Giant Gold Nanowire Vesicles Based Colorimetric and SERS Dual

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Giant Gold Nanowire Vesicles Based Colorimetric and SERS Dual Mode Immunosensor for Ultrasensitive Detection of Vibrio Parahemolyticus Zhiyong Guo, Yaru Jia, Xinxin Song, Jing Lu, Xuefei Lu, Baoqing Liu, Jiaojiao Han, Youju Huang, Jiawei Zhang, and Tao Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00292 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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Analytical Chemistry

Giant Gold Nanowire Vesicles Based Colorimetric and SERS Dual Mode Immunosensor for Ultrasensitive Detection of Vibrio Parahemolyticus Zhiyong Guo,∗a Yaru Jia,a,b Xinxin Song,a Jing Lu,a Xuefei Lu,b Baoqing Liu,b Jiaojiao Han,b Youju Huang,∗b,c Jiawei Zhang,b Tao Chen*b a.

Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, P.R. China

b.

Key Laboratory of Marine Materials and Related Technologies, Zhejiang Key Laboratory of Marine

Materials and Protective Technologies, Division of Polymer and Composite Materials, Ningbo Institute of Materials Technology & Engineering, Chinese Academy of Sciences, Ningbo 315201, P. R. China c.

∗

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128, Mainz, Germany

Corresponding Authors: E-mail: [email protected]; [email protected] and [email protected].

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Analytical Chemistry 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

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Abstract: Conventional methods for the detection of Vibrio parahaemolyticus (VP) usually need tedious, labor-intensive processes, and have low sensitivity, which further limits their practical applications. Herein, we developed a simple and efficient colorimetry and surface enhanced Raman scattering (SERS) dual-mode immunosensor for sensitive detection of VP, by employing giant Au vesicles with anchored tiny gold nanowires (AuNW) as a smart probe. Due to the larger specific surface and special hollow structure of giant Au vesicles, silver staining would easily lead to vivid color change for colorimetric analysis and further amplify SERS signals. The t-test was further used to determine if two sets of data from colorimetry and SERS are significantly different from each other. The result shows that there was no significant difference between data from two methods. Two sets of data can mutually validate and avoid false positive and negative detection. The designed colorimetry-SERS dualmode sensor would be very promising in various applications such as food safety inspection, personal healthcare and on-site environmental monitoring. Key Words: Dual-mode detection; Giant Au vesicles; In-situ label; gold label silver staining; colorimetric assay; SERS Analysis 1. Introduction Vibrio parahaemolyticus (VP), 1 a Gram-negative and halophilic bacterium, is usually found in coastal fish, zooplankton and shellfish.2 It has been one of the most significant food-borne pathogens in the United States and Asia,3 due to its widespread distribution in the marine waters. VP often grows very fast, up to 1000-fold at room temperature only in 2-3 h,4 resulting in lots of outbreaks of the food poisoning in China, Japan and several Southeast countries. The VP contaminated raw or undercooked seafood usually leads to a series of clinical disease, including abdominal cramps, low fever, headache and even bloody diarrhea.5 Therefore, it is critical to develop effective methods and strategies for sensitive and rapid detection of VP. Traditionally, various culture-based biochemical methods have been used for isolation and identification of VP strains such as enzyme-linked immune-sorbent assay (ELISA),6-10 DNA probe,11 loop-mediated isothermal amplification (LAMP),12-16 and electrochemistry (EC).17 But these methods 2 Environment ACS Paragon Plus

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are usually time-consuming with laborious steps. In order to shorten the analysis time and improve the detection efficiency, several strategies based on polymerase chain reaction (PCR)18-22 have been explored for targeting specific genes of VP. However, it is highly restricted by the requirement of special operators and equipment in practical applications. To overcome these limitations, various rapid testing methods such as colorimetric analytical methods 23 has been established. For example, Liu24 et al developed a robust colorimetric assay for sensitive and selective detection of VP in foods using magnetic beads-based sandwich immunoassay. The low detection limit (LOD) with 10 CFU mL-1 was achieved, but the line arrange was relatively narrow, just from 10 to 105 CFU mL-1, which is difficult to reach a quantitative detection in a wide range concentration of VP. Gold nanoparticles (AuNPs)25-29 display unique physical and chemical properties, and other promising features including rapid and easy synthesis, non-toxic, and desirable biocompatibility,30-40 which are widely used as attractive labels in the biomedical fields. For example, AuNPs was used in gold label silver staining (GLSS) technique for visible detection of DNA,41 proteins42 and bacteria.9 AuNPs in GLSS43-44 mainly act as nucleation sites for deposition of reduced silver atoms in the stained silver process, leading to obvious color change for further visual detection.42 This would provide probability of a highly sensitive, simple and visual method for detection VP in sea water and seafood. The morphology of AuNPs plays a significant role in improving the detection efficiency and sensitivity in GLSS. Au nanowire vesicles (AuNW vesicles) have rich sharp tips and larger specific surface area,4546

which favor the fast catalytic reduction of silver ions into silver atoms and deposition onto the

surfaces of Au wires, making clear color change and high sensitivity. However, the colorimetric assay can only quantitatively detect VP in a narrow concentration range. While, surface-enhanced Raman scattering (SERS)47-51 can provide “fingerprint” feature of analytes, thus allowing for the detection of the probe molecule sensitively. AuNW vesicles52 provide the high density of sharp tips and tip-to-tip nanogaps for significantly improving SERS sensitivity, and their array would overcome the common issues such as instability, non-uniformity and low reproducibility of SERS signals. Combined silver



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staining and SERS method, a dual mode sensing would be more convincible than single approach sensing, and more feasible for various practical applications. Herein, we develop a colorimetric-SERS dual mode sensor for the detection of VP based on AuNW vesicles. The high amount of VP would capture the high amount of AuNW vesicles-detection Antibody (dAb), leading to the deeper color and lower sensitivity in GLSS. Then by taking advantages of abundant sharp tips, tip-to-tip nanogaps and in-situ label MBA of AuNW vesicles, strong Raman signals can be used to act as an instruction of VP with a concentration range from 0-108 CFU mL-1. Combining colorimetry with SERS method, it is easy to achieve qualitative and quantitative detection of VP simultaneously. The t-test was further used to determine if two sets of data from colorimetry and SERS are significantly different from each other. The result shows that two sets of data can mutually validate, which can avoid false positive and negative detection effectively. 2. Material and methods 2.1 Materials. The Silver Enhancer Kit consisting of solution A (silver salt) and solution B (initiator), 25% glutaraldehyde solution, Poly (N-vinylpyrrolidone) (Mw 550,000), sodium citrate tribasic dihydrate (99.0%), L-ascorbic acid and 3-aminopropyltriethoxysilane (APTES), were purchased from Sigma-Aldrich (St. Louis, USA). 2,2-azobisisobutyronitrile (AIBN) was provided by Aladdin company in Shanghai, China. Chloroauric acid (HAuCl4·4H2O, 99.9%) was obtained from the Alfa Aesar. Polyclonal Vibrio parahaemolyticus (VP) antibody, bovine serum albumin (BSA), capture Antibody (cAb) and detection Antibody (dAb), were obtained from Sangon Biotech Co., Ltd. (Hangzhou, China). Styrene was distillated to refine under reduced pressure and AIBN was recrystallized three times in methanol before using (analytical grade). Other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd in Shanghai, China and used as received. Milli-Q-grade water (18.2 MΩ·cm) was used for all experiments. 2.2 Characterizations. The morphologies of different particles in the experiment were characterized by scanning electron microscopy (SEM), which was conducted on the JEOL S4800 electron microscope. Raman spectra were recorded by Renishaw in Via Reflex Confocal micro Raman spectrometer from 4 Environment ACS Paragon Plus

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Renishaw. The silver stain signals on the slide were scanned into grayscale images with a resolution of 600 dpi. Then silver spots were picked up by Photoshop CS6 and these data of separate spots were processed by Matlab 6.0 using custom-made program. The average grey value of silver stain spot was calculated as the summation of grey value. The ANOVA test and Tukey’s post hoc test (SPSS (version 19.0), Chicago, IL, USA) were used to analyse data in the recovery test. A P < 0.05 was defined as the standard criterion for statistical significance. 2.3 Preparation of Au NPs. The Au nanoparticles were prepared according to the Frens method.53 Briefly, 90 mL of 2.5×10–4 M HAuCl4 solution was heated to 120°C in an oil bath under vigorous stirring for 30 min. Subsequently, 10 mL 1% sodium citrate solution was added into the above solution with continuous boiling. After 20 min, the color of the boiled solution changed to ruby red, indicating the formation of Au NPs in the solution. Then the solution was stored at 4℃ for further use. 2.4 Preparation of AuNW vesicles. Au nanowires54-55 were grown from the polystyrene (PS) substrate coated with Au seeds according to our previous report.52 The specific steps as follows: 100 µL of seedadsorbed microspheres were added into a reaction solution containing 4-mercaptobenzoic acid (MBA) with a pre-determined concentration, HAuCl4 (1.7 mM) and L-ascorbic acid (4.1 mM). The mixture of ethanol/water was used to remove the free MBA. Finally, the vesicles was obtained by removing the PS template using tetrahydrofuran (THF) and the Au vesicles solution was stored in fridge for further application. 2.5 Preparation and silanization of glass substrate. The whole assay was performed on glass slide, which was functionalized for subsequent interaction with cAb. These details56 as follows: first, all glass slides were washed in ultrapure water and pretreated with piranha solution (a 7 : 3 mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide) at 80 °C for 30 min; Then, the slides were washed three times with ultrapure water and soaked into 1% (v/v) APTES ethanol solution overnight at room temperature; Finally the glass slides were rinsed with ethanol and dried in a N2 atmosphere with a following heating at 120 °C for 1 h in vacuum environment to obtain the silanized slides. Compared to



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bare glass slide, a well-defined spot could be obtained when detection antibody solution was dropped because of the hydrophobic surface. 2.6 Colorimetric and SERS detection of VP. The silanized slides were used for detection of VP using a sandwich immunoassay. Firstly, 5 µL 2.5 % glutaraldehyde (C5H8O2) solution was dripped onto the surface of silanzied slide for 30 min to construct a test zone. Then, 5 µL 10-1 mg mL-1 cAb was added to the test zone and incubated for 30 min. To avoid the non-specific binding process, test zone was incubated with 1% BSA and the excess solution was washed. Secondly, 5 µL VP with different concentrations (5, 101, 102, 103, 104, 105, 106, 107, 108 CFU mL-1) were incubated over the glass substrate to react with cAb for 30 min and the ultrapure water was used to remove excess VP solution. Then, 5 µL dAb-AuNW vesicles / AuNPs were incubated onto VP for 30 min. Finally, GLSS process was carried out: Equal amounts of silver enhancer solution A and solution B were well mixed to make the enhancer-solution and 5 µL mixture was dripped on the test zone above-mentioned. Then, the slide was immediately placed in the dark to react for 20 min. After that, the slide was washed with ultrapure water to terminate the reaction and air-dried at room temperature. At last, Matlab 6.0 was used to value the grey spots of 1.5 cm in diameter cutouting from the reaction ones, and the Raman spectra of in-situ label MBA were collected by a Raman spectrometer from Renishaw using a HeNe laser source (633 nm). Exposure time and laser power were 1 s and1.7 mW, respectively. 50× aperture (NA = 0.75) was used for all spectra. Then the SERS characteristic peak of MBA at 1077 cm-1 corresponding to the ringbreathing mode was chosen to evaluate the sensitivity and reproducibility. The LOD was estimated by the IUPAC standard method (LOD= yblank+ 3 × SDblank), in which yblank is the average SERS intensity when VP was absent and SDblank is the standard deviation of the blank measurement. 3. Results and Discussion 3.1 Fabrication and Characterization of the dual-mode immunosensor. The fabrication process of colorimetric and SERS dual-mode immunosensor for the detection of VP is illustrated in Scheme 1. Initially, the glass slide was treated with piranha solution and functionalized with APTES to promote the 6 Environment ACS Paragon Plus

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subsequent interaction with cAb, VP and dAb-AuNW vesicles. The sandwiched structure was formed via glutaraldehyde between the glass slide and cAb. As shown in Fig S1, after treated with APTES, a larger contact angle could be achieved from 43±3° to 74±2°, which ensure the formation of a sharply defined spot to provide specific reaction point. Fig 1A/B showed the morphology of VP and AuNW vesicles respectively. VP was about 1.8 µm in length and 400 nm in width, respectively. While AuNW vesicles consist of anchored tiny gold nanowires. The length and width of gold nanowires were 1.0 µm and 16.7 nm, respectively. Fig 1C showed that the dAb-AuNWs were captured successfully by VP. After the gold label silver staining, Au nanowires (Fig 1D) turned thicker. Fig 1E/F clearly shows an about 3 nm increase in the width of Au nanowires. EDS and the elemental mapping (Fig S2) experiments were further used to confirm the success of silver staining process.

Scheme 1. Schematic illustration of immunosensor construction and GLSS colorimetric/SERS doublemode detection of VP.



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Figure 1. SEM of VP (A), Au vesicles (B), Au vesicles adsorbed on VP via the dAb before (C, E) and after (D, F) silver staining.

3.2 Optimization of assay conditions. In general, the average grey value was greatly influenced by (i) the labeling pH, (ii) silver staining time, (iii) reaction time between VP and dAb (incubating time) and (v) the concentration of cAb (CcAb). In order to find the optimal experimental condition, 106 CFU mL-1 of VP was used for the detection. The average grey value is usually defined as the value in the presence of VP. However, nonspecific adsorption still occurs even there was no VP. The background signal should be deducted. Therefore, the reduced average grey value (∆AGV) was defined as the average grey value, by the value in the presence of VP subtracting the one in the absence of VP. Fig 2A shows the effect of pH value on the antibody activity and coupling efficiency. When the labeling pH was set at 6.0, 7.0, 7.5, 8.0, 8.5 and 9.0, the ∆AGV of immunosensor were about 40, 80, 65, 53, 49, 50, respectively. It is clear to see that pH value at 7.0 was the best condition for the antibody activity and coupling efficiency. While, the increase of silver staining time first enhanced ∆AGV, then had slight effect on the value of ∆AGV (Fig 2B). The reaction time between VP and dAb showed the similar effect trend on ∆AGV (Fig 2C and Fig S5). As can be seen in Fig 2D and Fig S6, ∆AGV increased with increasing concentration of cAb and then reached a constant value at 0.1 mg mL-1. 8 Environment ACS Paragon Plus

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Figure 2. The optimization of (A) labeling pH, (B) silver staining time, (C) incubating time, (D) concentration of cAb.

3.3 Sensitivity and working curve. The sensitivity and working curve were established under optimal experimental conditions. As shown in Fig 3A, after the silver staining, the color of the reaction spots gradually changed from nearly colorless to dark at the time of 20 min when the concentrations of VP increased from 0 to 108 CFU mL-1 (the images from a to i). Then, the specific average values were read out by Matlab 6.0, which was demonstrated by histogram in Fig 3B. The corresponding working curve was shown in Fig 3C. LOD for colorimetric method was calculated to be 10 CFU mL-1, which was much lower than the maximum level of VP by the US Food and Drug Administration (FDA) 2 and the subsequent state/federal authorities statement following the 1998 outbreaks.57 For comparison, traditional silver staining based on AuNPs functionalized with MBA (LOD of 103 CFU mL-1) (Fig 4A/B) was also conducted. As shown in Fig S7A/B, the replacement of sodium citrate by ligand MBA didn’t affect the morphology and dispersibility of AuNPs, and AuNPs-MBA were adsorbed onto VP through the anti-VP (Fig S7C/D). The obtained LOD was 103 CFU mL-1. Compared with solid gold spheres, giant Au vesicles contain the rich sharp tips from Au nanowire, the formed dense tip-to-tip gaps, the larger specific surface area and special hollow structure. These special features of giant Au vesicles 9 Environment ACS Paragon Plus


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favor the fast catalytic reduction of silver ions into silver atoms and homogeneous deposition onto the surfaces of gold nanowire vesicles in the process of silver staining, making clear color change and the much lower sensitivity for colorimetric analysis.

Figure 3. The spot images from a-i is corresponding to 0, 10, 102, 103, 104, 105, 106, 107, 108 CFU mL-1 (A), average grey value of logarithm (B, C) and Raman Intensity (D, E) at different VP concentrations by AuNW vesicles derived method.

In the fabrication process of AuNW vesicles, the strong ligand MBA probe molecules were strongly attached on the surfaces of AuNW vesicles, which can be in situ used as the label for Raman test. As shown in Fig 3D, the increase of VP amount will lead to a stronger Raman signal. It was obvious that more dAb absorbed on the Au vesicles will be captured, that is to say, more MBA molecule was included, resulting in higher Raman Intensity. In order to prove that the metallic silver formed in the silver staining process can further improve the surface enhancement Raman scattering, AuNW vesicles was exposed into the silver enhancement solution for 20 min in the darkness. As shown in Fig S8, compared with AuNW vesicles, bimetallic vesicles show higher Raman Intensity, which lead to the lower LOD of VP to some extent. In Fig 3E, it is clear to see that even when the concentration of VP 10 Environment ACS Paragon Plus

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was as low as 5 CFU mL-1, quite weak Raman signal can also be detected. The most prominent peak in the SERS spectra of MBA at 1077 cm-1 corresponds to the ring-breathing mode,58 and it was chosen to evaluate the quantitative property. Apparently, compared with AuNPs, giant Au vesicles can provide large-volume hot spots because of sharp tips formed the tip-to-tip structure and abundant gaps, thus enhancing the electromagnetic intensity for SERS performance. In the process of silver staining, silver atoms and Ag small particles were deposited onto the surfaces of gold nanowire vesicles, which further amplify the enhancement of Raman signal because Ag particles results in much larger electromagnetic enhancements in the visible (at least up to 600-650 nm). The amplified SERS signals would improve significantly LOD of VP.

Figure 4. The spot images from a-i corresponding to 0, 10, 102, 103, 104, 105, 106, 107, 108 CFU mL-1 (A) average grey value of logarithm (B, C) and Raman Intensity (D, E) at different VP concentrations by AuNPs derived method.

3.4 Specificity, stability and reproducibility of the immunosensor. To examine the specificity of the colorimetric and SERS assay for VP, a series of control experiments were conducted using potential interfering substance, including Enterobacter cloacae (EC) Vibrio vulnificus (VV), Vibrio harveyi (VH) and Shewanella marisflavi (SM) at a high concentration of 106 CFU mL-1, while the concentration of VP was kept at 102 CFU mL-1. As illustrated in Fig 5 and Fig S9, the separate addition of these interfering 11 Environment ACS Paragon Plus


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substances without VP do not exhibit any significant response in the average grey value and Raman intensity respectively, indicating that VP is highly specific to the colorimetry- SERS dual-mode sensor. As for the stability of the proposed immunosensor, AuNW vesicles solution was stored in refrigerator for a month at 4℃. The average grey value for the detection of VP at 106 CFU mL-1 is similar to the initial one with a proximate of 93.7±5.2% in 5 times, and Raman Intensity has a similarity of 95.3± 5.9% to the original one. The 106 CFU mL-1 of VP was analyzed for ten times to assess the reproducibility. The relative standard deviation (RSD) of the measurements was 6.5%, demonstrating the high reproducibility.

Figure 5. The specificity of the assay. (A) Histogram of the average grey value corresponding to spot images in (B); (B) spot images of VP at the concentration of 102 CFU mL-1, interfering bacteria (including VH, EC, SM, VV) at the concentration of 106 CFU mL-1 and blank sample.

3.5 Real Sample Analysis In order to test the practical performance of the dual-mode immunosensor, eight levels of VP ranging from 10 to108 CFU mL-1 were spiked to tap water. As shown in Table 1, for colorimetric detection, the 12 Environment ACS Paragon Plus

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satisfactory recovery from 95.2 to 109.7% were obtained, and the RSD were ranging from 3.1 to 6.3%. For SERS detection, the recovery and RSD were in the range of 92.7 to 108.7% and 4.6 to 6.6% respectively. The t-test was further used to determine if two sets of data from colorimetry and SERS are significantly different from each other. The obtained P value is 0.16, much bigger than 5%, indicating that there was no significant difference between data from two methods. Two sets of data can mutually validate and avoid false positive and negative detection. In addition, as shown in Fig 6, when detectable concentration by colorimetry was used as x axis, and the concentration by SERS was used as y axis, a good linear relationship was achieved. Herein, the dual mode detection of VP was feasible, which was highly unified in the spiked tap water. Table 1 Recovery tests for VP in spiked tap water ( x ± s , n = 5) Method

Samples

Added (CFU mL-1)

Recovered (CFU mL-1)

RSD (%)

Recovery (%)

Tap water 1

101

(1.097± 0.034) × 101

3.1

109.7

Tap water 2

2

10

(0.973 ± 0.046) × 10

2

4.7

97.3

Tap water 3

103

(1.074 ± 0.058) × 103

5.4

107.4

Tap water 4

104

(1.085 ± 0.069) × 104

6.3

108.5

Tap water 5

5

10

(0.952 ± 0.047) × 10

5

4.9

95.2

Tap water 6

106

(1.034 ± 0.054) × 106

5.2

103.4

Tap water 7

107

(1.021 ± 0.037) × 107

3.6

102.1

Tap water 8

8

10

(0.983 ± 0.054) × 10

8

5.5

98.3

Tap water 1

101

(0.992 ± 0.051) × 101

5.1

99.2

Tap water 2

102

(1.035 ± 0.048) × 102

4.6

103.5

Tap water 3

103

(1.069 ± 0.052) × 103

4.8

106.9

Tap water 4

104

(1.054 ± 0.062) × 104

5.8

105.4

Tap water 5

105

(0.927 ± 0.048) × 105

5.2

92.7

Tap water 6

6

10

(0.986 ± 0.054) × 10

6

5.4

98.6

Tap water 7

107

(1.087 ± 0.072) × 107

6.6

108.7

Tap water 8

108

(1.048 ± 0.063) × 108

6.0

104.8

Colorimetric

SERS



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Figure 6. Correlation of the detection results of VP in the recovery test between the developed colorimetric and SERS method.

4. Conclusions In summary, we have developed a novel dual-mode sensing system based on AuNW vesicles for the assay of VP. Benefiting from the larger specific surface and distinctive structure of AuNW vesicles, the sensing assay exhibits remarkable colorimetric response to VP through the naked-eye observation. Giant Au vesicles can provide large-volume hot spots because of sharp tips formed the tip-to-tip structure and abundant gaps, thus enhancing the electromagnetic intensity for SERS performance. Silver staining would further amplify the enhancement of Raman signal for the much lower detection limit. Compared with those commercially available techniques such as traditional carrier like AuNPs, the proposed colorimetric-SERS dual-mode detection method is highly sensitive, stable and selective especially in reliability. It would be very promising in various applications such as food safety inspection, personal healthcare and on-site environmental monitoring. Two sets of data form dual-mode sensing can mutually validate and avoid false positive and negative detection. The designed colorimetry- SERS dual-mode sensor would be very promising in various applications such as food safety inspection, personal healthcare and on-site environmental monitoring. 14 Environment ACS Paragon Plus

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Acknowledgments We gratefully acknowledge the Natural Science Foundation of China (Grants 51473179, 51603219, 41576098, 81773483), the Bureau of Frontier Science and Education of Chinese Academy of Sciences (QYZDB-SSW-SLH036),

Fujian

Province-Chinese

Academy

of

Sciences

STS

project

(2017T31010024), Innovation Promotion Association of Chinese Academy of Science (2016268 and 2017337). Supporting Information Available: Static WCA measurements for the glass before and after treated with APTES, EDS and the elemental mapping of Au nanowire vesicle before and after silver staining, SEM of AuNPS before and after functioned with MBA, SEM of AuNPs adsorbed on VP via the dAb before and after silver staining, the spot images about the optimal of conditions. These materials are available free of charge via the Internet at http://pubs.acs.org.

References

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Giant Gold Nanowire Vesicles Based Colorimetric and SERS Dual

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