Journal of Nanomaterials

Journal of Nanomaterials / 2012 / Article
Special Issue

Advanced Nanohybrid Materials: Surface Modification and Applications

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Review Article | Open Access

Volume 2012 |Article ID 206069 | 8 pages | https://doi.org/10.1155/2012/206069

Topical Review: Design, Fabrication, and Applications of Hybrid Nanostructured Array

Academic Editor: Hui Wang
Received20 Jul 2011
Accepted31 Aug 2011
Published15 Nov 2011

Abstract

Nanohybrid materials have been widely used in the material chemistry research areas. In this paper, we mainly discussed the hybrid nanostructures used for nanobiosensor applications. It is one of the most promising and rapidly emerging research areas in nanotechnology field. Design, fabrication, and applications of hybrid nanostructures are reviewed, respectively. Finite difference time domain (FDTD) methods are applied to design different materials of hybrid nanostructures. Nanosphere lithography (NSL) is used to fabricate our designed hybrid nanostructures. Moreover, protein A and staphylococcal enterotoixn B (SEB), an enterotoxin, are detected by our designed hybrid nanostructures. From all the experiment results, we can see that our designed hybrid nanostructures are one of important nanohybrid materials. They have many potential applications in the nanobiosensor in the future.

1. Introduction

Localized surface plasmon resonance- (LSPR-) based nano-biosensors are of great interest in various applications such as environmental protection [35], biotechnology [68], and food safety [8]. It is well known that LSPR can be excited when the incident photon frequency is resonant with the collective oscillation of the conduction electrons. It is named as the LSPR effect. Transmission peaks of LSPR-related spectra are sensitive to the electric medium on surface of metal films. The LSPR-based nano-biosensor is a refractive index-based sensing device which relies on the extraordinary optical properties of noble metal (e.g., Ag, Au, and Cu, etc.) nanoparticles [9, 10]. The sensing capability of the LSPR sensor can be modified by tuning shape, size, and material composition of the nanoparticles. The nanoparticles are effective for quantitative detection of chemical and biological targets [4]. The sensing principle employed in these experiments relies on the high sensitivity of the LSPR spectrum of the noble metal nanoparticles due to an absorbate-induced change occurring in the dielectric constant of the surrounding environment. The local environment that surrounds the nanoparticles can be modified by means of binding of the biological molecular. The extinction spectrum of the nanosensor can be derived using a spectrophotometer [11].

Many biological and chemical agents such as bacteria, algae, fungi, viruses, and toxins are capable of extensively affecting humans and animals [1215]. Staphylococcus aureus enterotoxin B (SEB), a small protein toxin [12, 16], was selected as a typical small protein toxin in our experiments. SEB with 28.4 kDa protein toxin is one of a group of five major serological types of related proteins with molecular weights ranging from 26 kDa to 29.6 kDa. SEB is an incapacitating toxin, but it is rarely lethal. Detection and quantification of SEB in buffer were demonstrated using the LSPR-based nano-biosensor. Theoretically, the detection concentration of SEB can reach to nanogramme per milliliter level. The methods used to detect SEB include enzyme-linked immunosorbent assays (ELISAs) [17], light addressable potentiometric sensors (LAPSs) [18], array biosensors [19], immunomagnetic separation electro-chemiluminescence and fluorescence procedures (IMS-ECL and IMS-FCL) [20] or rapid chromatographic assays (RCA) [21], and Surface Plasmon Resonance (SPR) sensors [22]. In our previous research work, we have designed and explored some nanohybrid materials to detect the different refractive index of chemical materials and the various kinds of biomolecules [1, 2, 2327]. In this paper, we reviewed the design, fabrication, and applications of hybrid nanostructured array. The paper is divided into the following three parts: (1) finite-difference and time-domain (FDTD) algorithm was used to design the hybrid nanostructured array. (2) Nanosphere lithography was employed to fabricate our designed array. (3) The functionalized nanostructures are used for detection of proteins A and SEB.

2. Design Methods for Hybrid Nanostructures

There are many design methods to aid in deciding the parameters of the nanostructured array. For example, Mie scattering theory [28] discrete dipole approximation (DDA) [29], finite integration technique (FIT) [30], finite difference time domain (FDTD) [31] method, and the finite element method [32] are widely used for the design of the nanoparticles. FDTD is a commercial software to design arbitrary shapes, sizes and periods of nanostructures. The following parts are our design model and method. The triangular hybrid Au-Ag nanostructure array was proposed as a sensitive cell of the LSPR-based nano-biosensor. Using FDTD algorithm [33], we designed and calculated the refractive index sensitivity of the hybrid nanostructures. The corresponding model of the triangular hybrid Au-Ag nanostructure array is shown in Figure 1(a). A cross-section of a single particle labeling the materials of the substrate and particle and their thicknesses is shown in Figure 1(b).

The out-of plane heights of the Ag nanostructures under the Au layer is 50 nm and the top Au nanostructures is 5 nm only. The in-plane widths of each nanostructures are 100 nm. The period of the nanostructure array is 400 nm. In order to investigate the effectiveness of mediums with different refractive index surrounding the hybrid nanostructures, we selected the air ( 𝑛 1 = 1 . 0 ) and Protein A (Protein A: PBS (0.01 M, pH = 7.4) = 1 1 0 0 , 𝑛 2 = 1 . 3 3 5 2 ) surrounding the nanostructures. When the refractive index of the mediums surrounding this hybrid nanostructures is 1.0 and 1.3352, respectively, the FDTD algorithm-based calculation results can be obtained, as shown in Figure 2. From the results, we can calculate the sensitivity of the hybrid Au-Ag triangular nanostructure array as 𝑆 = ( 𝜆 m a x 1 𝜆 m a x 2 ) / ( 𝑛 2 𝑛 1 ) = ( 5 5 1 . 6 3 8 4 8 4 . 5 1 3 ) / ( 1 . 3 3 5 2 1 . 0 ) = 2 0 0 n m / R I U . It can realize a detection of SEB with higher sensitivity.

3. Fabrication of Nanostructures

An NSL technique [34] was employed to create the surface-confined hybrid Au-Ag triangular nanostructures supported on a glass substrate (see Figure 3). NSL process begins from the self-assembly of size-monodisperse nanospheres into a two-dimensional (2D) colloidal crystal. As the solvent of the nanosphere solution evaporates, capillary forces draw the nanospheres together, thereby crystallizing them into a hexagonally close-packed pattern on the substrate. Following self-assembly of the nanosphere mask, some silver and gold metals are deposited onto the nanosphere-coated substrate, respectively. Metal deposition parameters and vacuum condition are listed as follows: vacuum 2 × 𝑒 6  Torr, deposition rate 0.4 A/sec. After the metals deposition, the nanosphere mask is removed via sonication in ethanol resulting in surface-confined nanostructures with triangular footprints. The nanostructures have out-of plane heights of silver nanostructures ~50 nm and the upper gold nanostructures ~5 nm in thickness, and ~100 nm in plane widths of each nanostructure and ~400 nm period of the nanostructure array as measured using JSM-5900 LA scanning electron microscope (SEM). In order to approve the existence of Au cap layer, we used EDX to analyse the materials of hybrid nanostructured array. Figure 4 shows the EDX results for all the elements for the hybrid nanostructured array. EDX analysis results are based on the same materials as the hybrid Au and Ag film. From these results, we can see that there are Au in the cap layer in the hybrid materials. More details for NSL fabrication information are listed in [1, 35].

4. Applications of Hybrid Nanostructures

There are many applications for the hybrid nanomaterials. They are widely used as polymers [36, 37]. Polymers can be made into various bulk forms and thin films and widely applied to optical and electronic materials, biomaterials, catalysis, sensing, coating, and energy storage, and so forth fields. Here, we only focus on our research groups’ applications such as protein A and SEB detection based on our designed nanostructured array.

4.1. Detecting Protein A

The LSPR-based nanobiosensors are extremely sensitive to variation of refractive index (RI) within a few hundred nanometers of gold surface. Capture of the target analyte (Protein A, from Sigma-Aldrich) by the specific reaction between the metal Au and the Protein A. Protein A bound to the sensing face changes the apparent RI due to solution displacement by the analytes of higher refractive index. To test the detection capability of the hybrid Au-Ag nano-biosensors, experiments were performed using solutions of Protein A in PBS buffer (1 : 100, 0.01 M, pH 7.4, from Jinshan Chemical Analyte Pte. Ltd.), and the refractive index of Protein A (1.3352) was detected by Abbe refractometer ZWA-J (temperature 20°C, Δ 𝑛 = ± 0 . 0 0 0 2 ). All the buffer used in the experiments was prepared using double glass-distilled water.

Resonant wavelength 𝜆 m a x of the bare hybrid Au-Ag nanostructures (see Figure 5, black line) was measured to be 575.99 nm. Exposure to 1 : 100 Protein A resolution resulted in 𝜆 m a x = 5 5 6 . 3 1  nm (see Figure 5, red line), corresponding to a −19.68 nm shift. It should be noted that Δ 𝜆 m a x = 1 9 . 6 8  nm is smaller than the calculated result of the FDTD. It attributes the experiment defects caused by the NSL fabrication technique. The blue shift is caused by the scattered radiation from the incident radiation ( 𝜔 ) less than the vibrational frequencies of the molecules ( 𝜔 v i b ).

4.2. Detecting SEB

For SEB detection, Our experiments were carried out using home-made SEB prepared by our collaborator (Chinese Academy of Chemical Defence). Monoclonal mouse anti-SEB was purchased from Chemicon; 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCI (EDC) and Sulfo-Nhydroxysuccinimide (S-NHS) from Pierce; ethanolamine from Sigma-Aldrich; dithiodiglycolic acid from Sigma; and phosphate buffer solution (PBS, 0.01 M, pH7.4) from Jinshan Chemical analyte Pte. Ltd. The buffer used in the experiments was prepared using double glass-distilled water.

Functionalization of the sensor is a multistep process that prepares the surface for specific detection applications. Prior to each experiment, the triangular hybrid Au-Ag nanostructure array was cleaned and prefunctionalized according to the following protocol: (1) the hybrid nanostructures were cleaned by sonicating the sample in ethanol in 3 min.; (2) dithiodiglycolic acid (2 mM) aqueous solution was dropped to the surface of the samples and reacting in 30 min.; (3) the carboxyl groups on dithiodiglycolic acid were activated in 30 min. using the same volume of EDC (0.4 M) and S-NHS (0.1 M); (4) the samples were thoroughly rinsed with 0.01 M PBS buffer and then dried by N2 blowing with high-pressure; (5) monoclonal mouse anti-SEB IgG antibodies were coupled to the surface of the nanostructures by dropping 10 μg/mL solution in PBS buffer through chemical reaction between amidogen beside the alkaline aminophenol (Arg and Lys) of IgG and the active carboxyl; (6) redundant active ester groups were enclosed by 1 M ethanolamine aqueous solution. Finally, the samples were rinsed by 0.01 M PBS buffer and a hybrid Au-Ag SEB nano-biosensor was formed. A schematic illustration of the prefunction for the nanostructures surface and a description (with figure) of the LSPR-sensor are shown in Figures 6 and 7, respectively.

The LSPR nano-biosensors are extremely sensitive to a change of refractive index (RI) within the range of a few hundred nanometers from the Au surface. Capturing of the target analyte (SEB) by homologous antibody (monoclonal mouse anti-SEB antibody) bound to the sensing surface varies the RI significantly due to a solution displacement by the analytes with higher refractive index. To test the detection capability of the hybrid Au-Ag triangular particle-based nano-biosensors, experiments were carried out on the basis of the SEB solutions of various concentrations in PBS buffer. Three independent experiments were performed for the SEB solutions in different concentrations of 10 μg/mL, 1 μg/mL, and 100 ng/mL SEB, respectively. In this study, the LSPR spectra for the specific binding signals were measured using the integrated LSPR biosensor (see Figure 8(a)). The LSPR 𝜆 m a x after monoclonal mouse anti-SEB antibody attachment (see Figure 8(a), black line) was measured to be 611.66 nm. Exposure to 10 μg/mL SEB, corresponding peak wavelength of LSPR 𝜆 m a x = 5 2 8 . 1 9  nm (see Figure 8(a), red line), which corresponds to a Δ 𝜆 m a x = 8 3 . 4 7  nm peak shift. Hereinafter, “ + ” denotes a red-shift and “ ” a blue-shift. When the concentration of the SEB solution is changed to be 1 μg/mL and 100 ng/mL, the corresponding peak shifting of LSPR is Δ 𝜆 m a x = 6 8 . 3 3  nm (see Figure 8(b)), and Δ 𝜆 m a x = 2 5 . 6 2  nm (see Figure 8(c)), respectively. The main absorbance peak for the anti-SEB drifting from sample to sample (Figures 8(a)8(c)) attributes to the changing of effective refractive index of the surrounding medium due to binding of SEB and anti-SEB. It should be noted that Δ 𝜆 m a x = 2 5 . 6 2  nm is greatly larger than the resolving power of the integrated LSPR sensor (spectrum resolving power of the spectrometer is 1.7 nm). Therefore, it is reasonable to believe that even the SEB solution in lower concentration can be detected by the integrated LSPR biosensor. These will be performed in our next research project. All the extinction measurements were collected at atmosphere environment. To further explore detection performance of the SEB sensors, the concentration of SEB as low as 1 ng/mL was applied on the biochips. Figure 9 is the measured spectra of the LSPR-based hybrid Au-Ag nano-biosensor for the specific binding detection with detection concentration of 1 ng/mL SEB. It can be seen that the peak wavelength blue shifts are Δ 𝜆 m a x = 5 3 9 . 8 3  nm 5 7 2 . 2 3  nm = 3 2 . 4  nm. Actually, the differences of the resonance peak position are caused by different refractive index materials around the Ag nanostructures. The refractive index of materials changed can influence the resonance peak position. When the concentration of the SEB changed, the peak shifts will change. The red and blue shifts are caused by the scattered radiation from the incident radiation ( 𝜔 ) by an amount that corresponds to vibrational frequencies of the molecules ( 𝜔 v i b ). The frequency emitted by the photo 𝜔 𝑅 = 𝜔 ± 𝜔 v i b . If 𝜔 > 𝜔 v i b , it causes red shift; if 𝜔 < 𝜔 v i b (smaller SEB concentration), it leads to blue shift.

Our Ag-Au composite structures can be verified by our binding detection results, as shown in Figure 8. If the structure does not involve Au, dithiodiglycolic acid (see Figure 6) cannot bind with the nanostructures because it binds with Au only [38]. Dithiodiglycolic acid derivatives have a thiol group that reacts with Au atoms. Therefore, the immobilization process of dithiodiglycolic acid derivatives on the Au surface occurred spontaneously. The pure Ag particles bind with –OH– only [11]. The binding detection spectra cannot be obtained without the hybrid structure with Au capped on the Ag particles. Compared to conventional SPR systems, at the same detection level, our presented LSPR-based biosensor is far simpler than the traditional SPR systems (e.g., Biacore system, etc.). The LSPR system is cost-effective, small volume, light weight, and portable because some subsystems such as temperature control, pressure control, and precise incident angle control are not necessary. The important advantages of our approach include that: (1) it can provide good selectivity and sensitivity without the labeling process. Our LSPR biosensor assay can detect SEB at approximately 1 ng/mL rapidly within 1 min; (2) spatial resolution of our detect approach is a single nanostructure while SPR sensors require at least a 1 0 × 1 0 μm area for sensing experiments; (3) our LSPR nanobiosensor does not have temperature control, which can reduce the weight and the volume of the detected spectrometric system; (4) The detected spectrometric system costs only 1/30 of the commercialized SPR instruments.

5. Summary

We reviewed the design, fabricate, and applications of our proposed hybrid Au-Ag triangular nanostructures. The refractive index sensitivity of hybrid Au-Ag triangular nanostructures is calculated by FDTD method. And we detected the sensitivity in experiment using protein A. This hybrid Au-Ag nanostructures are used to detect SEB solution. The detection sensitivity for SEB is nanogramme per milliliter level. All the design and experiment results show that the hybrid nanostructures are useful in the nanobiosensor research field. It has many potential applications in chemical material research fields.

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Copyright © 2012 Shaoli Zhu and Wei Zhou. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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