Nanoshell-Enhanced Raman Spectroscopy on a Microplate for

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Nanoshell-Enhanced Raman Spectroscopy on a Microplate for Staphylococcal Enterotoxin B Sensing Wenbin Wang,†,‡ Weiwei Wang,§ Liqiang Liu,†,‡ Liguang Xu,†,‡ Hua Kuang,†,‡ Jianping Zhu,†,‡ and Chuanlai Xu*,†,‡ †

State Key Laboratory of Food Science and Technology and ‡School of Food Science and Technology, Jiangnan University, Wuxi, JiangSu 214122, People’s Republic of China § Cereals & Oils Nutrition Research Group, Academy of Science & Technology of State Administration of Grain, Beijing 100037, People’s Republic of China S Supporting Information *

ABSTRACT: A sensitive surface-enhanced Raman scattering (SERS) immunosensor based on the Au nanoparticle (Au NP) shell structure was developed to detect staphylococcal enterotoxin B (SEB) on a microplate. Au NPs modified with 4-nitrothiophenol (4-NTP) and coated with Ag shell of controlled thickness at 6.6 nm exhibited excellent SERS intensity and were used as signal reporters in the detection of SEB. The engaged 4-NTP allowed the significant electromagnetic enhancement between Au NPs and the Ag shell and prevented the dissociation of the Raman reporter. More importantly, 4-NTP-differentiated SERS signals between the sample and microplate. The SERS-based immunosensor had a limit of detection of 1.3 pg/mL SEB. Analysis of SEB-spiked milk samples revealed that the developed method had high accuracy. Therefore, the SERS-encoded Au@Ag core−shell structure-based immunosensor is promising for the detection of biotoxins, pathogens, and environmental pollutants. KEYWORDS: SERS, Au@Ag, core−shell, SEB, antibody

1. INTRODUCTION Staphylococcal enterotoxins (SEs) are toxic proteins secreted by Staphylococcus aureus. SEs (molecular weight, 28−30 kDa) are stable in a wide range of pH values and temperatures and resistant to enzymatic digestion.1,2 It has been reported that SErelated food poisoning is the second most common foodborne illness worldwide.3 SEs associated with food poisoning include SEA, SEB, SEC, SED, and SEE;4 however, novel SEs have recently emerged. 5 Among the SEs, SEB has drawn considerable attention as a potential biological weapon due to its availability and toxicity;6,7 the consumption of 0.4 ng of SEB per kg of body weight is sufficient to cause food poisoning in humans.8 Therefore, sensitive SEB detection methods are of utmost importance in both food safety and clinical diagnosis. Current SEB detection methods include sandwich enzymelinked immunosorbent assay (ELISA)9 and lateral flow assay.10 These methods are robust, portable, and effective; however, they have limited sensitivity. To overcome this limitation, biosensors such as quartz crystal microbalance sensors,11 electrochemical sensors,6 and fluorescence resonance energytransfer aptasensors12 have been developed; however, they are quite complex to use and are considerably costly. Surface-enhanced Raman scattering (SERS), which is based on the enhancement of Raman scattering on metal surfaces, improves detection sensitivity and has great potential applications in biosensing.13,14 The SERS intensity of Raman © 2016 American Chemical Society

scattering on single nanoparticles (e.g., nanoparticles, nanorods, and nanostars) is low, but increases when the nanoparticles are aggregated due to the trapped Raman reporter in the hot spot between two metal layers.15 It has been reported that biosensors using aggregated nanomaterials,16 hollow spheres,17 assemblies,18−21 and core−shell structure nanoparticles22 contribute to high detection sensitivity. However, the aggregation of nanomaterials cannot be controlled, leading to poor reproducibility. Hollow spheres and assemblies are promising in SERS-based biosensors; however, these materials are relatively hard to prepare.23 Recently, bimetallic core−shell nanoparticles have been utilized in cell imaging24,25 and biosensors26 because of their excellent SERS properties. Individual Au@Ag core−shell nanoparticles, modified with the Raman reporter, generate SERS signals that are approximately 2 orders of magnitude stronger than those of Au and Ag nanoparticles (NPs).27 The use of these nanomaterials as SERS substrates for the label-free detection of pesticides,28 pathogens,29 and pigments17 and for the competitive detection of heavy metals30 has been thoroughly investigated. Label-free detection is usually limited to analytes with inherent SERS signals,31,32 while competitive Received: March 8, 2016 Accepted: May 19, 2016 Published: May 19, 2016 15591

DOI: 10.1021/acsami.6b02905 ACS Appl. Mater. Interfaces 2016, 8, 15591−15597

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ACS Applied Materials & Interfaces

Figure 1. Scheme of the Au@Ag core−shell structure-based SERS immunosensor for SEB. solution and boiled for 15 min. The solution, which turned red wine in color, was allowed to cool to room temperature and stored at 4 °C. 2.4. Preparation of 4-NTP-Modified Au NPs. Au NPs were centrifuged (6200g, 15 min) and concentrated twice in 10 mM phosphate buffer saline (PBS). Aqueous 4-NTP (200 μM, 25 μL) was mixed with 1 mL of Au NPs and allowed to react at room temperature for 6 h. Unbound 4-NTP was removed by centrifugation (6200g, 15 min). Modified Au NPs were dispersed in PBS (10 mM, 500 μL). 2.5. Synthesis of Au-4-NTP@Ag NPs. To the 4-NTP-modified Au NPs (50 μL), we added polyvinylpyrrolidone (10%, 50 μL), different volumes of 2 mM AgNO3 (0, 5, 10, 15, and 20 μL), and ascorbic acid (10 mM, 10 μL). The solution was allowed to react for 10 min at room temperature and centrifuged (6900g, 15 min). The resulting precipitate was dispersed in PBS (10 mM, 100 μL). Average diameter and Ag shell thickness of the synthesized Au-4-NTP@Ag NPs were obtained by counting 100 particles on the TEM image and calculating the mean value. 2.6. Preparation of mAb-Labeled Au-4-NTP@Ag NPs. Au-4NTP@Ag NPs were modified with mAb 1B3. Briefly, mAb 1B3 (2 mg/mL, 10 μL) was added to Au-4-NTP@Ag NPs (1 mL) and allowed to react for 2 h at room temperature under constant stirring. To block the redundant binding site, BSA (50 mg/mL, 10 μL) was added and allowed to react for 2 h. Finally, mAb-labeled Au-4-NTP@ Ag NPs was centrifuged (6900g, 15 min), and the sediment was resuspended in PBS (10 mM, 1 mL) containing 0.1% Tween 20. 2.7. SERS-Based Immunoassay of SEB. The SERS-based immunoassay was performed on a microplate. Initially, the microplate was coated with mAb 4F2 (4 μg/mL in 0.05 M sodium carbonate coating buffer or CB, 100 μL/well) and incubated at 37 °C for 2 h. The plate was subsequently washed three times with washing buffer (0.01 M PBS, 0.05% Tween). Blocking buffer (0.05 M CB containing 0.2% gelatin, 200 μL/well) was added and incubated for 2 h at 37 °C. Following another wash, 100 μL of sample was added to the plate and incubated for 1 h at 37 °C. After washing, 100 μL of mAb-labeled Au4-NTP@Ag NPs was added and incubated for 1 h at 37 °C. After being washed four times, the plate was air-dried. SERS spectra were obtained in a LabRam-HR800 Raman spectrometer with an acquisition time of 60 s. Each sample was scanned six times at different areas of the microplate. 2.8. Sensitivity and Specificity. SEB, diluted to 0, 2, 5, 10, 20, and 100 pg/mL in PBS containing 0.1% Tween, was added to the plate to assess the sensitivity of the immunoassay. SEA, SEC, SED, SEE, BSA, and OVA at 50 ng/mL and SEB at 100 pg/mL in dilution buffer were used to measure the specificity of the immunoassay. Limit of detection (LOD) was defined as the corresponding concentration of the average signal of the negative samples plus 3 times the standard deviation of the blank. 2.9. Analysis of Milk Samples. Pure milk was purchased from a local market and confirmed to be free of SEB by Jiangsu Entry-Exit Inspection and Quarantine Bureau (Nanjing, China). The milk was

detection is not suitable for large molecules such as pathogens and proteins. Raman reporter-embedded Au@Ag core−shell structures are self-supported SERS nanomaterials due to the significant electromagnetic enhancement by the Raman molecules between the Au and Ag layers.33,34 These materials coupled to antibodies are desirable to improve the detection sensitivity of highly toxic agents in the environment and of biomakers in clinical research.35 In this study, we developed a novel 4-nitrothiophenol (4NTP)-encoded Au@Ag core−shell NP-based SERS immunosensor to detect SEB. The highly SERS-active 4-NTP-encoded Au@Ag core−shell NPs was easily synthesized. Au NPs core was modified with the Raman reporter. An Ag shell of controlled thickness was reduced on the Au NPs with ascorbic acid and AgNO3. The SERS-active nanostructures were coupled to an SEB monoclonal antibody (mAb) as a signal reporter in the sandwich-based immunosensor on the microplate. The embedded Raman reporter between the metal layers of controlled shell thickness improved both the sensitivity and reproducibility of the method.

2. EXPERIMENTAL SECTION 2.1. Materials. Chloroauric acid (HAuCl4), sodium citrate, 4-NTP, ascorbic acid, polyvinylpyrrolidone, silver nitrate, bovine serum albumin (BSA), and ovalbumin (OVA) were purchased from Sigma−Aldrich (Shanghai, China). Staphylococcal enterotoxins (SEA, SEB, SEC, SED, and SEE) were acquired from the Academy of Military Medical Sciences (Beijing, China), and mAb4F2 and mAb1B3 against SEB were prepared in our laboratory. All other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The 96-well microplate was obtained from GuoSheng Bio-Engineering Co., Ltd. (Wuxi, China). 2.2. Instruments. A UV−vis spectrophotometer (Evolution 60S, Thermo Fisher Scientific, Waltham, MA) was used to monitor the absorption of Ag on Au NPs. Transmission electron microscopy (TEM) (JEM-2100, JEOL) allowed the visualization of the Ag shell on the Au NPs. Dynamic light scattering (DLS, Zetasizer Nano ZS system, Malvern Instruments, Malvern, U.K.) was used to measure the hydrodynamic diameter of the resulting products. Raman spectrometer (LabRam-HR800, HORIBA Jobin Yvon, Edison, NJ) was utilized to characterize the SERS intensity of the samples, with a holographic grating of 600 g mm−1, an air-cooled He−Ne laser of 632.8 nm, and an excitation of ∼8 mW. The Raman spectrum resolution was ≤0.65 cm−1, and the diameter of the laser spot was 7720.16 nm. 2.3. Synthesis of Au NPs. Au NPs of 18 nm in diameter were prepared using a seed-mediated growth method.36 Briefly, HAuCl4 (0.01%, 100 mL) was boiled for 10 min under constant magnetic stirring. Trisodium citrate (10%, 180 μL) was quickly added to the 15592


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Figure 2. Typical TEM images (a−e) and photograph (f) of Au@Ag NSs with increasing shell thickness by addition of 0, 5, 10, 15, and 20 μL of 2 mM AgNO3. (g) Thickness of the Ag shell with different volumes of 2 mM AgNO3. (h) UV−vis spectra of Au@Ag NSs with different volumes of 2 mM AgNO3. spiked with SEB at 5, 10, and 20 pg/mL. Following centrifugation at 7000g for 10 min, 100 μL of the SEB-spiked milk samples were analyzed by the developed SERS-based immunoassay. Each of the samples was measured in six duplicate.

3.2. Preparation of Au-4-NTP@Ag NPs with High SERS Intensity. The TEM image in Figure 2a shows uniform and dispersed Au NPs with a diameter of 18 nm. This diameter is stable and provides a relatively large specific area. 4-NTP was used as the Raman reporter because its characteristic SERS signal at 1333 cm−1 differs from that of the sample. 4-NTPmodified Au NPs were confirmed by Raman spectra (Figure 3). The deposition of Ag shell on 4-NTP-modified Au NPs was characterized by TEM, UV−vis spectra, and DLS. With increasing AgNO3, the average diameter of Au NPs in TEM increased from 18 to 27 nm (Figure 2a−e) and the wine red color of the AuNP changed into saffron yellow (Figure 2f). The average Ag shell thickness obtained by counting was 0, 1.1, 3.2, 6.6, and 9.1 nm with 0, 5, 10, 15, and 20 μL of 2 mM AgNO3

3. RESULTS AND DISCUSSION 3.1. Principle of the SERS-Based Immunoassay for SEB. Au NPs were first modified with 4-NTP and coated with Ag shells of different thicknesses (Figure 1). Au-4-NTP@Ag NPs with maximum SERS intensity were synthesized as Raman tags and attached to mAb 1B3. The microplate was coated with mAb 4F2. SEB in contaminated samples would be captured by mAb 4F2 and bound to the mAb 1B3-labeled Au-4-NTP@Ag NPs. Therefore, SERS intensity would be positively correlated with the sample SEB concentration. 15593


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Au NPs and that the thickness was dependent on AgNO3 amount. The thickness of the Ag shell on the 4-NTP-modified Au NPs plays a key role in the SERS intensity of the Au@Ag core− shell structure. Figure 3a showed the SERS spectra of Au-4NTP@Ag NPs at different thicknesses. The SERS signal of 4NTP-modified Au NPs (3200) at 1333 cm−1 was substantially enhanced with 5 μL of 2 mM AgNO3 (9500), reaching a maximum (15 600) with 15 μL of 2 mM AgNO3 (Figure 3b). The low SERS intensity of Au@Ag-4-NTP with a Raman label on the Ag surface was observed (Figure S2). Meanwhile, the significant increase in SERS intensity of Au-4-NTP@Ag NPs was attributed to the embedded Raman reporter between Au NPs and the Ag shell.34,37 In addition, unlike the conventional method where the Raman reporter is modified on the outer sphere,38 engaged 4-NTP prevented the dissociation of the Raman reporter from the surface, thereby improving the stability of the Raman tag. However, with further increasing shell thickness at 9.1 nm, the SERS intensity of the Au-4NTP@Ag NPs decreased. This is because thickness of the Ag shell on the 4-NTP-modified Au NPs affected both the electromagnetic enhancement of the Au−Ag layer and the optical availability of the Raman reporter inside the nanospheres. Further increased Ag shell make it hard for the laser (excitation of 632.8 nm) to penetrate the shell and interact with the Raman reporter. Therefore, Au-4-NTP@Ag NPs with maximum SERS intensity were successfully synthesized by controlling the thickness of the Ag shell on the 4-NT-modified Au NPs at 6.6 nm, with 15 μL of 2 mM AgNO3. Recently, Lim et al.,39 Oh et al.,40 and Song et al.41 reported nanogaped core−shell structures with various interior nanogaps from 1 to 11 nm and found that sub-2 nm or ∼1 nm nanogap could lead to the plasmonic hot spot with highest electromagnetic field. The core−shell structure in our text was synthesized with no gap because of the absence of modified DNA or polymer with relatively large molecular size. The enhancement of SERS intensity of the embedded Raman label in the synthesized core−shell structure could be attributed to chemical enhancement (sandwiched Raman label with the two linkages to metal surfaces) and electromagnetic enhancement between the Au core and Ag shell (Raman labels caused cavities between the metal layers).37,42,43 The core−shell structure

Figure 3. SERS spectra (a) and SERS intensity at 1333 cm−1 (b) of Au@Ag NSs with different volumes of 2 mM AgNO3.

(Figure 2g); similar results were obtained by DLS (Figure S1 in the Supporting Information). Furthermore, the maximum absorption wavelength of Au on the UV spectra blue-shifted from 522 to 499 nm, and the absorption of Ag at 400 nm increased with increasing AgNO3 (Figure 2h). The results confirmed that the Ag shell was formed on the 4-NTP-modified

Figure 4. (a) SERS spectra of Au@Ag NSs on the plate with different concentrations of SEB (signal of the plate was deducted). (b) Standard curve of SEB based on the SERS intensity of 4-NTP at 1333 cm−1. 15594


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ACS Applied Materials & Interfaces without gap is more readily available and paves another way to generate Raman tags with high SERS properties for biosensing. SERS tag that was coated with either a silica shell or methoxy polyethylene glycol thiol has been useful to prevent the abscission of the Raman reporter and improve the stability.44,45 SERS labels embedded bimetal core−shell structure both generated high SERS intensity and good stability after being functionalized with bioligand.14 In this paper, Au@Ag core− shell nanospheres were fabricated with high Raman intensity and functionalized by an antibody as a SERS tag for the sensing of big molecular (SEB) on the microplate, which is promising for high-throughout detection of large samples such as tumor markers and pathogens. 3.3. Sensitivity and Specificity of the SERS-Based Immunoassay. The mAb 1B3-labeled Au-4-NTP@Ag NPs were characterized by UV−vis spectra (Figure 2h). The red shift of the maximum absorption wavelength of Au from 502 to 508 nm revealed the increased diameter following effective conjugation. The SERS intensity of 4-NTP on the plate increased at 2− 100 pg/mL SEB (Figure 4a). As shown in Figure 4B, SERS intensity at 1333 cm−1 was linearly correlated with the logarithmic function of SEB concentration, which was probably due to the Raman enhancement of the embedded Raman reporter 4-NTP with the Au@Ag nanospheres, and this was consistent with the reported studies.46−48 The standard curve had a wide linear range at 2−100 pg/mL and an excellent correlation coefficient (R2) of 0.993. The typical SEM images of the SERS tag on the microplate corresponding to different concentrations of SEB are shown in Figure S3. The LOD was calculated to be 1.3 pg/mL, approximately 25 times lower than that of the sandwich ELISA based on the same pair (Figure S4) and comparable to that previously reported6,49 (Table S1). 4-NTP as a commonly used Raman label50,51 could greatly improve the sensitivity of detection. First, 4-NTP was modified on the surface of Au NPs by an Au−SH bond. The relatively low Raman signal of 4-NTP at the Au NPs surface was significantly enhanced between the Au NPs and the Ag shell, which makes the nanoshell structure suitable as a Raman tag for the sensitive detection of SEB; second, the 4-NTP-modified Au NPs was found to be more stable than Raman reporter like 4ATP used in our experiments, which could prevent the aggregation of Au NPs (negative charge). The third is that the distinctive SERS signal of 4-NTP at 1333 cm−1 differs from the signal of the sample from that of the microplate. And this low background could also decrease the noise/signal ratio and enhance the sensitivity. The absorption of the microplate was taken into account because the microplate has the benzene ring structure and strong characteristic peaks, especially at 1080 cm−1. The original Raman spectra with different SEB concentrations are presented in Figure S5. Scanning of the SERS spectra of the sample six times at different areas of the microplate found the coefficient of variations with the result for each concentration ranging from 1.33% to 5.06%. This may be because of the welldispersed coating antibody on the microplate and the banded Raman tag of Au-4-NTP@Ag NPs with the presentation of SEB. As shown in Figure 5, Raman intensity was very high for SEB at 100 pg/mL and low for SEA, SEC, SED, SEE, BSA, and OVA even at 50 ng/mL. Therefore, the developed method had high sensitivity and specificity for SEB.

Figure 5. Specificity of the SERS-based immunoassay for detection of SEB. Concentration of SEA, SEC, SED, SEE, BSA, and OVA was at 50 ng/mL and SEB was at 100 pg/mL.

3.4. Analysis of Milk Samples. The developed SERSbased immunoassay was used to detect SEB-spiked pure milk samples. As shown in Table 1, the SEB recoveries from spiked pure milk ranged from 88.2% to 92.36%, with a coefficient of variation of 6.64%−7.85%. Therefore, the developed method had high accuracy. Table 1. Accuracy of the Analysis of Milk Samples with the SERS-Based Immunosensor pure milk

spiked concentration (pg/mL)

detected concentration (pg/ mL, mean ± SD, n = 6)

recovery (%) (mean ± SD, n = 6)

no. 1 no. 2 no. 3

5 10 20

4.41 ± 0.33 9.24 ± 0.73 18.33 ± 1.57

88.2 ± 6.64 92.36 ± 7.33 91.67 ± 7.85

4. CONCLUSION We developed a novel and sensitive SERS immunosensor for SEB on a microplate based on the 4-NTP-encoded Au@Ag NPs structure. The encoded 4-NTP in the Au@Ag NPs at controlled shell thickness of 6.6 nm not only enables significant enhancement between Au NPs and the Ag shell but also allows the differentiation of SERS signals between the sample and microplate. With the highly SERS-active 4-NTP-encoded Au@ Ag NPs used as Raman tags, the method had a LOD of 1.3 pg/ mL for SEB. The analysis of SEB-spiked pure milk samples revealed that the method had high accuracy. The 4-NTPencoded Au@Ag NPs with strong and stable SERS signals are promising in a wide range of biosensing applications.

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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/acsami.6b02905. DLS data of the Au@Ag NSs with increasing shell thickness; standard curve of the sandwich ELISA methods for SEB; original SERS spectra of Au@Ag NSs on the plate with different concentrations of SEB; review of recent works in detection of SEB (PDF) 15595


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.X.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by Natural Science Foundation of Jiangsu Province, MOF and MOE (BK20150145, BE2013613, BE2013611, 201513006).

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Nanoshell-Enhanced Raman Spectroscopy on a Microplate for

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