Giant Gold Nanowire Vesicle-Based Colorimetric and SERS Dual

Apr 27, 2018 - The low detection limit (LOD) with 10 CFU mL–1 was achieved, but the linear ... silver process, leading to obvious color change for f...
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Article Cite This: Anal. Chem. 2018, 90, 6124−6130

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Giant Gold Nanowire Vesicle-Based Colorimetric and SERS DualMode 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*,‡ †

Faculty of Materials Science and Chemical Engineering, Ningbo University, Ningbo 315211, P.R. China 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 § Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany ‡

S Supporting Information *

ABSTRACT: Conventional methods for the detection of Vibrio parahemolyticus (VP) usually need tedious, laborintensive 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 were significantly different from each other. The result shows that there was no significant difference between data from the two methods. Two sets of data can mutually validate each other 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.

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ibrio parahemolyticus (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 in only 2−3 h,4 resulting in lots of outbreaks of food poisoning in China, Japan, and several Southeast countries. The VP contaminated raw or undercooked seafood usually leads to a series of clinical diseases, 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 However, these methods are usually time-consuming with laborious steps. In order to shorten the analysis time and improve the detection efficiency, several strategies based on the 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 © 2018 American Chemical Society

overcome these limitations, various rapid testing methods such as colorimetric analytical methods23 have been established. For example, Liu24 et al. developed a robust colorimetric assay for sensitive and selective detection of VP in foods using a magnetic-bead-based sandwich immunoassay. The low detection limit (LOD) with 10 CFU mL−1 was achieved, but the linear range was relatively narrow, just from 10 to 105 CFU mL−1, which makes it difficult to achieve quantitative detection for a wide range of concentrations of VP. Gold nanoparticles (AuNPs)25−29 display unique physical and chemical properties, and other promising features including rapid and easy synthesis, nontoxicity, and desirable biocompatibility,30−40 which are widely used as attractive labels in the biomedical fields. For example, AuNPs were used in gold label silver staining (GLSS) for visible detection of DNA,41 proteins,42 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 a probability of a highly sensitive, simple, and visual method for detection of VP Received: January 19, 2018 Accepted: April 27, 2018 Published: April 27, 2018 6124

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6.0 using a custom-made program. The average gray value of silver staining spot was calculated as the summation of gray values. The ANOVA test and Tukey’s post hoc test (SPSS (version 19.0), Chicago, IL, U.S.A.) were used to analyze data in the recovery test. P < 0.05 was defined as the standard criterion for statistical significance. 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 of 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 °C for further use. 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 seed-adsorbed microspheres were added into a reaction solution containing 4-mercaptobenzoic acid (MBA) with a predetermined concentration, HAuCl4 (1.7 mM), and Lascorbic acid (4.1 mM). The mixture of ethanol/water was used to remove the free MBA. Finally, the vesicles were obtained by removing the PS template using tetrahydrofuran (THF), and the Au vesicle solution was refrigerated for further application. Preparation and Silanization of Glass Substrate. The whole assay was performed on a glass slide, which was functionalized for subsequent interaction with cAb. The details56 are 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 an N2 atmosphere followed by heating at 120 °C for 1 h in vacuum to obtain the silanized slides. Compared to bare glass slide, a well-defined spot could be obtained when detection antibody solution was dropped because of the hydrophobic surface. Colorimetric and SERS Detection of VP. The silanized slides were used for detection of VP using a sandwich immunoassay. First, 5 μL of 2.5% glutaraldehyde (C5H8O2) solution was dripped onto the surface of silanzied slide for 30 min to construct a test zone. Then, 5 μL of 10−1 mg mL−1 cAb was added to the test zone and incubated for 30 min. To avoid the nonspecific binding process, the test zone was incubated with 1% BSA, and the excess solution was washed. Second, 5 μL of 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 of dAb− AuNW vesicles/AuNPs were incubated onto VP for 30 min. Finally, the 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 of the mixture was dripped onto the above-mentioned test zone. 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. Finally, Matlab 6.0 was used to value the gray spots of 1.5 mm in diameter by cutting them out from the reaction spots, and the Raman spectra of in situ labeled MBA were collected by a Raman

in seawater 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,45,46 which favor the fast catalytic reduction of silver ions into silver atoms and deposition onto the surfaces of Au wires, making color change and high sensitivity clear. However, the colorimetric assay can only quantitatively detect VP in a narrow concentration range. Meanwhile, surface-enhanced Raman scattering (SERS)47−51 can provide a “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 common issues such as the instability, nonuniformity, and low reproducibility of SERS signals. By combining silver staining and the SERS method, a dual-mode sensing can be achieved, which would be more convincing than single approach sensing and more feasible for various practical applications. Herein, we developed 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 vesicle−detection antibody (dAb), leading to the deeper color and higher sensitivity in GLSS. Then, by taking advantages of abundant sharp tips, tip-to-tip nanogaps, and in situ labeled MBA of AuNW vesicles, strong Raman signals can be used as a guide to determine VP in a concentration range from 0 to 108 CFU mL−1. Combining colorimetry with the 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 each other, which can avoid false positives and negative detection effectively.

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MATERIAL AND METHODS 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 3aminopropyltriethoxysilane (APTES), were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). 2,2-Azobis(isobutyronitrile) (AIBN) was provided by Aladdin in Shanghai, China. Chloroauric acid (HAuCl4·4H2O, 99.9%) was obtained from Alfa Aesar. Polyclonal Vibrio parahemolyticus (VP) antibody, bovine serum albumin (BSA), capture antibody (cAb), and detection antibody (dAb) were obtained from Sangon Biotech Co., Ltd. (Hangzhou, China). Styrene was distilled to refine under reduced pressure, and AIBN was recrystallized three times in methanol before use (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. Characterizations. The morphologies of the 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 using the Via Reflex Confocal micro Raman spectrometer from 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 the data of these separate spots were processed by Matlab 6125

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nanowires were 1.0 μm and 16.7 nm, respectively. Figure 1C showed that the dAb−AuNWs were captured successfully by VP. After the gold label silver staining, Au nanowires (Figure 1D) turned thicker. Figure 1E,F clearly shows an ∼3 nm increase in the width of the Au nanowires. EDS and the elemental mapping (Figure S2) experiments were further used to confirm the success of the silver staining process. Optimization of Assay Conditions. In general, the average gray 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 conditions, 106 CFU mL−1 of VP was used for the detection. The average gray value is usually defined as the value in the presence of VP. However, nonspecific adsorption still occurs even when there was no VP. The background signal should be deducted. Therefore, the reduced average gray value (ΔAGV) was defined as the average gray value, by the value in the absence of VP subtracted from the one in the presence of VP. Figure 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 ΔAGV of immunosensor was about 40, 80, 65, 53, 49, 50, respectively. It is clear to see that the pH value at 7.0 was the best condition for the antibody activity and coupling efficiency. Meanwhile, the increase of the silver staining time first enhanced ΔAGV but then had only a slight effect on the value of ΔAGV (Figure 2B). The reaction time between VP and dAb showed the similar trend on ΔAGV (Figures 2C and S5). As can be seen in Figures 2D and S6, ΔAGV increased with increasing concentration of cAb and then reached a constant value at 0.1 mg mL−1. Sensitivity and Working Curve. The sensitivity and working curve were established under optimal experimental conditions. As shown in Figure 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− i)). Then, the specific average values were read out by Matlab 6.0, which is demonstrated by a histogram in Figure 3B. The corresponding working curve is shown in Figure 3C. The LOD for the colorimetric method was calculated to be 10 CFU mL−1, which was much lower than the maximum level of VP recommended 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) (Figure 4A,B) was also conducted. As shown in Figure S7A/B, the replacement of sodium citrate by ligand MBA did not affect the morphology and dispersibility of AuNPs, and AuNPs−MBA were adsorbed onto VP through the anti-VP (Figure 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 the special hollow structure. These special features of giant Au vesicles 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 color change clear and the much lower sensitivity for colorimetric analysis. In the fabrication process of the AuNW vesicles, the strong ligand MBA probe molecules were strongly attached onto the surfaces of AuNW vesicles, which can be used in situ as the

spectrometer from Renishaw using a HeNe laser source (633 nm). The exposure time and laser power were 1 s and 1.7 mW, respectively. A 50× aperture (NA = 0.75) was used for all spectra. Then, the characteristic SERS peak of MBA at 1077 cm−1 corresponding to the ring-breathing 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.

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RESULTS AND DISCUSSION 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 Scheme 1. Schematic Illustration of Immunosensor Construction and GLSS Colorimetric/SERS Double-Mode Detection of VP

piranha solution and functionalized with APTES to promote the 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 Figure S1, after treatment with APTES, a larger contact angle could be achieved from 43 ± 3 to 74 ± 2°, which ensured the formation of a sharply defined spot to provide specific reaction point. Figure 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. Meanwhile, AuNW vesicles consist of anchored tiny gold nanowires. The length and width of the gold

Figure 1. SEM of VP (A), Au vesicles (B), and Au vesicles adsorbed on VP via the dAb before (C,E) and after (D,F) silver staining. 6126

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

Figure 3. Spot images from a−i corresponding to 0, 10, 102, 103, 104, 105, 106, 107, and 108 CFU mL−1 (A), average gray value of logarithm (B,C), and Raman intensity (D,E) different VP concentrations by AuNW vesicle-derived method.

label for the Raman test. As shown in Figure 3D, the increase of the VP amount will lead to a stronger Raman signal. It was obvious that more dAb absorbed onto the Au vesicles will be captured, that is to say, more MBA molecules were 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 were exposed onto the silver enhancement solution for 20 min in darkness. As shown in Figure S8, compared with AuNW vesicles, bimetallic vesicles show higher Raman intensity, which leads to the lower LOD of VP to some extent. In Figure 3E, it is clear to see that even when the concentration of VP was as low as 5 CFU mL−1, a quite weak Raman signal can also be detected. The most prominent peak in the SERS 6127

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

Figure 5. Specificity of the assay. (A) Histogram of the average gray 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.

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 the sharp tips that 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 small silver particles were deposited onto the surfaces of gold nanowire vesicles, which further amplify the enhancement of Raman signal, because Ag particles result in much larger electromagnetic enhancements in the visible range (at least up to 600−650 nm). The amplified SERS signals would improve the LOD of VP significantly. 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 substances, including Enterobacter cloacae (EC), Vibrio vulnificus (VV), Vibrio harveyi

(VH), and Shewanella marisf lavi (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 Figures 5 and S9, the separate addition of these interfering substances without VP do not exhibit any significant response in the average gray 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 vesicle solution was stored in a refrigerator for a month at 4 °C. The average gray value for the detection of VP at 106 CFU mL−1 is similar to the initial gray value with approximately 93.7 ± 5.2% over 5 measurements, and the Raman intensity has a similarity of 95.3 ± 5.9% to the original value. The 106 CFU mL−1 VP was analyzed ten times to assess the reproducibility. The relative standard deviation (RSD) of the measurements was 6.5%, demonstrating the high reproducibility. Real Sample Analysis. In order to test the practical performance of the dual-mode immunosensor, eight levels of 6128

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Analytical Chemistry Table 1. Recovery Tests for VP in Spiked Tap Water (x̅ ± s, n = 5) method colorimetric

SERS

added (CFU mL−1)

samples tap tap tap tap tap tap tap tap tap tap tap tap tap tap tap tap

water water water water water water water water water water water water water water water water

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

recovered (CFU mL−1)

1

10 102 103 104 105 106 107 108 101 102 103 104 105 106 107 108

(1.097 (0.973 (1.074 (1.085 (0.952 (1.034 (1.021 (0.983 (0.992 (1.035 (1.069 (1.054 (0.927 (0.986 (1.087 (1.048

VP ranging from 10 to 108 CFU mL−1 were spiked into tap water. As shown in Table 1, for colorimetric detection, satisfactory recovery from 95.2 to 109.7% was obtained, and the RSD ranged 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 larger than 5%, indicating that there was no significant difference between data from two methods. Two sets of data can mutually validate each other and avoid false positives and negative detection. In addition, as shown in Figure 6, when

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.034) 0.046) 0.058) 0.069) 0.047) 0.054) 0.037) 0.054) 0.051) 0.048) 0.052) 0.062) 0.048) 0.054) 0.072) 0.063)

× × × × × × × × × × × × × × × ×

RSD (%)

recovery (%)

3.1 4.7 5.4 6.3 4.9 5.2 3.6 5.5 5.1 4.6 4.8 5.8 5.2 5.4 6.6 6.0

109.7 97.3 107.4 108.5 95.2 103.4 102.1 98.3 99.2 103.5 106.9 105.4 92.7 98.6 108.7 104.8

101 102 103 104 105 106 107 108 101 102 103 104 105 106 107 108

Giant Au vesicles can provide large-volume hot spots because of sharp tips forming the tip-to-tip structure and abundant gaps, thus enhancing the electromagnetic intensity for the 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. Two sets of data from dual-mode sensing can mutually validate each other and avoid false positives 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.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b00292. Static WCA measurements for the glass before and after treatment with APTES; EDS and the elemental mapping of the 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 for determination of the optimal condition (PDF)

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

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detectable concentration by colorimetry was used as the x axis, and the concentration by SERS was used as the y axis, a good linear relationship was achieved. Herein, the dual-mode detection of VP was feasible and was highly unified in the spiked tap water.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.G.) *E-mail: [email protected] (Y.H.) *E-mail: [email protected] (T.C.)

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ORCID

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 a remarkable colorimetric response to VP through naked-eye observation.

Youju Huang: 0000-0001-5815-9784 Jiawei Zhang: 0000-0002-3182-9239 Tao Chen: 0000-0001-9704-9545 Notes

The authors declare no competing financial interest. 6129

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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), the Fujian Province−Chinese Academy of Sciences STS project (2017T31010024), and the Innovation Promotion Association of Chinese Academy of Science (2016268 and 2017337).

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DOI: 10.1021/acs.analchem.8b00292 Anal. Chem. 2018, 90, 6124−6130