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We demonstrate a novel in vivo sampling device for preparation-free quantitative detection of residual malachite green in fish with depth profiles. Th...
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Gold Nanoparticle-Decorated Silver Needle for Surface-Enhanced Raman Spectroscopy Screening of Residual Malachite Green in Aquaculture Products Binbin Zhou, junda shen, Pan Li, meihong ge, dongyue lin, Yangyang Li, Jian Lu, and LiangBao Yang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.9b00262 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Gold Nanoparticle-Decorated Silver Needle for Surface-Enhanced Raman Spectroscopy Screening of Residual Malachite Green in Aquaculture Products Binbin Zhou,†,‡ Junda Shen,‡ Pan Li, † Meihong Ge,† Dongyue Lin,† Yang Yang Li,§Jian Lu,‡,∥,⊥ Liangbao Yang *,† †Center

of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031,

China ‡Department

of Mechanical Engineering, City University of Hong Kong, Hong Kong, China.

§Department

of Materials Science and Engineering City University of Hong Kong, Kowloon, Hong Kong, China

∥Hong

Kong Branch of National Precious Metals Material Engineering Research Centre, Department of Material Science and Engineering, City University of Hong Kong, Hong Kong, China.

⊥Center

for Advanced Structural Materials, City University of Hong Kong Shenzhen Research Institute, Shenzhen, China

Corresponding Author

*L.B.Y. ([email protected]). Fax: (+86)551-65592420. Phone: (+86)551-65592385

ABSTRACT: We demonstrate a novel in vivo sampling device for preparation-free quantitative detection of residual malachite green in fish with depth profiles. The device is a surface-enhanced Raman scattering (SERS)-active acupuncture silver needle carved with an array of micron-sized grooves for trapping polyvinylpyrrolidone (PVP)-stabilized Au nanoparticles (Au NPs). The microgrooves not only prevented the absorbed Au NPs being erased during insertion, but also provide observation/locating convenience under the optical microscope of the Raman spectrometer. Moreover, the Au NPs anchored onto the Ag needle help improve the sensitivity of the SERS measurements, while the plasmon coupling between Ag needle and Au NPs greatly amplifies the SERS signal. Furthermore, the PVP on the surface of the Au NPs serves as an inherent internal calibration standard leads to reduced SERS signal fluctuation, holding promising for quantitative SERS detection. The fabricated Au NPs-groove-trapped Ag needles (Au-G-AgNs) are further applied for in vivo detection of malachite green, a prohibited cancerogen commonly found in aquaculture products that are expensive and complicated to detect for current techniques. The result shows that in vivo detection of malachite green at an ultralow concentration of 0.1 nM is achieved, demonstrating the capability of the as SERS sampler for ultrasensitive in vivo detection with minimal invasion. KKEYWORDS: in vivo detection, silver acupuncture needle, minimally invasive, groove, quantitative SERS

■ INTRODUCTION Quantitative detecting trace target analytes in vivo with minimal invasion is of fundamental importance to biological detection. Among various analytical techniques, surface-enhanced Raman scattering (SERS) technique hold potential for non-destructive1 and ultrasensitive characterization.2-3 The most commonly used SERS strategy in vivo is based on the injection of SERS tags since the target information always suffer from the interferences of complicate background signals.4 However, the SERS signals of these studies can acquired indirectly from SERS tags, but not the target molecule itself.5 Moreover, only a few SERS tags can reached target position in the body, which led to the poor efficiency of the SERS tags, and greatly limits the application of SERS tags in vivo detection. 6 The challenge is amplified when the potential toxicity of SERS tags is considered in vivo. In recent years, Raman spectroscopy take advantage of optical fibres, miniaturised lasers, the advanced optical microscopy technologies and other photonic devices, to improve label free diagnostic depth and performance.78 For example, Van Duyne and co-worker placed a metal frame in an incision to collect SERS signals of target molecule to avoid attenuation in the skin.5 However, most of these methods obtain the target information in vivo by reform the equipment,9-10 which are time consuming and relatively costly. Hence, it is need to develop a simply extracted strategy, which are capable of excellent SERS performance and minimal invasion in vivo. 11

Taking inspiration from traditional Chinese acupuncture, scientists have used needle-based sensors for both single-cell and in vivo detection. 12-15 Liu at el developed microprobes-based sensor for probing proteins in singe cell and acupuncture needlebased sensor for living bodies.12 However, in the early research, the needles are only shown as the carriers, without any SERS enhancement. As we know, silver is one of the most widely used Raman enhancing platforms. Recently, our group firstly modified the Au NPs on the Ag acupuncture to improve the sensitively and stability of the used needle-based SERS sensors.15 However, the stability of needle-based SERS sensors is also a great challenge, especially for the in vivo detection. Dong at el prevented the Au nanoshells being erased during insertion by modifying the polystyrene on the needle. 13However, polymer coating on the surface could reduce SERS enhancement, which increases the difficulties of trace analyte detection. The challenge is amplified when quantitative SERS analysis are considered in vivo. Herein, we propose and fabricate a novel SERS sampler, which composed of Au NPs in carefully designed groove array on the silver needle. The groove, with  m size, is not only to protect the absorbed Au NPs from being erased during insertion, but also can be found by optical microscopy conveniently. The analytes absorbed in different grooves in the surface of the needle were taken out with the SERS sampler pulled out. The traditional Ag needle is modified by PVP- Au NPs to improve the sensitivity and repeatability in SERS measurement. What is more, the PVP

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can be acted as stable internal standard, to reduce the fluctuation of the signals, which illustrates the great potential for quantitative analysis. Consequently, SERS detection at groove array can provide reproducibility of SERS signals, and quantitative different depth information in tissues. ■ EXPERIMENTAL SECTION Reagents. Hydrogen tetrachloroaurate (HAuCl4·4H2O), hydroxylamine hydrochloride (NH2OH•HCl), polyvinylpyrrolidone (PVP, MW = 55 000) and sodium citrate (NaCitrate) and were purchased from Shanghai Reagent Co., Ltd. Ag acupuncture needles with diameter of 0.40 mm were produced by Zhenjiang xincheng Acupuncture Instruments Co. Ltd. (China). Fish were purchased from local supermarket. Apparatus. SERS spectrum were acquired on a Raman system (LabRAM HR800), with a 633 nm laser. The scanning electron microscopy (SEM) images were acquired by a focused ion-beam (FIB) SEM, equipped with X-ray energy dispersive spectroscopy (EDS) capabilities. Moreover, the FIB-SEM instrument equipped with a gallium-ion beam, mill a series of the same size of the groove on the Ag needles. Furthermore, four of the same size grooves were mill in the same distance from the needle tip. Therefore, the reproducibility of Au-G-AgNs can be evaluate by the SERS signal from the grooves in the same distance from the needle tip. X-Ray photoelectron spectroscopy (XPS) analysis used X-ray photoelectron spectroscopy (Thermo ESCALAB 250Xi) analyses. Preparation of PVP-stability Au NPs. Au NPs seed were synthesized based on a modified literature procedure.32 Briefly, 2 mL of HAuCl4 (1% w/v) in 198 mL ultrapure water was heated to boiling and then adding 10 mL of Na-Citrate (1% w/v) and keeping boiling for 25 min. 22 mL of the mixture solution A, containing 20 mL NH2OH solution (0.02% w/v), 1 mL of Na-Citrate (1% w/v) and 1 mL PVP solution (1% w/v), were added separately in the 25 mL Au NPs seed solution at room temperature. Then, 20 mL HAuCl4 solution (0.1% w/v) was injected at room temperature.4 The size of the resulting particles was about 55 nm. The obtained PVP-stability Au NPs were centrifuged for further application. Fabrication of Au-G-AgNs. Commercial Ag acupuncture needles with diameter of 0.40 mm were ultrasonic cleaning with acetone and ethanol for three times, respectively. What is more, the Ag needles were dried under nitrogen. After drying, the Ag needles were fixed on the FIB-SEM specimen mount carefully. Gallium ion beam from the FIB-SEM was used to mill a series of the grooves on the Ag needles. Milling process was conducted at 30 kV and 600 pA in 100 seconds. After milling, we soaked the milled Ag needles in the concentrated PVP-stability Au NPs (Scheme 1).

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Stability of the Au-G-AgNs. Firstly, the Au-G-AgNs were incubated in 10-5 M of MG solution for 10 min, and then, the AuG-AgNs were inserted into the fish. SERS spectrum were acquired in every groove on the Au-G-AgNs while it was pulled out. Different spots including ones in the grooves and ones on the original needle surface, respectively. Furthermore, in order to obtain more credible experimental results, above experiments were repeated for ten times. In Vivo Malachite Green Measurement by Au-G-AgNs. To obtain the depth-resolution SERS detection in living systems. Fish were incubated in solutions with 10-5 M of MG solution for 60 min after that, the skin of the fish were rinsed in water repeatedly to avoid the SERS signal interference form the fish skin. The Au-GAgNs were then inserted into the fish and held for 10 min. SERS detection were execute immediately with laser power of 0.89 mW and 3 s acquisition time after the Au-G-AgNs were pulled out. The carefully designed grooves, can be found under optical microscopy, allowing us to monitor SERS in situ. The information from grooves in different position, corresponding to depth-resolution SERS detection in fish. ■ RESULTS AND DISCUSSION Characterization of Au-G-AgNs. Most reported SERS substrates have been shown the colloid or micro fabricated nanostructures on the flat solid surface.16 Here, we used acupuncture Ag needle to fabricate SERS-active structure. It is not only because the Ag is one of the most widely used Raman enhancing platforms, but also Ag needles can insert into the body with minimal invasion. In order to increase the contact area and fixed detection site of the Ag needles in the body, grooves were designed on the surface of Ag needles. However, due to threedimensional curved surface structure of the Ag needle, lithography is not applicable to this process. Here, FIB was used to mill the microgrooves in the Ag needles. What is more, FIB-SEM can be used to observe the change of the surface morphology and element distribution in the process of preparing Au-G-AgNs (Figure S1). Compared to traditional stainless steel needle, silver needle is more easily for etching (Figure S2). As shown in the Figure 1a, the size and depth of grooves can be accurately milled by FIB. Four of the same grooves in every 1 mm are designed in the Ag needles. The design showed that SERS measurement based on the different grooves along with the needle length direction is capable of monitoring the concentration profile of target molecules in living systems. Moreover, the repeatability of signals can be assessed by the grooves along with the needle width direction. After milling, we soaked the milled Ag needles in the concentrated Au NPs. As shown in Figure 1b, a uniform array of PVP-stability Au NPs was obtained in the grooves due to the steric and adhesiveness of PVP. Apart from the ion beam for milling, the electron beam in the FIBSEM is also used for elemental analysis and mor-phology characterization.17 As shown in Figure S3, the vast majority composition in the commercial needles is Ag elements, which avoid the interference of other elements. Ga elements, most common ion source, is obvious after the Ag needles accurately milled by the ion beam. More importantly, only a few Ga elements content in Au-G-AgNs, the effect for SERS detection can be negligible (Figure S4).

Scheme 1. Schematic images of Au-G-AgNs synthesis. Reproducibility and Sensitivity of the Au-G-AgNs. To obtain the reproducibility and sensitivity of the Au-G-AgNs, firstly, the Au-G-AgNs immersed in MG solutions for 10 min, and then SERS measurements were acquired with laser power of 0.89 mW and 3s of integration time.

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Figure 1. (a) SEM image of Au-G-AgNs. (b) SEM image of PVPAu NPs in the groove. Evaluation of Au-G-AgNs. Since its discovery on the rough surface of the silver electrode, SERS has been applied to many analyses, such as biochemical,18-20 drugs,21-22 solar cell23 and pesticide.24 The SERS signal on the commercial Ag needles with poor repeatability is due to the random distribution of nanocoarse structure (Figure S5). Ag needles cannot directly use as a SERS structure limited in the poor repeatability. As we have known, the enhancement of the Raman signal is depending on the localized surface plasmons within metallic nanostructures, SERS is a kind of surface effect. In our study, Ag needle was considered as “film”, the thickness of PVP is about 1-3 nm (Figure S6), which was considered as “gap”, therefore, we try to build a nanoparticles-filmgap system (Figure 2a).25 PVP-stabilized Au NPs hold great promise in this issue for extremely stability26 and the excellent monodisperse properties.27 Figure 2b presented a 30 SERS spectra of 10-8 M MG collected randomly from grooves. The relative standard deviations (RSD) of the main vibrations (1169 cm-1) less than 10 % (Figure 2c). The values of the RSD obviously decreased when the Au NPs was modified on the Ag needles. In other words, the repeatability of SERS spectra is greatly improved when the Ag needle modified by the Au NPs. More significantly, when the Au NPs is modified in the Ag grooves, the limit of detection (LOD) of the MG arrived at 0.1nM (Figure 2d), Table S1 compares the LOD of MG in the Au-G-AgNs with other existing methods. The detection limit of the MG for the Au-G-AgNs was significantly lower than the two individual metal structures with MG (Figure 2e). The enhancement of Au-G-AgNs is promoted because of the multiple plasmonic couplings including the Au-gap-AgNs coupling and Au NPs-Au NPs coupling.

Figure 2. The repeatability and sensitivity of the Au-G-AgNs. (a) Schemetic drawing of the fabricated Au-G-AgNs. (b) 2D presentation of 30 SERS spectrum of 10-8 M MG collected randomly from grooves. (c) Histogram of SERS intensity at 1169 cm-1 of the 30 spectra of MG on the Ag needle and the Au-G-AgNs. (d) SERS spectrum obtained at different concentrations of MG on the Au-G-AgNs. (e) Comparison of enhancement effect of Au NPs, Ag Needle and Au-G-AgNs. In order to optimize the reaction time, UV−vis spectra were used to characterize the effect of the Ag needle for MG molecule adsorption over time. The UV−vis spectra result shown adsorption MG molecules on the needle get to balance in 10 min. In our experiment, all the sampling time is10 min (Figure S7). In addition to the reproducibility and sensitivity, stability is also the key issue of the SERS substrate especially when the Au-G-AgNs insert into the living body. As shown in Figure 3, the grooves have been accurately milled by FIB in the Ag Needles. The groove, 10 m × 10 m × 1m, which is visible under optical microscope, allow us to monitor by in situ SERS. The Au-G-AgNs were incubated in 10-5 M of MG solution for 10 min before inserting into the fish. And the SERS measurements were carried out immediately on the Au-G-AgNs after it pulled out. The most signal of MG received in the groove, even the experiment repeats ten times (Figure 3a). On the contrary, the MG signal out of the groove on the surface of the Au-G-AgNs sharp decrease after its insertion into the fish (Figure 3b). The intensity of SERS signal in the grooves is higher than that out of the grooves, especially when the Au-G-AgNs applied in vivo measurement (Figure 3c). The SERS signal out of the grooves attenuation sharply after the Ag needles inserted into the fish because the Au NPs on the Ag needle was easily erased during insertion.

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Figure 3. Comparison of SERS spectra of MG analyzed in the groove and out of the groove while the Au-G-AgNs insert the fish with 10 times. (a) SERS spectra analyzed in the groove. (b) SERS spectra analyzed out of the groove. (c) Comparison of stability between in the groove and out of the groove. The inset shows optical image and SEM of grooves on the Au-G-AgNs, the scale bar is 10 m. On the contrary, the SERS signal in the grooves is stability after the process, even after10 times of replication. The result shows that the grooves can not only prevent the pAu NPs coming off while insertion but also allowing us to monitor by in situ SERS. In conclusion, Au-G-AgNs holds great promise in vivo measurement for remarkably reproducibility, sensitivity and stability. Au-G-AgNs for Quantitative Detection. As mentioned above, an Au-G-AgNs has been fabricated for ultrasensitive label-free detection MG. Nevertheless, it is difficult to use this nanosampler for quantitative detection since SERS is vulnerable to interference of the microenvironment.28-32 Interestingly, we found that PVP can use as the internal standard to reduce SERS signal variability Whether N or S in PVP is combined with precious metals is controversial in early reports. 33Here, XPS technique was used to characterize the binding form of PVP in Au-G-AgNs. The O 1s peak from pure PVP appears at 530.8 Ev (Figure S8a), while the peak form the Au-G-AgNs at higher binding energy position (532.5 eV). On the other hand, there is no significant change in the binding energy position of N 1s peak from pure PVP and Au-G-AgNs (Figure S8b). This result reveals that carboxyl oxygen (C=O) is more likely to directly combine with Au NPs than N. What is more, theoretical calculations predicting the stretching vibrations modes of the Au−O band at 224 cm-1, which is consistent with other’s research.34 It's worth noting that 224 cm−1 , without any overlap of the Raman peaks from MG, is an ideal internal standard for SERS quantitative detection. SERS spectrum of 10-4 M MG from independent 8 times SERS measurements (Figure 4a). The repeatability of the spectrum was very poor until we used Au-O band as the internal standard (Figure 4b). An idea internal standard can reduce the disturbance of experiment and improve the accuracy of quantification.28-29 The SERS spectra of different concentrations of MG is obtained on the Au-G-AgNs (Figure 4c). As shown in Figure 4d, the correlation coefficient between the concentration and intensity of the Raman peak improved from 0.57 to 0.99 by using PVP as the internal standard. According to our measurement results, Au-G-AgNs held potential for quantitative SERS analysis.

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Figure 4. (a) SERS spectra of 10-4 M MG from independent 8 times SERS measurements (b) absolute intensity and relative intensity of MG corresponds to part c, the red line was the relative intensity with Ag-O band as internal standard while blue was the intensity of the 224 cm-1 and 1169 cm-1. (c) SERS spectra of different MG concentration. (d) Linear fitting of the SERS peak intensity with the corresponding MG concentration, the red line was the relative intensity with Ag-O band as internal standard while blue was the intensity of the 1169 cm-1. Au-G-AgNs Applied MG Quantitative Measurement in Vivo. In addition to be a commonly used Raman probe, MG is widely used as biocide due to its high efficacy against fungal infections. However, due to its carcinogenic properties and genotoxicity,35 MG is not approved by several countries. Various analytical techniques exist that can be used for MG detection, such as fluorescent36 and SERS37-39. In recent years, SERS has become the most common analytical techniques to detect the MG, due to nondestructive data acquisition and ultrasensitive characterization. However, varieties of SERS substrates, such as flexibility SERS substrates38 and portable SERS-enabled micropipettes39 are confined to detect MG molecule on the surface of the fish. So far, it is still a challenge to quantitative detect residual MG molecule in the fish, which has more significance in the field of food safety. Here, Au-G-AgNs, without discernible SERS peak (Figure S9), is inserted into the fish to detect residual MG molecule in the fish (Figure 5). Firstly, the fish were incubated in solutions with 10-6 M of MG solution for 60 min, and then, the fish was rinsed repeatedly to avoid the SERS signal interference form the fish surface. When the Au-G-AgNs were inserted into the fish (Figure 5a), grooves at different positions of the Au-G-AgNs can contact interstitial fluids at different depths in the fish. In our research, SERS spectra were obtained from different grooves on the Au-G-AgNs in vitro, after Au-G-AgNs inserting into the fish for 10 min (Figure 5b). Figure 5c shows the SERS spectra from different, the 1614 cm-1 band intensity distribution and reproducibility of MG at different depth in the fish is shown on Figure 5d. As shown in Figure 5e, the depth information is from the grooves along with the needle length direction, while reproducibility information from the wide direction.

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ACS Applied Nano Materials Fax: (+86)551-65592420. Phone: (+86)551-65592385 ORCID Liangbao Yang: 0000-0002-6559-6947 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (No.2018ZX09J18112), Innovation and Technology Commission of HKSAR (PJ. ITS/453/17 and through Hong Kong Branch of National Precious Metals Material Engineering Research Center).

Figure 5. Au-G-AgNs applied in the fish. (a) Digital photos of AuG-AgNs insert into the fish. (b) Digital photos of SERS signal acquisition on the Au-G-AgNs, the SERS signal from different groove is obtained by moving the objective table. (c) The SERS spectra obtained at different depth in the fish. (d) The 1169 cm-1 band intensity distribution of MG at different depth in the fish. (e) Schematic representation the depth information and signal reproducibility can be obtained in the groove array at the same time. ■ Conclusions In summary, Au-G-AgNs has been fabricated by milling grooves on the Ag needles and absorbing Au NPs into the grooves, which offers high SERS activity and excellent stability. The high SERS activity is further enhanced because of the multiple plasmonic couplings, which including the Augap-AgNs coupling and Au NPs-Au NPs coupling. And the groove design also protects the absorbed Au NPs from being erased during insertion, improved the stability of the Au-GAgNs. Besides, the grooves are recognizable under optical micro-scope, this allows us to monitor SERS in situ. SERS signal obtained from different grooves gives vertically distributed Raman information on the needle, which provided us a depth profiles of analytes. More importantly, the PVP acted as internal molecules through Au-O covalent bond, which can calibrate the SERS signal, making quantitative measurements more reliable. Therefore, the Au-G-AgNs provides a promising approach for collecting information for life and SERS quantitative detection.

ASSOCIATED CONTENT Supporting Information Supplementary figures and experimental details: Characterization and of the preparation process of Au-GAgNs; the reproducibility of the Au-G-AgNs; the sensitivity of the Au-G-AgNs; the adsorption kinetics of MG on the AuG-AgNs; the background signal of the Au-G-AgNs. The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *L.B.Y. ([email protected]).

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