Article pubs.acs.org/ac

Fabrication of Gold Nanoparticle-Embedded Metal−Organic Framework for Highly Sensitive Surface-Enhanced Raman Scattering Detection Yuling Hu,* Jia Liao, Dongmei Wang, and Gongke Li* School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, Guangdong 510275, China S Supporting Information *

ABSTRACT: Surface-enhanced Raman scattering (SERS) signals strongly rely on the interactions and distance between analyte molecules and metallic nanostructures. In this work, the use of a gold nanoparticle (AuNP)-embedded metal− organic framework was introduced for the highly sensitive SERS detection. The AuNPs were in situ grown and encapsulated within the host matrix of MIL-101 by a solution impregnation strategy. The as-synthesized AuNPs/MIL-101 nanocomposites combined the localized surface plasmon resonance properties of the gold nanoparticles and the high adsorption capability of metal−organic framework, making them highly sensitive SERS substrates by effectively preconcentrating analytes in close proximity to the electromagnetic fields at the SERS-active metal surface. We discussed the fabrication, physical characterization, and SERS activity of our novel substrates by measuring the Raman signals of a variety of model analytes. The SERS substrate was found to be highly sensitive, robust, and amiable to several different target analytes. A SERS detection limit of 41.75 and 0.54 fmol for Rhodamine 6G and benzadine, respectively, was demonstrated. The substrate also showed high stability and reproducibility, as well as molecular sieving effect thanks to the protective shell of the metal−organic framework. Subsequently, the potential practical application of the novel SERS substrate was evaluated by quantitative analysis of organic pollutant p-phenylenediamine in environmental water and tumor marker alpha-fetoprotein in human serum. The method showed good linearity between 1.0 and 100.0 ng/mL for pphenylenediamine and 1.0−130.0 ng/mL for alpha-fetoprotein with the correlation coefficients of 0.9950 and −0.9938, respectively. The recoveries ranged from 80.5% to 114.7% for p-phenylenediamine in environmental water and 79.3% to 107.3% for alpha-fetoprotein in human serum. These results foresee promising application of the novel metal−organic framework based composites as sensitive SERS-active substrates in both environmental and clinical samples.

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However, many other molecules show low affinity toward the metal, thus limiting the use of SERS techniques in their detection. One of the biggest challenges is to guide target analytes to the places where the surface plasmon resonance can be enhanced. Therefore, a lot of research was focused on functionalization of nanoparticles aiming to improve affinity of the analyte to the metal surface. For this purpose, a great number of molecule modified metal nanoparticles was investigated for their enhancement of SERS, such as Au or Ag nanoparticles functionalized with dithiocarbamate calix[4]arene,6 viologen dications,7,8 cyclodextrin,9 alkanethiolate tri(ethylene glycol),10 and cysteine.11 However, the metallic colloids are easily aggregated. The aggregation state is inherently unstable and tends to precipitate from solution, resulting in loss of SERS signals.12 In recent years, great efforts have been devoted to developing a strategy to protect metal nanoparticles with a stabilizing matrix, and the most common

urface enhanced Raman spectroscopy (SERS) has emerged as a promising spectroscopic technique for biological and chemical sample screening because of its unique advantages including high sensitivity with a potential of single molecule detection and highly informative spectra characteristics.1−3 The SERS phenomenon observed is mainly attributed to two main mechanisms (i) the electromagnetic fields generated at or near nanostructured surfaces and (ii) the physical or chemical adsorption of the analyte to a surface to produce charge-transfer states between the metal and adsorbate.4 In this sense, both the electromagnetic and chemical mechanism enhancement require a close proximity of the analyte toward the metal surface. The engineering of substrates to generate high-quality SERS signals from specific analytes is still a hot topic. Metallic colloids were widely employed as SERS substrates due primarily to ease of preparation and large SERS signal enhancement.5 However, interactions between suspended colloids and analytes rely on slow diffusive transport from the bulk solution to the surface of metal nanoparticles (NPs) to facilitate molecule−metal interactions. In general, the molecules which are SERS-active display a certain affinity for adsorption on a metal surface. © 2014 American Chemical Society

Received: January 18, 2014 Accepted: March 19, 2014 Published: March 19, 2014 3955

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the network so that they closely approach the guest molecules in the pores; (4) the inherent water stability of the host matrix allows the AuNPs/MIL-101 composites to work in aqueous solution. The excellent SERS activity was tested by measuring the Raman signals for a variety of model analytes. We show the great potential of the fabricated AuNPs/MIL-101 for trace analysis of p-phenylenediamine in environmental samples and alpha-fetoprotein (AFP) in clinical samples.

approach is to coat metal nanoparticles with either organic or inorganic shells, such as polymers,13−15 transition-metal materials,16,17 carbon,18 and mesoporous silica.19 These protective shells improved the mechanical stability of nanoparticles and better signal reproducibility. In particular, the use of silica shells offers some advantages including good stability and biocompatibility and was termed shell-isolated nanoparticle-enhanced Raman spectroscopy.19 However, the silica shells are composed of disordered and amorphous structure, and the diffusion of molecules to the metal core is limited. It would thus be advantageous to develop a SERS detection element with excellent stability while simultaneously offering enhanced analyte−metal interactions by preconcentrating target molecules in close proximity to the surface of metal core. Metal−organic frameworks (MOFs) are a new class of highly porous crystalline materials, prepared by ordered assembly of metal ions and organic ligands linked together through a coordination interaction. MOFs are typically characterized by high surface areas, tailorable chemistry, and uniform and tunable nanostructured cavities. These unique characteristics make them very promising for diverse applications in analytical chemistry, for instance, as sorbents for sampling,20,21 stationary phases for chromatography,22−24 magnetic solid-phase extraction,25,26 solid-phase microextraction,27,28 and enantioselective separation.29,30 Additionally, MOFs are expected to serve as a stabilizing matrix of nanoparticles. The encapsulation of nanoparticles within MOFs attracts much attention because of the benefits of novel chemical and physical properties exhibited by certain classes of nanoparticles, yielding nanocomposites with hybrid functions.31−33 We conceived the idea that the excellent characteristics of MOFs would also make them leading candidates for the SERS technique by embedding noble metal nanoparticles within the porous structures. The property of large specific surface area and porous structure of MOFs can preconcentrate analytes proximal to the metal surface. In addition, the unique configuration of porous MOFs provides 3-D structures to accommodate much more metal nanoparticles of SERS activity. The adsorption properties can be controlled through a choice of precursors and synthetic conditions, yielding tunable chemical selectivity based on molecular sieving effects, hydrogen bonding, electrostatic interaction, etc.34 To our knowledge, there are few reports about the construction of SERS substrates based on a metal− organic framework.35−37 In these works, MOF-5 was used as the supporting shell. Consequently, the application of the material in aqueous solution, commonly encountered for practical SERS detection, is limited due to the susceptible characteristics of MOF-5 to water. Additionally, no trials for application to real sample analysis were explored. In this work, we demonstrate the fabrication of a sensitive SERS substrate by embedding gold nanoparticles (AuNPs) within MIL-101, one of the most fascinating MOFs that possess high specific surface area, large pore diameter, and extremely high water and solvent stability, 38,39 by the solution impregnation strategy. MIL-101 serves as stabilizing host material providing a confined space as well as adsorptive domains for enrichment of analytes in proximity to the embedded metal surface. The resulting nanocomposites are unique in that: (1) the embedding of gold nanocrystals in the MIL-101 skeleton retains the advanced nanoporosity of the network structure; (2) the dispersible and discrete AuNPs/ MIL-101 are readily amenable to enrichment of analytes; (3) the SERS-active sites of the gold nanocrystals are imposed on

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EXPERIMENTAL SECTION Materials and Reagents. All chemicals and reagents used were at least of analytical grade. Cr(NO3)3·9H2O was bought from Kermel Chemical Reagent Co. Ltd. (Tianjin, China). Terephthalic acid, chloroauric acid, p-phenylenediamine, benzidine, 4,4′-bipyridine, diphenylamine, 4-aminothiophenol, and Rhodamine 6G were purchased from Jinchun Reagent (Shanghai, China). Poly(4-vinylpyridine) was obtained from Sigma-Aldrich (MW 60 000, USA). N,N′-Dimethylformamide (DMF), ethanol, hydrofluoric acid, hydrogen peroxide, trisodium citrate dehydrate, sodium hydrogen phosphate, and sodium dihydrogen phosphate were obtained from Guangzhou Chemical Regent Factory (Guangzhou, China). The commercial AFP enzyme-linked immunosorbent assay (ELISA) Kit was purchased from Zhengzhou Bosai Biotechnique Academe. Alpha-fetoprotein (AFP) antigen, monoclonal anti-AFP antibody (anti-AFP) coated microtiter plate, and horseradish peroxidase (HRP)-labeled anti-AFP (HRP-anti-AFP) were also included in the AFP ELISA Kit. Fabrication of SERS-Active AuNPs/MIL-101 Nanocomposites. MIL-101 was synthesized according to the method described by Férey.39 For the embedding of AuNPs, 50 mg of MIL-101 was suspended in 30 mL of aqueous solution of chloroauric acid with different concentrations (0.04, 0.06, 0.08, 0.1, 0.12% (w/v)) and was kept under continuous stirring for 2.5 h at 45 °C. The solution was then heated to vigorous boiling, followed by quick injection of 220 μL of sodium citrate (4%, 6%, 8%, 10%, 12% (w/v)) and further stirring for 40 min at boiling. After cooling down, the assynthesized material was separated by centrifugation and redispersed in 300 μL of deionized water. General Characterization Methods. Transmission electron microscopy (TEM) images and energy-dispersive X-ray spectroscopy (EDS) were performed on an FEI Tecnai G2 Spirit and FEI Tecnai G2 F30 instrument (FEI, Netherlands), respectively. Scanning electron microscopy (SEM) images were recorded on S-4300 SEM instrument (HITACHI, Japan). The thermogravimetric analysis (TGA) curve was recorded using a thermogravimetric (TG) analyzer (TG-209, Netzscha, Germany) from room temperature to 900 °C in flowing nitrogen at a heating rate of 10 °C/min. X-ray diffractometry (XRD) was obtained on D-MAX 2200 VPC (RIGAKU, Japan). Nitrogen sorption isotherms were measured at 77 K with gas sorption analyzer ASAP-2020 (Micromeritics, USA). UV−vis spectra were conducted on a CARY 300Conc UV spectrophotometer (Varian, American). ELISA was measured on an iMark 1681135 microplate reader (Bio-Rad, USA) for contrast. Raman spectra were performed on a battery-powered Raman spectrometer (model Inspector Raman, diode laser, excitation wavelength λex = 785 nm) in the range of 200−2000 cm−1 (DeltaNu, USA). SERS Measurements Using the AuNPs/MIL-101. In a typical process, 4 μL of analyte solution was mixed with 2 μL of AuNPs/MIL-101 nanocomposite suspension and were then 3956

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Figure 1. (A) SEM images of the as-synthesized AuNPs/MIL-101; (B) TEM images of the as-synthesized AuNPs/MIL-101; (C) the energydispersive X-ray spectroscopy of AuNPs/MIL-101; (D) the selected area electron diffraction pattern recorded from the gold nanoparticles in AuNPs/MIL-101.

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RESULTS AND DISCUSSION Preparation and Characterization of the AuNPs/MIL101 Nanocomposites. Loading nanoparticles inside MOFs are usually based on postsynthesis by chemical vapor deposition,40 solution impregnation,41 and solid grinding.42 In this study, MIL-101 was synthesized and adopted as the host material for solution infiltration of gold mother solution. Because of the dramatically large surface area of the MIL-101, chloroauric acid would infiltrate into the hydrophilic zeolite-like solid frame, which greatly minimizes the deposition of Au in mother solution. Then, after the addition of the reductant, nucleation takes place within the solid matrix for in situ growth of AuNPs. The suspension immediately turned its color from light green to purple. The reduction of chloroauric acid with different amounts of sodium citrate can prepare gold nanoparticles with different particle diameters. The loading ratio of Au nanoparticles could be easily controlled by changing the initial concentration of chloroauric acids. In Sada’s report,37 gold nanorods/MOFs were prepared by growing MOF-5 crystals with modified gold nanorods as seeds. However, we found that the AuNPs are prone to aggregation by introducing mother components of MIL-101. This may result from the change of the dielectric constant of the local environment of the nanoparticles.31 The morphology and structure of AuNPs/MIL-101 were first examined by SEM and TEM. As shown in the SEM image (Figure 1A), the smooth crystal surface of the composites revealed that AuNPs were mostly encapsulated within the MIL101 crystals and not at the outer surface, which favor the stable

deposited on a silicon substrate for SERS analysis. The typical exposure time for each measurement was 1 s with 3 accumulations. Nine measurements from three samples were used to calculate the relative standard deviations unless specified. The sewage samples and river samples were collected from Guangzhou. These samples were directly used for SERS measurement without a sample preparation procedure. SERS-ELISA Detection of Alpha-Fetoprotein Using the AuNPs/MIL-101. First, 100 μL of different concentrations of the AFP standard solution or the serum samples was added to each well of the antibody-coated microplates and incubated at 37 °C for 30 min. Then, 100 μL of washing buffer was added and shaken for 5 s. Afterward, the wells were rinsed with deionized water three times. Then, 100 μL of HRP-anti-AFP was added to each well, incubated at 37 °C for 30 min, and rinsed as above. Following this step, 100 μL of 0.02 mol/L PBS buffer (pH 6.75), 60 μL of 1 × 10−4 mol/L H2O2, and 30 μL of 2 × 10−7 mol/L benzidine were added to each well. After shaking for 5 s, the wells were incubated at 37 °C for 35 min. Then, 4 μL of reaction solution was mixed with 2 μL of AuNPs/MIL-101 nanocomposite for SERS detection. Clinical serum samples were obtained from healthy volunteers. For comparison, the serum samples were also measured by a commercial ELISA kit with microplate reader. The procedures exactly followed the protocols suggested by the commercial kit. The optical density at the wavelength of 450 nm was detected. 3957

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Figure 2. (A) XRD pattern of the MIL-101 (black) and the AuNPs/MIL-101 (red). (B) The UV−vis absorption spectra of the aqueous dispersion of MIL-101 and AuNPs/MIL-101 with the increase of chloroauric acid concentration (0.04%, 0.06%, 0.08%, 0.1%, 0.12% (w/v)).

combination of the two components. The TEM image clearly shows that the octahedral crystal structure of MIL-101 was not destroyed and the AuNPs were homogeneously distributed and embedded inside MIL-101 (Figure 1B). The density of AuNPs could be easily controlled by just changing the amounts of chloroauric acid for incubation with MIL-101 and increased along with the increase of the chloroauric acid concentration. However, excessive precursor would lead to inhomogeneous size distribution and formation of the nanoparticles outside of MIL-101 (Supporting Information, Figure S1). The corresponding energy dispersive X-ray spectroscopy of AuNPs/MIL101 shows the peaks of C, O, Au, and Cr elements, confirming the existence of AuNPs (Figure 1C). From the selected area electron diffraction pattern obtained from the embedded AuNPs, the hexagonal symmetry of the pattern spots is clearly observed, indicating the nanocrystalline nature (Figure 1D).43 The size distribution of AuNPs revealed an average diameter of about 50 nm with relatively narrow particle size distribution (Supporting Information, Figure S2). In addition, the TGA further supported that the loading density of AuNPs increased with increasing concentration of chloroauric acid (Supporting Information, Figure S3). The X-ray diffraction (XRD) pattern of the as-synthesized AuNPs/MIL-101 is shown in Figure 2A. Although the encapsulation of AuNPs caused the relatively lower intensity of its XRD pattern as compared with the bare MOFs, the obvious diffraction peaks assignable to MIL-101 remained and matched well with previously published XRD patterns of MIL101, clearly indicating that the crystalline framework of MIL101 is mostly maintained. Moreover, the obvious diffraction peaks at 38.1°, 44.4°, 64.6°, and 77.5° exactly match with the reference values for the Au (111), Au (200), Au (220), and Au (311) lattice planes, respectively,44 which also indicates that AuNPs exist in the crystalline state. The decrease in the amount of nitrogen adsorption and the pore volume of AuNPs/MIL101 revealed that the cavity of MIL-101 were partially occupied by highly dispersed AuNPs; however, the BET surface area remained as high as 2070 m2/g (Table 1 and Supporting Information, Figure S4), which guarantees the inclusion of functional components while maintaining the high adsorption capability of the metal−organic framework. Figure 2B shows the UV−vis absorption spectra of the aqueous dispersion of MIL-101 and AuNPs/MIL-101. After embedding AuNPs inside MIL-101, there is evidently a new peak at about 600 nm. This is the typical characteristic of AuNPs due to the surface plasmon absorption. The intensity of absorption peak increased along with the loading density of AuNPs controlled by chloroauric

Table 1. Surface Area Measurement of the Pure MIL-101 and AuNPs/MIL-101

a

sample

BET surface area (m2/g)

Langmuir surface area (m2/g)

pore volume (cm3/g)

pore size (Å)

MIL-101 AuNPs/MIL-101a

3019 2070

4117 2784

1.45 0.98

23.36 21.44

Prepared with the chloroauric acid concentration of 0.1%.

acid concentration, while the suspension of the bare MIL-101 does not show any peaks at that wavelength. On the basis of these results, it can be concluded that successful inclusion of AuNPs inside MIL-101 was obtained. SERS Activity of the AuNPs/MIL-101 Nanocomposite. To quantify the enhancement of the AuNPs/MIL-101 nanocomposites, the Raman signal of the common SERS model analyte Rhodamine 6G (R6G) was preliminarily evaluated. The Au colloids with about 50 nm and the bare MIL-101 were used as controls. After loading R6G solutions (0.21 μmol/L) on the AuNPs/MIL-101 composites, intense scattering signals were detected at multiple Raman shifts characteristic of R6G (Figure 3A), whereas no R6G Raman signals were detected on virgin MIL-101 without embedding AuNPs, even when a significantly higher R6G concentration was used. Signals with lower intensity was also observed with Au colloids as SERS substrate; however, the concentration of R6G used was 20-fold higher (4.18 μmol/L). These significant differences may arise from the difference of local concentration of the analyte adjacent to the surface of the SERS-active sites. In this work, metal−organic framework (MOF) was used as a shell of SERS-active nanomaterials. Owing to the well-known powerful adsorption capability of the metal−organic framework, analytes can be preconcentrated by the porous MIL-101 via physical adsorption and chemical interactions mainly based on the electrostatic interactions and the intrinsic coordination property, which greatly accelerates the analytes to approach the AuNPs embedded in the solid matrix and thus helps in the significant enhancement of SERS intensity. In addition, the unique configuration of porous MOFs provides 3-D structures to accommodate much more available SERS-active sites, which also helps to enhance the Raman signal. To further verify the excellent SERS activity of the AuNPs/MIL-101 and the contribution from the synergetic effect, a series of aromatic amine compounds was tested for their Raman responses (Figure 3B−F). Similar to that of R6G, all of these selected chemicals exhibited a huge SERS enhancement with the 3958

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Figure 3. SERS spectra of Rhodamine 6G (A), benzidine (B), 4-aminothiophenol (C), diphenylamine (D), 4,4-bipyridine (E), and pphenylenediamine (F) on the AuNPs/MIL-101 substrate (blue lines), Au colloids substrate (red line), and virgin MIL-101 (black line). The concentration of (A−F) for detection is 0.21 μmol/L, 0.27 nmol/L, 0.80 μmol/L, 0.59 μmol/L, 0.64 μmol/L, and 0.92 μmol/L for AuNPs/MIL-101 and 4.18, 10.97, 0.80, 11.82, 12.81, and 18.49 μmol/L for Au colloids and MIL-101, respectively.

AuNPs/MIL-101 substrate. Among these compounds, benzidine exhibited the highest enhancement effect even at very low concentration (0.27 nmol/L), while no visible Raman signal of benzidine (10.97 μmol/L) was observed on the Au colloids. For the controlled experiments with Au colloids, only 4aminothiophenol exhibited similar enhancement effect as compared with the AuNPs/MIL-101 substrate. The result is easy to understand because that 4-aminothiophenol can strongly adsorb on the surface of Au colloids via the thiol group. However, lack of this specific interaction for other compounds with Au colloids would limit the enhancement effect as compared with AuNPs/MIL-101, which can offer enhanced analyte−metal interactions by preconcentrating target molecules in close proximity to the surface of metal core. The estimated molar detection limits were measured for Rhodamine 6G and benzidine and were found to be 41.75 and 0.54 fmol, respectively. The influence of embedded AuNPs density on the SERS effect was investigated by measuring Raman intensity of R6G on AuNPs/MIL-101 prepared with different chloroauric acid

concentration and with different incubation times (Supporting Information, Figure S5). The Raman intensity increased with increasing the concentration of chloroauric acid up to 0.1%, which may be attributed to the resulting higher density of Au nanoparticles embedded within the MIL-101 framework. However, the Raman intensity began to decrease while the concentration of chloroauric acid was as high as 0.12%, owing to the loss of surface area of the nanocomposites. Furthermore, the Raman intensity increased with increasing incubation time, indicating the sufficient diffusion of mother solution through the supports. Sieving Effect of the SERS Measurement. It is wellknown that MOFs had a molecular sieving effect owing to the nanoscopic pore dimensions.45 To investigate the size selectivity of the SERS measurement using the AuNPs/MIL101 nanocomposites, we chose 4,4′-bipyridine and poly(4vinylpyridine), two pyridine derivatives with different molecular size, as model analytes.37 The AuNPs/MIL-101 nanocomposites were added to respective 0.64 μmol/L pyridine derivative solution, and their SERS signals were collected. It can be clearly 3959

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molecules by efficiently removing the interference from those macromolecules larger than the pore size of the substrate. Stability and Reproducibility. One limitation of using suspension of Au colloids for SERS detection is the inherent instability arisen from the change of detection environment (pH, ion strength, etc.), which would certainly influence the reproducibility for quantitative detection. The colloids may even tend to precipitate from solution, resulting in loss of SERS signal. Unlike the colloids, the metal nanoparticles in the AuNPs/MIL-101 were stabilized and protected by the metal− organic framework shell, which offered advantages of stable localized surface plasmon resonance property. To investigate the stability of the AuNPs/MIL-101 substrates, the SERS performance of both AuNPs/MIL-101 and AuNPs colloids was measured with aqueous solutions of benzidine at different pH values (Figure 5A,B). The SERS activity of AuNPs/MIL-101 was stable while the pH varied from 2 to 12. In contrast, the Raman intensity of AuNPs colloids was significantly different with the variation of pH value, which was due to the irregular aggregation verified by the UV−vis method (Supporting Information, Figure S6). Even after centrifugation and redispersion, the UV−vis absorbance spectrum of the AuNPs/MIL-101 did not change, indicating the ease of storage for the novel SERS substrate (Supporting Information, Figure S7). We then further studied the reproducibility of the assynthesized AuNPs/MIL-101 substrate in the same and between different batches (Figure 5C,D). The coefficients of variation for SERS performance were less than 10% for one batch of 40 repeated tests and below 20% for 25 different batches tested. Therefore, it can be concluded that the hybrid AuNPs/MIL-101 SERS substrate not only provides good

observed from Figure 4 that much stronger SERS signals were readily detected for 4,4′-bipyridine while almost no Raman

Figure 4. Raman intensity plotted against time at 1030 and 1292 cm−1 for 0.64 μmol/L 4,4′-bipyridine (black square) and poly(4-vinylpyridine) (red circle), respectively.

response was observed for poly(4-vinylpyridine), which was not able to diffuse into nanopores of the MIL-101 shell and to interact with the embedded AuNPs. In addition, 4,4′-bipyridine showed the time-dependent SERS signals which reached a stable value within about 4 min, indicating the evidence of the diffusion process. In conclusion, the above results of distinct Raman response dependent on the molecular size of analytes should be attributed to the sieving effect of the MIL-101 crystals. The micropore structure discriminates guest molecules by their size and would thus improve selectivity for small

Figure 5. Stability investigation of the AuNPs/MIL-101 substrate. Raman intensity of benzidine on AuNPs/MIL-101 (A) and AuNPs colloid (B) at different pHs; the reproducibility of the as-synthesized AuNPs/MIL-101 substrate within the same (C) and between different batches (D). 3960

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sensitivity but also processes high stability and good reproducibility. SERS Detection of p-Phenylenediamine in Environmental Water. Pharmaceuticals and personal care products (PPCPs) are a diverse group of environmental chemicals that have increasingly gained attention due to their ubiquitous occurrence in the environment and their potential to cause undesirable ecological effects. Development of a rapid, simple, and in situ method for monitoring PPCPs is of great importance. In the following, we applied the novel AuNPs/MIL-101 SERS substrate in combination with the portable Raman spectrometer for the rapid determination of p-phenylenediamine, one of the PPCPs of special concern, in environmental water samples. The p-phenylenediamine with electron donor property allows electrostatic and coordination interactions with the MIL-101, which then demonstrates preferential adsorption of the analyte, and thus promotes the analytes adjacent to the metal nanoparticles surface. The Raman signals were significantly enhanced and were much higher than that using the Au colloids thanks to the synergistic effect of MIL-101 and the embedded Au nanoparticles. The effect of the experimental conditions, including concentration of AuNPs/MIL-101, incubation time of analytes, and AuNPs/MIL-101 were investigated (Supporting Information, Figure S8). A stable and sensitive Raman response was obtained with incubation time of 4 min while the as-synthesized AuNPs/MIL-101 substrate was concentrated in 300 μL. Figure 6A shows the SERS spectra of p-phenylenediamine and the calibration curve for the SERS intensity versus logarithm concentration at the Raman peak of 1167 cm−1. Satisfactory linearity between 1.0 and 100.0 ng/mL was obtained with correlation coefficients of 0.9950. The limits of detection (LOD) were calculated considering the lowest p-phenylenediamine concentration leading to a SERS intensity which produces a signal/noise ratio higher than 3 and was 0.10 ng/mL, which was remarkably lower than some of the previous reports.46−48 The method was then applied to the analysis of pphenylenediamine in the sewage water and river water, respectively (Figure 6B). The background SERS signal of the AuNPs/MIL-101 was also measured and showed no visible signal at the Raman shift of the characteristic peaks. No sample preparation procedure was necessary prior to the SERS detection. The p-phenylenediamine was quantified with concentration of 1.3 ng/mL in sewage water and was not detectable in river water (Table 2). The samples were then spiked with standard solutions of p-phenylenediamine to evaluate the recoveries, which ranged from 80.5% to 114.7% for sewage water and river water, respectively. SERS-ELISA Based Detection of Alpha-Fetoprotein in Human Serum. AFP is a widely used tumor marker of hepatocellular carcinoma (HCC) which has roused a public concern. It is of great importance to detect serum levels of AFP for early screening and clinical diagnosis of HCC. ELISA is a common bioassay method for the detection and quantification of important biomarkers, using the spectroscopic detection of a colored reagent. Considering the diverse characteristics of analytes, it is important to broaden the read-out technique for ELISA.49,50 In this work, we use SERS detection as a new detection technique for ELISA because of its high sensitivity and capability of providing unique vibrational fingerprints for distinguishing structurally similar molecules.

Figure 6. (A) SERS spectra obtained in the presence of different concentrations of p-phenylenediamine (the concentrations from bottom to top (a−i) are 1.0, 3.0, 5.0, 8.0, 10.0, 30.0, 50.0, 80.0, and 100.0 ng/mL). Inset: the calibration curve for the SERS intensity versus logarithm of the concentration at the Raman peak of 1167 cm−1. (B) SERS detectection of p-phenylenediamine in sewage water.

Table 2. SERS Analysis of p-Phenylenediamine in Environmental Water sample

found (ng/mL)a

sewage water

1.3

river water

N.D.b

added (ng/mL)

recovery (%)

RSD (%)c

1.0 5.0 1.0 5.0

83.4 85.7 114.7 80.5

7.7 4.0 6.8 4.6

Quantitative data at 1167 cm−1. bN.D., not detected. cn = 9, quantitative data from nine different spots of three samples.

a

On the basis of the sensitive SERS response of benzidine on the AuNPs/MIL-101, which has been clearly demonstrated above, we propose here an ELISA-SERS method based on the benzidine−H2O2−HRP system for detection of AFP. In brief, the oxidation reaction of the chromogenic substrate benzidine with hydrogen peroxide is efficiently catalyzed by HRP attached to antibody (HRP-anti-AFP) that specifically binds with antigen AFP. The concentration of AFP can be estimated by observing SERS signals of the unreacted benzidine. The high adsorptive capability of the metal organic framework MIL-101 makes the Raman reporter molecules reside in close proximity to the embedded Au nanoparticles, leading to the enormous enhancement of Raman scattering (Supporting Information, Figure S9). Different experimental parameters that influence the ELISA reaction including pH of PBS buffer, concentration of hydrogen 3961

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significant improvement of sensitivity for Raman detection is possible. The method was applied to the analysis of AFP in human serum from four healthy volunteers (Figure 7B). As shown in Table 3, AFP was detected with the concentration ranging from 2.7 to 17.1 ng/mL, which was in good agreement with that determined by the commercial ELISA kit with the microplate reader. When the given amounts of AFP were added into the human serum samples, the results showed satisfied recoveries in the range of 79.3−107.3%. The developed ELISASERS method provides an interesting alternative tool for detection of tumor biomarkers in clinical samples.

peroxide, reaction temperature, and time were investigated (Supporting Information, Figure S10). Then, the SERS-ELISA detection was carried out using a series of AFP standards under the optimum conditions. Figure 7A shows the Raman response

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CONCLUSIONS This work presents fabrication and application of the highly sensitive, stable, and reproducible SERS substrate, which was synthesized by embedding nanoparticles inside the solid matrix of metal−organic framework. The water-stable MIL-101 used in this work provides permanent high porosity enabling effective loading of AuNPs and prevents the migration of the nanoparticles, together with large surface areas for preferential adsorption of the analyte close to the SERS-active sites at the metal surface, leading to a dramatic enhancement of Raman intensity. The potential applications of the novel AuNPs/MIL101 SERS substrate were tested both for environmental and clinical samples. SERS measurement was performed by detecting the characteristic Raman scattering of p-phenylenediamine in sewage water and river water. The sensitivity of the SERS detection was greatly improved with limits of detection as low as 0.1 ng/mL. Then, a SERS-ELISA based method for detection of alpha-fetoprotein in serum samples was established, indicating the novel SERS substrate was also applicable to clinical samples. Considering the diversity structures and the controllable chemical functionality of MOFs, we anticipate that this new type of the AuNP embedded metal−organic framework nanostructure will find more potential applications in sensing, bioanalytical, and biomedical assays.

Figure 7. (A) SERS spectra obtained in the presence of different concentrations of AFP (the concentrations from top to bottom (a−h) are 1.0, 5.0, 10.0, 30.0, 50.0, 80.0, 100.0, and 130.0 ng/mL). Inset: the calibration curve for the SERS intensity versus logarithm of the concentration at the Raman peak of 1197 cm−1. (B) SERS-ELISA detection of AFP in serum samples.

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ASSOCIATED CONTENT

S Supporting Information *

Figures about characterization of the material, effect of the experimental conditions, scheme of the principle of the SERSELISA system, and additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

of the solution with the variation of the AFP concentration. It can be observed that the Raman intensity decreased with the increase of the AFP concentration. The intensity of the band at 1197 cm−1 versus logarithm concentration of the AFP was plotted with good linearity in the range of 1.0−130.0 ng/mL. The correlation coefficient (R2) was calculated to be −0.9938. By using the novel AuNPs/MIL-101 as SERS substrate,

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AUTHOR INFORMATION

Corresponding Authors

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Table 3. SERS-ELISA Analysis of Alpha-Fetoprotein in Human Serum

a

sample

SERS-ELISAa (ng/mL)

added (ng/mL)

recovery (%)

RSDb (%)

microplate reader (ng/mL)

serum 1

4.2 2.7

serum 3

5.9

serum 4

17.1

81.8 84.7 92.9 79.3 107.3 80.3 100.1 92.3

6.3 3.9 4.3 7.1 10.8 6.8 9.1 8.8

4.7 ± 0.8

serum 2

5.0 20.0 5.0 20.0 5.0 20.0 5.0 20.0

2.3 ± 0.4 6.8 ± 0.9 18.8 ± 1.4

Quantitative data at 1197 cm−1. bn = 9, quantitative data from nine different spots of three samples. 3962

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work was supported by the National Natural Science Foundation of China (Nos. 21277176, 21275168, and 21127008), the National Key Scientific Instrument and Equipment Development Project (No. 2011YQ03012409), and the Guangdong Provincial Natural Science Foundation of China (No. S2013010012091), respectively.

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