as the capping agent. The pH of this reaction needs to be maintained at nearly 7.5 and this can be achieved by the addition of NaOH to the reaction mixture. Apart from pH maintenance, NaOH also helps in the reduction of Au. It is noted that the sequence of NaOH addition has a great influence in the ease of formation of stable spherical pegylated gold nanoparticles. Also, gold nanoparticles functionalized with PEG are stable for months at room temperature and have potential use for in vivo and in vitro applications. Different spectroscopic techniques like UV-Visible and IR spectrometer are used for the characterization of the nanoparticle.

Keywords : biocompatibility, drug delivery agent, UV-Visible spectrophotometer, Infrared spectrophotometer


AuNPs Gold nanoparticles
PEG Polyethylene glycol
SPR Surface Plasmon Resonance
DDL Diffuse double layer
FDA Food and Drug Administration
Ctr3 -  Citrate ion
ACDC2− Acetone dicarboxylate ion


Nanoparticles have found wide applications in the field of catalysis, biomedicine, optics and electronics because of their high surface-to-volume ratio and their unique size-dependent optical, electrical and magnetic properties. The optoelectronic properties of gold nanoparticles related to the occurrence of surface SPR band make them different from other nanoparticles. Biocompatibility of AuNPs and their endocytotic fate inside the cell compartment ensures that it can be utilized as an effective drug delivery agent.

AuNPs were first synthesized by Turkevich by the citrate reduction method [1]. AuNPs synthesized in water and linked to the biomolecules have many applications like gene transfer, bioprobes in cell and tissue analysis, drug-delivery and study of biological processes at nanoscale. But one of the drawbacks of using AuNP for biomedical purposes is its relative instability in biological fluids due to its interaction with the biomolecules. Biological fluids, with high ionic strength, can interact with nanoparticle which eventually leads to the aggregation. So, the stability of gold nanoparticle is an important factor to look into. It has been found that the stability of colloids can be achieved by either utilizing electrostatic repulsion or steric stabilization. Stabilization by electrostatic repulsion for instance, capping with citrate ligands is ineffective in high ionic strength fluids. Therefore, electrostatic stabilization is not an effective method for the stabilization of NPs in biological media. Generation of physical barriers on NP surfaces (steric stabilization) by functionalizing the nanoparticles with polymers like polyethylene glycol (PEG) or polyvinyl pyrrolidone (PVP), increases the NP stability in suspensions. The hydrophilic nature of these polymers induces extra stabilization through the short range repulsive hydration forces. Thus steric stabilization is more efficient for a biological system [2].

    Figure 1 : Strategies to acheive stable NPs in bio-fluids

    Polyethylene glycol is one of the widely used functionalizing agent because of its ability to increase the colloidal stability and biocompatibility in biological fluids. This polymer’s hydrophilicity provides excellent steric stabilization and short range repulsive hydration layer around the nanoparticle surface that imparts extra stability in high salt concentrations and extreme pH. PEG is a principal component of most of the therapeutic drugs like pegfiber powder, pegclear oral solution, GlycoLax etc. It is innocuous, versatile and FDA (Food and Drug Administration) approved for clinical use. PEG terminated with functional groups like methoxy, thiol, and carboxylate are known to have high stability towards biological media. The thiol terminated pegylated gold nanoparticles improve in vivo stability and they avoid the uptake by a reticular endothelial system.

    Objectives of the Research

    ✯ To synthesise PEG functionalized gold nanoparticles.

    ✯ To characterize PEG functionalized gold nanoparticles.

    Literature review

    Synthesis of pegylated gold colloids

    For the synthesis of pegylated gold nanoparticles, chloroauric acid is treated with a reducing agent and a stabilizing agent. Different types of synthetic methods are mentioned in prior literature. Out of those, the commonly used methods are:

    1) Two step synthesis: Initially, citrate capped gold nanoparticles are produced. Then the citrate ligand is replaced with PEG.

    2) One step synthesis: PEG functionalized AuNPs are produced by treating chloroauric acid with NaOH and PEG.

    Reduction can be done by different kinds of reducing agents like sodium citrate, sodium hydroxide, cetyl trimethyl ammonium bromide (CTAB), sodium borohydride, ascorbic acid etc. Stabilizing agents are also called capping agents, for example polyethyelenimine (PEI), PEG, PVP etc. PEG is one of the commonly used capping agents.

    Role of sodium citrate

    Sodium citrate can act as both a reducing and a stabilizing agent. Sodium citrate, on addition to chloroauric acid, reduces Au from +3 to 0 oxidation state and itself undergoes oxidation to acetone dicarboxylate (ACDC2-), a ligand that complexes Au(III) [3].


    1.  Oxidation of sodium citrate to acetone dicarboxylate.

    2AuCl4- + 3Ctr3- → 2Au + 3ACDC2- + 3CO2 ↑ + 8Cl− + 3H+

    2.  Degradation of acetone dicarboxylate to acetate at ~100 °C.

    ACDC2-+ 2H2O → acetone + 2CO2 + OH−

    3.  If the ratio of sodium citrate to chloroauric acid is less than 1.5 then there may be the possibility for ACDC2− or its degradation products to take part in a redox reaction. This involves the reduction of auric chloride by carboxylate and other degradation products leading to the complete conversion of Au(III) to Au0.

    4AuCl4 + 6H2O + acetone → 4Au + 3CH2O + 3CO2 + 8Cl− + 3H+

    4. Summing up the above equations;

    2AuCl4 + Ctr3 - → 2H2O → 2Au + 3CH2O + 3CO2 + 8Cl− + 3H+

    5.  At room temperature, ACDC2− undergoes slow oxidation in the presence of oxygen.

    22AuCl4+ 6ACDC2− + 24H2O → 22Au + 6Au + 3 HCOOH + 21 CO2 + 88 Cl−  + 54 H+

    6.  Summing the equations 1 and 5;

    6Ctr3 -  + 26 AuCl4 + 24H2O → 26Au + 6CH2O + 3HCOOH + 27CO2 + 104 Cl−  + 60H+

    Ctr3− and ACDC2- are carboxylate and their byproducts, due to degradation, oxidation, and reduction also produce carboxylates. This shows that there is an attachment of carboxylate moiety regardless of the exact species involved in AuNP capping.

    Role of NaOH

    NaOH is a reducing agent which is required for the reduction of gold. For the stable pegylated gold nanoparticle formation, the pH needs to be maintained at around 7.8. This can be achieved by the addition of NaOH. Initially, the colour of the reaction mixture is pale yellow. Later, on addition of NaOH, the color slowly changes to colourless due to the formation of hydroxy-gold (III) derivative [4].

    AuCl4- + nOH[AuCl4-n (OH)n] + nCl-

    Due to this complex formation, the intensity of metal to ligand charge transfer transition decreases and finally losses the yellow colour of the complex due to the blue shift in the absorption spectra. The order of NaOH addition also impacts the formation of nanoparticles.

    Role of PEG

    PEG is a polymer, also known as polyethyelene oxide or polyoxy ethylene depending on its molecular weight. On adding PEG to citrate capped gold nanoparticles under vigorous stirring condition, the citrate ligands were replaced by PEG ligands. This provides the gold nanoparticles more stability in in vivo and in vitro applications ​[5]. They are also known to increase the circulation time of biomedicines in bloodstream. By using PEG of different molecular weight, the surface charge on gold nanoparticles can be tuned. The length of the PEG also contributes to the stability of the nanoparticles. Here is a comprehensive summary of the differences between citrate and PEG coatings (Table 1).

    Table 1 Summary of the differences between citrate and PEG coatings
      Citrate PEG
    Binding strength to np surface Low Very Strong
    Ability to modify surface Very Easy Hard
    Coating Thickness ~1 nm >2 nm
    Stabilizing factor Charge Steric
    Zeta Potential < -15 mV -5 - +5 mV
    Cytotoxicity No No

      The characterization of gold nanoparticles can be done by UV-Visible spectrophotometer, FTIR spectrophotometer, TEM analysis, Zeta potential measurements etc.

    For the TEM analysis, a drop of nanoparticle solution is placed on a copper grid. The droplet is dried and is used for TEM analysis. This give detailed information about the shape and size of the nanoparticles.

    PEG with a different molecular weight can be used to tune the surface charge on the gold nanoparticles. Zeta potential measurement will help in finding out the surface charge on nanoparticles.

    UV-Visible spectroscopy is carried out in a UV-Visible spectrophotometer. According to the literature, citrate capped AuNPs shows a peak at 519.4nm and PEG functionalized AuNP shows the peak at 519.5 – 520.6nm for PEG 5000 [6]. This shows that there won’t be much difference in the spectra of both.

    FTIR is a more effective way of characterization in the ligand exchange method. PEG terminated with functional groups like methoxy, thiol etc will give more peaks than citrate ligands.


    UV-Visible spectroscopy

    Characterization of AuNPs can be done by using UV-Visible spectrophotometer. UV-Visible spectroscopy is an absorption spectroscopy which involves the transitions in the electronic energy levels. AuNP have electrons on the surface, collectively known as plasmons. If there is a resonance between the frequencies of the incident electromagnetic radiation and the vibration of Plasmon, which will lead to the appearance of SPR band in UV –vis spectra, resonance occurs [7].

    UV-Visible spectrophotometers can be single beam or double beam. In single beam spectrophotometer, all the light passes through the sample cell which is then detected by the detector (figure 2). 

      Figure 2 : Single beam spectrophotometer


      A double beam spectrophotometer consists of a light source, a monochromator, reference and sample cells, a detector and data output. The UV light from the light source is passed through the monochromator which allows light of only a particular wavelength to pass through. The monochromatic beam is then split into two beams and is allowed to pass through the reference and the sample cell. The intensity of transmitted light through the reference cell is taken as I0 and the intensity of transmitted light through the sample cell is taken as I. It is then passed through the detector where light signals are converted to electrical signals. The detector is connected to an operating system which gives the output data (figure 3).

        Figure 3 : Double beam spectrophotometer

        UV-Visible spectroscopy is based on the principle of Beer-Lambert’s Law which states that:

        “When a monochromatic beam of light is passed through a solution of absorbing medium, the decrease in the intensity of incident light is proportional to the intensity of transmitted light and the concentration of the absorbing medium.”

        A = log (I0/I) = ε C l

        where, A = absorbance, I0 = incident intensity, I = transmitted intensity, ε = molar extinction coefficient, C = concentration of the absorbing medium, l = path length (usually taken as 1cm).

        By knowing the value of A, ε and l, the concentration of AuNPs can be found out. The broadness of the UV-Visible spectra depends on the monodispersity of the AuNPs. Depending upon the size of the nanoparticles, the peak position also changes. As the size increases, a red shift in wavelength is observed. Past studies show that pegylated gold nanoparticles with different molecular weight also produces a shift in the wavelength.

        FTIR Analysis

        Fourier-Transform Infrared Spectroscopy is a technique used for the characterization of chemical species. It involves the scanning of wavelength and the transitions of vibrational energy levels. The infrared region is divided into three: Near-IR region (12500cm-1 – 4000cm-1), Mid-IR region (4000cm-1 – 600cm-1) and Far-IR region (600cm-1 – 50cm-1). Most of the molecules absorb infrared radiation in the mid-IR region. This absorption gives an idea about the nature of bonds present in the molecule. The IR spectra of each molecule are unique. Therefore, IR spectra can be called as the fingerprint of a molecule. If the IR spectras of two molecules are the same, we can say that the molecules are identical.

        PEG terminated with methoxy, carboxylic, amine group will give more specific peaks and are easy to characterize. It will give peak at 2,850–3,000 cm−1(−CH2 stretching), 1,631 cm−1 (N–H bending), 1,660 cm−1 (C=O stretching), 1,380 cm−1 (C–H bending; −CH2 and −CH3), 1,100 cm−1 (C–O–C stretching) and 600- 900 cm−1 (N–H wagging) and confirms the presence of bound PEG [6].

        Project Review

        Experimental set up (figure 4)

          Figure 4 : Experimental set up


          All the glassware are washed with aqua regia (mixture of concentrated HCl and concentrated HNO3 in the ratio 3:1) and are thoroughly dried in the oven. Chloroauric acid (25millimolar) is the precursor of the gold used. Sodium citrate, citric acid and PEG 10000 (figure 5) were obtained from Sigma Aldrich. 2% wt/vol of sodium citrate and 0.1% wt/vol of citric acid solution were made using ultrapure water.

            Figure 5 : Polyethylene glycol


            The experiment is carried out by two different methods as mentioned in the literature. Initially, NPs are synthesized by the ligand exchange method.

              24.75 mL of ultrapure water and 0.25mL of 25 millimolar choloroauric acid are taken in a round-bottomed flask. The reaction mixture is refluxed to 100 °C with constant stirring and then 500µL of sodium citrate and 500µL of citric acid solution are added. The heating and stirring are continued for 10-12 minutes. The formation of citrate capped gold nanoparticles is confirmed by the change in colour from pale yellow to ruby red colour (figure 6).

              Figure 6 : Citrate capped AuNPs

              The solution is then allowed to cool to the room temperature. To this solution, approximately 0.9mg of PEG is added and is stirred continuously for two hours [6]. Since the binding strength of the citrate ligand on the nanoparticle surface is very weak, it can be easily replaced by any other ligand. Thus here citrate ligands of gold nanoparticles get replaced by the PEG.

                Here chloroauric acid is the precursor of gold (Au) in which Au is in +3 oxidation state. Citric acid is the reducing agent which reduces Au from +3 to 0 oxidation state (figure 7).

                Figure 7 : Reduction of Au in chloroauric acid

                Sodium citrate can act both as a reducing and a capping agent and it stabilizes the formed gold nanoparticles. On addition of citric acid and sodium citrate, the surface of gold nanoparticles gets surrounded by a negatively charged citrate ligand (figure 8). The electrostatic repulsion between the adjacent nanoparticles prevents agglomeration and makes them stable and monodisperse [8].

                cit aunp.PNG
                  Figure 8 : Citrate stabilized AuNP

                  The second time, the synthesis is carried out by the latter mentioned method in the literature.

                  The materials required for this synthesis are chloroauric acid, NaOH as the reducing agent and PEG as the capping agent. 18 mL of ultrapure water and 2.4g of PEG10000 is taken in a round bottomed flask. The reaction mixture is allowed to reflux till the temperature reaches 50 °C under vigorous stirring conditions. To this, 0.3mL of NaOH is added and the mixture is slowly heated to 80°C. A wine red coloration indicates the formation of pegylated AuNPs. By using PEG of different molecular weight, one can tune the surface charge on gold nanoparticles [9].

                  Purification of gold colloids

                  The unbounded PEG and citrate ligands can be removed from the gold colloid by centrifugation. For centrifugation, the PEG functionalized gold colloids are taken as 1mL batches and centrifuged for 15 minutes at an rpm (rotation per minute) of 12500. As a result, the gold nanoparticles will settle down at the bottom of the centrifuging tube. Once the supernatant is clear, it is discarded and the nanoparticle pellet remains at the bottom of the centrifuging tube. The centrifuging tube is then refilled with 0.9mL of ultrapure water and the process is repeated three times. The gold colloid is thus purified.

                  Results and Discussion

                  Different sizes of gold nanoparticles exhibit different colours. Ruby red colour indicates that the gold nanoparticles formed were within a size range of 50 nm. The surface modification with PEG depends on so many factors like pH, temperature, stirring rate, chain length of PEG etc. PEG with smaller a molecular weight forms a layer over gold nanoparticle and thereby provides stability to the Au-NPs. At the same time, PEG with a high molecular weight, suffers repulsion, and the surface charge is inadequate to provide stability to Au-NPs.

                  The absorption spectra of AuNPs were recorded in Shimadzu UV-2600 UV-Visible spectrophotometer (figure 9) in the wavelength range 200-800 nm. Initially, the baseline correction is done by taking 1mL of ultrapure water in both the quartz cuvettes (sample and reference cuvettes) and the analysis is carried out with a medium scan speed. Then, the water in the sample cuvette is replaced by 1mL aqueous solution of Au-NPs and the UV-Vis spectra is recorded.

                    Figure 9 : UV-Visible spectrophotometer

                    The discussion of characterization of AuNP produced by the first method is given below.

                    Trial 1:

                    UV-Visible spectra for citrate capped gold nanoparticle (figure 10)

                      Figure 10 : Citrate capped AuNP

                      Capture 111.PNG
                        Figure 11 : UV-Visible spectra of citrate capped AuNP

                        The spectrum gives a narrow peak at 525 nm with an absorbance value of 0.203 (figure 11). This shows that the nanoparticles formed were monodispersed.

                        UV-Visible spectra of PEG functionalized gold nanoparticles.

                        Capture 2.PNG
                          Figure 12 : UV-Visible spectra of PEG AuNP

                          Pegylated gold nanoparticles gives a sharp peak at 525nm with an absorbance value of 0.325 (figure 12).

                          Comparing the graphs (figure 13)

                          Capture 3.PNG
                            Figure 13

                            Trial 2:

                            UV-Visible spectra of citrate capped gold nanoparticle.

                            capture 14.PNG
                              Figure 14 : UV-Visible spectra of citrate capped AuNP

                              The obtained spectra has a sharp peak at 521nm with optical density (absorbance) value as 0.983 (figure 14).

                              UV-Visible spectra of PEG functionalized gold nanoparticles

                              Capture 15.PNG
                                Figure 15 : UV-Visible spectra of PEG AuNP

                                The obtained spectra has the same value for wavelength and absorbance as that of citrate capped gold nanoparticles (figure 15).

                                Comparing the graphs (figure 16).

                                Capture 16.PNG
                                  Figure 16

                                  Trial 3:

                                  UV-Visible spectra of citrate capped gold nanoparticle.

                                  Capture 17.PNG
                                    Figure 17 : UV-Visible spectra of citrate capped AuNP

                                    The spectra has a peak at 519nm with an absorbance value at 0.967 (figure 17).

                                    UV-Visible spectra of PEG functionalized gold nanoparticles

                                    Capture 18.PNG
                                      Figure 18 : UV-Visible spectra of PEG AuNP

                                      The pegylated gold nanoparticles shows narrow peak at 519nm as same as that of citrate capped gold nanoparticles with an absorbance value of 0.768 (figure 18).

                                      Comparing the graphs (figure 19).

                                      Capture 19.PNG
                                        Figure 19

                                        Trial 4 (figure 20) :

                                        Capture 20.PNG
                                          Figure 20

                                          Both the citrate and PEG capped gold nanoparticles show a sharp peak at 524 nm. But the absorbance of citrate capped is higher than that of the PEG functionalized (figure 20).

                                          It is clear from the above trials that the nanoparticles obtained were not of single size range as the wavelength ranges from 519-524nm. From the spectras obtained we can say that the citrate capped nanoparticles formed with an average size around 15nm [10]. The UV-Vis spectra of citrate capped and PEG capped resemble each other. Therefore, UV-Visible spectrophotometer is not an effective characterization method. It doesn’t give the confirmation of the complete exchange of the citrate ligands by PEG.

                                          For the nanoparticles synthesized by the second method, a dark colored solution is obtained after long heating than the mentioned temperature in the literature.

                                          Capture 21_1.PNG
                                            Figure 21 : UV-Visible spectra of PEG AuNP

                                            The UV-Vis spectra obtained shows a broad peak around 543 nm which indicates that the nanoparticles formed were not monodispersed (figure 21). This result is far from the one that is mentioned in prior literature. The experiment was tried thrice and the results obtained show the poor reproducibility of the method.

                                            FTIR Analysis is carried out in Perkin Elmer Spectrum One FTIR Spectrometer. It is an effective analysis for the characterization of ligand exchange method. Initially, background correction is carried out without the sample. Then, the samples of citric acid, PEG, citrate capped AuNP and PEG capped AuNP were analyzed. The sample is first taken in the glass plate and dried in the desiccator for 10 minutes. It is then kept in the FTIR instrument in the path of IR radiation.

                                            Capture 22.PNG
                                              Figure 22 : FTIR spectra of citric acid

                                              The FTIR spectrum of citric acid shows a sharp peak in the wave number range of 1700 -1725cm-1. This indicates the presence of carboxylic carbonyl group in the compound (figure 22).

                                              Capture 23_2.PNG
                                                Figure 23 : FTIR spectra of citrate capped AuNP

                                                FTIR spectra of citrate capped AuNP shows a sharp peak in the wavelength range of 1500 -1700cm-1. This indicates that there is a shift in the carbonyl peak due to its adsorption on the AuNP. Also, a peak at 1465cm-1 indicates the –CH2 bending (figure 23).

                                                Capture 24_1.PNG
                                                  Figure 24 : FTIR spectra of PEG

                                                  A strong peak just below 3000cm-1 indicates the presence of C-H stretch. C-O bond is confirmed by the presence of peak in the wavelength range 1000-1300cm-1 (figure 24).

                                                  Capture 25.PNG
                                                    Figure 25 : FTIR spectra of PEG AuNP

                                                    The C-O peak is shifted to 1300-1500cm-1 due to its capping with AuNP. The strong peak in the range of 1500-1700cm-1 indicates the presence of carbonyl group formed by the oxidative transformation of PEG. Broad peak in the wavenumber range of 3300-3500cm-1 indicates the presence of H bonded O-H group in the sample (figure 25).


                                                    In this work, gold nanoparticle synthesis is carried out with two different methods. The second method is found to be a failure. By the first method, I have successfully synthesized and characterized pegylated gold nanoparticles. Gold nanoparticles have a great potential to act as a drug delivery agent. The stability of AuNPs in biological fluids can be increased by surface modification [11]. PEG is an excellent capping agent that can sterically protect the gold nanoparticles and stabilize them [12]. Characterization by the UV-Visible spectroscopy is not an effective method as both the citrate and PEG capped AuNP spectra appear at a particular λ max. Characterization by FTIR technique gives much detailed information about the capping. Hence, FTIR is found to be an effective method of characterization for ligand exchanging method.


                                                    I would like to acknowledge all those people who have made this research work possible, an experience that I will cherish forever.

                                                    First of all, I am indebted to the Indian Academy of Sciences, Bangalore for giving me this opportunity to work at Indian Institute of Science, Bangalore, one of the top institutes for scientific research and higher education in India.

                                                    I owe my sincere thanks and the deepest sense of gratitude to my guide, Prof. Puspendu K Das, who has been the source of inspiration and support throughout the period of the project, for his guidance and constant encouragement. I have been extremely fortunate to have worked under his supervision.

                                                    I would like to extend my heartiest thanks to Prof. Puspendu K Das’s Research group, especially Ms. Karthika (Ph.D scholar), Ms. Kamini Mishra (Ph.D scholar), Mr. Sourav Saikia (Ph.D scholar), Ms. Akriti (Ph.D scholar), Mr. Bedabyas Behera (Ph.D scholar) and Sreepriya (Project assistant) without whom it would have been difficult for me to complete the work within the stipulated time, for their inspirational guidance, timely help, valuable suggestions and discussions throughout my project.

                                                    I thank all the supporting staff of AuthorCafe for providing such an excellent space to write my report and collaborate with my guide.

                                                    I would also like to convey my thanks to the Head of the Department, Dr. T. Ramachandran and all the Faculty members of the Department of Science, Amrita Vishwa Vidyapeetham, Coimbatore, Tamil Nadu.

                                                    Last but not the least, I am deeply obliged to my parents, grandparents, teachers and friends for their love, care and everlasting support towards me.


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