Carbon Nanodot-Decorated Ag@SiO2 Nanoparticles for Fluorescence

Research Article www.acsami.org

Carbon Nanodot-Decorated Ag@SiO2 Nanoparticles for Fluorescence and Surface-Enhanced Raman Scattering Immunoassays Xianfeng Zhang†,‡ and Xuezhong Du*,† †

Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, Collaborative Innovation Center of Chemistry for Life Sciences, and School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210023, P. R. China ‡ Department of Applied Chemistry and Environmental Engineering, Bengbu College, Bengbu, Anhui 233030, P. R. China S Supporting Information *

ABSTRACT: A novel immunoassay protocol was demonstrated by the combination of fluorescent carbon nanodots (CNDs) and Ag@SiO2 surface-enhanced Raman scattering (SERS) tag nanoparticles into ensembles for a bifunctional nanoplatform. The CND-decorated Ag@SiO2 nanoparticles were constructed for sensitive fluorescence and SERS immunoassays. The silica shell thickness and amount of Ag@SiO2 nanoparticles were optimized for availability of strong fluorescence emission. The considerably large Raman scattering cross section of in situ-generated actual Raman reporter, 4,4′-dimercaptoazobenzene, from the apparent reporter p-aminothiophenol modified on the surfaces of Ag nanoparticles upon illumination of laser compensated for the reduction of SERS signals resulting from silica coating to a great degree. The antibody-modified bifunctional nanoparticles were captured by antibody-modified quartz slides in the presence of antigens in the sandwich structures for fluorescence and SERS immunoassays. The bifunctional nanoparticles could be used not only as bimodal probes for biodetection but also as bimodal tracers for bioimaging. KEYWORDS: carbon nanodot, core@shell nanoparticle, fluorescence, immunoassay, SERS

1. INTRODUCTION Functional nanostructured materials with excellent optical properties can be used for future biomedical applications, including biodetection, bioimaging, and disease diagnosis. A combination of different nanomaterials will be beneficial to the development of multifunctional nanoplatforms. Recently, extensive research has focused on multifunctional nanomaterials that are constructed from different nanoparticles for highthroughput biodetection and multimodal imaging. These nanoparticles primarily include silver nanoparticles (AgNPs),1,2 gold nanopartilces (AuNPs),3,4 and quantum dots (QDs)5 that are encapsulated with various types of polymers and inorganic shells, which endow the nanoplatforms with unique optical properties for high-throughput detection and multimodal imaging. Fluorescent carbon nanodots (CNDs) are oxygenous carbon nanoparticles with lateral sizes of less than 10 nm. CNDs have drawn much attention in the fields of optoelectronic devices, biolabeling, and biomedicine.6−9 Compared with traditional semiconductor QDs and organic dyes, CNDs are promising alternatives due to high aqueous solubility, robust chemical inertness, low toxicity, good biocompatibility, easy functionalization, and high resistance to photobleaching.10−14 Surface-enhanced Raman scattering (SERS) spectroscopy has attained considerable prominence as a nondestructive and © 2015 American Chemical Society

highly sensitive analytical technique even down to the singlemolecule level,15−18 based on the localized surface plasmon resonance (LSPR) effects of AgNPs and AuNPs. Currently, the Raman-label detection is the commonly used approach for protein assays because Raman reporters with large Raman scattering cross sections can yield strong characteristic SERS signals in comparison with the label-free detection.19−22 The AgNPs and AuNPs labeled with the Raman reporters are coated with biocompatible silica shell (core@shell nanostructures) not only to improve stability of the nanoparticles against oxidation and aggregation20,23−25 but also to facilitate functionalization of the nanoparticles with proteins, DNA, and cells.26−30 Herein, fluorescent CNDs were covalently attached to the surfaces of Ag@SiO2 SERS tag nanoparticles into biocompatible ensembles for bifunctional biodetection and bioimaging (Scheme 1). Owing to the inherent absorption and emission properties of CNDs available so far, there is a small overlap between the absorption band of CNDs and the LSPR band of AgNPs, while a relatively large overlap between the emission band of CNDs and the LSPR band of Ag@SiO2 nanoparticles Received: November 25, 2015 Accepted: December 21, 2015 Published: December 21, 2015 1033

DOI: 10.1021/acsami.5b11446 ACS Appl. Mater. Interfaces 2016, 8, 1033−1040

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ACS Applied Materials & Interfaces

Scheme 1. Illustration of Construction of Bifunctional CND-Decorated Ag/PATP@SiO2 Nanoparticles for Fluorescence and SERS Immunoassays

Tween-20 (PBST) was prepared by mixing PBS solution with Tween 20 (0.05%). 2.2. Apparatus and Characterizations. UV−vis spectra were collected on a LAMBDA-35 spectrophotometer (PerkinElmer) with quartz cells of 1 cm optical path length, and FTIR spectra of CNDs were on a VECTOR 22 FTIR spectrophotometer (Bruker) with a pellet method. Fluorescence spectra of aqueous solutions were obtained on a RF-5301 PC spectrophotometer (Shimadzu), and fluorescence spectra of modified quartz slides were on an Edinburg FLS 920 spectrofluorometer with the source Xe900. The transmission electron microscope (TEM) images of CNDs, AgNPs, Ag@SiO2, and Ag/PATP@SiO2/CND nanoparticles were acquired on a JEOL JEM2100 microscope at an acceleration voltage of 200 kV, and corresponding scanning electron microscope (SEM) images were on a Hitachi S-4800 microscope. Twenty microliters of aqueous CND solution, AgNP hydrosol, Ag/PATP@SiO2 in absolute ethanol, and Ag/PATP@SiO2/CND hydrosol were dropped onto carbon-coated copper grids to leave in the air for drying followed by TEM observations, respectively, and 20 μL of AgNP or Ag/PATP@SiO2 hydrosol was dropped onto aluminum slices to leave in the air for drying followed by SEM observations, respectively. SERS spectra were recorded on a LabRAM Aramis HJY Raman spectrometer with a CCD detector at the excitation wavelength of 532 nm. The exposure times for the Raman measurements were 0.5 s, and SERS spectra were collected by coaddition of 8 scans. Zeta potentials were obtained from a Nano-Z potential instrument (MALVERN). The zeta potential of AgNP hydrosol was measured after the synthesized AgNPs were centrifuged, and double-distilled water was used as a solvent during the zeta potential measurements. 2.3. Synthesis of AgNPs. AgNPs were synthesized according to the method in the literature with a slight modification.33 The glassware used was thoroughly cleaned with aqua regia and then washed with water followed by drying for use. AgNO3 (35 mg) was dissolved in 200 mL of water, and the aqueous solution was heated for boiling. Four milliliters of 1% sodium citrate was added, and the aqueous solution was kept boiling for 1 h. The as-synthesized AgNP hydrosol was greenish yellow. 2.4. Preparation of Ag/PATP@SiO2 Nanoparticles. Larger nanoparticles were removed from the as-synthesized AgNP hydrosol under centrifugation at 500 rpm for 10 min. Five microliters of PATP in ethanol (1 mM) was added dropwise to 10 mL of AgNP hydrosol under vigorous stirring for 20 min. If the total amount of added PATP

with thick silica shells occurs. It is essential to integrate the bifunctional ensembles with the optimized silica shell thickness for both strong emission of CNDs and strong signals of the Ag@SiO2 SERS tags for biodetection. The SERS tags were prepared from AgNPs, p-aminothiophenol (PATP), and thin silica shells. PATP was used as an apparent Raman reporter and underwent a photocoupling reaction to generate 4,4′dimercaptoazobenzene (DMAB) upon illumination of laser during the SERS measurements, with considerably strong characteristic Raman shifts different from PATP.31,32 The in situ-generated DMAB, with a remarkably large Raman scattering cross section, functioned as an actual Raman reporter to improve the sensitivity of detection. The CND-decorated Ag@SiO2 ensembles were used for fluorescence and SERS immunoassays and have potential applications in the field of biodetection and bioimaging.

2. EXPERIMENTAL SECTION 2.1. Chemical and Reagents. Silver nitrate (AgNO3), tetraethylorthosilicate (TEOS, 99%), 3-mercaptopropyltrimethoxysilane ( M P T M S ) , p - a m i n o t h i o p h e n o l ( P A TP ) , 1 - e t h y l - 3 - ( 3 (dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and 3-aminopropyltrimethoxysilane (APTMS) were purchased from Sigma−Aldrich. Bovine serum albumin (BSA), antibody (anti-β-actin mouse monoclonal antibody), and antigen (goat-antimouse IgG) were purchased from Beijing CoWin Bioscience Co., Ltd. (China). Ascorbic acid, glutaraldehyde (GA, 25%), and cupric acetate (Cu(Ac)2) were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Trisodium citrate dehydrate (99%), aqueous ammonia (NH3·H2O), anhydrous ethanol, hydrogen peroxide (H2O2, 30%), sulfuric acid (H2SO4, 98%), sodium hydroxide (NaOH), sodium chloride (NaCl), and Tween-20 were obtained from Nanjing Chemical Reagent Co., Ltd. (China). All of the reagents were used as received. The aqueous solutions used were prepared with double-distilled water. The phosphate buffered saline (PBS, 10 mM, pH 7.4) was prepared by dissolving sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4) in double-distilled water containing 150 mM NaCl, and phosphate buffer (PB, 10 mM, pH 7.4) was prepared without NaCl. The phosphate buffered saline containing 1034


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modified Ag/PATP@SiO2/CND nanoparticles were redispersed in 5 mL of aqueous BSA solution (1%) under gentle stirring for 2 h to block excess surface amino groups and nonspecific binding sites. After centrifugation at 9000 rpm for 5 min and washing with PBS twice, the final precipitate was dispersed in 1 mL of PBS solution (pH 7.4). 2.9. Preparation of Antibody-Modified Quartz Slides. Quartz slides (2.0 cm × 2.0 cm) were sonicated in water and ethanol, respectively, and then were cleaned with freshly prepared piranha solution (98% H2SO4/30% H2O2 = 3:1, v/v) for 10 min followed by washing with water. (CAUTION: “Piranha” solution reacts violently with organic materials; it must be handled with extreme care.) After drying with a nitrogen stream, the slides were treated in an oven at 80 °C for 4 h. The cleaned quartz slides were immersed in 0.05% APTMS solution in ethanol at room temperature in the dark for 24 h. After that, the slides were washed with ethanol and then sonicated in ethanol for 5 min to remove unimmobilized APTMS followed by rinsing with water and drying with a nitrogen stream. The aminofunctionalized quartz slides were further dried in an oven at 120 °C for 30 min and then immersed in aqueous GA solution (2.5%) for 3 h followed by washing with water and drying with a nitrogen stream. Ten microliters of anti-β-actin mouse monoclonal antibodies (100 μg/ mL in PBS) was pipetted onto the GA-modified quartz slides followed by incubating in a refrigerator at 4 °C for 12 h. The antibody-modified slides were washed with PBST buffer and rinsed with water three times to remove excess antibodies and then were immersed in aqueous BSA solution (1%) for 3 h to block nonspecific binding sites on the slide surfaces. Finally, the antibody-modified quartz slides were washed with PBST buffer and dried with a nitrogen stream. 2.10. Sandwich Immunoassays. The antibody-modified Ag/ PATP@SiO2/CND nanoparticles were used for quantitative sandwich immunoassays of antigens. Goat-anti-mouse IgG of various concentrations (0.01, 0.1, 1, 10, and 100 μg/mL) were pipetted onto different antibody-modified quartz slides and incubated at room temperature for 2 h. After the slides were washed with PBST buffer and dried with a nitrogen stream, 50 μL of the antibody-modified Ag/PATP@SiO2/ CND hydrosol was pipetted onto the antibody-modified slides and incubated at room temperature for 2 h. After washing with PBST buffer and water followed by drying with a nitrogen stream, the treated quartz slides were used for fluorescence and SERS measurements.

was smaller than the required amount for a full monolayer of PATP (average molecular area of 0.25 nm2)34,35 on the AgNP surfaces, it was conjectured that the amount of modified PATP on the AgNP surfaces was equal to the added amount. The surface coverage (θ) of PATP on the AgNP surfaces was calculated on the basis of the total surface area (Stotal) of AgNPs and the amounts (n) of added PATP, θ = 0.25nNa /Stotal = 0.25n/(CAgVπd 2) where Na is the Avogadro constant, CAg is the concentration of AgNPs, V is the volume of AgNP hydrosol, and d is the average diameter of AgNPs. The concentration of AgNPs in the hydrosol could be estimated on the basis of the Beer’s law using UV−vis spectroscopy with a molar extinction coefficient of 3 × 1011 M−1 cm−1.36 A surface coverage of approximately 50% on the AgNP surfaces was estimated (see details in the Supporting Information). Then, 5 μL of MPTMS in ethanol (1 mM) was added dropwise for another 20 min to modify the unoccupied surfaces for silica coating. To encapsulate AgNPs with silica shells, the method reported previously37 was used with a small modification. Ethanol (40 mL) was added to 10 mL of the PATP/ MPTMS-modified AgNP hydrosol under stirring, and 0.7 mL of aqueous ammonia (28%) was added to the colloidal solution followed by stirring for 5 min. Various amounts (1.0, 1.1, 2.0, and 4.0 mL) of TEOS in ethanol (10 mM) were added to the colloidal solution to coat AgNPs with silica shells. The colloidal solutions were gently stirred at room temperature for 12 h and then left to age without stirring for 24 h. Eventually, each colloidal solution of Ag/PATP@ SiO2 nanoparticles was centrifuged at 7000 rmp for 10 min and washed with ethanol four times followed by redispersing in 10 mL of anhydrous ethanol. 2.5. Preparation of Amino-Functionalized Ag/PATP@SiO2 Nanoparticles. APTMS (1 μL) was added to 10 mL of anhydrous ethanol solution containing freshly prepared Ag/PATP@SiO2 nanoparticles, and the mixture was shaken at room temperature for 5 h followed by heating at 50 °C for 1 h. The amino-functionalized Ag/ PATP@SiO2 nanoparticles were separated by centrifugation at 7000 rmp for 10 min and redispersed with 10 mL of ethanol three times for purification. Finally, the obtained amino-functionalized Ag/PATP@ SiO2 nanoparticles were dispersed in 10 mL of water for characterizations and further covalent conjugation with CNDs. 2.6. Synthesis of CNDs. CNDs were synthesized as reported recently38 with a slight modification. Ascorbic acid (0.176 g) was added to 19 mL of water followed by adding 1 mL of aqueous Cu(Ac)2 solution (0.1 M). The mixture was stirred at room temperature for 10 min and then heated at 90 °C in an oil bath under stirring for 5 h. After that, the reaction mixture was cooled to room temperature and then centrifuged at 8000 rpm to collect the supernatant. The supernatant was dialyzed with the semipermeable membrane (MWCO 1000) overnight, and the obtained aqueous CND solution was stored in a refrigerator at 4 °C prior to use. 2.7. Preparation of CND-Decorated Ag/PATP@SiO2 (Ag/ PATP@SiO2/CND) Nanoparticles. To activate the carboxyl groups on the CND surfaces, 1 mL of EDC solution (20 mg/mL) and 1 mL of NHS solution (20 mg/mL) were added to 5.0 mL of aqueous CND solution at 0 °C, and the mixture was stirred for 15 min. After that, 1 mL of amino-functionalized Ag/PATP@SiO2 hydrosol was added, and the mixture underwent continuous stirring at 0 °C for 2 h and then at room temperature for 10 h. Unimmobilized CNDs were removed by centrifugation at 9000 rpm for 10 min followed by washing with water twice. The resulting Ag/PATP@SiO2/CND nanoparticles were dispersed in 1 mL of water, and the Ag/PATP@SiO2/CND hydrosol was diluted to 3 mL for fluorescence measurement. 2.8. Preparation of Antibody-Modified Ag/PATP@SiO2/CND Nanoparticles. Freshly prepared EDC (20 mg/mL, 0.1 mL, pH 7.4) and 0.2 mL of NHS (20 mg/mL, pH 7.4) were mixed with 1 mL of Ag/PATP@SiO2/CND hydrosol under shaking at 25 °C for 15 min; then, 0.5 mL of anti-β-actin mouse monoclonal antibodies in 10 mM PBS (20 μg/mL, pH 7.4) was added, and the mixture was kept shaking at 25 °C for 2 h. Unimmobilized antibodies were removed by centrifugation at 9000 rpm for 10 min twice to obtain the antibodymodified Ag/PATP@SiO2/CND nanoparticles. Finally, the antibody-

3. RESULTS AND DISCUSSION 3.1. Construction of Bifunctional Nanoparticles. CNDs were synthesized through “polymerization” and “carbonization” steps under the experimental conditions, and the appearance of the orange reaction solution suggested the formation of some aromatic compounds and oligosaccharides, namely, the “polymerization” step.38,39 The addition of Cu(Ac)2 as a catalyst largely shortened the reaction time by accelerating the oxidization of ascorbic acid.38 The synthesized CNDs appeared as quasispherical nanoparticles with good monodispersity and had the average size of 3.2 ± 0.7 nm calculated from one hundred nanoparticles in the TEM image (Figure 1a). A broad infrared absorption band overlapped with a couple of sharp peaks at 3527, 3408, and 3321 cm−1 was due to the OH stretching vibrations of surface hydroxyl groups and carboxylic acid moieties as well as adsorbed water (Figure 1b). The bands at 1756 and 1676 cm−1 were assigned to the CO stretching vibrations of the ester and carboxylic acid groups conjugated to aromatic carbons, respectively. The bands at 1350−1000 cm−1 were due to the C−O stretching modes of these groups. The aqueous CND solution showed an absorption maximum at 292 nm, contributed from the n−π* transition of the CO bonds and the π−π* transition of the conjugated CC bonds (Figure 1c). CNDs displayed a clear dependence of photoluminescence wavelength and intensity on excitation wavelength11 and had an emission maximum at 448 nm at the excitation of 371 nm (Figure 1d). 1035


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Figure 3. Zeta potentials of AgNPs, Ag/PATP@SiO2, aminofunctionalized Ag/PATP@SiO2, CNDs, and Ag/PATP@SiO2/CND.

Figure 1. (a) TEM image (inset shows the size distribution histogram of CNDs), (b) FTIR spectrum, (c) UV−vis spectrum, and (d) fluorescence spectra of the synthesized CNDs at different excitation wavelengths from 330 to 410 nm.

Figure 2. TEM images of Ag/PATP@SiO2 nanoparticles with different silica shell thicknesses (nm): (a) 0; (b) 6; (c) 9; (d) 20; (e) 35. (f) TEM image of Ag/PATP@SiO2/CND with the silica shell of 9 nm. Arrows indicate the covalently attached CNDs.

Figure 4. (a) UV−vis absorption spectra of Ag/PATP@SiO2/CND (silica shell of 9 nm) hydrosol and corresponding PATP@SiO2/CND hydrosol after the dissolution of the AgNP cores with NaCl. (b) Fluorescence spectra of Ag/PATP@SiO2/CND (silica shell of 9 nm) hydrosol and corresponding PATP@SiO2/CND hydrosol after the dissolution of the AgNP cores with NaCl: excitation wavelength, 371 nm; slit width, 10 nm.

AgNPs with an average diameter of 60 ± 8 nm were synthesized as the cores (Figure 2a) and then modified with the apparent Raman reporter PATP at a surface coverage of approximately 50% available for strong SERS signals and subsequently with the coupling agent MPTMS to obtain vitreophilic surfaces for coating of complete silica shells.25 The Ag/PATP@SiO2 nanoparticles with silica shells of different thicknesses (6, 9, 20, and 35 nm) were clearly observed by TEM since there was a strong contrast between the black cores and gray shells (Figure 2b−e). The CNDs of the same size were covalently attached to the surface of Ag/PATP@SiO2

nanoparticles with the silica shell of 9 nm (Figure 2f) for comparison. The morphologies of the AgNPs and the Ag/ PATP@SiO2 nanoparticles could be further observed by SEM (Figure S1). The SEM images also showed that AgNPs were coated with silica shells of different thicknesses. The LSPR band of the AgNP hydrosol apperared at 412 nm, and the band did not show a significant change upon modification of PATP on the AgNP surfaces (Figure S2). However, an obvious red 1036


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Figure 5. (a) Fluorescence spectra of anti-β-actin mouse monoclonal antibody-modified Ag/PATP@SiO2/CND (silica shell of 9 nm) nanoparticles captured by antibody-modified quartz slides in the presence of goat-anti-mouse IgG of different concentrations. (b) Plot of fluorescence intensity at the maximum emission wavelength as a function of the logarithm of goat-anti-mouse IgG concentration.

Figure 6. (a) SERS spectra of anti-β-actin mouse monoclonal antibody-modified Ag/PATP@SiO2/CND (silica shell of 9 nm) nanoparticles captured by antibody-modified quartz slides in the presence of goat-anti-mouse IgG of different concentrations. (b) Plot of the logarithm of the SERS intensity at 1435 cm−1 as a function of the logarithm of goat-anti-mouse IgG concentration.

shift of the LSPR band of the Ag/PATP@SiO2 nanoparticles was observed from 412 to 442 nm with the increase of silica shells30,40 (Figure S2), owing to the increase in local refractive index of silica shells around AgNPs. Upon addition of CNDs to the hydrosols of aminofunctionalized Ag/PATP@SiO2 nanoparticles, the fluorescence of CNDs was significantly quenched in the presence of bare AgNPs (without silica coating) due to the direct contact with the bare AgNPs,41 but the fluorescence rapidly intensified with the increase of silica shell thickness of the Ag/PATP@SiO2 nanoparticles until a maximum luminescence intensity was observed for the silica shell of 9 nm (Figure S3A). However, a gradual decrease in fluorescence intensity was observed upon further increase of silica shell thickness (Figure S3B). Since the LSPR bands of the Ag/PATP@SiO2 nanoparticles with thick silica shells appeared at long wavelength up to 442 nm, there was a large spectral overlap between the LSPR bands of the Ag/ PATP@SiO2 nanoparticles and the emission bands of CNDs at 448 nm, resulting in the quenching of CND fluorescence to a great degree. Thus, the Ag/PATP@SiO2 nanoparticles with the silica shell of 9 nm were used for covalent immobilization of CNDs to develop nanostructured ensembles available for strong fluorescent emission and SERS signals. Although the Ag/PATP@SiO2 tags were reduced in SERS intensity with the increase of silica shell thickness,42 the intensities of the SERS tags in the case of 9 nm silica shell were considerably strong,

due to the large Raman scattering cross section of in situgenerated Raman reporter DMAB and were clearly able to meet the detection sensitivity. When the amino-functionalized Ag/PATP@SiO2 nanoparticles (silica shell of 9 nm) were added, the fluorescence of aqueous CND solution was slightly reduced in intensity with the increase of the amount of amino-functionalized Ag/ PATP@SiO2 hydrosol (Figure S4). Upon addition of 0.2 mL of amino-functionalized Ag/PATP@SiO2 hydrosol, the fluorescence intensity remained almost unchanged. The amount was used for covalent immobilization of CNDs on the surfaces of the amino-functionalized Ag/PATP@SiO2 nanoparticles via the amide linkages to meet two requirements: strong fluorescence emission and a large amount of immobilized antibodies. The covalent immobilization of CNDs was monitored by zeta potential measurements (Figures 3 and S5). The zeta potential of CNDs was −12.3 mV, which supports the above FTIR result that there were a large amount of residual carboxyl and hydroxyl groups on the CND surfaces.43 The zeta potential of the synthesized AgNPs with citrate coating was −37.2 mV after centrifugation followed by dispersing in double-distilled water. Upon encapsulation of AgNPs with silica shells, the zeta potential was −25.8 mV due to the silanol groups on the surfaces. Upon further modification of amino functionalities on the Ag/PATP@SiO2 nanoparticles, 1037


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fluorescence or SERS immunoassays to confirm the advantages of the Ag/PATP@SiO2/CND ensembles. The fluorescence intensities using Ag/PATP@SiO2/CND and Ag@SiO2/CND nanoparticles as the probes were almost identical (Figure S7). For the SERS immunoassay, the SERS intensities of the Ag/ PATP@SiO2/CND nanoparticles as the probes were slightly lower than those of the Ag/PATP@SiO2 counterparts, owing to both the increase of optical path throughout the decorated CNDs and the scattering of excitation laser by the decorated CNDs (Figure S8).

the zeta potential increased to 7.29 mV. After CNDs were covalently attached to the Ag/PATP@SiO2 surfaces, the zeta potential of the CND-decorated Ag/PATP@SiO2 nanoparticles was −1.57 mV. The obtained Ag/PATP@SiO2/CND nanoparticles showed a strong fluorescence emission at 445 nm at the excitation of 371 nm (Figure S6). The effect of the AgNP cores on the fluorescence emission of the covalently attached CNDs was further investigated by the dissolution of the AgNP cores of the Ag/PATP@SiO2/CND nanoparticles with NaCl.42 The Ag/PATP@SiO2/CND nanoparticles (silica shell of 9 nm) showed an absorption band at 426 nm, primarily due to the LSPR absorption of the AgNP cores, and the band disappeared after the dissolution of the AgNP cores of the Ag/PATP@SiO2/CND nanoparticles with NaCl (Figure 4a). The fluorescence emission of the Ag/ PATP@SiO2/CND nanoparticles was observed to decrease to a small extent, concomitant with a red shift after the dissolution of the AgNP cores (Figure 4b). This indicates that the fluorescence of the covalently attached CNDs was slightly enhanced by the AgNP cores with a spacing of 9 nm silica shell, owing to the small overlap between the absorption band of CNDs and the LSPR band of AgNPs. 3.2. Fluorescence and SERS Immunoassays. The Ag/ PATP@SiO2/CND nanoparticles were conjugated with anti-βactin mouse monoclonal antibodies and could be captured by the anti-β-actin mouse monoclonal antibody-modified quartz slides in the presence of goat-anti-mouse IgG in the sandwich structure. The intensities of fluorescence emission (Figure 5) and SERS peaks (Figure 6) of the captured bifunctional nanoparticles increased with increasing concentration of goatanti-mouse IgG from 0.01 to 100 μg/mL. Distinct fluorescent emissions were obtained when the concentration of goat-antimouse IgG was higher than 0.1 μg/mL; however, the fluorescent peak became very weak and was barely detected at 0.01 μg/mL (Figure 5a). The fluorescent intensity of the Ag/ PATP@SiO2/CND ensembles at the maximum emission wavelength as a function of the logarithm of goat-anti-mouse IgG concentration was shown in Figure 5b. It is noted that the observed SERS signals did not result from PATP but from its photocoupling product, 4,4′-dimercaptoazobenzene DMAB, which was generated in situ on the AgNP surfaces upon illumination of laser during the SERS measurements.31,32 It is shown that the Raman scattering cross section of DMAB is more than 3 orders of magnitude higher than that of benzenethiol derivatives with a synergic effect of resonance Raman and binding effect to AgNPs.44,45 The SERS peak at 1435 cm−1 was assigned to the NN stretching mode of DMAB,31,45 and the SERS intensity was observed to increase with increasing goat-anti-mouse IgG concentration ranging from 0.01 to 100 μg/mL (Figure 6a); there was an almost linear relationship between the logarithm of SERS intensity and the logarithm of goat-anti-mouse IgG concentration (Figure 6b), with the detection limit of as low as 0.0025 μg/mL (2.5 ng/mL) (S/N = 3). The large Raman scattering cross section of the in situ-generated Raman reporters compensated for the reduction of SERS intensity resulting from the silica coating of the Ag/PATP@SiO2 nanoparticles to a great degree, so that the SERS sensitivity was significantly improved. It is obvious that the SERS immunoassays were not disturbed by the photocoupling reaction of PATP; on the contrary, the SERS sensitivity would be considerably improved. Further control experiments with Ag@SiO2/CND or Ag/PATP@SiO2 nanoparticles with the silica shell of 9 nm were performed in the

4. CONCLUSIONS The fluorescent CNDs and Ag@SiO2 SERS tag nanoparticles were combined into a nanoplatform for sensitive fluorescence and SERS immunoassays. The thickness of silica shell and amount of immobilized CNDs were optimized to obtain strong fluorescence emission. The considerably large Raman scattering cross section of the in situ-generated Raman reporters (DMAB) from PATP compensated for the reduction of SERS intensity resulting from a silica shell of 9 nm to a great degree; thus, the SERS sensitivity was significantly improved in general. This protocol was demonstrated to be facile and effective and could be used not only as bimodal probes for biodetection but also as bimodal tracers for bioimaging.

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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.5b11446. Calculation details of surface coverage, SEM images, UV−vis and fluorescence spectra, zeta potentials, and SERS spectra. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +86-25-89687761. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 21273112 and No. 21503004) and Anhui Provincial Natural Science Foundation (No. 1508085QB32).

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REFERENCES

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