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We have developed an optical system designed for detecting colored nanomaterials in aqueous solutions, using the concept of evanescent-field-coupled waveguide-mode sensors. In this study, we found that the waveguide modes induced in the sensor are intrinsically sensitive to a change in optical absorption, or a ‘change in color’. The system detects less than one gold nanoparticle (diameter: 20 nm) adsorbed per square micrometer. It is also demonstrated that significant signal enhancement due to adsorption of molecules is achieved using a dye. The developed sensor rarely suffers from a drawback of impurity adsorption. The system is expected to be applied as an effective sensing tool for metal colloids, nanoparticles, and colored biomolecules in solution.
©2010 Optical Society of America
1. Introduction
Highly sensitive and accurate tools for the detection, observation, and measurement of nanomaterials are required in various fields such as nanomanufacturing technology, medical technology, and environmental sciences. For characterizing properties of nanomaterials, such as the shape, structure, size, and weight, many methods have been developed employing technologies that utilize electron beams [1], X rays [2–4], atomic force [5], resonators [6,7], and so on. However, some of the methods are unsuitable for characterizing nanomaterials dispersed in a solution because of the presence of the solution itself, and some of the methods are rendered unsuitable by the viscosity of the solution [8].
Sensors that detect molecules in aqueous solutions by observing changes in the dielectric environment due to specific adsorption of molecules using near-field optics, such as sensors using surface plasmon resonance [9–13], optical waveguide modes [13–19], or ring resonators [20–23], are well known. These sensors can quantitatively analyze molecular adsorption in real time and their sensitivity is extremely good. However, since these sensors are basically developed for detection of transparent biomaterials, they are not suitable for detection of colored materials having optical absorption at the wavelength of incident light used in the detection. This is because the incident light significantly decays due to the optical absorption. Further, they have drawbacks that their detection ability is strongly affected by the temperature of surroundings because of perturbation of refractive indices of aqueous solutions by temperature [24] and that they cannot distinguish adsorption of target materials from non-specific adsorption of impurities. Here, we developed an optical system designed for detecting colored nanomaterials using the concept of a monolithic sensing plate of evanescent-field-coupled waveguide-mode (EFC-WM) sensors [19].
2. Simulation
Figure 1(a) shows a setup of the EFC-WM sensor. The setup geometry is based on the Kretschmann configuration [25], which is one of the typical optical setups of EFC-WM sensors. The monolithic sensing plate comprises a SiO2 glass substrate, a single-crystalline Si layer, and a thermally grown SiO2 waveguide. Figure 1(b) shows a cross-sectional view of the monolithic sensing plate. A waveguide mode is excited by passing light through the prism at an incident angle greater than the critical angle of total internal reflection at the waveguide surface. At this incident angle, the reflectance decreases; this decrease appears as a dip in the plot of the reflectance versus the incident angle.
Fig. 1 (a) Sketch of the setup of an EFC-WM sensor. A prism, a sensing plate, and a Teflon cuvette that supports samples to be examined are mounted on a goniometer. Light illuminates the sensing plate and the reflected light is detected by a photodiode. (b) Sketch of a cross sectional view of the monolithic sensing plate. The plate has a layered structure of a SiO2 glass substrate, single-crystalline Si layer, and thermally grown SiO2 waveguide layer, on which molecular probes that specifically capture analytes are formed. A prism (SiO2 glass) will be placed on the SiO2 glass substrate.
Now, capture of target materials on the waveguide surface causes a change in the complex refractive index (n + ki) near the waveguide surface. Figures 2(a) and 2(b) show the simulated reflectance property of an EFC-WM sensor. The Reflectance of the EFC-WM sensors was calculated using the Fresnel equation. For the calculation, a transfer-matrix technique for stratified media was used [26]. The sensing plates assumed in the calculation are monolithic sensing plates having a waveguide with a thickness of 500 nm. The thickness of the Si layer is in the range of 10–80 nm in Fig. 2(a) and 100–160 nm in Fig. 2(b). In the calculation, the incident light and the prism are assumed to be an s-polarized beam with a wavelength of 632.8 nm and an isosceles prism (SiO2 glass) having a vertex angle of 30°, respectively. The solid curves show the reflectance calculated by assuming that the waveguide is soaked in water. The dotted curves show the reflectance calculated by assuming that a material with n = 3, k = 3, and the thickness t = 0.01 nm is placed on the waveguide. These values of n and k are comparable to those of metals. Figure 3 shows correlation between the thickness of the Si layer and a change in the reflectance (ΔR) caused by the colored material; ΔR is defined as the difference between the reflectance values at the bottom of the dips with and without the colored material layer in Figs. 2(a) and 2(b). A large ΔR implies high sensitivity. The thickness of the Si layer significantly affects the sensitivity. The result shown in Fig. 3 indicates that the optimal thickness of the Si layer is ~40 nm and the expected ΔR is more than 0.3. As can be seen in Fig. 2, a sharp dip is obtained if the thickness is optimal. This is because the Si layer with the optimal thickness induces a sharp optical resonance, which results in the good sensitivity. As increase in the thickness of the Si layer, the sensitivity decreases. This is due to increase in optical absorption of light propagating in the waveguide by the Si layer. The thickness of 0.01 nm of the colored material is impractical, i.e. completely theoretical; nevertheless, the result indicates that the designed sensor is extremely sensitive to colored materials.
Fig. 2 Reflectance of EFC-WM sensors calculated using the Fresnel equation. The sensing plates assumed in the calculation are monolithic sensing plates having a waveguide with a thickness of 500 nm. The thickness of the Si layer is in the range of 10–80 nm in (a) and 100–160 nm in (b). The thicknesses shown on top of the figures indicate those of the Si layers. The incident light is an s-polarised beam with a wavelength of 632.8 nm. The solid curves show the reflectance calculated by assuming that the waveguide is soaked in water. The dotted curves show the reflectance calculated by assuming that a colored material with n = 3, k = 3, and the thickness t = 0.01 nm is placed on the waveguide.
Fig. 3 Correlation between the thickness of the Si layer and the change in reflectance (ΔR) caused by the colored material. The values of ΔR are defined as the difference between the reflectance values at the bottom of the dips with and without the colored material layer in Fig. 2.
Figure 4(a) shows the calculated reflectance property of the EFC-WM sensor comprising the optimized monolithic sensing plate, where the thicknesses of the Si layer and waveguide are set to 40 and 500 nm, respectively. The conditions used in the calculation are same as those in the previous calculation. The black curve is the reflectance obtained by assuming that the waveguide is soaked in water. The red curve in Fig. 4(a) shows the reflectance of the sensor for a material with n = 1.45, k = 0, and t = 10 nm placed on the waveguide. These values of n, k, and t are comparable to those of a protein. In this case, the dip shifts towards the higher angle side. The blue curve in Fig. 4(a) shows the reflectance of the sensor for the material with n = 3, k = 3, and t = 0.01 nm placed on the waveguide. Figures 4(b), 4(c), and 4(d) show the simulated amplitudes of the electric fields of the waveguide modes excited at the incident angles corresponding to the bottoms of the dips seen in the black, red, and blue curves in Fig. 4(a), respectively. The profiles shown in Figs. 4(b) and 4(c) are very similar with each other. This implies that adsorption of a thin transparent material on the waveguide causes a change in the excitation condition of a waveguide mode but scarcely affects the propagation condition of the waveguide mode. As shown in Fig. 4(d), the adsorption of a colored material significantly weakens the electric field in the waveguide. This is due to the absorption of incident light power by the colored material. As a result, the reflectivity at the resonant position, i.e. at the dip, is significantly weakened. This is the underlying mechanism due to which the adsorption of the colored material can be detected sensitively.
Fig. 4 (a) Reflectance of the EFC-WM sensor designed for the detection of change in k. The reflectance values are calculated using the Fresnel equation. Black curve: reflectance obtained by assuming that the waveguide is soaked in water. Red curve: reflectance for a material with n = 1.45, k = 0, and t = 10 nm placed on the waveguide. Blue curve: reflectance for a material with n = 3, k = 3, and t = 0.01 nm placed on the waveguide. (b), (c), (d), Simulated amplitudes of electric fields of the waveguide modes at the incident angles corresponding to the bottoms of the dips seen in the black, red, and blue curves in (a), respectively. A transfer matrix technique for stratified media was used in the calculation. The substrate, the Si layer, and the waveguide are in line from left to right. Each boundary is indicated by a green line. The illuminating plane wave propagates in the positive z-direction. The strength of the field is indicated by the color bar.
3. Experimental Details
On the basis of the above-mentioned calculation, we constructed a system that enabled the detection of colored nanomaterials. For fabrication of the sensing plate, we used a silicon-on-quartz (SOQ) substrate (Shin-Etsu Chemical, Japan) comprising a 270-nm thick single-crystalline Si (100) layer on a 1.2-mm thick SiO2 glass substrate. The SOQ substrate was cut into plates measuring 25 × 25 mm and thermally oxidized in an electric furnace in an atmosphere of O2 containing water vapor at 1000 °C at ambient pressure for 65 min. The oxidation process turned the surface of the Si layer into a SiO2 waveguide layer. Figures 5(a) and 5(b) show cross-sectional views of the substrate before and after the thermal oxidation, respectively. The fabricated sensing plate had a Si layer and a waveguide with thicknesses of 35 nm and 520 nm, respectively.
Fig. 5 (a) Cross-sectional view of the SOQ substrate used for fabricating the monolithic sensing plate. The substrate consists of a single-crystalline Si layer on a SiO2 glass substrate. The thickness of the Si layer was 270 nm. (b) Cross-sectional view of the monolithic sensing plate. The waveguide layer with a thickness of 520 nm was formed by thermal oxidisation of the Si layer of the SOQ substrate. The thickness of the remaining Si layer was 35 nm.
An s-polarized He–Ne laser beam (632.8 nm) was used as the light source. An isosceles prism (SiO2 glass) having a vertex angle of 30° was optically matched with the substrate of the sensing plate, and a Teflon cuvette was connected to the waveguide side of the plate. The prism, sensing plate, and cuvette were mounted on a goniometer [see Fig. 1(a)]. A liquid sample containing target materials to be examined was injected in the Teflon cuvette.
On the waveguide of the sensing plate, an amino base was introduced by immersing the waveguide in a 0.2 wt.% solution of 3-aminopropyltriethoxysilane (3APT) in ethanol for 24 h, and then biotin was introduced onto it by immersing the 3APT-modified waveguide in a 0.5 mM solution of biotin-(AC5)2-Sulfo-OSu in a 1/15 M PBS buffer (pH 7.4). Gold nanoparticles (diameter: 20 nm) with streptavidin (immunogold conjugate, Funakosi, Japan) dissolved in Tris-buffered saline (TBS) were used as the target material to be detected. We also performed the detection of colored proteins, where streptavidin dyed using Coomassie Brilliant Blue G-250 (CBBG) contained in a Bio-Rad protein assay solution was used. For the preparation of the streptavidin-dye complex, streptavidin was mixed with the Bio-Rad protein assay solution diluted as 1:4 in phosphate-buffered saline (PBS) and stored for 1 h at room temperature. Uncomplexed free dye molecules were separated from the complex using a centrifugal filter with a molecular weight cut-off of 10,000 Da. The biotin introduced onto the waveguide surface worked as a molecular probe to capture the target materials.
4. Results and Discussion
The black curve in Fig. 6(a) shows the original reflectance property of the system, measured by soaking the waveguide surface in the TBS buffer. The red curve in this figure shows the reflectance measured 20 h after injecting a sample containing 1 nM of the immunogold conjugate into the cuvette. There is a significant decrease in the reflectance. Figure 6(b) shows an image of the waveguide surface after the 20-h reaction, captured using a scanning electron microscope (SEM, Hitachi High-Technologies, S4800). Before carrying out the SEM observation, the sensing plate was gently rinsed with distilled water in order to remove the salt dissolved in the buffer. By measuring the reflectance before and after the rinsing process, we confirmed that almost all the gold nanoparticles remained adsorbed on the surface after rinsing. The average number of gold nanoparticles observed by the SEM is ~30/μm2.
Fig. 6 (a) Reflectance of the system measured by soaking the waveguide surface in the buffer (black curve) and reflectance measured 20 h after injecting a solution containing 1 nM of immunogold conjugate (red curve). Dotted blue curve is reflectance calculated using the Fresnel equation by assuming that a single layer (thickness: 20 nm) with a complex refractive index of 1.3556 + 0.0038i is formed on the waveguide. The inset shows a schematic of adsorption of immunogold conjugate on biotin probes. (b) SEM image of the waveguide surface after the 20-h reaction of 1 nM of immunogold conjugate.
The inset of Fig. 6(a) shows a schematic of the adsorption of the immunogold conjugate on the biotin probe. By assuming that the adsorbed immunogold conjugate forms a single layer (thickness: 20 nm) of a mixture containing the buffer and the immunogold conjugate and by applying the Maxwell–Garnett effective medium expressions [27] to the layer, the average complex refractive index of the layer is calculated to be 1.3556 + 0.0038i. The dotted blue curve in Fig. 6(a) shows the reflectance calculated using the Fresnel equation by assuming that the layer is formed on the waveguide. The calculated curve is almost identical to that obtained experimentally. The slight difference would be due to the approximation in the calculation of the average complex refractive index.
The solid curve in Fig. 7(a) shows the original reflectance property of the system, and the dotted curve shows the reflectance measured 20 h after injecting a sample containing 10 pM of the immunogold conjugate into the cuvette. The adsorption of the immunogold conjugate appeared as a decrease of 0.016 in the reflectance. Figures 7(b) and 7(c) show the images of the waveguide surface observed after the 20-h reaction, captured using the SEM. Even though, only a few gold nanoparticles could be seen, and therefore, it was difficult to accurately determine the average number of nanoparticles per unit area, we confirmed that at the most a single nanoparticle was adsorbed per square micrometer. These results indicate that the developed system can detect such a small areal density of gold nanoparticles. According to the above-described calculations of average complex refractive indices and reflectance, the reflectance is expected to decrease by 0.01 by the adsorption of 0.75 gold nanoparticle (diameter: 20 nm) per square micrometer. The experimental results agree well with the calculation.
Fig. 7 (a) Reflectance of the system measured by soaking the waveguide surface in the buffer (solid curve) and reflectance measured 20 h after injecting a solution containing 10 pM of immunogold conjugate (dotted curve). (b), (c) SEM images of the waveguide surface after the 20-h reaction. The insets in (b) and (c) are enlarged view of areas surrounded by the white circles in (b) and (c). The white dots are gold nanoparticles.
Figure 8 shows change in reflectivity in the case of detection of dyed streptavidin. The black curves in Figs. 8(a), 8(b), and 8(c) show the reflectance measured before molecular adsorption. There is a slight difference in the positions of the bottoms of the dips, which is due to experimental error in the fabrication of the waveguide layers. The red curve in Fig. 8(a) shows the reflectance measured 1 h after the sample containing 500 nM of non-colored streptavidin was injected. The observed shift in the peak position is due to a change in n with the adsorption of streptavidin. The red curves in Figs. 8(b) and 8(c) show reflectance spectra measured 1 h after the injection of the sample containing 100 pM of dyed streptavidin and 20 h after the injection of the sample containing 10 pM of dyed streptavidin, respectively. Even though the analyte in Fig. 8(b) is 5000 times more diluted than that used in the experiment shown in Fig. 8(a), the change in reflectance shown in Fig. 8(b) is more than that shown in Fig. 8(a). The sensor is capable of detecting more diluted analytes [see Fig. 8(c)]. Figure 9 shows the optical absorption spectrum of the dyed streptavidin dissolved in the PBS buffer (25 nM). An optical absorption band is observed at around 600 nm, which is rather close to the wavelength of the incident light. The detection sensitivity is expected to improve further by optimization of the wavelength of incident light and selection of appropriate dye.
Fig. 8 Change in reflectance with adsorption of dyed streptavidin. (a) Reflectance before (black curve) and after (red curve) adsorption of streptavidin without dye. The red curve was measured 1 h after the injection of a sample containing non-colored streptavidin dissolved in PBS (500 nM). (b) (c) Reflectance before (black curves) and after (red curves) adsorption of dyed streptavidin. The red curve in (b) was measured 1 h after the injection of a sample containing 100 pM of dyed streptavidin. The red curve in (c) was measured 20 h after the injection of a sample containing 10 pM of dyed streptavidin.
In the present detection scheme, target materials are detected by a change in the depth of a dip in reflectance. Therefore, the detection is not affected by a change in the refractive index of a solvent with temperature and adsorption of impurities having no optical absorption at the wavelength of incident light, since these factors merely shift the angular position of the dip and do not change the depth of the dip.
The present detection mechanism is similar to that of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The new system combines the ATR-FTIR mechanism with the high sensitivity waveguide multiple reflection, thus creating a superior detection system. Sensitivities of sensors used for molecular detection are often measured by the lowest weight or smallest size of molecules that can be detected. However, the detection limit of the developed sensor is not determined by these factors but by k—the darkness of color—of analytes. The present detection method can be applied for detection of biomolecules, such as proteins or viruses, by attaching colored nanoparticles to the molecules. In such a case, higher sensitivity can be easily obtained by using bigger or darker nanoparticles. However, some information about properties of the molecules might be lost in this case. Further, in principle, the present system can detect any kinds of metals by choosing an appropriate wavelength of the incident light, indicating that the system is applicable for detection of heavy metal pollution. Quantitative analysis of adsorbed molecules is also feasible if the complex refractive index of an analyte is known beforehand.
5. Conclusions
We succeeded in the development of the sensor that sensitively detects colored nanomaterials in solutions. The sensor can quantitatively analyze adsorption of colored nanomaterials. In addition, the ability is scarcely affected by a change in temperature and adsorption of impurities. The sensor will be a strong tool for detection and observation of colored nanoparticles, metal colloids, and dyed biomolecules in solution.
Acknowledgements
We would like to thank the Advanced Functional Materials Research Center, Shin-Etsu Chemical Co., Ltd., for supplying the SOQ substrate. This study was partly supported by Industrial Technology Research Grant Program in 2009 from New Energy and Industrial Technology Development Organization, Japan (NEDO). A part of this study was conducted at the AIST Nano-Processing Facility and was supported by the ‘Nanotechnology Support Project’ of the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). One of the authors, K.N., thanks the Japan Society for the Promotion of Science (JSPS) for a Grant-in-Aid for JSPS Young Fellows.
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