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Stable graphene-isolated-Au-nanocrystal for accurate and rapid SERS analysis Yin Zhang, Yu-Xiu Zou, Fang Liu, Yi-Ting Xu, Xue-Wei Wang, Yunjie Li, Hao Liang, Long Chen, Zhuo Chen, and Weihong Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02958 • Publication Date (Web): 07 Oct 2016 Downloaded from http://pubs.acs.org on October 9, 2016

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Analytical Chemistry

Stable graphene-isolated-Au-nanocrystal for accurate and rapid SERS analysis Yin Zhang†, Yuxiu Zou†, Fang Liu†, Yiting Xu†, Xuewei Wang†, Yunjie Li†, Hao Liang†, Long Chenǁ,*, Zhuo Chen†,*, and Weihong Tan†,* †

Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Innovation Center for Molecular Engineering and Theranostics, Hunan University, Changsha 410082, China. ǁ

Faculty of Science and Technology, University of Macau, E11, Avenida da Universidade, Taipa, Macau, China.

Abstract: Various interferences from measurement conditions and substrate inhomogeneity are well-known confounding factors for poor reproducibility which is a challenge in SERS quantification. To address these issues, novel substrates and versatile internal standards have been designed and the repeatability is improved to some degree. However, these internal standards are either complex or unstable enough to resist harsh environments such as acid and oxidation. Graphene-isolated-Au-nanocrystal (GIAN) has unique properties and been applied for cell multimodal imaging and chemotherapy but not for SERS quantification analysis yet. Herein, we chose GIANs to improve the accuracy of SERS analysis. GIAN integrates the SERS effect and internal standard into a simple nanoparticle, and is proved to be an ideal platform for SERS analysis given its superior properties: (1) chemical stability, it remains stable in strong acid and oxidation, even mimic bio-environment; (2) a simple core-shell structure, with a thin graphitic shell which is not only a protector that avoiding inner Au catalysis unnecessary reaction but also an internal standard to eliminate the interference during the Raman detections; (3) the big-Π structure can absorb target molecule thus achieve an enrichment effect and quench background fluorescence. Laser power, focus and substrate fluctuations, as well as coexist substance interferences were investigated and the accuracy was improved greatly with the introduction of 2D band internal standard in Raman silent region with less background. Moreover, GIAN was applied for crystal violet determination directly on fish muscle and scale which was rapid and convenient without complex extraction process. All these results indicate GIAN is an optimum choice for SERS analysis in complex systems.

Surface enhancement Raman scattering (SERS) has received increasing attention in various fields for its unique and superior advantages as a powerful detection method.1-4 Now SERS is capable of providing more valuable information by its specific fingerprint and it is sensitive enough down to the level of a single molecule.5-9 Furthermore it is noninvasive, and only a few treatments are needed for sample preparation. However, reproducibility remains a challenge in SERS quantification to date. 10,11 More specifically, it is difficult to acquire a reliable result based on the interference of such factors as small difference in focusing depth, laser fluctuation, and inhomogeneity of substrate.12 Numerous strategies have been tried to tackle this problem and some success has been achieved, notably those efforts involving substrate fabrication or the introduction of an internal standard. In fact, various novel substrates with different morphology and properties including homogeneity, sensitivity and stability have emerged, such as gold rods,13-15 gold cubes,16 silver stars17 and most recently, metal-organic frameworks (MOFs).18,19 Simultaneously, versatile internal standards have been rationally designed and reported, but, at the same time, they have inherent drawbacks. For example, the selfassembled monolayer (SAM),20 as an internal standard may prevent the chemical absorption and decrease the competition between target molecule and internal standard. Nevertheless, the enhancement effect of substrate is limited owing to the long distance between analyte and substrate. The isotope edited internal standard (IEIS)21,22 is another example. Although it narrowed the difference of enhancement factor to

some degree, the Raman spectrum between analyte and isotopically labeled internal standard must differ considerably, thus limiting types of analytes that can be tested. Organic molecules are frequently used as internal standards such as 4aminothiphenol,23 p-thiocresol24 and cyano-containing compounds20 etc. However, using conventional organic molecules has its problems since they are not steady in an acidic or strong oxidation environments. Zhao25 used silicon as an internal standard to annihilate variables from instrumentbearing variables, such as laser power and focus, and obtained desirable results. Ren10 designed a new structure namely the core-molecule-shell (CMS) which has an embedded internal standard, and achieved success in SERS quantification. However, the structure was complicated and extra organic molecules needed to be introduced and the control of internal standard was not so easy. Chen utilized gold coated graphitic magnetic nanocapsules26 with a low background internal standard for SERS quantification which was another progress for SERS quantification, similarly, its structure is quite complicated, so the substrate-making is quite time consuming and labor intensive. Graphene, which is a honeycomb lattice consisting of a single layer carbon atoms which is ubiquitous and has been widely used in all fields based on its excellent chemical and physical properties.27-32 The Raman signal of graphene is quite simple and unique, especially the 2D band located in the Raman silent region with less backgrounds. Moreover, it possesses the anti-photobleaching and anti-photocarbonization capabilities. Based on these characteristics, we rationally chose

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graphene-isolated-Au-nanocrystal (GIAN)33 to addressing the shortcomings of other published internal standards, as noted above. GIAN has a protective shell that can resist to oxidation and powerful laser irradiation, as well as avoid inner Au catalysis of unnecessary reactions and photocarbonization.34 Graphene can also improve the Raman signal which has been reported as GERS,35-37 through chemical enhancement. With its huge specific area, the graphitic shell of GIAN can absorb organic molecules by π-π interaction and thus realize an enrichment effect simultaneously quench background fluorescence while the plasmonic Au core dramatically improves not only the Raman signal of target molecule but also internal standards. When GIAN was utilized as a SERS substrate with the 2D band as the low background internal standard for quantification, it demonstrated improved accuracy through effectively eliminating the laser power, focus and substrate fluctuations, as well as coexist substance interferences. Moreover, when GIAN was applied for the detection of crystal violet (CV), a triphenylmethane dye widely utilized in breeding industry, on the surface of fish muscle and scale, an easily achieved, rapid result was obtained.

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Zeta sizer Nano ZS90. Raman detection was accumulated by Renishaw’s InVia Raman system under laser irradiation at 532 nm. Stability Characterization of Material and Internal Standard. A total of 4.79 mg R6G powder were dissolved in 10 mL water as a stock solution, and diluted to appropriate concentration as needed. Ten µL of 2.5 µM R6G were mixed with 15 µL concentrated AuNP39 and GIAN respectively. Then 10 µL of the mixtures were dropped on glass and dried at room temperature (RT). Both mixtures were exposed for 1 min to irradiation of 10% 532 nm laser and then applied for Raman analysis. A total 4.3 mg of p-Toluenethiol were dissolved in 10 mL C2H5OH as a stock solution. A moderate amount of AuNP were added to p-Tolunenethiol solution and diluted to 20 µL; then 30% H2O2 was introduced. Equal GIAN (same plasmonic absorbance as AuNP) was diluted as AuNP, and H2O2 was added as well. After 30 min, Raman spectra were obtained. One mM CV stock solution was attained by dissolving 2.04 mg CV powder in 5 mL H2O, followed by diluting to 40 nM, 60 nM, 100 nM, and 140 nM. Various interferences including isometric HCl, NaOH, cell culture medium, and PBS, were added to 1 µM CV solution respectively and diluted to the same volume. Preparation for Fast Determination of CV On Site. First, we obtained fish extraction containing CV by the conventional method.40 A fresh fish was purchased from local supermarket. The slice of fish muscle and scale was washed with ultrapure water for three times. In order to test the applicability of detection on fish directly, GIAN was firstly mixed with CV and then dropped on fish muscle and scale. Another detection method involved immersion of fish muscle and scale in CV solution for 30 min followed by application of GIAN after drying at RT. All the samples were fixed on glass for SERS analysis.

Figure 1: GIAN fabrication and characterization. (a) Schematic diagram of GIAN; ( b) UV-vis spectrum of GIAN and Photo of GIAN dissolved in water inserted; (c) Raman spectrum of GIAN under 532 nm laser; (d) TEM image of GIAN; scale bar, 50 nm; (e) HR-TEM image; scale bar, 5 nm.

EXPERIMENTAL SECTION Reagents: Chloroauric acid (HAuCl4·4H2O, 99.9%) and R6G (C28H31NO3Cl, 99%) were purchased from Changsha Chemical Reagents Company, and p-Toluenethiol (C7H8S, 99%) was purchased from Energy Chemical. Polyoxyethylene stearyl ether was obtained from Yarebio Company. Millipore Milli-Q-grade ultrapure water of Type 1 (18.2 MΩ·cm-1) was used in all experiments. Synthesis of GIAN. GIAN was synthesized by chemical vapor deposition (CVD).26,33,38 Briefly, we sonicated fumed silica (1.00 g) dissolved in methanol and chloroauric acid (10.81 mL, 0.1%) mixed with 80 mL methanol for 2 h. After removal of the methanol and dried the mixture, 0.25 g of the powder was introduced into a tube furnace for CVD growth. The product was finally treated with HF to remove silica. A JEOL 3010 microscope was used to acquire the TEM images. The size and Zeta potential of GIAN were attained by Malvern

RESULTS AND DISCUSSION Synthesis and Characterization of GIAN. Stable GIAN was synthesized by CVD method. Graphene was intact deposited on AuNP which was a protective shell, as well as providing an internal standard, as shown in Figure 1a. In order to obtain better solubility, polyoxyethylene stearyl ether molecule was introduced to functionalize GIAN. Consequently GIAN was adequately dispersed in water which became transparently red as shown in Figure 1b (inserted photo). The plasmonic resonance absorbance of GIAN located in 538 nm (Figure 1b), which overlapped with the excitation of 532 nm and showed a resonance effect which could dramatically promote the Raman signal. The average size of GIAN was around 45 nm (Figure S1a), which was consistent with the TEM results (Figure 1d). Through HR-TEM (Figure 1e), the structure of GIAN was quite clear, with deposition of a few layer graphene on AuNP. GIAN was slightly negatively charged (Figure S1b; ζ=- 4 mV), according to Zeta potential measurement, which might have been induced during the process of HF etching. Raman spectrum by GIAN was shown in Figure 1c, and two evident bands could be observed at 1352 cm-1 (designated as D band) and 1600 cm-1 (G band) respectively, both of them are special vibration modes of graphitic carbon shells. Another prominent peak could also be observed around 2700 cm-1 (2D band), which is in the Raman silent region with low background interference. The 2D band


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of GIAN is unique and strong and thus suitable for accurate SERS quantification.

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SERS substrate (Figure 2c). After H2O2 treated for 30 min, the Raman signal of the p-Tolunenethiol was dramatically decreased indicating possible Au-S destruction by H2O2, while for the 2D band (2700 cm-1) of GIAN, the Raman signal was in concordance with before adding of H2O2 (Figure 2d). Further characterization of the stability of the 2D band in lysozyme (Figure S5), which mimics biotic factors in the environment, was conducted. Almost no obvious changes of the 2D band intensity could be observed. Above all, these results show the superior stability of GIAN by its resistance of harsh environment to such as strong oxidation and biological enzyme.

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Figure 2:Stability of GIAN. (a), (b) Raman spectra of Au-R6G and GIAN-R6G exposed to 10% 532 nm laser power at 0 min (red) and 1 min (orange); (c) Raman Spectra of pTolunenethiol with H2O2 at 0 min (dark red) and 30 min (gray); (d) Raman spectra of GIAN with H2O2 at 0 min (black) and 30 min (red). Stability of GIAN for Raman Analysis. A desirable substrate should be stable, homogenous, and reproducible, all goals which remain challenging in SERS analysis. Previous reports have demonstrated that graphene, as an isolated layer for metal, integrated superior properties of graphene and noble metal.41-49 The Graphene shell can absorb target molecule through π-π interaction and simultaneously quench fluorescence, while the noble metal core acts as the enhancement substrate. The GIAN can enhance the Raman signal of CV more than 2000 times at the concentration of 0.25 µΜ. Spectra from 17 randomly selected sites were collected, as shown in Figure S2. The Raman signal of CV was rather steady with no fluctuation as indicated by the repeatability. For comparison, different enhancement materials (AuNP and GIAN) were investigated. Figure 2a, b showed 40 µM R6G was exposed to 10% laser irradiation for 1 min. The signal from R6G with AuNP was severely reduced by the strong laser with strong laser and no prominent peak (775 cm-1, 1649 cm-1) could be observed, whereas the peaks of R6G with GIAN (772 cm-1, 1648 cm-1) still remained clear. CV with GIAN and AuNP were also exposed to 10% laser for 1 min (Figure S3a, b) and demonstrated the similar result. This can be explained by GIAN's graphitic shell which can transfer the laser heat faster50-54 and thus avoid inner Au catalysis of unnecessary reactions and photocarbonization, properties which protect against deterioration of target molecule otherwise induced by strong laser irradiation. More data were collected with laser exposure for 1 min as shown in Figure S4. Both 775 and 772 cm-1 were chosen to analyze the variation tendency, and as expected, the decline of Au-R6G was fairly drastic whereas GIAN-R6G was much less so. Owing to the good stability of the graphitic nanomaterials, the Raman bands of the GIAN also exhibited superior stability. Especially, the stability of the GIAN 2D band which could be utilized as the internal standard was investigated and compared with the pTolunenethiol, a conventional internal standard. Both 1076 and 1594 cm-1 bands stem from p-Tolunenethiol with Au as the

Raman Analysis with GIAN as an Internal Standard. An internal standard is implemented to calibrate the deviation caused by experimental factors, such as laser power and focusing, as well as substrate fluctuation during Raman quantification analysis. Using GIAN as the internal standard, Raman analysis was performed to investigate the effects of these interferences. Figure 3a showed the original Raman spectra of CV with exposure to 10% laser power for different times. The 913 and 2700 cm-1 bands belonging to CV and GIAN were enlarged as respectively shown in Figure 3b respectively. The intensity of CV sometimes strengthened then weakened, as time elapsed. The height, i.e., intensity of CV, varied significantly with no regularity. However, when the 2D band was introduced as the internal standard, the ratio (I913/I2700) remained at the same level (Figure 3c), demonstrating that an internal standard can reduce the deviation from the effect of laser and improve the accuracy of quantification. The influence of the laser focusing was also investigated and showed in Figure 3d. Without the internal standard the signal fluctuated irregularly; however, when 2D band was used as internal standard, the signal was comparatively steady. Since aggregation state of nanoparticles is a significant factor for SERS detection, we collected spectra of CV up to five rounds with the original spectrum given in Figure 3e by a waterfall plot. As indicated in Figure 3f, the height fluctuated dramatically, while the ratio was comparatively more stable and precise. Therefore, GIAN as an internal standard can decrease the errors caused by fluctuation in measurement conditions and improve accuracy.


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Figure 3: Applicability of GIAN in quantification. (a) Raman spectra of CV exposed to 532 nm laser for different seconds; (b) Zoom in peaks 913 and 2700 cm-1 in (a); (c) Histogram of different exposure time, Height (=intensity; black) means intensity and Ratio (I913/I2700) (red), data of height and ratio are normalized by the first data respectively; (d) Histogram of different focusing depth. Red and black indicate ratio and height respectively; (e) Original Raman spectra of five test rounds; (f) Histogram of different rounds corresponds to (e).

Precise Raman Quantification Analysis with GIAN. Apart from interferences of measurement condition, coexist substances in the measurement system can change the state of SERS substrates and thus affect the accuracy of Raman analysis. Therefore, different interferences, such as acid, base and extra protein in the detection system were investigated to verify the capability of the GIAN for quantification analysis. As shown in Figure 4a, the height results of the measurements dramatically fluctuated and with an introduction of internal standard (ratio), the result was much more accurate, indicating that GIAN was an ideal enhancement material for SERS quantification. Extra ions were also introduced as the interferences, and the results also indicated the superiority of the GIAN (Figure S6). Various concentrations of CV dissolved in water were determined with the GIAN for signal enhancement. The results after linear fitting were shown in Figure 4b, the correlation coefficient (R) with height was 0.982 which was not as good as using the ratio (R= 0.995) indicating that an internal standard could achieve a better quantification effect even if when no adscititious interference was in system. Then, to spike in interference, cell culture medium which was severely affect the accuracy of detection implied in Figure 4a was added into the CV solution, and the Raman spectra were shown in Figure 4c. The correlation coefficient was significantly improved from 0.778 to 0.996 by using GIAN as the internal standard (Figure 4d). Given these results, GIAN is demonstrated to be an optimum selection for

Figure 4:GIAN for SERS quantification. (a) 0.1 µM CV was added to interferences including PBS, acid (HCl), base (NaOH), BSA, and cell culture medium, and diluted to a certain volume; (b) Various CV concentrations without any interference; Linear fitting of CV without internal standard (black) and with internal standard (blue); (c) Raman spectra of different CV concentration in diluted cell culture medium as an interference; (d) Linear fitting of CV corresponds to (c), the black line with an internal standard while blue without internal standard.

Using GIAN for Raman Analysis of CV on Fish Muscle and Scale. Having established that GIAN is capable of accurately detection small molecule in complex system, we further applied GIAN to a real bio-sample. Crystal violet is a triarylmethane dye. It is typically used as a histological stain in Gram’s method of classifying bacteria, but it is also used as a fungicide.55,56 However, to a certain extent, it is also carcinogenic when accumulated in the body through food chains. Methods for CV detection such as HPLC, ELISA, Fluorescence, both are time-consuming, and a tedious extraction process is needed. GIAN, however, was utilized to detect CV directly on the surface of fish muscle and scales, and no complicated extraction was required. Here, two methods was investigated. One method first mixed CV and GIAN, and then dropped the solution on fish muscle and scale (Figure S7a and b). The 913 cm-1 band from CV on fish muscle and scale were acquired. Another method was illustrated in Figure 5a, when fish muscle and scale was immersed in CV solution firstly then added GIAN for SERS analysis. Fluorescence background is a primary hindrance for bio-sample analysis. However, with the superb fluorescence quenching ability of the graphitic nanomaterials,57,58 Raman signals of CV from fish or scale were easily detected with the GIAN. As shown in Figure 5b, c and d, which represent fish muscle, fish scale and extraction of fish respectively, in the absence of GIAN, the fluorescence was quite evident especially from 1800-2700 cm-1 with a steep rising curve and no peaks could be observed. On the other hand, with the GIAN, the 913 cm-1 band from CV on fish muscle and scale were


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obtained as clearly shown in Figure 5b and c. To compare results, Raman signal of CV using the conventional extraction method was also explored (Figure 5d). Since the fluorescence has been overcome, GIAN for SERS detection has high potential for such biomedical application.

Procedures and additional data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

[email protected]; [email protected]; [email protected].

ACKNOWLEDGMENT This work was financially supported by the National Key Basic Research Program of China (No. 2013CB932702), the National Natural Science Foundation of China (No. 21522501, 61673405), the Science and Technology Development Fund of Macao S.A.R (FDCT, 067/2014/A, 097/2015/A3).

REFERENCES

Figure 5: (a) Schematic illustration of CV detection on site. (b) Spectra of CV detection on fish muscle, M indicates fish muscle; (c) Spectra of CV detection on fish scale, S indicates fish scale; (d) Spectra of extraction containing CV, E indicates fish extraction.

CONCLUSIONS We utilized an ideal Raman enhancement material, GIAN, which integrates the superior properties of both graphene and Au for Raman analysis. The GIAN has unique D, G and 2D bands from the graphitic shell, and superior SERS performance in enhancing signal from the Au core. The graphitic shell of GIAN can transfer the laser heat faster and thus avoid inner Au catalysis of unnecessary reactions and photocarbonization induced by strong laser irradiation during the Raman detection. GIAN also demonstrated high stability, as manifested in its resistance to such harsh environments as strong oxidation and biological enzyme, making it ideal for Raman biochemical analysis. The 2D band, as an internal standard which located in Raman silent region with less background could decrease errors in detection, such as laser power, focusing, and substrate fluctuation, and improve quantification accuracy. Coexist interferences, such as acid, base and extra protein in the detection system which could affect the accuracy of Raman analysis were effectively eliminated with utilizing the GIAN analysis platform. The correlation coefficient was significantly improved from 0.778 to 0.996 by using GIAN as the internal standard for the samples with cell culture medium as the interference. Furthermore, GIAN was applied for the detection of CV directly on the surface of fish and demonstrated superior biosample detection capability. Above all, GIAN is indicated to be a preferred enhancement substrate for precise quantification and biochemical Raman analysis.

ASSOCIATED CONTENT Supporting Information

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For TOC:


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Supporting Information Stable graphene-isolated-Au-nanoparticles for accurate and rapid SERS analysis Yin Zhang†, Yuxiu Zou†, Fang Liu†, Yiting Xu†, Xuewei Wang†, Yunjie Li†, Hao Liang†, Long Chenǁ,*, Zhuo Chen†,*, and Weihong Tan†,*

1

Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Innovation Center for Molecular Engineering and Theranostics, Hunan University, Changsha 410082, China.

2

Faculty of Science and Technology, University of Macau, E11, Avenida da Universidade, Taipa, Macau, China.

*Email: [email protected]; [email protected]; [email protected].


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Figure S1. (a) Size distribution of GIAN in water (b) Zeta potential of GIAN.

Figure S2. Random selected 17 points for CV-GIAN in one round under 532 nm 10% laser power.

a)

b) 6000

1617cm

CV-GIAN

6000 10% laser for 1 min -1

4500

913 cm

3000 10% laser 1 min

1500

Intensity (a.u.)

-1

Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CV-AuNP

-1

1617 cm

5000 -1

913 cm

4000

10% laser for 1 min

3000 2000 1000

600

900

1200

1500

1800

-1

Raman shift (cm )

600

900

1200

1500

1800

-1

Raman shift (cm )

Figure S3. (a), (b) Raman spectra of CV-GIAN and CV-AuNP exposed to 10% 532 nm laser power at 0

min (red) and 1 min (black)


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Figure S4. (a),(b) Raman spectra of Au-R6G and GIAN-R6G exposed to 1% 532 nm laser for 1 min, 45 data were collected during 1min; dark blue (a) and red (b) indicate 0 min, sky blue (a) and pink (b) indicate irradiation 1 min later. (c) The intensity of Au-R6G and GIAN-R6G by the 775 and 772 cm-1 respectively, zoom out is normalized intensity by the first data for both Au-R6G and GIAN-R6G.

Figure S5. Raman spectra of GIAN added lysozyme as an interference at 0 min (black) and 30 min (red).

Figure S6. 0.1 µM CV was added to interferences including Ca2+, Na+, and diluted to a certain volume

Figure S7. Capability of determination CV on fish muscle (a) and fish scale (b), M indicates fish muscle, S indicates fish scale.


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Stable Graphene-Isolated-Au-Nanocrystal for ... - ACS Publications

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