Buildup of gold nanoparticle multilayer thin films based on the covalent-bonding interaction between boronic acids and polyols

Journal of Colloid and Interface Science 295 (2006) 583–588 www.elsevier.com/locate/jcis

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Buildup of gold nanoparticle multilayer thin films based on the covalent-bonding interaction between boronic acids and polyols Ying Ma, Lei Qian, Haizhen Huang, Xiurong Yang ∗ State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China Received 2 April 2005; accepted 11 May 2005 Available online 18 January 2006

Abstract A novel method for the fabrication of gold nanoparticle multilayer films based on the covalent-bonding interaction between boronic acid and polyols, poly(vinyl alcohol) (PVA), was developed. The multilayer buildup was monitored by UV–vis absorbance spectroscopy, which showed a linear increase of the film absorbance with the number of adsorbed Au layers and indicated the stepwise and uniform assembling process. The atomic force microscopy (AFM) image showed that a compact gold multilayer thin film was successfully assembled. The residual boronic acid group on the surface of thin film could incorporate glycosylated-protein horseradish peroxidase (HRP), and good catalytic activity for H2 O2 could be observed.  2005 Published by Elsevier Inc. Keywords: Boronic acid; Poly(vinyl alcohol); Nanoparticles; Self-assembly; Biosensors

1. Introduction Nanostructure science and technology now form a common thread that runs through all physical and materials sciences and is emerging in industrial applications. Metallic nanoparticles exhibit unique optical, electrical, catalytic, or magnetic properties stemming from their high surface-tovolume ratio and their ability to couple with surface plasmons of neighboring metal particles or with electromagnetic waves. At a scale where a particle has dimensions comparable to those of a supramolecule, chemical interaction play a dominant role in directing the assembly of inorganic nanosized building blocks and the layer-by-layer self-assembly technique is one of the most appropriate layering techniques to form multilayer assemblies of such objects [1,2]. Controlled assembly of nanoparticles based on supramolecular chemistry, i.e., noncovalent bonding [3], is a general strategy leading to well-organized gold nanoparticle materials. Thus, approaches have been reported using covalent-bonding [4], * Corresponding author. Fax: +86 4315689711.

E-mail address: [email protected] (X. Yang). 0021-9797/$ – see front matter  2005 Published by Elsevier Inc. doi:10.1016/j.jcis.2005.05.031

π–π [5], host–guest [6], van der Waals [7], electrostatic [8], charge-transfer [9], and antigen–antibody [10], and hydrophilicity–hydrophobicity interaction and so on. These results provide powerful methods for employing preprogrammed materials with the potential for multidimensional ordering for the creation of well-defined structures at a molecular level. In recent years, the interaction between boronic acids and diols has been proposed as an alternative to covalent bonding to keep synthetic molecular receptors bound to their guest moleculars. Boronic acids can rapidly form cyclic esters with diols in both nonaqueous and aqueous media at room temperature [11]. They can form reversible bonds with 1,2or 1,3-diols to generate five- or six-membered cyclic complexes [12]. These diol complexes include carbohydrates such as glucose, fructose, mannitol, catechol derivatives such as dopamine, and some polymers such as poly(vinyl alcohol) and so on. A number of sensing strategies using this covalent interaction, including direct pH measurements [13], fluorescence [14], UV–vis [15], near-IR [16], and surface plasmon resonance spectroscopy [17], potentiometry [18], and quartz crystal microbalance measurements [17], have

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been developed. This approach could also been used to immobilize glycosylated enzymes such as glucose oxidase, horseradish peroxidase, and lactate dehydrogenase [19–21], and the enzyme immobilized in this way maintains its biological activity. In this paper, we describe a new method to assemble gold nanoparticles into three-dimensional structures based on the covalent-bonding interaction between boronic acid and PVA. The Au nanoparticles modified by dithiodipropionic acid (DTPA)–3-aminophenylboric acid (APBA) conjugate (DTPA–APBA) could effectively incorporate PVA and the residual hydroxyl group of PVA could in turn incorporate the DTPA–APBA-modified Au nanoparticles. Gold nanoparticle multilayer films could be constructed by repeating this procedure. The film constructed in this way could incorporate glycosylated-enzyme HRP since the residual boronic acid group could form covalent bonds with the mannose among the sugar residues in HRP molecules. The gold nanoparticle multilayer film could also assist the electron transfer between the redox protein and the bulk electrode surface, and good catalytic reduction activity to H2 O2 was observed.

2. Experimental

2.3. Synthesis of DTPA–APBA modified gold nanoparticles The sodium citrate-protected gold nanoparticles were synthesized according to the Frens method [23]. Simply, 100 ml sample of aqueous HAuCl4 (2.5 × 10−4 M) was brought to a boil under stirring, and 1.2 ml of 1% aqueous sodium citrate solution was added. The mixed solution was refluxed under stirring for 30 min and cooled to room temperature. The Au nanoparticles were all spherical in shape and had an average nanoparticle diameter of about 16 nm as measured by transmission electron microscopy (TEM). DTPA–ABA modified gold nanoparticles were synthesized as in [22] using the ligand-exchange reaction. Simply, 1 mM DTPA–APBA (dissolved in 0.2 M NaOH) 200 µl was added to 40 ml gold colloid solution and the mixed solution was stirred for 12 h at room temperature. The surface coverage of the gold particles with the boronic acid components was estimated to be 204 boronic acids for a 16-nm gold particle. 2.4. The formation of gold nanoparticle/PVA (Au NP/PVA) multilayer film

2.1. Reagents HAuCl4 ·3H2 O, 3-aminophenylboronic acid, dithiodipropionic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide–HCl (EDC), PVA, (3-mercaptopropyl) trimethoxysilane (3-MPTMS), and HRP were purchased from Sigma. DTPA–APBA was synthesized according to the reported method [22]. All the other chemicals were reagent grade and used without further purification. Water used for preparation of aqueous solution was purified using Millipore Milli-Q water purification system. 2.2. Apparatus UV–vis absorption spectra were collected by a Cary 50 UV–vis–NIR spectrophotometer (Varian, USA). The AFM measurements were performed with a Digital Nanoscope IIIa multimode system (DI, Santa Barbara, CA). The images were acquired in a tapping mode. All electrochemical measurements were carried out with an Autolab PG30 electrochemical analyzer system (Eco Chemie B.B.Netherlands). All the potentials were recorded and reported versus Ag/AgCl/saturated KCl reference electrode. The working electrode used was an indium–tin oxide (ITO) slide (the surface area was about 28.3 mm2 ) and the counterelectrode was a large platinum foil. All the electrolytes were purged with high-purity nitrogen prior to and blanked with nitrogen during the electrochemical experiments.

The self-assembly procedure of a gold nanoparticle multilayer film is depicted in Scheme 1. The DTPA–APBA modified nanoparticles resulted in a boronic acid tailored surface, allowing alternating deposition with PVA based on the covalent-bonding interaction between the boronic acid group on the surface of Au nanoparticles and the diol group of PVA. The ITO substrate was ultrasonicated for 20 min in each of the following solvents: soapy water, water, acetone, and methanol. After thorough cleaning, the ITO substrate was immersed in 2% (V:V) 3-MPTMS ethanol solution for 1 day, resulting in an HS-tailored surface. After exhaustive rinsing with ethanol and water, the 3-MPTMS-modified ITO was first immersed in the DTPA–APBA modified nanoparticle solution for 60 min, adding one layer of Au nanoparticles. After rinsing with water, the substrate was transferred to 2 mg ml−1 PVA water solution for 60 min. The gold nanoparticle multilayer film could be constructed by repeating the last two steps. 2.5. The adsorption of HRP on the ITO electrode The ITO electrode modified with 9 layers of Au NP/PVA film was immersed in 5 mg ml−1 HRP solution for 2 h and rinsed with buffer solution to remove the weakly adsorbed enzyme. The resulting electrode was stored at 4 ◦ C when not in use.

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Scheme 1. The procedure of Au NP/PVA multilayer formation.

3. Results and discussion 3.1. UV–vis absorption spectra and AFM image UV–vis absorbance spectroscopy was used to monitor the fabrication of multilayer film. Fig. 1 shows the UV– vis absorbance spectra of the Au NP/PVA multilayer film with different numbers of bilayers on an ITO slide. When the Au nanoparticles are adsorbed on the ITO surface, absorption in the visible region emerges. The absorbance of nanoparticles increases with the increased number of bilayers, illustrating successful assembly of DTPA–APBA Au nanoparticles into the film. But this absorption band could not be clearly observed until three absorption cycles were completed. The possible reason is that the nanoparticle film assembled was too thin to be detected for the first two layers. The maximum of the surface plasmon band shifts to 535 nm after assembly on the ITO slide. The red shift of the surface plasmon band is due to the reduced nanoparticle– nanoparticle distance in the film compared with nanoparticles dispersed in water solution [24]. Furthermore, the peaks become broader with the increased number of bilayers, which could be ascribed to the aggregation of nanoparticles. This aggregation was due to the softness of PVA, which could make the nanoparticles form dense structures. A linear increase of the absorbance with the number of bilayers from three to nine bilayers (shown in the inset of Fig. 1) was observed, indicating that the stepwise and uniform Au NP/PVA multilayer film was successfully assembled on the ITO slide. The morphology of the Au NP/PVA hybrid film was investigated by tapping mode AFM. Fig. 2 depicts AFM images of a multilayer film. A dense, rough gold multilayer

film can be observed in this image. The surface coverage of the Au nanoparticle multilayer film was estimated to be 5.5 × 1012 particles cm−2 . 3.2. Amperometric biosensing of hydrogen peroxide The presence of mannose among the sugar residues in HRP molecules provides good affinity toward boronic acid adsorbed on the gold nanoparticles surface. The enzyme could be covalently immobilized on the electrode surface based on this covalent interaction. The enzyme immobilized in this way also shows good biological activity [21]. The electrocatalytic behavior of the enzyme electrode toward the electrochemical reduction of H2 O2 was studied using cyclic voltammetry. Fig. 3 shows cyclic voltammetric plots of different ITO electrodes in pH 7.4 buffer solutions. Only a weak reductive current could be observed for ITO electrodes modified with an Au NP/PVA multilayer film (ITO/Au NP/PVA) in the presence of 2.5 mM H2 O2 , while an obvious catalytic current was observed after adsorption of HRP (ITO/Au NP/PVA/HRP) in the presence of equal molar H2 O2 , which illustrates that the enzyme immobilized in this way shows good catalytic activity. A good linear relationship for H2 O2 between 28 µM and 7.3 mM could be observed (shown in the inset of Fig. 3); the correlation coefficient is 0.996 and the limit of detection reaches 7 µM. The Au NP/PVA multilayer film plays dual roles in this biosensor: immobilizing the enzyme on the electrode and allowing efficient electron tunneling and assisting the electron transfer between the HRP and the electrode surface [25]. The repeatability of the current response of enzyme electrode to 2.5 mM H2 O2 was studied and the relative standard deviation (RSD) was 2% for 10 successive assays. The stability

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Fig. 1. The UV–vis spectra of multilayer films. From the lower to upper curves, the numbers of Au NP/PVA bilayers are 3, 4, 5, 6, 7, 8, and 9. Inset: linear relationship between the absorption intensity and number of bilayers.

Fig. 2. AFM image of nine bilayers of Au NP/PVA multilayer film on ITO slide.

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Fig. 3. The cyclic voltammograms of different ITO electrodes in 50 mM pH 7.4 PB solutions: (a) ITO/Au NP/PVA in the absence and (b) in the presence of 2.5 mM H2 O2 ; (c) ITO/Au NP/PVA/HRP in the presence of 2.5 mM H2 O2 . Inset: the calibration curve of the biosensor; the applied potential is −0.25 V. Scan rate: 50 mV s−1 .

of the enzyme electrode has also been investigated and the enzyme electrode retained 96% of its initial response to the reduction of 2.5 mM H2 O2 after 1 week.

Basic Research Development Project “Research on Human Major Disease Proteomics” (No. 2001CB5102).

References 4. Conclusions A gold nanoparticle multilayer film based covalent-bonding interaction between boronic acid and PVA was developed. UV–vis absorbance spectroscopy and AFM was used to characterize the multilayer film. HRP could be immobilized on this film and good catalytic activity to H2 O2 was also observed. The multilayer gold thin film constructed in this way could also be used as a surface-enhanced Raman spectroscopy (SERS) or surface plasmon resonance (SPR) substrate. This film can also recognize other glycosylated enzymes, antigens, antibodies, and so on.

Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 20075027) and the National Key

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