Chitosan-Luminol Reduced Gold Nanoflowers: From One-Pot

Jun 27, 2008 - By reducing HAuCl4 with chemiluminescent (CL) reagent luminol in the presence of hydrophilic polymer chitosan, three-dimensional flower...
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J. Phys. Chem. C 2008, 112, 10759–10766

10759

Chitosan-Luminol Reduced Gold Nanoflowers: From One-Pot Synthesis to Morphology-Dependent SPR and Chemiluminescence Sensing Wei Wang and Hua Cui* Department of Chemistry, UniVersity of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China ReceiVed: March 7, 2008; ReVised Manuscript ReceiVed: May 7, 2008

By reducing HAuCl4 with chemiluminescent (CL) reagent luminol in the presence of hydrophilic polymer chitosan, three-dimensional flowerlike gold nanostructures (AuNFs) were synthesized via a convenient onepot method. As-prepared stable and monodisperse AuNFs were consisted of smaller-sized nanodots according to subsequent characterizations by high-resolution transmission electron microscopy, scanning electron microscopy, and powder X-ray diffraction. The size and morphology of AuNFs could be tailored by varying the amount of luminol or chitosan, which further influenced their surface plasmon resonance (SPR) properties in both visible and near-infrared regions. On the basis of the characterizations, a chitosan-assisted secondgrowth mechanism was proposed to explain their formations and morphology evolutions with the amount of reactants. Moreover, an electromagnetic simulation method, discrete dipole approximation, was introduced to calculate the morphology-dependent extinction spectra of geometrically irregular AuNFs. The simulations were consistent with the experimental results. Finally, because luminol was attached on the surface of AuNFs, as-prepared AuNFs could react with H2O2 to generate chemiluminescence. The functionalized AuNFs were immobilized on the solid supports by virtue of the film-forming property of chitosan solution to fabricate a reagent-free CL sensor for the determination of H2O2. Due to their shape-dependent SPR properties and specific surface structures, these AuNFs might also have great potential for the applications in biomedicine and surface enhanced Raman scattering. 1. Introduction Gold nanomaterials are now of intense interests in both fundamental researches and practical applications in material and life science due to their promising optical properties, catalysis, and biocompatibility.1–4 Because the optical properties and surface reactivity of gold nanomaterials were greatly influenced by their size and morphology, the synthesis of gold nanomaterials of controllable shape was a general routine to prepare gold nanomaterials with valuable properties.5–7 In recent years, synthesis and shape-dependent properties of anisotropic gold nanostructures, such as nanorod, nanocube, nanocage, nanopolygon, and nanopolyhedron, were intensely studied all over the world.8–16 Generally, surfactants were used in the synthesis of anisotropic gold nanomaterials to induce the preferential growth along some specific directions. However, the introduction of surfactants also complicated the separation and purification for their further applications. Therefore, the effort was also made to use some other structure-directing agents such as hydrophilic polymers,9,10,17–19 metal chelators20 and inorganic precipitators11 in the synthesis of anisotropic gold nanomaterials. Besides the shape-control, the modification of surface ligands was another effective way to design functionalized gold nanomaterials.21 To improve the performance of surface modification, one-pot synthesis of AuNPs, which directly took functional molecules as stabilizing reagents and/or reductants,22–27 was proposed instead of complex postmodifications. Up to date, many compounds such as biomolecules22–25 (amino acids, peptides or proteins), optical probes26,27 and polymers2 have been utilized in the one-pot synthesis of gold * Corresponding author. Tel: +86-551-3606645. Fax: +86-551-3600730. E-mail: [email protected].

nanomaterials. Convenient attachments of functional molecules thus offered as-prepared nanomaterials corresponding properties for practical applications. Considering the two aspects mentioned above, it was worthwhile to make efforts on the synthesis via an intelligent method, in which the introduction of ligands could not only functionalize nanomaterials but also influence their morphology. In our previous studies, luminol, one of the most commonly used chemiluminescent (CL) reagents, successfully reduced HAuCl4 to produce spheric gold nanoparticles (AuNPs) with CL activity.26,27 They were subsequently immobilized on the surface of gold electrode by virtue of a bridge molecule cysteine to fabricate an electrochemiluminescence sensor for the determination of H2O2. However, the long-term stability of asfabricated sensor was limited due to the relatively poor performance of electrostatic interaction-based immobilization.26 It was realized that the film-forming polymer might be an attractive candidate to improve the performance of AuNPs immobilization. Chitosan is one kind of deacetylated derivatives of chitin, and it has been widely used in drug delivery, food industry, and biosensors due to its polycationic, biocompatible, and film-forming properties.28 Chitosan is selected in the present work because of three reasons. First, chitosan solution is easy to form a stable and biocompatible film on the solid supports such as glassy carbon and glass, which has been utilized in the development of biosensors.29–31 Second, hydrophilic chitosan is also compatible with gold nanoparticles, and chitosan was reported to produce AuNPs by acting as stabilizer and/or reductant by virtue of its abundant amino and hydroxyl groups.31–37 Finally, though the influence of some polymers such as PVP,10,17,18 PVA,19 and PEG9 on the morphology of nano-

10.1021/jp802028r CCC: $40.75  2008 American Chemical Society Published on Web 06/27/2008

10760 J. Phys. Chem. C, Vol. 112, No. 29, 2008 materials has been investigated, there are few reports on the similar structure-directing effect of chitosan. Because chitosan is a hydrophilic polymer with flexible molecular conformations, it might also act as a structure-directing agent to affect the morphology of AuNPs via the interaction between the active groups of chitosan and gold. Zhu reported the synthesis of onedimensional (1D) chainlike gold nanoaggregates in the presence of low molecular weight chitosan in a very recent work.38 Consequently, it is a valuable subject to investigate the onepot synthesis of AuNPs in the simultaneous presence of functional molecules luminol and chitosan, and their applications in luminol immobilization. In the present work, luminol was used to reduce HAuCl4 in the presence of chitosan. It was found that blue colloids containing well-dispersed gold nanoflowers (AuNFs) were obtained, which exhibited strong morphology-dependent absorption in both visible and near-infrared (NIR) regions. The effects of chitosan and luminol concentrations on the morphology of AuNFs and the surface plasmon resonance (SPR) properties were investigated by high-resolution transmission electron microscopy (HRTEM), visible-NIR absorption spectroscopy (vis-NIR), and scanning electron microscopy (SEM). The growth mechanism of this flowerlike nanostructure was proposed based on the fluorescence (FL), powder X-ray diffraction (XRD) results, and a second-growth experiment. Moreover, a discrete dipole approximation (DDA) method was further introduced to simulate the extinction spectra of AuNFs and their evolution trends with morphology and size. A partially random bottomup method was proposed to describe the shape of geometrically irregular nanoflowers, which was an indispensable request for performing DDA calculation. Finally, as a practical application, AuNFs with CL activity were immobilized on the surface of quartz glass by virtue of the film-forming property of chitosan solution. The CL sensing of this functional film to H2O2 were also investigated by using a flow-injection system. 2. Experimental Section 2.1. Chemicals and Solutions. A 0.01 mol/L stock solution of luminol was prepared by dissolving luminol (Sigma) in 0.1 mol/L NaOH solution without further purification. A HAuCl4 stock solution (0.2% HAuCl4, w/w) was prepared by dissolving 1.0 g of HAuCl4 · 4H2O (Shanghai Reagent, Shanghai, China) in 412 mL of purified water and stored at 4 °C. Chitosan (Mv ) 1000 kDa, degree of deacetylation ) 90%) was purchased from Shanghai Reagent (Shanghai, China) and used as received. Working solutions of H2O2 were prepared fresh daily from 30% (v/v) H2O2 (Xinke Electrochemical Reagent Factory, Bengbu, China). All other reagents were of analytical grade. Ultrapure water was prepared by a Millipore Milli-Q system and used throughout. 2.2. Synthesis and Characterization of Gold Nanoflowers. All glasswares used in the synthesis were cleaned in a bath of freshly prepared 3:1 (v/v) HNO3-HCl and rinsed thoroughly prior to use. Gold colloids were prepared via the reduction of HAuCl4 by luminol in the presence of chitosan. In a typical procedure, 0.20 g chitosan was dissolved in 50 mL acetic acid solution (HAc, 2% v/v). After adding 1.0 mL of luminol stock solution, the mixture was diluted to 95 mL and heated to boiling. While stirring vigorously, 5.0 mL of HAuCl4 stock solution was added rapidly. The solution was maintained at the boiling point for 30 min, during which time a color change from pale yellow to dark brown was observed before a purple or blue color was reached. The heating source was removed, and the colloid was kept at room temperature for another 20 min and then stored at

Wang and Cui 4 °C. The weight of chitosan or the volume of luminol was varied to synthesize gold nanomaterials of different shapes and sizes. The total volume of the reaction solution was controlled to be 100 mL, so that the weight of chitosan or the volume of luminol and HAuCl4 could represent their corresponding concentrations in the following context. To avoid degradation of chitosan in HAc solution during storage, chitosan powder was freshly dissolved prior to synthesis. As-synthesized colloidal solutions were characterized by visNIR absorption spectroscopy (Shimadzu UV-365 spectrophotometer, Japan) and HRTEM (JEOL-2010, Japan). For the SEM analysis of AuNFs, the colloids were centrifuged, redispersed in water to avoid the influence of chitosan film, and subsequently dropped on a piece of quartz glass and dried under an infrared lamp, which was measured by SEM (JEOL JSM-6700F, Japan). The glass was carefully processed by piranha solution and washed by water and ethanol prior to use, respectively. For XRD measurement, proper amount of colloids was centrifuged and dried under vacuum at room temperature. The obtained dry powders were characterized by using a Philips X’Pert Pro Super diffractometer with Cu KR radiation (λ ) 1.54 Å). The FL measurements were performed by a fluorometer (Hitachi F-7000, Japan). 2.3. Fabrication and Chemiluminescence of AuNF-Chitosan Film. The AuNF-chitosan film was fabricated on the surface of a piece of as-processed quartz glass by dropping gold colloids and drying under an infrared lamp. The surface morphology of AuNF-chitosan film was characterized by SEM. The drop-and-dry operation was repeated five times to thicken the film for CL application. The CL detection was conducted on a flow injection CL system (Remax, China) consisting of a model IFIS-D peristaltic pump, a model RFL-1 CL detector, and a CR-105 photomultiplier tube (PMT). The glass modified by AuNF-chitosan film was fixed in a CL cell, and its surface could be easily soaked and mixed by the injected liquids. Water was used as a carrier for the H2O2 working solution containing 0.1 mol/L carbonate buffer with pH 10.0. The H2O2 solution was first mixed with 1 × 10-6 mol/L Co2+, then passed through the CL cell and generated light emission with the immobilized luminol. The CL signals were monitored by the PMT adjacent to the flow CL cell. 3. Results and Discussion 3.1. Synthesis and Characterizations of Gold Nanoflowers. When HAuCl4 was reduced by luminol26 or chitosan32,37 solely, red-colored gold colloids containing spherical AuNPs were obtained. However, when HAuCl4 was reduced by the mixture of luminol and chitosan, blue colloids were obtained instead of red ones. Subsequently, vis-NIR absorption spectra of gold colloids synthesized by varying the amount of luminol from 0 to 2.0 mL were measured as shown in Figure 1. The inset shows the corresponding pictures of as-prepared colloids taken by a commercial digital camera. In the absence of luminol (curve a), a wine-red colloid with maximum absorption wavelength, λmax, at 522 nm was obtained, which was consistent with the previous report.37 However, a remarkable red shift of λmax from 522 to 663 nm was observed by increasing the amount of luminol, which was accompanied by a color change from winered, purple to blue and gray green. Besides, strong NIR absorption was observed if the amount of luminol was above 0.5 mL (curves d∼f), indicating the deviation of AuNPs morphology from sphere.4,5,7 This point was subsequently confirmed by HRTEM and SEM images, respectively. According to Figure 2, the spherical AuNPs with average diameter 18

Chitosan-Luminol Reduced Gold Nanoflowers

Figure 1. Vis-NIR absorption spectra of AuNFs synthesized by varying the volumes of luminol in the presence of 0.20 g chitosan. Luminol: 0 (a), 0.1 (b), 0.3 (c), 0.5 (d), 1.0 (e), and 2.0 (f) mL. Inset shows the corresponding pictures of as-prepared colloids.

( 5 nm were observed in the absence of luminol (image A). However, the diameters of nanostructures greatly increased to approximate 90 nm in the presence of luminol. Moreover, an obvious evolution of shape from quasi-spheres (image B) to multipods (image C) to flowerlike nanostructures (images D∼F) was observed by increasing the amount of luminol. To have a clear view about the 3D shape of as-prepared nanomaterials, AuNFs in Figure 2E were further characterized by SEM as shown in Figure 3. Plenty of nanoflowers of approximate 90 nm diameter were separately distributed on the surface without aggregation, which was very consistent with the HRTEM results. On the other hand, only spherical AuNPs were formed in the absence of chitosan according to our previous work.26 The effects of chitosan concentration on the SPR properties and morphology of AuNFs were thus examined as shown in Figures 4 and 5, respectively. The presence of chitosan led to the formation of AuNFs instead of spherical AuNPs. Besides, there was a great red shift from 635 to 818 nm with the decreasing amount of chitosan (Figure 4), which was accompanied by an obvious increase in size of AuNFs (Figure 5). However, the size and morphology of as-prepared AuNPs in the absence of chitosan (Figures 4a and 5A) were quite different with our previous results, in which spherical AuNPs with diameters ranging from 14 to 35 nm were observed.26 This difference was due to the 1% HAc solution in the present work, which greatly decreased the pH of reaction system. The lower pH increased the oxidation activity of AuCl4- and made the nucleation and growth process faster and less controllable, which led to the worsening in both size and morphology distribution. This point was supported by the fast color change in several seconds in the presence of 1% HAc, while it took approximate 20 min to achieve a stable red color in the absence of HAc. From HRTEM and SEM images above, one can summarize three trends on the morphology evolution of AuNFs as follows. First, all the nanoflowers were consisted of many nanodots with approximate diameters in the range of 10∼20 nm. The word “nanodots” was used to exclusively represent the smaller-sized spherical nanoparticles, which were formed in the initial reduction of HAuCl4 by luminol in the present work. Second, the size of such nanodots decreased with increasing amount of luminol, while the size of nanoflowers did not obviously change (Figure 2D∼F). Third, the diameter of AuNFs decreased from 225 to 100 nm with increasing amount of chitosan (Figure

J. Phys. Chem. C, Vol. 112, No. 29, 2008 10761 5B∼D). To confirm the nanodots in these flowerlike nanostructures, powder XRD was used to calculate the average crystal size, which was compared with the HRTEM observations. According to the Debye-Scherrer formula, the full-width at half-maximum (fwhm) indirectly indicated the crystal size in nanomaterials. Five samples were measured and all the samples exhibited similar diffraction patterns of different fwhm values as shown in Figure 6. Obtained diffraction peaks were very consistent with the gold crystal of face-centered cubic (fcc) structure. The measured fwhm of (100) peak and the calculated sizes of the crystal are listed in Table 1. It was found that the crystal sizes ranged from 12.9 to 21.2 nm. Moreover, the size actually decreased with increasing amount of either luminol or chitosan. Both the size and their evolution were well consistent with those of the nanodots in HRTEM images. Either luminol26 or chitosan32,37 could reduce HAuCl4 to form AuNPs and stabilize the AuNPs solely. Their roles were thus investigated by monitoring the fluorescence of reaction products when both of them were simultaneously used in the present work. The oxidation product of chitosan was considered to be fluorescent, because FL emission (λex/λem ) 310/390 nm) increased by five times in the supernatant of gold colloids after the synthesis by solely using chitosan. Besides, it was validated that the oxidation of luminol by HAuCl4 would generate fluorescent product, 3-aminophthalate (APA).27 When the mixture of luminol and chitosan was used to synthesize gold colloids, both the emission bands of APA and the oxidized product of chitosan were observed in the supernatant, indicating that both chitosan and luminol acted as reductants. This point was also supported by the XRD results in Table 1, in which the size of nanodots decreased with increasing amount of either luminol or chitosan. It was well known that the increasing amount of reductant could lead to a decrease in size of nanoparticles. However, from the reaction rate aspect, luminol was a stronger reductant than chitosan and the reaction of luminol with HAuCl4 was much faster than that of chitosan according to the color change during the reaction, suggesting that luminol was the major reductant compared with chitosan. 3.2. Growth Mechanism. Due to their shape-dependent optical and chemical properties, flowerlike nanostructure has been one of the most attractive anisotropic nanomaterials.9 Though the heterogeneous synthesis of flowerlike gold nanostructures on the surface of solid substrates via chemical or electrochemical methods has been reported by several groups,39–42 the homogeneous synthesis of gold nanoflowers was limited because gold usually possesses a highly symmetric fcc structure due to the intrinsic properties of gold crystals.11 Recently, Yang reported the synthesis of AuNFs of strong NIR absorption,9 in which strict control on reagent concentrations was requested. Another synthesis and electrocatalysis applications of flowerlike gold nanostructures at room temperature were achieved by Jena.43,44 However, the mechanism of the template-free growth was not clearly discussed, and the strong SPR absorption still remained at 532 nm with a shoulder in NIR region. In the present work, as-prepared AuNFs exhibited strong and morphology-dependent NIR absorption. Besides, the excellent biocompatibility and film-forming property made such colloids to be promising materials for the applications in biosensors.45 Moreover, they might be useful in surface-enhanced Raman scattering (SERS) due to abundant junctions, edges, and corners on the surface, which are generally considered as “hot spots” for SERS.40–42,46 Therefore, the studies on the growth mechanism might be helpful for the synthesis and shape control of novel

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Figure 2. HRTEM images of AuNFs. Conditions for the synthesis of AuNFs in images A-F are the same as the descriptions in the caption of Figure 1 panels a-f, respectively. Calculated diameters are 18 ( 5 (A), 72 ( 12 (B), 85 ( 15 (C), 91 ( 9 (D), 100 ( 16 (E), and 89 ( 16 (F) nm, respectively, by accounting about 50 units.

Figure 3. Typical SEM image of as-prepared AuNFs.

AuNFs, which is of significance for both fundamental interests and practical applications. The mechanism of AuNFs formation was clarified by a designed second-growth experiment, which utilized a batch addition of HAuCl4 during the synthetic procedure of quasispherical AuNPs in Figure 1c. In detail, after adding 2.5 mL of HAuCl4 instead of 5.0 mL of HAuCl4, blue colloid was obtained and remained stable as shown in Figure 7a. Half an hour later, the addition of another 1.0 mL of HAuCl4 led to a color change from blue to purple (image b). When the last 1.5 mL of HAuCl4 was added after another half an hour, a purple-red colloid was finally obtained (image c). During this process, λmax was blueshifted from 620 to 545 nm. Corresponding HRTEM images in Figure 8 also confirmed the morphology evolution from multipods (Figure 8A) to quasi-spherical (Figure 8C) AuNPs. Meanwhile, their diameters were similar (approximate 50 nm), indicating the comprehensive second-growth on the surface of multipods gold nanostructures with the addition of gold source (HAuCl4). On the basis of the results above, the growth mechanism of AuNFs is proposed as follows (Scheme 1). The reduction of

Figure 4. Vis-NIR absorption spectra of AuNFs synthesized by varying the amounts of chitosan in the presence of 1.0 mL luminol. Chitosan: 0 (a), 50 (b), 100 (c), and 200 (d) mg.

HAuCl4 by luminol first produced many nanodots. Since both luminol and chitosan were considered to act as reductants, the

Chitosan-Luminol Reduced Gold Nanoflowers

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Figure 7. Vis-NIR absorption spectra during the second-growth experiment by using 0.20 g of chitosan and 0.3 mL of luminol. The amount of HAuCl4 is 2.5 (a), 3.5 (b) and 5.0 (c) mL, respectively.

Figure 5. HRTEM images of AuNFs. Conditions for the synthesis of AuNFs in images A-D are the same as the descriptions in the caption of Figure 4a-d, respectively. Calculated diameters are 105 ( 25 (A), 225 ( 45 (B), 152 ( 24 (C), and 100 ( 16 (D) nm, respectively.

Figure 6. Typical XRD pattern of AuNFs.

TABLE 1: Relationship between the Amount of Reductants and the Size of Nanodots CTS (g)

luminol (ml)

fwhm (degree)

sizea (nm)

CTS (g)

luminol (ml)

fwhm (degree)

sizea (nm)

0.20 0.20 0.20

0.5 1.0 2.0

0.483 0.624 0.734

21.2 15.6 12.9

0.05 0.10 0.20

1.0 1.0 1.0

0.495 0.600 0.624

20.5 16.3 15.6

a The K value was 0.89 and the instrumental broadening (0.09 degree) was subtracted in the calculation.

size of the nanodots was affected by the concentration of either luminol or chitosan (Figure 2 and Table 1). Subsequently, initial AuNFs were formed by the limited aggregation of nanodots and then protected by chitosan via the interaction between these primary nanodots and the flexible chitosan molecules. Therefore, the size of AuNFs was mainly affected by chitosan concentration. Less chitosan led to weaker protection and larger size (Figure 5). Then, chitosan would further reduce the residual HAuCl4 on the surface of initial AuNFs to form stable AuNFs via a slow second-growth process. If the amount of luminol was not enough for the great consumption of HAuCl4 in the first stage, the considerable growth would cover the surface of AuNFs to form quasi-spherical AuNPs (Figure 8). This process implied that enough reductant and limited HAuCl4 concentration facilitate the formation of AuNFs. It is worthwhile to mention that the SPR absorption of asprepared AuNFs exhibited only one dominant band around 630 nm. This feature was different from many previous reports on

the SPR bands of gold nanoaggregates, in which two bands are usually observed.9,43,44 One of them was located in the range of 520∼600 nm, which represented the characteristic SPR band of individual nanoparticle. And another one was located at the longer wavelength, showing that of nanoaggregates.47 Zhong et al. studied the optical properties of nanoaggregates via a twostage process induced by a tridentate thioether ligand. The SPR band at 520 nm could be clearly resolved. The different SPR features might be caused by the different formation mechanisms of nanoaggregates in these cases. In the present work, because chitosan is also an effective reductant for gold salt, a secondgrowth occurred on the surface of nanoaggregates after they were formed, resulting in complex 3D structures instead of simple aggregates and assemblies. Therefore, the characteristic SPR band of individual nanoparticle became a shoulder peak and only one dominant band at longer wavelength was observed. This phenomenon was consistent with a recent work reported by Zhao et al.11 in which the surface growth also led to the formation of SPR shoulder at 540 nm and exhibited only one SPR band in NIR region. Such SPR properties indicated the formation of complex structures in the as-prepared AuNFs, instead of simple 3D assembly of smaller-sized nanoparticles. 3.3. Electromagnetic Simulations. It was well known that the SPR properties of gold nanostructures were highly dependent on their size and morphology. For many geometrically regular shapes, such as nanosphere, nanorod, and nanoshell, the general relationships between size and SPR properties have been well established from both experimental1,7 and theoretic aspects.4,48 Theoretic calculation of SPR was generally based on the electromagnetic interactions between AuNPs and the incident light. DDA15,49–51 was a commonly used computational procedure to calculate the SPR properties of AuNPs. By representing the object as a cubic lattice of polarizable points, the DDA method was supposed to be able to simulate the SPR of arbitrarily shaped gold nanostructures. As-prepared AuNFs exhibited a strong absorption in both visible and NIR regions, which was also greatly influenced by their size and morphology. Therefore, DDA was introduced to simulate the SPR properties of AuNFs and their evolutions with size and morphology. A portable Fortran implementation of DDA, the program DDSCAT 6.1, has been developed by Draine and Flatau52 and was utilized to perform the electromagnetic simulations. The dielectric function of gold was obtained from literature.53 The first challenge in DDA calculations was the shape description of geometrically irregular AuNFs. According to the second-growth mechanism above, a method was therefore

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Figure 8. HRTEM images of AuNFs during the second-growth experiment described in the caption of Figure 7.

SCHEME 1: Chitosan-Assisted Second-Growth Mechanism for the Formation of AuNFs

proposed to describe the AuNFs via a partially random bottomup strategy, which was elicited and modified from a top-down method for describing the etching of gold nanoshell.54 In detail, N spherical nanoparticles (representing nanodots) with a uniform diameter d (representing the size of nanodots) were generated and their centers were randomly positioned in a spherical space with a diameter D (representing the size of nanoflowers), in which the dipole distance was defined as 1. Above operation represented the generation of primary nanodots. Subsequently, the inner core of nanoflowers began to grow by adding new dipoles around its center, i.e., second-growth. This process was controlled by an expanding coefficient a, which meant that a dipole would be added if its distance to the center was smaller than aD/2. Therefore, the generation and morphology evolution could be approximately described by above parameters N, d, D, and a. Because the positions of primary nanodots were generated randomly to weaken the random disturbance, 60 independently generated shapes and their corresponding extinction spectra were calculated and averaged for each set of parameters. All the extinction spectra below were the average results of 60 spectra. It was found that the size of nanodots decreased with increasing amount of luminol whereas the size of nanoflowers did not change obviously (Figure 2). Therefore, the effect of luminol on the morphology of AuNFs could be simulated by decreasing d and correspondingly increasing N. Figure 9B illustrates three typical 3D views in 60 shapes generated under each set of parameters (see caption for details). By comparing the 3D views with HRTEM observations, one can find that the

similarity in their shapes and evolution was acceptable. By decreasing d and increasing N, a red shift of the maximal extinction wavelength from 588 to 640 nm was indeed observed from the simulations, which was very consistent with the experimental results (Figure 1). Furthermore, the effect of chitosan could be simulated by varying the geometrical diameter of AuNFs from 100 to 200 nm. If the specific morphology (Figure 9B2) was selected, simulations exhibited a great red shift from 601 to 837 nm with increasing the diameter of AuNFs. These simulations were also consistent with the experimental results (Figure 4). 3.4. Chemiluminescence Sensing. According to our previous report,26 luminol molecules could attach to the surface of AuNPs via Au-N interactions. The chemiluminescence property of asprepared AuNFs was subsequently investigated. After two times of centrifugation and washing, both the supernatant and the separated AuNFs exhibited CL with regular oxidants such as H2O2 and K3Fe(CN)6. Since the as-prepared AuNFs were dispersed in chitosan solution, a film could be formed by volatilization of solvents and HAc under an infrared lamp. This film-forming property thus offered us an opportunity to immobilize luminol and develop a reagent-free CL sensor. Figure 10A shows SEM image of chitosan film containing AuNFs. The colloid containing AuNFs in Figure 1E was selected in the CL experiments. Ninety nanometers of AuNFs were evenly distributed on the relatively smooth surface without aggregation, indicating the good compatibility of AuNFs with the chitosan film. An obvious difference in contrast of these AuNFs might imply the different depths of AuNFs embedded in the film. Because the thick polymer membrane on the glass could not suffer from the intense electron attacks during SEM measurements, only lower resolution images could be obtained in the presence of chitosan with high concentration. When the concentration of chitosan decreased, the amplified image (Figure 10B) demonstrated clearly the view of AuNFs partially embedded in the polymer film with different depths. It is well known that the oxidation of luminol by various oxidants such as H2O2 leads to the formation of the excited state 3-aminophthalate ion, which emits light when returning to the ground state. The CL mechanism and analytical application of luminol CL has been well documented in a recent review.55 Considering that luminol was bound to the surface of AuNFs embedded in the chitosan film, its response to H2O2 was subsequently studied. It was found that H2O2 could react with immobilized luminol to generate CL in the presence of Co2+. Co2+ is a commonly used catalyst of luminol-H2O2 CL reaction.56 In the present work, the CL intensity was enhanced by five times in the presence of 1 × 10-6 mol/L Co2+. To exclude the influence of free luminol molecules in the film, 20

Chitosan-Luminol Reduced Gold Nanoflowers

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Figure 9. (A) Calculated extinction spectra under various set of morphology parameters (N, d, D, a) and AuNF radius (R). R ) 50 nm for (a-c) with [N d D a] ) [60 8 20 1.1] (a), [100 4 20 1.1] (b), and [200 3 20 1.1] (c), respectively. For scans b, d and e, [N d D a] ) [100 4 20 1.1] and R ) 50 (b), 75 (d), and 100 (e) nm, respectively. (B) 3D views of three generated shapes under various morphology parameters. The parameters for B1∼B3 are the same as those for scan a∼c, respectively.

Figure 11. CL profiles by continuously injecting H2O2 solution with various concentrations. The inset shows the CL peak intensities during 140 times of continuous injections of 1 × 10-3 mol/L H2O2 solution.

Figure 10. SEM images of AuNF-chitosan film with original chitosan concentration (A) and with chitosan diluted by 10 times (B).

min washing by purified water was performed until stable light emission was obtained by injecting 1 × 10-4 mol/L H2O2. As a contrast experiment, no light emission could be observed after 20 min washing when 0.2% chitosan solution containing 1 × 10-5 mol/L luminol was used, indicating the effective immobilization of luminol by AuNFs. After the equilibrium was achieved, working solutions of H2O2 with increasing concentration were injected into the flow cell and the CL signals were recorded as shown in Figure 11. The CL intensity increased linearly with the H2O2 concentration in the range of 3 × 10-5 ∼ 3 × 10-3 mol/L, and regression equation was I ) 0.77 +

39 600C (r ) 0.99, n ) 5). The detection limit for H2O2 (S/N ) 3) was 1 × 10-5 mol/L. The relative standard deviation with the H2O2 concentration of 1 × 10-3 mol/L was 2.6% during 11 times consecutive injections. Though luminol was consumed in the CL reactions, 80% CL intensity still remained after 140 times of injections as shown in the inset of Figure 11, indicating the consumed amount of luminol during one injection time was actually limited if the concentration of luminol and H2O2 were quite low. The proposed method was convenient, fast, and easy to control, which thus offered the potential of this luminolimmobilized film for developing disposable sensors. 4. Conclusion In the present work, flowerlike gold nanostructures were synthesized by reducing HAuCl4 with the mixture of hydrophilic polymer chitosan and CL reagent luminol via a convenient onepot method. On the basis of vis-NIR, HRTEM, FL, and XRD results, the formation mechanism of as-prepared AuNFs was proposed. Chitosan-assisted aggregation of primary nanodots and the subsequent growth were responsible for the formation of stable and monodisperse AuNFs in aqueous solution.

10766 J. Phys. Chem. C, Vol. 112, No. 29, 2008 Moreover, the relationship between morphology and size of AuNPs and their SPR properties were investigated from both experimental and electromagnetic simulation aspects, and the calculations well matched the experimental results. Finally, the AuNFs with chemiluminescence activity were immobilized on a piece of glass by virtue of the film-forming property of chitosan solution to fabricate a CL sensor for the determination of H2O2. The sensor was applicable for the detection of H2O2 in the range of 3 × 10-5 mol/L ∼ 3 × 10-3 mol/L with the detection limit of 1 × 10-5 mol/L (S/N ) 3). The present work offered new insights into the growth mechanism and shapedependent optical properties of AuNFs, which might be helpful for the controllable synthesis of such flowerlike nanostructures. The obtained flowerlike nanostructures may be of great application potentials in sensors, SERS, and biomedicine fields. Furthermore, the introduction of chitosan was the key point for the formation of AuNFs. The roles of chitosan were summarized as follows. First, it acted as a structure-directing agent by virtue of its active groups and flexible molecular conformations. It also directly affected the size of AuNFs and corresponding SPR properties. Second, it was also a reductant and was responsible for the second-growth on the surface of AuNFs. Third, the chitosan in HAc solution could easily form a film on the surface of solid supports to immobilize the functionalized AuNFs. Meanwhile, the role of luminol in the formation of AuNFs was relatively simple that it acted as proper reductant and was responsible for the fast formation of nanodots during the initial stage. However, luminol was not a conventional reductant in the synthesis of nanostructures, which made the present synthesis lack of generality. Efforts are currently made to explore other reductants to develop a general method for the synthesis of AuNFs. Further work is under investigations. Acknowledgment. The support of this research by the National Natural Science Foundation of Poeple’s Republic of China (Grant 20625517 and 20573101) and the Overseas Outstanding Young Scientist Program of Chinese Academy of Sciences are gratefully acknowledged. W.W. thanks Mr. Bo Shang for kindly offering hardware platform for DDA simulations. References and Notes (1) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293. (2) Shan, J.; Tenhu, H. Chem. Commun. 2007, 44, 4580. (3) Guo, S.; Wang, E. Anal. Chim. Acta 2007, 598, 181. (4) Hu, M.; Chen, J.; Li, Z. Y.; Au, L.; Hartland, G. V.; Li, X.; Marquez, M.; Xia, Y. Chem. Soc. ReV. 2006, 35, 1084. (5) Ghosh, S. K.; Pal, T. Chem. ReV. 2007, 107, 4797. (6) Pileni, M. P. J. Phys. Chem. C 2007, 111, 9019. (7) Eustis, S.; El-Sayed, M. A. Chem. Soc. ReV. 2006, 35, 209. (8) Skrabalak, S. E.; Chen, J.; Au, L.; Lu, X.; Li, X.; Xia, Y. AdV. Mater. 2007, 19, 3177. (9) Yang, Z.; Lin, Z. H.; Tang, C. Y.; Chang, H. T. Nanotechnology 2007, 18, 255606. (10) Zhang, X.; Tsuji, M.; Lim, S.; Miyamae, N.; Nishio, M.; Hikino, S.; Umezu, M. Langmuir 2007, 23, 6372. (11) Yuan, H.; Ma, W.; Chen, C.; Zhao, J.; Liu, J.; Zhu, H.; Gao, X. Chem. Mater. 2007, 19, 1592. (12) Schwartzberg, A. M.; Olson, T. Y.; Talley, C. E.; Zhang, J. Z. J. Phys. Chem. B 2006, 110, 19935. (13) Sanchez-Iglesias, A.; Pastoriza-Santos, I.; Perez-Juste, J.; RodriguezGonzalez, B.; Abajo, F. J. G.d.; Liz-Marzan, L. M. AdV. Mater. 2006, 18, 2529. (14) Nehl, C. L.; Liao, H.; Hafner, J. H. Nano Lett. 2006, 6, 683.

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