Rapid Synthesis of Carbon Dots by Hydrothermal

materials Article

Rapid Synthesis of Carbon Dots by Hydrothermal Treatment of Lignin Wenxin Chen 1 , Chaofan Hu 2 , Yunhua Yang 3 , Jianghu Cui 4 and Yingliang Liu 4, * 1 2 3

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Department of Chemistry, Jinan University, Guangzhou 510632, China; [email protected] College of Mechanics, Taiyuan University of Technology, Taiyuan 030024, China; [email protected] Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangzhou 510070, China; [email protected] Department of Applied Chemistry, College of Science, South China Agricultural University, Guangzhou 510642, China; [email protected] Correspondence: [email protected]; Tel.: +86-20-3733-8319

Academic Editor: Lioz Etgar Received: 24 November 2015; Accepted: 23 February 2016; Published: 9 March 2016

Abstract: A rapid approach has been developed for the fluorescent carbon dots (CDs) by the hydrothermal treatment of lignin in the presence of H2 O2 . The as-synthesized CDs were found to emit blue photoluminescence with excellent photostability. Moreover, the CDs displayed biocompatibility, low cytotoxicity, and high water solubility properties. Finally, the as-resulted CDs were demonstrated to be excellent probes for bioimaging and biosensing applications. Keywords: carbon dots; lignin; H2 O2 ; hydrothermal synthesis; bioimaging

1. Introduction Carbon dots (CDs) are an interesting class of carbon nanoparticles, which are being investigated for various applications due to their favorable optical stability, low toxicity, biocompatibility, and ease of functionalization [1–3]. Many researchers have studied the synthesis methods and photoluminescence properties of CDs. A variety of synthesis approaches such as laser ablation, electrochemical exfoliation, pyrolysis, incomplete combustion oxidation, acidic oxidation, hydrothermal treatments and microwave synthesis have been developed to prepare CDs [4–11]. Various raw material including graphite oxide, citric acid, glycerol, coffee grounds, soy milk, grass and egg have been used in the synthesis of CDs [12–16]. However, it is still desirable to rapidly synthesize high-quality CDs by an easy and environmentally benign method with low-cost and readily available starting materials. One such suitable raw material is lignin, which is an abundant natural organic polymer, and an excellent source of carbon. There is an increasing interest in using lignin to prepare new carbon-based materials [17–19]. However, it is quite difficult to degrade lignin and its derivatives due to strong carbon to carbon linkages in their molecular structure. Amongst various techniques, the hydrothermal carbonization process is a promising approach for the synthesis of novel carbon-based materials, especially CDs [20]. Herein, we report a rapid route to synthesize highly luminescent CDs by the hydrothermal treatment of lignin with the assistance of H2 O2 . It is well-known that H2 O2 can be dissociated into hydroxyl radicals (¨ OH) under the photoassisted catalysis Fe3+ /Fe2+ in water, and the resulting OH radical is an extremely powerful oxidizing species [21]. The synthesis approach was simple and environmentally friendly. It was demonstrated that the as-prepared CDs exhibit good luminescence property, good water solubility, narrow particle size distribution and low cytotoxicity. The CDs also showed excellent bioimaging capabilities in Hela cells. This work provides a new approach for the preparation of CDs from natural materials, and also demonstrates the potential of CDs in bio-imaging applications. Materials 2016, 9, 184; doi:10.3390/ma9030184

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Materials 2016, 9, 184

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Materials 2016, 9, 184 Materials 2016, 9,of 184 CDs preparation

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2 ofin 8 from natural materials, and also demonstrates the potential of CDs bio‐imaging applications. preparation of CDs from natural materials, and also demonstrates the potential of CDs in bio‐imaging applications. 2. Results and Discussion 2. Results and Discussion 2. Results and Discussion Figure 1a indicates the pyrolysis products of lignin at 10, 20, 30, 40, 50 and 60 min, respectively. Figure 1a indicates the pyrolysis products of lignin at 10, 20, 30, 40, 50 and 60 min, respectively. It could be seen that the color of the initial product solution became pale as the time increased. It could be seen that the color of the initial product solution became pale as the time increased. The Figure 1a indicates the pyrolysis products of lignin at 10, 20, 30, 40, 50 and 60 min, respectively. The residual lignin was retained on the filter paper (Figure 1b). The synthesis yields of the CDs were residual lignin was retained on the filter paper (Figure 1b). The synthesis yields of the CDs were It could be seen that the color of the initial product solution became pale as the time increased. The 12.06%, 10.2%, 6.67%, 2.3%, 1.05%, 0.8% corresponding to 10, 20, 30, 40, 50 and 60 min, respectively. 12.06%, 10.2%, 6.67%, 2.3%, 1.05%, 0.8% corresponding to 10, 20, 30, 40, 50 and 60 min, respectively. residual lignin was retained on the filter paper (Figure 1b). The synthesis yields of the CDs were The optical images of the CDs solution exhibited blue luminescence under UV light excitation The optical images of the CDs solution exhibited blue luminescence under UV light excitation 12.06%, 10.2%, 6.67%, 2.3%, 1.05%, 0.8% corresponding to 10, 20, 30, 40, 50 and 60 min, respectively. (Figure 1c) and the pyrolysis time of 40 min exhibited the maximum brightness. The CDs solution (Figure 1c) and the pyrolysis time of 40 min exhibited the maximum brightness. The CDs solution The optical images of the CDs solution exhibited blue luminescence under UV light excitation remains transparent for half a year without precipitation. remains transparent for half a year without precipitation. (Figure 1c) and the pyrolysis time of 40 min exhibited the maximum brightness. The CDs solution remains transparent for half a year without precipitation.

Figure 1. Pyrolysis results for (a) the initial product solution; and (b) the residual lignin after 10, 20,

Figure 1. Pyrolysis results for (a) the initial product solution; and (b) the residual lignin after 10, 20, 30, 30, 40, 50 and 60 min hydrothermal reaction; and (c) the corresponding filtrate image under 365 nm Figure 1. Pyrolysis results for (a) the initial product solution; and (b) the residual lignin after 10, 20, 40, 50 and 60 min hydrothermal reaction; and (c) the corresponding filtrate image under 365 nm UV UV lamp irradiation. 30, 40, 50 and 60 min hydrothermal reaction; and (c) the corresponding filtrate image under 365 nm lamp irradiation. UV lamp irradiation.

Transmission electron microscopy (TEM) and high‐resolution TEM (HRTEM) performed on Transmission and high-resolution TEM (HRTEM) performed on the the CDs, and the results are shown in Figure 2. The size of the CDs ranged from 2 to 10 nm. The Transmission electron electron microscopy microscopy (TEM) (TEM) and high‐resolution TEM (HRTEM) performed on CDs, and the results are shown in Figure 2. The size of the CDs ranged from 2 to 10 nm. The HRTEM HRTEM images indicated that the carbon dots have crystalline structure and the lattice spacing the CDs, and the results are shown in Figure 2. The size of the CDs ranged from 2 to 10 nm. The images indicated that the carbon dots have crystalline and the lattice spacing distance was distance was about 0.21 nm, close to that of the graphite (100) plane [22]. HRTEM images indicated that the carbon dots have structure crystalline structure and the lattice spacing about 0.21 nm, close to that of the graphite (100) plane [22]. distance was about 0.21 nm, close to that of the graphite (100) plane [22].

Figure 2. (a) Transmission electron microscopy (TEM); and (b) high‐resolution TEM (HRTEM) images of the carbon dots (CDs) prepared by hydrothermal treatment of lignin. Figure 2. (a) (a) Transmission Transmission electron microscopy (TEM); and high-resolution (b) high‐resolution TEM (HRTEM) Figure 2. electron microscopy (TEM); and (b) TEM (HRTEM) images images of the carbon dots (CDs) prepared by hydrothermal treatment of lignin. of the carbon dots (CDs) prepared by hydrothermal treatment of lignin.


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From the wide-scan X-ray photo-electron spectroscopy (XPS) spectrum depicted in Figure 3, From the wide‐scan X‐ray photo‐electron spectroscopy (XPS) spectrum depicted in Figure 3, two strong peaks at 285.5 and 532.0 eV were attributed to oxygen and carbon, respectively. two strong peaks at 285.5 and 532.0 eV were attributed to oxygen and carbon, respectively. The The elemental components of the as-prepared CDs were C (82.58%) and O (17.42%). The deconvoluted elemental components of the as‐prepared CDs were C (82.58%) and O (17.42%). The deconvoluted 2 ) carbon C1s spectrum (Figure 3b) showed threethree components, which could be assigned as graphite (sp(sp 2) C1s spectrum (Figure 3b) showed components, which could be assigned as graphite 3 carbon at 3~286.1 eV, and carboxyl carbon at 288.6 eV. The O spectrum (Figure 3c) at ~283.2 eV, sp 1s The O1s spectrum carbon at ~283.2 eV, sp carbon at ~286.1 eV, and carboxyl carbon at 288.6 eV. exhibited three at 530.6, 533.2532.1 eV, which wereeV, attributed to the C=O, C-OH C-O-C (Figure 3c) peaks exhibited three 532.1 peaks and at 530.6, and 533.2 which were attributed to the and C=O, C‐OH and C‐O‐C groups, respectively [23,24]. groups, respectively [23,24].

Figure 3. (a) X‐ray photo‐electron spectroscopy (XPS); (b) C 1s spectra of the as‐prepared CDs. Figure 3. (a) X-ray photo-electron spectroscopy (XPS); (b) C1s1s; and (c) O ; and (c) O 1s spectra of the as-prepared CDs.

The CDs obtained after 40 min pyrolysis were characterized by Raman spectroscopy, as shown

The CDs obtained after 40 min pyrolysis were characterized by Raman spectroscopy, as shown −1, which corresponds to the in Figure 4a. Their Raman spectrum showed a strong D band at 1382 cm ´1 , which corresponds to −1, 1382 in Figure 4a. Their Raman spectrum showed a strongat D1578 band cmmatched sp3 defects in CDs. Also, a G band was observed cmat which well with the 3 defects in CDs. Also, a G ´ 1 , which matched well with the 2 the sp band was observed at 1578 cm disordered carbon and the sp clusters, indicating that there were aromatic groups inside CDs. It was observed that the CDs have an I /IG ration of 0.91, which might be due to oxygen‐rich edges of disordered carbon and the sp2 clusters,Dindicating that there were aromatic groups inside CDs. It was the CDs. spectra solution of CDs might showed at 282 and 348 nm, observed thatThe the UV‐Vis CDs have an IDof /Iaqueous ration of 0.91, which betwo duepeaks to oxygen-rich edges of the G indicating that there were different surface states present in the CDs solution. The fluorescence CDs. The UV-Vis spectra of aqueous solution of CDs showed two peaks at 282 and 348 nm, indicating spectra of the CDs40min were measured with an F‐4500 fluorescence spectrometer (HITACHI, Tokyo, that there were different surface states present in the CDs solution. The fluorescence spectra of the Japan), with a slit width of 10 nm for both excitation and emission beams. The excitation wavelength CDs40min were measured with an F-4500 fluorescence spectrometer (HITACHI, Tokyo, Japan), with a was varied from 280 to 500 nm, in 20 nm increments. The corresponding spectra are given in Figure slit width of 10 and nm for both photoluminescence excitation and emission beams. The was varied 4b. Bright colorful (PL) emissions were excitation observed wavelength from the CDs. The from emission 280 to 500 nm, in 20 nm increments. The corresponding spectra are given in Figure 4b. Bright maxima shifted as the excitation wavelength increased and exhibited a maximum PL and colorful photoluminescence (PL) emissions were observed from the CDs. The emission maxima intensity at an excitation wavelength of 320 nm and emission wavelength of 430 nm. The CDs shifted as the excitation wavelength increased and exhibited a maximum PL intensity at an excitation showed excellent photostability as the fluorescence intensity did not change, even after continuous excitation 150 W Xenon lamp. As shown in Figure 5, the of the fluorescein wavelength of under 320 nma and emission wavelength of 430 nm. The CDsfluorescence showed excellent photostability isothiocyanate (FITC) was quickly quenched within several minutes excitation and the as the fluorescence intensity did not change, even after continuous excitation under a 150 CdTe W Xenon lamp.quantum dots (QDs) were preserving 25% of the original PL intensity after 20 min excitation. The PL As shown in Figure 5, the fluorescence of the fluorescein isothiocyanate (FITC) was quickly intensity of the CDs that we synthesized retaining 95% of the initial intensity under ca. 100 min quenched within several minutes excitation and the CdTe quantum dots (QDs) were preserving 25% excitation. The result indicated that the PL of the CDs was much more stable than of the fluorescent of the original PL intensity after 20 min excitation. The PL intensity of the CDs that we synthesized FITC and the CdTe QDs. We considered that the formation of CDs and their surface functionalization retaining 95% of the initial intensity under ca. 100 min excitation. The result indicated that the PL of the CDs was much more stable than of the fluorescent FITC and the CdTe QDs. We considered that the formation of CDs and their surface functionalization take place simultaneously during the


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hydrothermal carbonizationduring process. presence ofcarbonization large number process. of carboxylic acids introduces take place simultaneously the The hydrothermal The presence of large several different surface defects. These defects behave as excitation energy traps, and are responsible number of carboxylic acids introduces several different surface defects. These defects behave as Materials 2016, 9, 184 4 of 7 for the different photoluminescence behaviors. In fact, several mechanisms have been proposed excitation energy traps, and are responsible for the different photoluminescence behaviors. In fact, to explain thesesimultaneously unique PLbeen properties, the size distribution ofproperties, the CDs, asuch distribution of take place during the such hydrothermal carbonization process. The presence of as large several mechanisms have proposed to as explain these unique PL the size different emissive trap sites and the formation of several different polyaromatic fluorophores within number of carboxylic acids introduces several different surface defects. These defects behave as distribution of the CDs, a distribution of different emissive trap sites and the formation of several the carbogenic core. However, the exact mechanism of the CDs’ PL behavior is still unclear and further excitation energy traps, and are responsible for the different photoluminescence behaviors. In fact, different polyaromatic fluorophores within the carbogenic core. However, the exact mechanism of the several mechanisms have been proposed to explain these unique PL properties, such as the size studies are required to understand this property in depth. CDs’ PL behavior is still unclear and further studies are required to understand this property in depth. distribution of the CDs, a distribution of different emissive trap sites and the formation of several different polyaromatic fluorophores within the carbogenic core. However, the exact mechanism of the CDs’ PL behavior is still unclear and further studies are required to understand this property in depth.

Figure 4. 4. (a) Raman absorbance and the is background given the dashed line; Figure (a) Raman absorbance and the background given by the is dashed line;by (b) photoluminescence

Figure 4. (a) Raman absorbance and the background is given by the dashed line; (b) photoluminescence (PL) spectra of the CDs at different excitation wavelengths. (PL) spectra of the CDs at different excitation wavelengths. (b) photoluminescence (PL) spectra of the CDs at different excitation wavelengths.

Figure 5. Photostability comparison of the fluorescent CDs, CdTe QDs and fluorescein isothiocyanate Figure 5. Photostability comparison of the fluorescent CDs, CdTe QDs and fluorescein isothiocyanate (FITC) in a fluorescence spectrophotometer with a 150 W Xe lamp under 360 nm excitation. (FITC) in a fluorescence spectrophotometer with a 150 W Xe lamp under 360 nm excitation. Figure 5. Photostability comparison of the fluorescent CDs, CdTe QDs and fluorescein isothiocyanate

(FITC) in a fluorescence spectrophotometer with a 150 W Xe lamp under 360 nm excitation. To investigate the feasibility of using CDs for bio‐imaging, A549 human lung adenocarcinoma To investigate the of using CDs for bio-imaging, A549 lung adenocarcinoma cells were used to feasibility evaluate the cytocompatibility of the CDs. The human cell viability of the CDs was cells were To investigate the feasibility of using CDs for bio‐imaging, A549 human lung adenocarcinoma used to evaluate the cytocompatibility of the CDs. The cell viability of the CDs was determined determined by a methylthiazoleterazolium (MTT) assay. As can be seen in Figure 6, the MTT assays cells were used to evaluate the cytocompatibility the inCDs. The cell MTT viability was of cell viability reports indicate that the CDs have very low cytotoxicity. This result confirms that by a methylthiazoleterazolium (MTT) assay. As can beof seen Figure 6, the assaysof ofthe cellCDs viability determined by a methylthiazoleterazolium (MTT) assay. As can be seen in Figure 6, the MTT assays CDs can be used for imaging or other biomedical applications. reports indicate that the CDs have very low cytotoxicity. This result confirms that CDs can be used for

of cell viability reports indicate that the CDs have very low cytotoxicity. This result confirms that imaging or other biomedical applications. CDs can be used for imaging or other biomedical applications.

Materials 2016, 9, 184 Materials 2016, 9, 184 Materials 2016, 9, 184

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Figure 6. Cytotoxicity evaluations test of A549 cells with different concentrations of CDs40min 40min after 24, after 24, Figure 6. Cytotoxicity evaluations test of A549 cells with different concentrations of CDs Figure 6. Cytotoxicity evaluations test of A549 cells with different concentrations of CDs40min after 24, 48 and 72 h incubation. 48 and 72 h incubation. 48 and 72 h incubation.

The obtained CDs from 40 min hydrothermal carbonization were introduced into the Hela cells, The obtained CDs from 40 min hydrothermal carbonization were introduced into the Hela cells, The obtained CDs from 40 min hydrothermal carbonization wereconfocal introduced into the Hela and their bio‐imaging capabilities were evaluated evaluated using in in vitro vitro microscopy test. cells, The and their bio‐imaging capabilities were using confocal microscopy test. The and their bio-imaging capabilities were evaluated using in vitro confocal microscopy test. The results results showed that the photoluminescent spots were observed only in the cell membrane and results showed that the photoluminescent spots were observed only in the cell membrane and showed that the photoluminescent spots were observed only in the cell membrane and cytoplasmic area cytoplasmic area of the cell, indicating that the CDs were able to easily penetrate into the cell (Figure 7). cytoplasmic area of the cell, indicating that the CDs were able to easily penetrate into the cell (Figure 7). of theobservation cell, indicating that theagreement with CDs were able toprevious easily penetrate the cell (Figure 7). This observation This was is in studies into on the the interaction of living living cells with with This observation was is in agreement with previous studies on interaction of cells was is in agreement with previous studies on the interaction of living cells with nanomaterials nanomaterials [25]. [25]. The The results results illustrate illustrate that that CDs CDs can can be be used used as as fluorescence fluorescence probe probe [25]. for nanomaterials for The results illustrate that CDs can be used as fluorescence probe for bio-imaging applications. bio‐imaging applications. bio‐imaging applications.

Figure 7. 7. (a) (a) A A confocal confocal fluorescence fluorescence microphotograph microphotograph of of Hela Hela cells cells labeled labeled with with the the CDs CDs Figure Figure 7. (a) A confocal fluorescence microphotograph of Hela cells labeled with the CDs (λex: 405 nm); (λex: 405 nm); (b) A bright field microphotograph of the cells; (c) An overlay image of (a) and (b). (λex: 405 nm); (b) A bright field microphotograph of the cells; (c) An overlay image of (a) and (b). (b) A bright field microphotograph of the cells; (c) An overlay image of (a) and (b).

3. Materials and Methods 3. Materials and Methods 3. Materials and Methods 3.1. Preparation of the Fluorescent CDs 3.1. Preparation of the Fluorescent CDs 3.1. Preparation of the Fluorescent CDs In a typical typical procedure, procedure, fluorescent fluorescent CDs CDs were synthesized synthesized as as follows: follows: 100 100 mg lignin lignin was was In In aa typical procedure, fluorescent CDs werewere synthesized as follows: 100 mg ligninmg was dispersed dispersed in 30 mL purified water and ultrasonicated for 10 min, then 2 mL H 2 O 2 was added, and dispersed in 30 mL purified water and ultrasonicated for 10 min, then 2 mL H 2O2 was added, and in 30 mL purified water and ultrasonicated for 10 min, then 2 mL H2 O2 was added, and the mixture the mixture was sealed into a 50 mL Teflon lined stainless steel autoclave, which was then placed in the mixture was sealed into a 50 mL Teflon lined stainless steel autoclave, which was then placed in was sealed into a 50 mL Teflon lined stainless steel autoclave, which was then placed in a muffle a muffle furnace followed by hydrothermal treatment at 180 °C for 10, 20, 30, 40, 50 and 60 min. After a muffle furnace followed by hydrothermal treatment at 180 °C for 10, 20, 30, 40, 50 and 60 min. After furnace followed by hydrothermal treatment at 180 ˝ C for 10, 20, 30, 40, 50 and 60 min. After the the reaction, reaction, the the autoclave autoclave was was cooled cooled down down naturally, and and the the obtained obtained yellow yellow solution solution was was the reaction, the autoclave was cooled down naturally,naturally, and the obtained yellow solution was filtered with a filtered with a a 0.22 0.22 μm μm membrane membrane filter filter (Millipore, Boston, Boston, MA, USA) USA) to to remove remove the the unreacted unreacted filtered 0.22 µm with membrane filter (Millipore, Boston,(Millipore, MA, USA) to removeMA, the unreacted lignin. The filtrate was lignin. The The filtrate filtrate was was subjected subjected to dialysis dialysis for for 2 2 days days using using a a 3500 3500 Da Da dialysis dialysis membrane membrane lignin. subjected to dialysis for 2 days using ato 3500 Da dialysis membrane (Spectrumlabs, Rancho Dominguez, (Spectrumlabs, Rancho Rancho Dominguez, Dominguez, CA, USA) USA) to to remove remove the the excess excess H H2O O22. . The The resulting resulting yellow yellow (Spectrumlabs, CA, USA) to remove the excess H2 O2CA, . The resulting yellow solution was 2freeze-dried to obtain the solution was freeze‐dried to obtain the final CDs. solution was freeze‐dried to obtain the final CDs. final CDs. 3.2. Characterization Methods 3.2. Characterization Methods Morphological features features of of the the CDs CDs were were using using a a transmission transmission electron electron microscopy microscopy (TEM, (TEM, Morphological Philips TECNAI 10, Amsterdam, Holland) and field emission electron microscope (JEOL JEM‐2100F, Philips TECNAI 10, Amsterdam, Holland) and field emission electron microscope (JEOL JEM‐2100F,


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3.2. Characterization Methods Morphological features of the CDs were using a transmission electron microscopy (TEM, Philips TECNAI 10, Amsterdam, Holland) and field emission electron microscope (JEOL JEM-2100F, JEOL, Tokyo, Japan). X-ray photo-electron spectroscopy (XPS, AXIS ULTRA DLD, Kratos, Manchester, British) was used to investigate the functional groups present on the surface of the CDs. The fluorescence spectra of the CDs were measured with a fluorescence spectrometer (F-4500, HITACHI, Tokyo, Japan), with a slit width of 10 nm and 10 nm for excitation and emission, respectively. The excitation wavelength increased by a 20 nm increment starting from 280 to 500 nm. 3.3. Fluorescence Imaging Experiments Hela cells were seeded in each well of a confocal dish (a coverglass-bottom dish) and cultured at 37 ˝ C for 24 h. An aqueous solution of the CDs (0.1 mg/mL) was passed through a 0.2 µm sterile membrane filter. The filtered fluorescent suspension (40, 50, and 60 µL) was mixed with the culture medium (200 µL) and then added to three wells of the confocal dish (the fourth used as a control) in which the Hela cells were grown. After an incubation period of 2 h, the medium was removed and the cells were washed thoroughly three times with phosphate buffered saline (PBS) and kept in PBS for the optical imaging. Cellular uptake of CDs by Hela cells was monitored by confocal microscopy under the excitation wavelength of 405 nm. 4. Conclusions In conclusion, we have demonstrated that a fast, efficient and green method to synthesize fluorescent carbon dots by the hydrothermal treatment of lignin under the action of H2 O2 . The resulting CDs were thoroughly characterized and showed excellent potential for applications in biological labeling and biosensors fields. Acknowledgments: This work was financially supported by the National Science Foundation of China (21401028, 21031001, 21571067 and 51372091), the higher school science and technology innovation project of Guangdong Province (cxzd1014), the youth fund of Guangdong Provincial Academy of Sciences (qnjj201406), and the Microbial Talents Cultivation Fund of Guangdong Institute of Microbiology. Author Contributions: Chen Wenxin contributed to the concept, supervised the entire research work and wrote the manuscript. Hu Chaofan and Yang Yunhua performed the core experimental works. Cui Jianghu measured the cell imaging experiment. Liu Yingliang contributed to the discussion of the results and conclusions and revised the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations The following abbreviations are used in this manuscript: CDs PL TEM XPS PBS

carbon dots photoluminescence transmission electron microscopy X-ray photo-electron spectroscopy phosphate buffered saline

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