Conference-ICSFS-16-Adsorption Reaction of L-Cysteine on Au Nanoparticle Prepared by Solution Plasma

S. Yagi Department of Quantum Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan, Synchrotron Radiation Center, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-0046, Japan, and EcoTopia Science Institute, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan (Received 15 July 2012; Accepted 31 December 2012; Published 2 February 2013)


I. INTRODUCTION
The gold (Au) materials possess the high reactivity with the molecule including thiol group.Recently, the Au nanoparticles (NPs) are studied for the applications to the drug delivery system [1], which is used for carrying the functionalized molecule to the objective tissue, and the oxidative stress biomarker [2], which is used for the detection of injury part.As those applications are utilized under water environment and the many amino acid molecule containing sulfur (S) such as L-cysteine (HSCH 2 CH(NH 2 )COOH) are existing in our body, it is important to reveal the adsorption reaction between the Au NPs and the sulfur-containing molecules under water environment.In the previous report, the study for the reaction between the Au NPs and the L-cysteine, which possesses thiol group, have been carried out under water environment [3].However, because the Au NPs have been fabricated by the chemical reduction method and the surfactant molecule has been used, there are possibilities that the surface of the Au NPs is poisoned and the adsorption behavior is affected by the surfactant molecule.Thus, we think that the fabrication of the NPs with clean surface and the understanding of the reaction between those NPs and the L-cysteine are necessary.
In our previous studies, the adsorption state of the Lcysteine on the palladium (Pd) NPs after promoting the adsorption reaction under water environment has been clarified [4,5].As the Pd NPs have been fabricated by the gas evaporation method, the Pd NPs possess a clean surface.However, since the Pd NPs are needed to take out from the fabrication chamber to react into the Lcysteine aqueous solution, the Pd NPs might be poisoned by air components.About the solution plasma method, because the surfactant molecule is not used and the fabricated NPs exist into the aqueous solution, it is thought that the NPs possess a clean surface under water environment [6,7].Thus, we can investigate the reaction between the Au NPs and the L-cysteine under ideal environment without the surfactant molecule by means of using the NPs fabrication technique with the solution plasma.In this study, we have decided the reaction conditions that the Au NPs are fabricated by the solution plasma method and the L-cysteine powder is added in the fabricated Au NPs colloidal solution.
We note the chemical states of the S element for Lcysteine in this study because the thiol group possesses the high reactivity for the Au NPs.The energy region of soft X-ray for using the S K-edge NEXAFS measurement is 2463-2493 eV.To carry out the NEXAFS measurement with keeping the water environment for the sample, the atmospheric pressure condition is necessary.Although the soft X-ray at S K-edge region is reduced by the air components, Yagi et al. have reported that the large reduction of soft X-ray is not occurred by using He-path NEXAFS measurement system [8].In the previous studies about the change for the color of the Au NPs colloidal solution, it has been reported that the particle diameter of the Au NPs affects the color of the solution, and the peak position and shape of the spectra obtained by UV-vis measurement [9].Furthermore, the value of the ζpotential also has reflected the particle diameter [10,11].Thus, we think that the relations among the color of the Au NPs colloidal solution, the value of the ζ-potential and the diameter of the Au NPs can be understood by using UV-vis, ζ-potential and AFM measurements.
The purpose of this study is to reveal the adsorption reaction between the L-cysteine and the Au NPs, which are fabricated by the solution plasma method, under water environment by using S K-edge NEXAFS measurement.Moreover, in the point of view for the change of the particle diameter for the Au NPs, we investigate the behavior of the Au NPs before/after reaction with the L-cysteine under water environment by means of AFM, UV-vis and ζ-potential measurements.

II. EXPERIMENTAL
The Au NPs with clean surface in the aqueous solution were fabricated by the solution plasma method [6,7].The Au rods (99.95%, 1 mmϕ) and KCl (≥99.95%) were purchased from Nilaco Co. and Wako Pure Chemical Industries Ltd., respectively.The Milli-Q water (≥18 MΩcm) was provided.Figure 1 shows the photographic views of the fabrication of Au NPs by solution plasma with reaction times (0, 5 and 30 min).Two Au rods faced each other are used for the following two reasons, which are an electrode and a source of Au NPs.The distance between rods was about 0.1-0.2mm.To control the electrical resistance of the water, the KCl was added to the Milli-Q water in small quantity.We used a power source produced by KURITA Seisakusho Co. Ltd. for AC-high voltage with the frequency of 20 kHz and the voltage region of 100-150 V.The solution was always agitated by the stirrer.The fabricated Au NPs colloidal solution is defined as sample (i) and called as "initial state".The photographic images for the sample (i) and several prepared samples, which are described below, are shown in Fig. 2.
The L-cysteine (HSCH 2 CH(NH 2 )COOH) powder was purchased from Sigma-Aldrich (≥98%).The L-cysteine powder of 0.035 mmol was added to the Au NPs colloidal solution of 5 ml at 300 K, and the adsorption reaction was promoted for (ii) 1 min, (iii) 4 min, (iv) 3 hours and (v) 24 hours.For the sample (iv), a slight precipitate is deposited on the bottom of the vial by the aggregation of the Au NPs, and the dark blue colored supernatant solution is observed.About the sample (v), the all visible NPs are deposited, and the black colored precipitate and the colorless supernatant solution occur.To rinse the Lcysteine without adsorption on the Au NPs surface, the supernatant solution in the sample (v) was extracted and subsequently the distilled water was added to the vial.Because the precipitate was diffused into the distilled water, the color of the solution became dark blue again.This dark blue colored solution is defined as sample (vi).After several days, the black colored precipitate was occurred again in the sample (vi).The precipitate under water environment is defined as sample (vii), and the precipitate dried in air on the polyethylene film is defined as sample (viii).
The Au NPs colloidal solutions after reaction under several conditions, which are (i), (iii), (iv) and (vi), were dropped and dispersed on the SiO 2 /Si(100) wafer by using spin coating method.The AFM observation for those sample substrates was carried out by NanoScope III-a (Veeco Instruments) with tapping mode.The diameters of the Au NPs were estimated by the AFM images for the height value of those NPs.The UV-vis measurement was carried out for the samples (i)-(vi) at 300 K in the range of 400-700 nm by means of spectrophotometer UV-2450 (SHIMADZU).The ζ-potential measurement was carried out for the samples (i), (iii), (iv) and (vi) at 300 K by means of SZ-100 (HORIBA).The Sulfur K-edge NEX-AFS measurements were carried out for the samples (iii)-(viii) by fluorescence X-ray yield method using the atmospheric XAFS measurement system with He-path at the BL-3 on Hiroshima Synchrotron Radiation Center (HSRC) [8,12,13].A purity of the He gas was ≥99.99% and a flow rate of the He was set about 10 µl/min.The incident X-ray energy was calibrated on the assumption that the first peak of K 2 SO 4 appears at 2481.7 eV.The fluorescence yield detection was employed using a gasflow type proportional counter with P-10 gas (10% CH 4 in Ar).The energy region of NEXAFS measurement and the energy step were 2463-2493 eV and 0.2 eV, respectively.The bubble-wrap, which was made of polyethylene film with about 12 µm thickness, was used as the cells for the samples (iii)-(vii).

A. AFM observation
Figure 3 shows AFM images and particle size distributions for samples (i), (iii), (iv) and (vi), respectively.About sample (vi), we show two images and distributions as samples (vi)-1 and (vi)-2.The size distributions for each sample are obtained by the diameter measurement of observed NPs in AFM images.The average diameter with standard deviation for the sample (i), which is defined as "initial state", is estimated to be 2.4±1.0 nm.We think that the sample (i) in the initial state includes the primary NP and secondary one, which is constructed from some primary NPs.In addition, because those NPs cannot be distinguished by AFM observation, we speculate that the size distribution for sample (i) becomes asymmetric Gaussian curve with tailing.About the sample (iii), the average diameter becomes slightly large compared to that for the sample (i) and is estimated to be 3.0±1.8nm.According to the NEXAFS measurement for the sample (iii) described in Sect.3.4.1, the L-cysteine adsorbs on the Au NPs surface as the L-cysteine thiolate.In our previous study, we have reported that the Pd NPs with L-cysteine thiolate adsorbate aggregate each other under water environment by forming the hydrogen bonding between each carboxyl groups and/or the amino and carboxyl groups, which are the functional groups of the L-cysteine thiolate [4].Thus, it is thought that the aggregation of the Au NPs for the sample (iii) is occurred by the hydrogen bonding of the L-cysteine thiolate on the Au NPs.When the adsorption reaction is promoted for 3 hours, the average diameter of Au NPs, which are included in the supernatant solution for the sample (iv), is estimated to be 1.9±0.6 nm.The values of both the average diameter and the standard deviation become smaller than those for the sample (i).The reduction of those values would represent following reasons.
1) The Au NPs for the initial state possess the wide distribution for the diameter.
2) The larger diameter particles such as secondary NPs can easily aggregate each other and deposit by the reaction with the L-cysteine.
3) On the other hand, because the smaller diameter particles such as primary NPs can not be aggregated to precipitating size, the particles still remain in the supernatant solution.
The average diameter for the sample (vi)-1 is estimated to be 3.0±1.5 nm, which are included both initial and aggregated NPs.Namely, the aggregated NPs are partly decomposed to initial NPs, and residual aggregated NPs exist.Because not only the initial but the aggregated NPs exist in sample (vi), we think that the hydrogen bonding keeps partly stably.In addition, the Au NPs with 2-dimensional beads line like are observed in AFM image for sample (vi)-2, and the height value of NPs is estimated to be 2.1±0.8nm.The value is similar to that of samples (i) and (iv).Thus, we speculate that the 2-dimensional beads line is constructed by the hydrogen bonding between initial NPs with L-cysteine adsorbates.Since these AFM sample substrates are prepared by using spin coating method, we think that the difference between the samples (vi)-1 and (vi)-2 is attributed to each distance between the solution drop point and AFM observation point.Therefore, the difference of the AFM images might occur by centrifugal force.

B. UV-vis measurement
Figure 4 shows the UV-vis spectra for the samples (i)-(iv).Those spectra are normalized by the total area of absorbance.The peak top about the sample (i) locates at 516 nm.When the L-cysteine is dissolved into the Au NPs colloidal solution, the color of the Au NPs colloidal solution is quickly changed from red to dark blue.The peaks of the spectra for the samples (ii) and (iii) are observed at longer wavelength side compared to that for sample (i), and it is found that the shift of peak to longer wavelength side occurs with promoting the reaction time.Taking into account the result for the NEXAFS measurement in Section III.D.1 because the L-cysteine chemisorbs on the Au NPs surface, we think that the peak shift is occurred by the back-donation of some electrons from the Au NPs surface to the thiol group of the L-cysteine.About the sample (iv), the precipitate of the Au NPs is observed small amount at the bottom of the vial.When the supernatant solution of the sample (iv) is measured, the peak top of obtained spectrum is shifted to long wavelength side further and observed at 604 nm.We think that the peak shift occurs by three reaction states as follows.
1) The amount of the L-cysteine adsorbate on the Au NPs increases.
2 3) The Au NPs with L-cysteine adsorbates aggregate each other by forming the hydrogen bonding of Lcysteine thiolate and/or L-cystine.
When we have carried out the UV-vis measurement of the colorless supernatant solution for the sample (v), not shown in Fig. 4, the absorption of the incident light is confirmed at the wavelength region of 530-630 nm.Since it is thought that the absorbance is originated from the L-cysteine adsorbed on the Au NPs, we speculate that the Au NPs exist in the colorless supernatant solution.The shape of the spectrum for the sample (vi), not shown in Fig. 4, is similar to that for the sample (iv).Thus, we suppose that the adsorption of the L-cysteine on the Au NPs is saturated after the reaction time for 3 hours and the aggregation and diffusion of the Au NPs occur easily by both forming and decomposing the hydrogen bonding.In addition, it is thought that the L-cysteine adsorbates stably exist on the Au NPs surface and are not desorbed from the Au NPs surface by rinsing.

C. ζ-potential measurement
The ζ-potential values for the samples (i) and (iii) are estimated to be −34.7 mV and −21.0 mV, respectively.Although the detailed discussions for the adsorption reaction between the L-cysteine and the Au NPs are described in Section III.D.1, it is revealed that the L-cysteine adsorbs on the Au NPs surface at the thiol group.We are speculating that the rapid change for the ζ-potential value is occurred by the adsorption of the L-cysteine on the Au NPs surface.He et al. have also reported about the same tendency, which is the reduction of the ζ-potential value for the Ni-Cu sulfide by the L-cysteine adsorption [14].Though Mocanu et al. have reported that the Au NPs in the colloidal solution aggregate and the ζ-potential value of the Au NPs is reduced by adding to the Lcysteine, the Au NPs are fabricated by using the sur- factant molecule [3].Thus, the relation between the adsorption reaction of the L-cysteine and the reduction of the ζ-potential value is unclear.According to the result of the AFM observation in Section III.A, the particle diameter for the sample (iii) becomes large compared to that for the sample (i).Therefore, the reduction of the ζ-potential value might occur also by the aggregation of the Au NPs.The ζ-potential value for the sample (iv) is estimated to be −16.4mV.Because the reaction time is much longer than that for the sample (iii) and the obvious aggregation has been observed in the sample (iv), we think that the further reduction of the ζ-potential value occurs by both the increase for the amount of L-cysteine adsorption and the promotion for the aggregation of the Au NPs.The ζ-potential value for the sample (vi) is estimated to be −12.6 mV.Since the sample (vi) is fabricated after reaction for 24 hours, the reduction of ζ-potential value might be occurred by increase of L-cysteine adsorbates on Au NPs.Moreover, although the particle size for sample (vi)-1 shown in Fig. 3 is estimated to be 3.0 nm and the particle size is the same as that for the sample (iii), the AFM image for the sample (vi)-2 is observed the forming shape of the Au NPs of 2-dimensional beads line like.Thus, it is supposed that the 2-dimensional aggregation also leads to the reduction of the ζ-potential value.

NEXAFS for samples (iii)-(v)
Figure 5 shows the S K-edge NEXAFS spectra for the Au NPs colloidal solution samples (iii)-(v).The spectrum for the L-cysteine aqueous solution is shown as a standard.To reveal the obvious broadening of peak at 2472.5 eV for the sample (v), we also show the subtracted spectrum, which is obtained by subtracting L-cysteine aqueous solution spectrum from Sample (v) one.The subtracted spectrum is as a reference and defined as "(Sample (v)) − (L-cysteine aq.)".All spectra are normalized by edgejump.The spectra for samples (iii)-(v) possess the peak at 2472.5 eV.Since the peak position at 2472.5 eV is equivalent to the peak position assigned to σ * (S-C) of the spectrum for the L-cysteine aqueous solution, the residual L-cysteine molecules without adsorption to the Au NPs are included in the prepared all Au NPs colloidal solution samples.However, the full width at half maximum (FWHM) value of the peak at 2472.5 eV of the spectrum for the sample (iii) becomes slightly wide in comparison with the FWHM one for the L-cysteine aqueous solution.The broadening of the FWHM is occurred by the superposition of the new component peak at lower energy side than 2472.5 eV.In our previous studies, when the Pd thin layer or the Pd NP has been reacted with the L-cysteine under water environment, the L-cysteine has adsorbed on those Pd surfaces and dissociated into the L-cysteine thiolate [4].The peak originated from the L-cysteine thiolate is observed at the photon energy region of 2470.5-2472.4eV.Therefore, it is found that the L-cysteine adsorbs on the Au NPs surface and is dissociated into the L-cysteine thiolate.
About the spectrum for the sample (iv), the FWHM value of the peak at 2472.5 eV becomes further wide com- pared to the FWHM one for the sample (iii).The broadening of the FWHM in this aspect is observed at higher energy side than 2472.5 eV.In our previous study, when the Pd NPs have reacted with the L-cysteine under water environment, it is speculated that the L-cystine is synthesized from two L-cysteine thiolate molecules and exists on the Pd NPs surface [4].In addition, according to the result described in Section III.D.2, because the spectrum for the L-cystine on the Au NPs surface by chemisorbing at S-S bonding possesses the shoulder structure assigned to σ * (S-S) at 2471.6 eV and the peak assigned to σ * (S-C) at 2473.0 eV, we conclude that the synthesized L-cystine exists on the Au NPs surface after the reaction for 3 hours.Moreover, the peak at 2470 eV appears in the spectrum for the sample (iv).Because the existence of the atomic S on the metal is exhibited as the peak at approximately 2470 eV [15], we suppose that a part of the L-cysteine thiolate on the Au NPs is dissociated into the atomic S after the reaction for 3 hours.
We have carried out the NEXAFS measurement for the colorless supernatant solution of the sample (v).The FWHM value of the peak at 2472.5 eV for the obtained spectrum becomes further wide compared to that after reaction for 3 hours and the shoulder structure is observed around 2470 eV.Taking into account the result for the UV-vis measurement described in Section III.B, it is thought that the Au NPs exist in the colorless supernatant solution.Thus, it is found that the shape of the http://www.sssj.org/ejssnt(J-Stage: http://www.jstage.jst.go.jp/browse/ejssnt/) e-Journal of Surface Science and Nanotechnology NEXAFS spectrum for the colorless supernatant solution originates from the Au NPs reacted with L-cysteine and shows the increase of the adsorbates on the Au NPs, which are L-cysteine thiolate, L-cystine and atomic S. In addition, because the (Sample (v)) − (L-cysteine aq.) spectrum has the residual peaks at higher and lower photon energy regions than 2472.5 eV, we think the spectrum for the sample (v) is constructed by the summation of the spectra originated from both L-cysteine aqueous solution and the adsorbates.This discussion will be possible to apply to spectra for samples (iii) and (iv).

NEXAFS for samples (vi)-(viii)
Figure 6 shows the S K-edge NEXAFS spectra for the samples (vi)-(viii).The spectra for the L-cystine powder and the K 2 SO 4 are also shown as standards.The spectrum for the sample (vii) possesses two peaks at the photon energy regions of 2470.5-2474.0eV and 2480-2485 eV.Firstly, we note the peak observed at the lower energy region.The FWHM of the peak becomes clearly wide in comparison with that of the peak assigned to σ * (S-C) at 2472.5 eV for the L-cysteine aqueous solution as a standard shown in Fig. 5.To prevent the reduction of the fluorescence X-ray emitted from the sulfur-containing molecules under water environment, the precipitate is dried in air (sample (viii)) and measured by the NEX-AFS.The spectrum at the photon energy region of 2470.5-2474.0eV exhibits a shoulder structure at 2471.6 eV and a peak at 2473.0 eV.The split value is 1.4 eV.On the other hand, the spectrum for the L-cystine powder possesses a peak of the σ * (S-S) at 2472.1 eV and a shoulder structure of σ * (S-C) at 2473.4 eV, and the split value is 1.3 eV.As those split values for the sample (viii) and the L-cystine powder are almost the same, it is speculated that the shoulder structure at 2471.6 eV and the peak at 2473.0 eV for the sample (viii) represent the σ * (S-S) and σ * (S-C) peaks originated from the L-cystine, respectively.The L-cystine is synthesized from two L-cysteine thiolate molecules on the Au NP.Although the peak intensity assigned to σ * (S-S) is larger than that assigned to σ * (S-C) in the spectrum for the L-cystine powder, the ratio of the peak intensity between σ * (S-S) and σ * (S-C) for the sample (viii) becomes opposite.Moreover, the energy positions of σ * (S-S) at 2472.1 eV and σ * (S-C) at 2473.4 eV for the L-cystine powder are shifted to 2471.6 eV and 2473.0 eV, respectively.We think that the reduction of the peak intensity for σ * (S-S) and the shift of those peaks are occurred by means of the back-donation of electrons from the Au NPs surface to σ * (S-S) of the L-cystine.According to the above results, it is speculated that the L-cystine chemisorbs on the Au NPs surface with S-S bonding part.Secondly, when we pay attention to the spectrum for the sample (vii) at the higher energy region, the peak located at 2481.7 eV is observed.The peak at 2481.7 eV indicates the existence of the SO 4 adsorbate because the peak of the spectrum for the K 2 SO 4 is shown at 2481.7 eV.As the several samples in this study are prepared under water environment, the oxidation of the sulfur-containing molecule is occurred only by bonding with the H 2 O molecule.About the result described in Section III.D.1, we have revealed that the atomic S on the Au NPs is produced by the dissociation of the L-cysteine thiolate after the reaction for 3-24 hours.The sample (vii) is maintained under water environment for several days.Thus, it is thought the coordination reaction between the atomic S and the oxygen of H 2 O molecule is promoted and the SO x (x = 3 or 4) is formed [16].Moreover, the peak intensity at 2481.7 eV is reduced considerably in the spectrum for the sample (viii), and a new shoulder structure and a small peak are observed at 2470 eV and 2480.2 eV, respectively.Since the sample (viii) is prepared by drying the precipitate, we speculate that the atomic S on the Au NPs appears again by means of the desorption of the coordinated H 2 O.The small peak at 2480.2 eV is observed at photon energy region between 2475 eV assigned to SO [17] and 2481.7 eV assigned to SO 4 .Nomoto et al. have reported that the peak at photon energy region of 2475-2481.70eV is assigned the adsorbate of SO 2 or SO 3 on the Rh/Al 2 O 3 /NiAl(100) surface [18] Therefore, the small peak is assigned to the adsorbate of SO x (x = 2 or 3).Therefore, we think that the SO x (x = 2 or 3) on the Au NPs is occurred by desorbing one or two H 2 O.
About the spectrum for the sample (vi), there are two peaks at 2471.6 eV and 2473.0 eV.We conclude those peaks are assigned to the σ * (S-S) and σ * (S-C) for the adsorbed L-cystine on the Au NPs by considering the above results.Moreover, the peak intensity at 2481.7 eV assigned to the SO 4 adsorbate becomes small in comparison with that for the sample (vii), and the two peaks are observed at the photon energy regions of 2468-2471 eV and 2475.5-2480eV.It is thought that those peaks indicate the adsorbate of the atomic S and the SO x (x = 2 or 3), respectively.Although the adsorbates existing on the Au NPs are different from the sample (vii), we can not clear the reason in this study.

IV. CONCLUSIONS
The Au NPs are fabricated under water environment by the solution plasma method.The particle diameter as fabricated is estimated to be 2.4±1.0 nm.When the L-cysteine is dissolved into the Au NPs colloidal solution, the color of the Au NPs colloidal solution is changed from red to dark blue.We reveal that the L-cysteine is adsorbs on the Au NPs surface as L-cysteine thiolate, L-cystine and atomic S by sulfur K-edge NEXAFS.Moreover, the Au NPs aggregate each other by the hydrogen bonding of the L-cysteine adsorbates on the Au NPs after several hours.It is clear that the change of the color for the Au NPs colloidal solution occurs by both the adsorption of the L-cysteine on the Au NPs with thiol group and the aggregation of the Au NPs with the hydrogen bonding.In addition, it seems that the hydrogen bonding keeps partly stably when the aggregated NPs are diffused again in distilled water.

FIG. 1 :
FIG. 1: Photographic views of the fabrication of Au NPs by solution plasma with reaction times (0, 5 and 30 min).

FIG. 2 :
FIG. 2: Photographic views for liquid color changes after mixing L-cysteine into Au NPs colloidal solution and precipitate.