Bonding of diatom frustules and Si substrates assisted by hydrofluoric acid

Abstract

Diatoms, with hierarchical micro/nanoscale porous silica structures, have promising application in micro-nanotechnology especially biochemical sensing. In order to explore their potential and prepare diatom based substrates for biochemical sensor application, a fabrication technology for bonding diatom frustules and Si substrates was developed. The bonding process was carried out at 75 °C and assisted by hydrofluoric acid (HF). The bonding mechanism was discussed and several bonding conditions were adjusted to keep the morphological integrity of diatom frustules after bonding. The bonding pressure was optimized from 2.0 × 10⁴ Pa to 3.0 × 10⁴ Pa and the HF concentration from 0.4% to 0.6%. And the optimal shear bonding strength achieved was 0.72 MPa. In addition, bonded diatom frustules were further used as masks to obtain nano gold pillar arrays for surface-enhanced Raman scattering (SERS) detection.

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Introduction

Diatoms are microscale unicellular photosynthetic microorganisms that are widely distributed in freshwater or oceans on Earth. Frustules, the rigid cell walls of diatoms, are transparent structures composed of SiO₂. They have regular arrays of hierarchical nanoscale/microscale through-pores, ranging from 40 nm to 2 μm.¹ As the biogenic silica nanostructures derived from diatoms have great potential in micro-nanotechnology application, more and more related research has been carried out over the last two decades,^1–4 such as modifying diatom frustule surfaces for photoluminescence^5–8 and electrochemical detection⁹ in the bio-chemical sensor field, using functionalized diatom frustules as gas sensors,^10,11 acquiring micro-nanoscale structures based on diatoms replication,^12,13 drug delivery with diatom frustules,^14,15 using frustules for solar cells and batteries.¹⁶

Most studies mainly focused on the application of a single diatom or numbers of disordered diatoms. Only a few researchers have bonded diatom frustules on substrates and applied the diatom based substrate in micro-nanotechnology especially biochemical sensing.^17–19 Bonding diatom frustules with Si substrates is of great importance. As semiconductor material, Si will greatly expand diatom applications in the biochemical detection field. For example, with Si substrates directly connected to diatom frustules, it is much easier to transmit any optical and electrical signal changes on diatom surfaces.⁹ In addition, we can use nanopore structured diatom frustules as masks to obtain nano gold pillar arrays. And these nano gold pillar arrays, which are bonded on the Si substrate, have a surface-enhanced Raman scattering (SERS) effect²⁰ and can be applied in the bio-chemical sensing field.

However, traditional bonding techniques for SiO₂ and Si flat substrates, such as direct bonding,^21,22 anodic bonding^23,24 and HF bonding,^25,26 cannot be applied for bonding micro-SiO₂ particles with Si flat substrates. And merely anyone has tried to bond diatom frustules with Si substrates. Only Losic has modified the Si substrate with polylysine and bonded diatom frustules with the substrate.²⁷ But he did not explore the bonding strength, and it might be quite weak. It has been demonstrated that microscale borosilicate glass spheres were bonded with the silicon cantilever through the fusion bonding method.²⁸ However, the 780 °C bonding temperature destroyed the structure of the glass sphere. And diatom frustules cannot resist such high temperature either.

In this paper, we present a HF assisted method for bonding diatom frustules and Si substrates with high bonding quality. This bonding technique introduced no impurities, and required low bonding temperature. The delicate nanostructures of diatom frustules were well preserved after bonding and the bonding strength was high enough for further treatment. The morphology and component changes of diatom frustules before and after bonding were analyzed. Bonding conditions like HF concentrations and applied pressures were well investigated. The bonded diatom frustules were used as masks to acquire nano gold pillar arrays, which were then used as SERS active substrates for SERS detection.

Experimental

Materials and instruments

Diatoms used in the bonding experiments were Coscinodiscus decrescens. diatom (Model: C292, Changbai Sailite Diatomaceous Earth Co., Ltd.). The diatom frustules consisted of 99% SiO₂ and 1% Na₂SiO₃. The Si substrate is 50 mm in diameter (Si Purity 99.9999%, SG 2506, Shaoguang Microelectronics Corp. China).

Scanning electronic microscopy (SEM) imaging and energy-dispersive X-ray spectroscopy (EDS) were performed using a Camscan Apollo 300 operated at 10–15 kV and fitted with a field-emission source. And the working distance was around 15 mm. Cleaned diatom frustules were coated with a thin platinum layer (around 5 nm). The model of the atomic force microscope (AFM) is Bruker Dimension FastScan. The constant of the AFM cantilever was 40 N m⁻¹.

Sample preparation

The integrated Coscinodiscus diatom frustules were separated from the original C292 samples by the settling method.²⁹ They were cleaned in H₂SO₄ (98%), purified using a centrifuge (2000 rpm, 5 min) and dried at 120 °C. The Si substrate was first washed with a detergent, and sequentially ultrasonically cleaned (Power: 500 W) in deionized water, acetone and alcohol for 10 min each. Finally, both diatom frustules and Si substrates were immersed in H₂SO₄ : H₂O₂ (3 : 1) for 24 hours to form the hydroxide radical on the surface.

Bonding procedure

The entire bonding process is shown in Fig. 1. The pretreated diatom frustules and Si substrates were washed with deionized water and dried at 75 °C. Then diatom frustules were added to HF solution. And the HF solution containing diatom frustules was dropped on the Si substrate. Later, a certain pressure was applied on diatom frustules vertically with the help of a direction guiding device. The pressure was calculated by the weight and the area of the substrate. During this process, a polyvinylchloride film (thickness: 2 mm) was placed between diatom frustules and the pressure. The flexible and corrosion-resistive polyvinylchloride film slightly deformed to comfort the curve surface of diatom frustules, so the pressure could be uniformly applied on almost every diatom. After baking at 75 °C for 2 h, the whole system was taken out of the oven and diatom frustules were bonded with the Si substrate. As the melting point of the polyvinylchloride film is around 135 °C, this film just began to soften but did not melt during the bonding process and diatom frustules would not stick or react with the film. In the end, the diatom based substrate was flushed by water to remove unbonded diatom frustules.

Fig. 1 The schematic of the bonding process and the nanogold pillar array acquisition.

Characterization of bonded diatom frustules

SEM and EDS were used to analyze the bonding quality and mechanism, while AFM was used to indirectly measure the bonding strength.³⁰ Bonding conditions like HF concentrations and applied pressures were well investigated. The physical and chemical properties of the diatom shells before and after the bonding process were analyzed using photoluminescence (PL) and infrared spectra scans. The bonding mechanism was discussed while the cross-section of the diatom based Si substrate was analyzed. The bonding strength was obtained with AFM calibrated glass needles, during which a bonded diatom was pushed with the glass needle and the deflection of the needle determined the shearing force.

Application of bonded diatom frustules

The diatom frustules could also be used as masks to acquire nano gold pillar arrays. The schematic process is shown in Fig. 1. After diatom frustules were bonded with Si substrates, gold was vertically sputtered on their surface with Cr as an adhesive interlayer. A gold layer was formed inside and outside the diatom nanopores. Later, the diatom based substrate was immersed into HF solution with 40% concentration for 5 min. HF would fully react with the diatom frustules, leaving nano gold pillar arrays on Si substrates. In this section, another kind of Coscinodiscus sp. diatoms (Diameter 100 μm, from the Diatom laboratory, Xiamen University, China) were used, because the Coscinodiscus sp. diatoms had more dedicated structures compared with the Coscinodiscus decrescens. diatoms.

After the nano gold pillar arrays were acquired, the SERS activity was demonstrated by modifying the gold arrays with an ethanol solution of crystal violet, whereby crystal violet was used as a probe molecule that could be adsorbed onto the nanoparticle surface as a monolayer. It was left to dry naturally in air. Then, the substrate was immersed into a 1 × 10⁻² mol L⁻¹ crystal violet ethanol solution overnight. After thoroughly rinsing with absolute ethanol several times to remove the free crystal violet molecules, the samples were then subjected to Raman characterization (LabRAM HR800, excitation wavelength 647 nm, magnification ×50). For comparison, the normal Raman spectra of solid crystal violet samples were also measured.

Results and discussion

Bonding quality evaluation

Morphology of diatom frustules before bonding. The Coscinodiscus decrescens. diatom frustule consists of two almost equal halves that fit together like a Petri dish, enclosing the organic materials inside. Each half consists of a valve (plate-shaped part) and a girdle (circular band of silica attached to the edge of the valve).¹⁴ The diameter of a diatom valve is around 50 μm. And the thickness of a valve is around 3 μm. After the organic matter of diatom was removed by acid, they split into separate valves and girdles. Since valves have more ordered structures for sensor application, we only used diatom valves for the bonding process. Organic materials and girdles were removed by the centrifugation and settling method.²⁹Fig. 3a shows the morphology of original Coscinodiscus diatom valves lying on the edge of a glass substrate, from which we can see almost all original Coscinodiscus diatom valves show a relatively large degree of concavity and convexity on their surface. If we take the girdle plane as the base level, the height of the concave or convex is around 2 μm. Fig. 2b and c show the outside and inside views of the valve, respectively. And the nano structures of diatom valves remained well after pretreatment.

Fig. 2 The morphology of (a) original Coscinodiscus diatom valves lying on the edge of a glass substrate, (b) the outside view and (c) the inside view of a diatom valve.

Morphology of bonded diatom frustules. Fig. 3 shows the bonding results under certain conditions (HF concentration: 0.5%, applied pressure: 2.5 × 10⁴ Pa). Numbers of diatom frustules were bonded with the Si substrate (Fig. 3a), and most diatoms broke into plate-like parts along the girdle band. The bonded diatom valves still showed a relatively large degree of concavity and convexity on their surface, and the height of the concave or convex was around 1.7 μm. The change of height before and after bonding might result from the slight deformation after the HF treatment. The outside (Fig. 3b) and inside (Fig. 3c) views of a single diatom valve were also provided. After bonding, nanopores over diatom valves were enlarged a little, and edges of valves slightly crushed. However, the main morphology of diatom frustules remained quite well after bonding. As the deformation of diatom frustules before and after bonding is in such a low degree, the bonded diatom frustules can still be used as kinds of bio-chemical sensors. Moreover, the 3D view of a valve (Fig. 3d) preliminarily showed that the edge of frustules has bonded with the Si substrate as a whole.

Fig. 3 Bonded Coscinodiscus diatom frustules, (a) the bonded diatom frustules, (b) the outside view and (c) the inside view of a diatom, (d) and (e) the bonding area of a diatom and the substrate.

Based on statistical experiments, the outside of a valve had the same opportunity to face upwards or downwards. The valve shaped like a plate, and the morphology of the inside and outside of valves was similar. The direction of valves did not remarkably influence the bonding result. Nevertheless, further study about directions controlling is still being undertaken based on the floating effect of diatoms.³¹

Analysis of the bonding mechanism. The bonding mechanism is similar to HF assisted bonding of Si substrates and SiO₂ substrates. After pretreatment of H₂SO₄ : H₂O₂, the surfaces of diatom frustules and Si substrates were both functionalised with the Si–OH group. The Si substrate and diatom frustules were brought into close contact with each other, and a thin HF solution layer was between them and reaction took place. During the bonding process, Si–OH groups gradually dehydrated with the help of HF, forming siloxane bonds and terminating with a condensation–polymerization reaction (Fig. 4).^25,32

Fig. 4 The dehydration and condensation/polymerization of Si–OH groups during bonding.

In addition, as diatom frustules were all made of SiO₂, HF would react with diatom frustules as follows.

SiO₂ + 4HF = SiF₄↑ + 2H₂O

As SiF₄ is in the gaseous state, it disappeared after the reaction was finished. With the disappearance of SiF₄ and evaporation of H₂O, the bottom of diatom first dissolved and then unified with the Si substrate as a whole. However, the concentration of the HF solution was lower than 1%. So the reaction would be relatively gentle and end before the diatom frustules crushed.

In order to further analyze the bonding quality and mechanism, the cross-section of the diatom based Si substrate was acquired. The Si substrate bonded with diatom frustules (HF concentration 0.5%, applied pressure 2.5 × 10⁴ Pa) was cut with glass cutter. The cutting force was exerted on the back of the substrate, and the diatom based substrate broke into two halves. With the cutting track through the center of a single diatom, the diatom broke off into two halves together with the substrate (Fig. 5a). The contacting area between diatom frustules and the Si substrate unified as a whole (Fig. 5b), while the nanopores and the cavum structures inside the diatom frustules remained undestroyed. In addition, EDS was used to analyze the bonding area. Table 1 shows the chemical composition of the star-marked area in Fig. 5c. Despite the systematic error of the spectrum analyzer, the bonding area was the combination of Si and SiO₂, which means that the bottom of diatom frustules actually dissolved and then unified with the Si substrate as a whole. It seemed that there was a flat intermediate layer between the diatom frustule and the substrate. This might be caused by the edge crushing when the diatom based substrate was cut into two half.

Fig. 5 The cross-sectional view of a bonded diatom, (a) schematic image of the cross-section, (b) the cross-sectional image of the bonded Coscinodiscus diatom, and (c) the bonding interface and marked area for EDS.

Table 1 Component (atom percentage) of the bonding area

Area	1	2	3
Si (%)	34.02	61.98	99.71
O (%)	65.98	38.02	0.29

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Later, the PL and infrared spectra scan experiments were carried out, indicating that there were merely any physical or chemical property changes of the valve before and after bonding. As a result, the surface of the valve could still be modified for photoluminescence and electrochemical detection in bio-chemical sensors.

Optimization of bonding conditions

Analysis of bonding strength. The bonded diatom frustules needed to resist kinds of gas or liquid flush in bio-chemical sensors application. Consequently, the bonding strength between diatom frustules and Si substrates needed to be analyzed to ensure that bonded diatom frustules would not be easily removed. The regular tensile test equipment was mostly designed for bonding between flat surfaces. A new method was required to characterize the bonding strength between diatom frustules and substrates.

As mentioned in our previous work,¹⁷ the glass needle and AFM could be used to precisely quantify the bonding strength. Firstly, a bonded diatom (0.5% HF concentration, 2.5 × 10⁴ Pa applied pressure) was pushed with the calibrated glass needle and the needle was deflected (Fig. 6a and b). When the glass needle deflected to a certain degree, the diatom crushed (Fig. 6c). The main structure of the diatom was broken and removed by the glass needle. But some remaining pieces of diatom frustules were still firmly bonded to the substrate (Fig. 6c), indicating that the bonding force was equal or even higher than the shearing force provided by the glass needle. In addition, the morphology of bonding areas was different from that of other areas, indicating that certain chemical reactions happened during the bonding process.

Fig. 6 Bonding strength acquisition, (a) and (b), a bonded diatom is pushed with the calibrated glass needles, (c) the crushed diatom.

The spring constant of our glass needle was 0.944 N m⁻¹. Fig. 6 shows the bonding result with a HF concentration of 0.5% and an applied pressure of 2.5 × 10⁴ Pa. With 900 μm deflection, the needle has resisted an applied force of 850 μN, which is also the bonding force between one diatom and the substrate. Mostly the valve is not completely flat and only about 60% of its surface can directly come into contact with the Si substrate. For this reason, the contact area of one diatom and the substrate can be taken as nearly 1178 μm.² As a result, the bonding strength of diatom frustules and the substrate is approximately 0.72 MPa.

Optimization. The bonding should be controlled to meet two demands. One, the bonding should not destroy the delicate three-dimensional structures and nanoscale porous for better use in bio-chemical sensors. Another, the bonding strength between diatom frustules and the substrate should be strong enough for further treatment, such as protein hybridization or gold sputtering. As a result, the bonding conditions were critical and should be optimized. Diatom frustules with the outside of their valves upward or downward had similar bonding strengths when they were under the same bonding conditions.

The bonding process took at least 24 hours at room temperature. In order to improve the bonding efficiency, all the bonding experiments were employed with a constant temperature of 75 °C for 2 hours. Since the applied pressure and the concentration of the HF solution were two most crucial factors affecting the bonding strength and the morphological integrity of the diatom, discussions about these two factors were done and the bonding strength was measured using glass needles respectively and shown in Fig. 7.

Fig. 7 The bonding strength variation with applied pressure and HF concentration.

The HF concentration was one important factor affecting the bonding strength. As shown in Fig. 7, when the HF concentration was lower than 0.4%, the diatom frustules could not be fully reacted with Si substrates and the bonding strength was relatively low. With the increase of the HF concentration, the bonding strength increased. When the HF concentration reached 0.5%, the highest bonding strength was obtained. However, if the HF concentration kept on increasing, diatom frustules fully reacted with HF and the main structure and nanopores of diatom frustules were severely damaged. Therefore, the bonding strength fell with the increase of HF concentration when the HF concentration was higher than 0.5%. Finally, the optimized HF concentration should be between 0.4% and 0.6% to achieve enough bonding strength while preserving the morphological integrity of diatom frustules.

In addition, the applied pressure was another crucial factor for the bonding strength. The bonding strength of diatom frustules and Si substrate was relatively low when the applied pressure during bonding was below 2.0 × 10⁴ Pa. With the increase of the applied pressure, the bonding strength increased. When the pressure reached 2.5 × 10⁴ Pa, we got the highest bonding strength. Afterwards the bonding strength fell with the increase of the applied pressure because more diatom valves crushed into pieces under such high pressure. Finally, the optimized pressure was found to be in the range of 2.0 × 10⁴ Pa to 3.0 × 10⁴ Pa with consideration of the balance of the bonding strength and damage to diatom frustules.

Applications of bonded diatom frustules

Nanogold pillar arrays. Since Si is a kind of semiconductor material which directly connects with diatom frustules, it is possible to obtain any optical and electrical signal changes of diatom frustules. And this will highly improve the detection sensitivity and efficiency compared with the original photoluminescence detection method used in the bio-chemical sensing field.³³ While in this paper, we would discuss the SERS application based on bonded diatom frustules.

As the diatom frustules still had nanoscale through pores regularly laid on the valves, they could be used as masks to acquire nano gold pillar arrays, which might have an SERS effect. Coscinodiscus sp. diatoms were also used in this part. And these pores showed a radicalized distribution pattern. Fig. 8a shows the top view of one Coscinodiscus sp. diatom frustule. The hierarchical microscale/nanoscale pores of diatom were radially distributed from the center of the frustule. For the nanoscale pores (diameter 80 nm), seven of them would form a heptamer structure (Fig. 8b).

Fig. 8 The nano gold pillar array, (a) the nano gold pillar array acquired with Coscinodiscus, the surface morphology of a Coscinodiscus sp. valve (b) before and (c) after it is sputtered with gold, (d) the nano gold pillar array acquired based on it.

The bonding process was gentle in this part (0.3% HF concentration, 1.5 × 10⁴ Pa applied pressure), because serious reaction would destroy the nanopore structures of Coscinodiscus sp. diatom frustules. And the sputtering in this part did not require high bonding strength.

After gold was sputtered, HF was used to remove the diatom frustules. Nano gold pillar arrays were acquired (Fig. 8c). The nano gold pillar had the same size as the nano pore. Seven nano gold pillars formed a symmetrical heptamer cluster, whose diameter was around 400 nm (Fig. 8d). This kind of heptamer structure has many applications in the SERS field.

SERS effect of the bonded diatom frustules. Fig. 9a shows the Raman spectrum obtained from the dry crystal violet powder at room temperature. So all peaks in the SERS spectra (Fig. 9b) were typically enhanced signals of the crystal violet on nano gold pillar arrays, instead of the contribution from nano gold pillar arrays themselves. The predominant features in the SERS spectra were at about 1179 cm⁻¹.

Fig. 9 (a) Raman spectrum of crystal violet, (b) SERS spectrum of crystal violet on diatom frustules.

To evaluate the magnitude of the enhancement factor (EF) for crystal violet on nano gold pillar arrays, the following equation was used.³⁴

I_SERS and I_bulk were the measured intensity of the SERS spectrum and the same mode of the normal spectrum of a solid sample. I_SERS and I_bulk were obtained from the experimental spectrum. N_bulk and N_ads were the number of crystal violet molecules under laser illumination for the solid and adsorbed on the SERS substrate, respectively. The sample volume of the solid was decided by the area of the laser spot (∼1.4 mm²) and the penetration depth of the focus laser (∼10 mm). On the basis of the density of bulk crystal violet, N_bulk was calculated to be 2.6 × 10¹⁰. The number of molecules adsorbed on the SERS substrate, N_ads, was obtained by multiplying the area of the illuminated focus spot size, the bonding density of crystal violet molecules on the nanopillars surfaces and the number density of nano gold pillar arrays. The number of probed molecules in the probed volume (N_ads) was calculated to be 4.5 × 10⁶. Considering the intensity ratios (I_SERS = 14 290, I_bulk = 1082) at 1179 cm⁻¹, the EF was estimated to be around 7.64 × 10⁴.

As there were thousands of diatoms species with thousands of different nano pores distributions, various nano gold pillar arrays could be acquired for different applications. This technique will be further discussed in the following research paper.

Conclusions

In summary, Coscinodiscus diatom frustules were bonded with Si substrates using a low temperature HF assisted bonding method. SEM and EDS were used to analyze the bonding area and bonding mechanism. The physical and chemical properties of diatom frustules almost remained unchanged after bonding. In order to achieve sufficient bonding strength while keeping the morphological integrity of the bonded diatom frustules, bonding conditions were optimized. The optimized pressure was between 2.0 × 10⁴ Pa and 3.0 × 10⁴ Pa, and the optimized concentration of HF is from 0.4% to 0.6%. The optimal shear bonding strength between Coscinodiscus decrescens. diatom frustules and Si substrates was around 0.72 MPa. The diatom based substrates may be used for improved photoluminescence and electrochemical detection in the bio-chemical sensor field in the future. And bonded diatom frustules were also used as masks to obtain nano gold pillar arrays having an SERS effect. This technique should be also useful for bonding of other micro silica particles with Si substrates, which will greatly contribute to the diatoms application in micro-nanotechnology.

Acknowledgements

The authors would like to thank Zhang Juan for lots of useful discussion. This work was supported by the National Natural Science Foundation of China (Grant No. 51075020, 51205012 and 51322503)

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