Au Nanoparticles-Based Resonant Raman

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Heterostructured ZnO/Au Nanoparticles-Based Resonant Raman Scattering for Protein Detection Guiye Shan,† Shuang Wang,† Xiaofang Fei,‡ Yichun Liu,*,† and Guoliang Yang§ Center for AdVanced Optoelectronic Functional Materials Research, Northeast Normal UniVersity, Changchun 130024, P. R. China, College of Life Science, Jilin UniVersity, Changchun 130012, P. R. China, and Department of Physics, Drexel UniVersity, Philadelphia, PennsylVania 19014 ReceiVed: May 24, 2008; ReVised Manuscript ReceiVed: October 10, 2008

A new method of protein detection was explored on the resonant Raman scattering signal of ZnO nanoparticles. A probe for the target protein was constructed by binding the ZnO/Au nanoparticles to secondary protein by eletrostatic interaction. The detection of proteins was achieved by an antibody-based sandwich assay. A first antibody, which could be specifically recognized by target protein, was attached to a solid silicon surface. The ZnO/Au protein probe could specifically recognize and bind to the complex of the target protein and first antibody. This method on the resonant Raman scattering signal of ZnO nanoparticles showed good selectivity and sensitivity for the target protein. 1. Introduction Detecting protein or other macromolecules with high sensitivity and specificity is essential for disease diagnostics, drug screening, and other applications.1-3 The development of labelfree optical biosensors for DNA and other biomolecules has the potential to impact life science as well as screening in medical and environmental sciences. Semiconductor nanoparticles in biological applications have increased dramatically since the first benchmark in 1998.4,5 Currently, the applications of semiconductor nanoparticles in biological labels mainly rely on fluorescent signal. However, fluorescent signal displayed strong background autofluorescence and fluorescence quench.6 The label-free detection of the smallest sample amounts by enhanced Raman spectroscopy is gaining considerable attention in biochemical and biophysical sciences.7-9 Raman signal shows more quality including narrow full width at half-maximum (fwhm), low backgroud autofluorescence, and high detection sensitivity. Many groups have successfully explored DNA or protein detection using the Raman signals of dye molecular, which showed higher detection sensitivity.10,11 However, it is wellknown that dye molecules as label materials show lower stability than semiconductor nanoparticles, including lower photostability and weaker chemistability. In our present work, an alternative detection strategy is employed, which makes use of Raman signal of semiconductor nanoparticles such as ZnO nanocrystals as efficient label. As a semiconductor material, ZnO nanoparticles are believed to be nontoxic, biosafe materials with bandedge energy of 3.3 eV. Biosensing applications of wide band gap ZnO have been paid more attention in typical biomolecular detection environments.12-17 ZnO quantum dots (QD) have characteristic resonance multiple-phonon Raman lines with the excitation wavelength of 325.0 nm. The unique optical properties of ZnO have been used to design a label-free biosensor. In this paper, we report the synthesis of ZnO/Au nanocomposites with strong resonant Raman scattering (RRS) signal for detection of * To whom the correspondence should be addressed. Tel: 86-43185099168. Fax: 86-431-85684009. E-mail: [email protected]. † Northeast Normal University. ‡ Jilin University. § Drexel University.

protein. In this paper, we study the physical mechanism of interaction between ZnO and Au. The morphology and resonant Raman intensity may be controlled by changing the ratio ZnO and Au, which is important to furthermore study on detection of protein. By comparison, the optimized ZnO/Au nanocomposite was chosen for detection of protein. The detection of protein by ZnO/Au nanocomposites presented the following advantage: it was simple, highly reproducible, and could be easily controlled. Importantly, few groups have reported the use of ZnO Raman signal as probe to detect protein since now. The Raman label of unknown biomolecule has been widely focused including analysis of proteins, nucleic acids in solution or quantitative detection of single proteins in sandwich-binding assays. The true potential of Raman probe for quantitative signal detection lies in the ability to accurately separate probe signals from background autofluoresence, which is particularly advantageous for low-abundant analytes and lowintensity signals. The approach obtains a label-free sensing platform for unambiguous detection of unknown protein using Raman signal. 2. Experimental Section Chemicals. AuCl3 · HCl · 3H2O(>99.9%), Zn(CH3COO)2 · 2H2O, LiOH · H2O, 3-aminopropyltrimethoxysilane, and Tween 20 were purchased from Sigma-Aldrich. Goat antihuman IgG, human IgG, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich. Ethanol (99.5%) and trisodium citrate were purchased from Shanghai Reagent Co. of Chinese Medical. All the other reagents in the experiments were used without further purification except ethanol which was distilled to obtain pure, dry ethanol. Synthesis of Heterostructured ZnO/Au Nanoparticles. Heterostructured ZnO/Au nanocomposites were synthesized by the growth of Au on the ZnO nanoparticles. The preparation procedure of ZnO nanoparticles was the same as that in the literature.18 Different ratios of ZnO/Au nanocomposites were synthesized by changing the amount of the precursor of Au. Here, the ratio of ZnO and Au was calculated by comparing the concentration of ZnO and gold ions. We provided procedure of ZnO/Au nanocomposites with a ratio of ZnO and Au at 4%.

10.1021/jp8046032 CCC: $40.75  2009 American Chemical Society Published on Web 01/12/2009

New Method of Protein Detection Six milliliters of synthesized 0.1 M ZnO nanoparticles in ethanol was precipitated by adding 25 mL of 0.15 mmol/L trisodium citrate solution. The precipitated ZnO nanocrystals were dispersed in 15 mL of 3 mmol/L trisodium citrate solution by ultrasound. Five milliliters of 5 mmol/L HAuCl4 solution was slowly dropped into the above solution while vigorously stirring for at least 48 h at room temperature. The solution turned wine from pale yellow and transparent, which indicated that heterostructured ZnO/Au nanoparticles have been formed. Preparation of ZnO/Au-Protein Probe. We compare the Raman scattering intensity and the morphology of ZnO/Au with different ratios of ZnO and Au. The ZnO/Au nanocomposite with ratio of ZnO and Au at 0.04 was chosen for formation of Raman probe. ZnO/Au-protein probes were prepared by addition of goat antihuman IgG to 5 mL of ZnO/Au solution and were incubated for 1 h on ice with periodic gentle mixing. It is well-known that pI of IgG is about 9.5 and positive charges were shown below this pH. In this experiment, the conjugated pH between ZnO/Au and protein was chosen at around 7. Ionic strength of solution is important for conjugating ZnO/Au and protein by electrostatic interaction. Ionic strength values were 0.45 M in this experiment. The conjugate was then divided into 1 mL fractions in 1.5 mL microcentrifuge tubes and centrifuged at 12 500g for 30 min. The clear to pink supernatant was removed. The precipitated stuff was redispersed in PBS buffer solution (pH 7.5). Conjugates can be stored at 4 °C for several days without loss of activity. Covalent Immobilization of Protein on Silicon Surface. A two-step procedure was used to covalently link antibody receptors to the surfaces of the silicon surface. First, silicon surface was cleaned by piranha solution (1:3 H2O2:H2SO4) for 15 min. Then, the fresh silicon was reacted with a fresh ethanol solution of 3-aminopropyltrimethoxysilane (APTES) for 1 h at room temperature. The modified silicon was followed by the rinsing with water and pure ethanol, respectively, three times and heated at 120 °C for 15 min. The silicon surfaces modified with APTES were reacted with a 2.5% solution of glutaraldehyde in PBS buffer solution for 2 h, followed by rinsing with PBS buffer. The glutaraldehyde surfaces were then placed into 0.1 mg/mL goat antihuman IgG solution with the addition of 1% Tween 20 at room temperature until the saturated protein layers were obtained. The surfaces were washed with PBS buffer solution, and the remaining aldehyde groups on the surfaces were deactivated with 1 M ethanolamine for 30 min. Quantitative Sandwich Assay. For quantitative analysis, sandwich immunoassays that detect human IgG were performed with respect to goat antihuman IgG modified silicon substrates. The human IgG (from 5 pM to 100 nM) was incubated with modified silicon substrates at 4 °C for 10 h, and unbound human IgG were then washed with 1% BSA PBS buffer. The ZnO/Au modified by goat antihuman IgG was added and incubated for 1 h. Then the silicon substrate was thoroughly washed with 0.05% BSA PBS buffer (0.05% Tween 20). 3. Results and Discussion Morphological Characterization of Heterostructured ZnO/ Au Nanocomposites. The transmission electron microscope (TEM) investigation of synthesized ZnO and ZnO/Au nanoparticles with different amounts of Au3+ are showed in Figure 1. The TEM images show that pure ZnO colloids are nearly monodispersed with a diameter ranging from 3 to 5 nm (panel a). Heterostructured ZnO/Au nanocomposites’ morphology could be changed with different amounts of Au3+ during the growth of ZnO/Au nanocomposites. As shown in Figure 1b,

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Figure 1. TEM image of pure ZnO nanoparticles (a), ZnO/Au nanocomposites with ZnO/Au molar ratio at 2% (b), 4% (c), and 5% (d).

the connected nanocomposites appear at molar ratio of ZnO to Au3+ as 0.02 and their average diameters are around 9 nm. On increasing the amount of Au3+ at molar ratio as 0.03, the particles were separated as shown in Figure 1c. However, the size distribution and shape of particles are not uniform. The maximal size is about 15 nm. After continuing to increase the amount of Au3+ at molar ratio of 0.05, ZnO/Au nanocomposites become more spherical as shown in Figure 1d and the measured average diameters are about 20 nm. The morphological changes observed by TEM images indicated that the amount of Au3+ is likely key for the morphology of ZnO/Au nanocomposites. A reaction between gold ions and ZnO nanoparticles should occur. Gold chloride ions reacted with ZnO to form metallic gold. Initially, a few Au nanoclusters were reduced onto specific sites of ZnO nanoparticls, resulting in the connected structure due to the aggregation of ZnO in aqueous solution. After increasing the amount of Au3+, reductions of Au nanoclusters on ZnO nanoparticles further fill these empty surface sites so that spherical nanocomposites appear. For ZnO/Au nanocomposites with molar ratio as 0.03 as shown in Figure 1c, the amount of Au could not occupy all the surface of ZnO, which results in the different size distribution and shape. When the amount of Au is enough, ZnO/Au nanocomposites with uniform shape and size may be observed. The control of the cover of Au on ZnO surface is important. On the one hand, we may control the optical properties of ZnO. On the other hand, it is important for further controlling bioconjugation. Optical Properties of ZnO/Au Nanocomposites. The ZnO/ Au nanocomposites exhibit strong absorption in the UV and visible region as shown in Figure 2. In comparison with the surface plasmon (SP) optical band for pure Au in solution, the characteristic gold plasmon peak in ZnO/Au composite was observed to red-shift. The shift of the SP band is characteristic changes in interparticle spacing and dielectric properties. For Au, the position of plasmon absorption is related to electron density of metal. When decreasing the electron density of metal, the plasmon absorption of metal was increased. In this experiment, the work function for the intrinsic ZnO and Au is generally taken as 5.3 and 5.1 eV, respectively.20 Thus, Au can

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Figure 2. Absorption spectra of Au (a), ZnO (b), and ZnO/Au nanocomposites with different ratio of ZnO and Au at 2% (c), 3% (d), and 4% (e).

Figure 3. Resonant (λexc ) 325 nm) Raman Scattering spectra of pure ZnO (a) and ZnO/Au nanocomposites with ratio of Au at 2% (b), 4% (c), and 5% (d).

donate electrons to ZnO readily until two systems reach a dynamic equilibration. The red-shift of plasmon peak of gold demonstrated that the electron density of Au was decreased. Furthermore, the local electrical field must be formed in the interface between ZnO and Au due to the transfer of electron from Au to ZnO. RRS experiment of ZnO and ZnO/Au nanocomposites further reveals that the effect of Au on ZnO is significant. ZnO belonging to C6V4 space group has the following optic modes: A1 +2B1 + E1 + 2E2 at the point of Brillouin zone, among which E1, E2, and A1 are the first-order Raman-active modes, and B1 is forbidden.21 Meanwhile, the E1 and A1 modes split into longitudinal optical (LO) and transverse optical (TO) components. A1, E1, and E2 mode are Raman-active. An intense multiphonon scattering of ZnO nanoparticles before and after coating Au with molar ratio at 0.02 and 0.04 was observed in RRS spectra under excitation at He-Cd laser (λ ) 325 nm) as shown in Figure 3. The peak at 571 cm-1 is first-order optical phonon mode of ZnO, which could be contributed to the superimposition of A1 (LO) and E1 (LO). The second, third, and fourth optical Raman scattering cross section was also found to increase remarkably with an increase of Au amount on the ZnO surface. It is well-known that the increase of intensity of Raman scattering is mainly due to the increasing electron-phonon interaction. Following Kaminow,22 the LO Raman scattering cross section includes contributions from both the Fro¨hlich potential and deformation potential. Fro¨hlich potential is brought from macroscopic electric field so that the intensity of LO phonon is greatly enhanced under an electric field. As discussed above for Figure 2, the interfacial electric field between ZnO

Shan et al.

Figure 4. Absorption spectra of (a) heterostructured ZnO/Au nanoparticles and (b) ZnO/Au-labeled goat antihuman IgG. Inset: corresponding photoluminescence spectra of (a) ZnO/Au nanocomposites and (b) ZnO/Au-labeled goat antihuman IgG.

and Au could appear after coating Au. The interfacial electric field increases the interaction of phonon and electron, which results in the increase of RRS intensity. Wang et al. has also observed the enhancement of Raman intensity.23 However, the Raman signal of ZnO may be decreased if the amount of Au continued to increase. In comparison, we found that the intensity of ZnO Raman scattering decreased at the ratio of ZnO and Au at 0.05 as shown in Figure 3d. The reason may be that the incident light was refracted by Au nanoparticles and electronhole creation was decreased, which results in the decrease of interfacial electric field. So, the amount of Au coating on the surface of ZnO is important for obtaining enhanced Raman scattering intensity. Stability Study of ZnO/Au-Protein Probe. Bare ZnO nanocrystals are unstable in aqueous solution due to the interaction of surface ligand and water. After capping the Au nanocrystal, ZnO could be stable in water solution for 5 months. The reason for that is due to the surface ligand on Au nanocrystals. The surface ligands on Au provide the bridge to bind the biomolecule. The two advantages of this strategy are (1) obtaining stable, water-soluble ZnO particles and (2) directly binding the biomolecule to the gold surface without complex modification of the surface. The probe for the target protein was constructed by binding the ZnO/Au nanoparticles to goat antihuman IgG protein by eletrostatic interaction. Figure 4 shows the absorption spectrum of ZnO/Au nanocomposites before and after binding protein. No significant broadening of the Au spectrum was observed after this step, indicating that the particles did not experience aggregation upon chemisorption of goat antihuman IgG protein. Inset shows the photoluminescence of ZnO/Au nanocomposites before and after binding to protein. Comparing with ZnO/Au without protein (a), the intensity of ZnO photoluminescence in visible region was remarkably decreased after connecting protein molecule (b). The broad green emission at about 530 nm is attributed to the surface defects. The change of defect amount has effect on the emission of defects. It has been reported that organic molecule (ligand) can be absorbed on the surface of ZnO nanoparticles.24 As these ligands are absorbed to the nanoparticles’ surface, the concentration of surface state of the defect is reduced. This process results in a decrease of the green defect emission. On ZnO/Au nanocomposite, a lot of defects still exist on the surface of ZnO as shown in inset (a) of Figure 4. The relative intensity of the defect emission decreased as protein concentration was increased, reflecting a significant reduction of the defect density

New Method of Protein Detection

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SCHEME 1: A Schematic of the Methoda

a Firstly, goat antihuman IgG was immobilized on the silicon surface. Secondly, human IgG was added to the system. Finally, ZnO-labeled goat antihuman IgG protein was added to the above system to construct antibody-based sandwich assay.

Figure 5. Resonant Raman scattering spectra on biofunctional silicon wafer (a) target protein and ZnO labeled secondary antibody, (b) BSAand ZnO-labeled secondary antibody, and (c) mouse antigoat IgG protein and ZnO-labeled secondary antibody. All spectra were performed on the Si substrate and the peak at 510 cm-1 is from Si.

in the ZnO nanostructure. This suggests that the ZnO defect site in the resulting heterostructured ZnO/Au nanocomposites must be blocked by protein molecular. Detection Assay of ZnO/Au-Protein Probe. To probe the antigen-antibody binding event using RRS, a general protocol was devised based on sandwich immunoassay on silicon substrate. The detection procedure of proteins is shown in Scheme 1. First, the first protein (goat antihuman antibody) was immobilized on silicon surface. Then, the human IgG protein was incubated on above antihuman IgG modified silicon surface. After immobilizing, the surface was flushed with a standard buffer so that nonspecifically bound material was removed. Subsequently, ZnO/Au probe continued to be incubated on modified protein silicon surface. Finally, we compare with the Raman scattering intensity and the morphology of ZnO/Au with different ratio of ZnO and Au. The ZnO/Au nanocomposite with ratio of ZnO and Au at 0.04 was chosen for formation of Raman probe. The RRS signals of ZnO could be detected from above silicon surface as shown in Figure 5a. The results show the characteristic fingerprint signal of ZnO including 1LO and 2LO phonon corresponding to the peaks at 571 and 1140 cm-1. The peak at 510 cm-1 corresponds to the Si Raman signal. To demonstrate the specificity of the experiment methods, different target proteins were chosen for control experiment on modified protein silicon surface. One array was incubated with BSA at the same concentration as human IgG protein, while the second array was probed with mouse antigoat IgG protein. Their surface signals were respectively detected as shown in Figure 5b,c, and no characteristic fingerprint signal of ZnO was observed. The results indicated that the provided detection method has good selectivity for specific protein.

Figure 6. RRS spectra of the human IgG protein at different concentrations on silicon surface: (a) 100 nM, (b) 1 nM, (c) 100 pM, (d) 50 pM, and (e) 5 pM. The 1LO phonon peaks of ZnO are shown and the peak at 510 cm-1 is from Si.

The sensitivity of the protein array is generally affected by the amount of the protein carriers (ZnO/Au), the types of the antibodies (monoclonal or polyclonal), and the detectors. In this study, we focus mainly on the effect of ZnO/Au probe concentration on the sensitivity. Sandwich-type immunoassay was performed to detect human IgG. The quantitative analysis is also performed with various concentrations of human IgG. The result shows the signal intensity of ZnO was decreased with changing the concentration of human IgG from 100 nM to 5 pM as shown in Figure 6 from (a) to (e). The peak at around 510 cm-1 is from the characteristic Si Raman signal. The 1LO phonons of ZnO were observed in this experiment. Figure 6 shows the change of Raman signal intensity of 1 LO phonons of ZnO with various concentrations of human IgG. It is seen that the RRS signal intensity decreased with the decrease of the concentration of protein. The result shows that the detection limit of the analyte, human IgG, is about 5 pM. We have succeeded in obtaining a label-free sensing platform for unambiguous detection of unknown protein using Raman signal of semiconductor nanoparticles. Relatively little work has been done thus far to develop Raman probes for biomolecular analysis. Unlike fluorescence emission from molecular fluorophores, Raman emission is insensitive to photobleaching. So, Raman probes may be furthermore applied in tissue analysis, which is important for multiplex analysis of protein expression. 4. Conclusion ZnO/Au nanocomposites have been synthesized on the growth of Au on the ZnO nanoparticles using the sol-gel methods, which shows the characteristic fingerprint signal of ZnO. ZnO/ Au nanocomposites are highly dispersible in aqueous solution because of the surface ligands and have been stable in aqueous

1472 J. Phys. Chem. B, Vol. 113, No. 5, 2009 solution without aggregation for 5 months. The optimized ZnO/ Au nanocomposites with good morphology and high Raman scattering signal intensity were chosen by changing the molar ratio of ZnO and Au. The ZnO/Au nanoparticle probe was designed for the studies of protein recognition. We have demonstrated the highly selective detection of different proteins utilizing RRS signal of ZnO as marker without any need for amplification. The results presented here also shows high detection sensitivity to 5 pM. Acknowledgment. This work was supported by the National Natural Science Foundation of China (No. 60701009 and No. 10704015), the Cultivation Fund of the Key Scientific, Technical Innovation Project, Ministry of Education of China (No. 70401F), the Science and Technology Development Foundation of Jilin Province, and the Ph.D. Station Foundation of Ministry of Education (20070200010). References and Notes (1) Sander, C. Science 2000, 287, 1977. (2) Lee, S.; Kim, S.; Choo, J.; Shin, S. Y.; Lee, Y. H.; Choi, H. Y.; Ha, S.; Kang, K.; Oh, C. H. Anal. Chem. 2007, 79, 916. (3) Nathaniel, L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547. (4) Chan, W. C. W.; Nie, S. M. Science 1998, 25, 2016. (5) Bruchez, M.; Moronne, M.; Gin, P.; Weiss, S.; Alivisatos, A. P. Science 1998, 281, 2013. (6) Kneipp, K.; Kneipp, H.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. ReV. 1999, 99, 2957.

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