Surface plasmon resonance (SPR) spectroscopy of the metal nanostructure (MN) is widely used in chemical and biological sensing. The biosensor characteristics of the MN are affected by the size, shapes, substrate, and so on. Especially, some studies about the mode of the MN show that the surface plasmon polaritons (SPPs) coupled mode excitation has a significant impact on the sensitivity of the MN biosensor. To reach the aim of obtaining the high sensitivity MN biosensor, the coupled mode of the gold elliptic nanohole array (GENA) was simulated and analyzed. It shows that the coupled mode was influenced by the refractive indexes of medium and substrate. The coupled mode resonance peak shows a higher sensitivity than that of other SPPs mode peaks. The GENA biosensor chips were fabricated and integrated with polydimethylsiloxane (PDMS) microfluidics. The sensitivity of the GENA is characterized by transmission spectra of GENA in deionized water and aqueous NaCl solution. The coupled mode peak sensitivity of the biosensor reaches 549 nm/RIU. An antigen-antibody interaction experiment was also adopted to verify the sensitivity of the surface binding reaction. The sensitivity of detected concentration of the AFP (α-fetoprotein) in our experiment has been reached 25 ng/ml closed to the clinic concentration. The GENA biosensor chip has potential application in label-free chemical and biomedical fields, especially, cancer biomarker testing.
© 2015 Optical Society of America
Surface plasmon resonance (SPR) is an optical charge carrier resonance that arises around noble metal nanostructure (MN) and could be excited by the light illuminating onto the nanostructure, whose frequency matches the natural frequency of the nanostructure . The SPR of the metal nanohole was discovered to produce extraordinary optical transmission (EOT) by Ebbesen and associates with a strongly enhanced transmission of light through the nanohole . Since the discovery of EOT in metal nanohole, a great deal of research activities has been carried out to make the applications of the MN in diverse fields of science including metamaterials, nonlinear photonics and biomedicine [3–7]. One of the most interesting application areas is label-free molecular biosensing based on the dependence of the molecular bonded with the nanostructure [8–10]. The biosensing characteristics of the MN depended on the shapes, sizes, compositions, and orientations which could be precisely controlled and tuned by the fabrication technology, such as electron beam lithography (EBL) [1, 11]. To improve the biosensing characteristics of the MN, many strategies have been adopted, including uniformly fabricating the nanostructure, making the metal film smoother, and design a new nanostructure [12–14]. All of these strategies could improve the sensing sensitivity by narrowing the sensing spectra and enhancing of the electric field intensity. Based on those efforts, MN is widely used in biosensor, such as detecting the clinical low-concentration cytokines and antigens [15, 16].
Nowadays, considerable progress has been made on the MN biosensor. Especially, the metal nanohole array (MNA) with high sensitivity and low detect limitation is adopted to be the MN biosensor. However, there are also some difficulties for using the MNA biosensor. The biosensing mechanism of the MNA is still needed carefully investigated to improve the sensitivity in the biomedicine diagnosis. Hereinto, studies on MNA have shown that the surface plasmon polaritons (SPPs) on the medium/MNA and substrate/MNA coupling could bring about modes coupling [17, 18]. The sensitivity of the MNA will be increased as the Fano resonance is excited by the coupled mode . The coupled mode of the MNA is affected by the refractive indexes of the medium and substrate. For the medium and substrate with different materials, the coupling intensity of the SPPs on the medium/MNA and substrate/MNA interfaces is different . Compared with the conventional round nanoholes, the elliptical nanoholes are more sensitive for biosensing, because of the enhancement of the transmission [20, 21]. Therefore, an analysis on the coupled mode of the gold elliptical nanohole array (GENA) is needed to operate the biosensor with a high sensitivity.
The focus of our research is to achieve a gold elliptical nanohole array (GENA) biosensor with high sensitivity. The exciting and coupling of the SPPs at both interfaces of the GENA are investigated by numerical simulation. It shows how the SPPs coupling of the GENA is influenced by substrate refractive index. The GENA structure is optimized for the biosensor application. The GENA transmission spectra are analyzed to show the effect of the SPP coupling strength on the sensitivity. The GENA are fabricated by the EBL technique. The polydimethylsiloxane (PDMS) microchannels are bonded onto the fabricated GENA. Then the refractive index sensitivity of the GENA are measured by injecting the deionized (DI) water and aqueous NaCl solution with different concentrations. Finally, the low-concentration AFP (α-fetoprotein) molecular is detected by using the pre-modified GENA biosensor chip is used to detect the low-concentration AFP.
2. Simulation and discussion
The sensing characteristics of the GENA were simulated by using the Finite Difference Time Domain (FDTD) software (Lumerical Inc., Vancouver, Canada). As illustrated in Fig. 1, the schematic of the GENA arranged in a two-dimension square lattice. The thickness of gold film is t. The period of nanohole array is P. The minor (x-direction) and major (y-direction) axes of nanohole are b and a, respectively. In our simulation model, anti-symmetric, symmetric and perfectly matched layer boundary conditions are adopted in the x, y and z boundaries in the simulation domain, respectively. A plane wave source polarizing along the minor axes of the GENA (x direction) is normally incident from the substrate of the GENA. The minimum mesh size is four nanometers. Above the GENA, a frequency domain profile monitor is used to collect the transmission spectrum through the GENA. The substrate is quartz (nsub = 1.463). Meanwhile, the refractive index of gold is modeled by fitting to the experimental values.
The refractive index sensitivity of the GENA biosensor was calculated by DI water and aqueous NaCl solution with different concentrations (5%, 10% and 15%). The refractive indexes of DI water and aqueous NaCl solution (5%, 10% and 15%) are 1.3333, 1.3418, 1.3505 and 1.3594, respectively. Figure 2(a) displays the GENA transmission spectra of the four testing samples, in which one resonance peak has obvious red-shifted from 793nm to 810nm. The peak wavelength shift is magnified in the insert of Fig. 2(a). In Fig. 2(b), the fitting curve of the wavelength data with medium refractive index demonstrates good linearity. It is used for calculating the sensitivity of the biosensor, i.e., the value of the variation of the peak wavelength divided by the variation of the refractive index is a constant coefficient S (, here is the wavelength shift of transmission peaks with the refractive index changing of medium). Then the S of the transmission peak is 625 nm/RIU.
In order to analyze the modes in transmission spectra of the GENA, firstly, the simulation model is modified to meet the condition that the GENA is surrounded by the same medium, i.e., without the substrate. The structures surrounded by the DI water (nd = 1.333) and quartz (nd = 1.463) are analyzed in the simulation model. In Fig. 3(a), the red and black lines correspond to the medium of DI water and quartz, respectively. Three obvious troughs are observed in the two transmission spectra. Based on the surface plasmon polaritons bloch wave (SPP-BW) theory, the three troughs are considered as SPP-BW (1, 0) water (quartz)/gold, (1, 1) water (quartz)/gold and (2, 0) water (quartz)/gold order modes, respectively . The blue line in the Fig. 3(a) is the transmission spectrum of the GENA with quartz substrate in DI water. Four troughs are observed in the spectrum. There are three troughs located between the SPP-BW modes of the red line (DI water) and the black line (quartz) in the Fig. 3(a), respectively. These SPP-BW modes are excited on the water/gold and quartz/gold interfaces of the GENA with quartz substrate. Therefore, six SPP-BW modes in the transmission spectrum should be excited. However, the excitation wavelengths of the same order SPP-BW mode of the two interfaces are close to each other and appear as one, such as the (1, 0) water/gold and (1, 0) quartz/gold modes form the (1, 0) GENA mode. This is the reason only three SPP-BW order modes existing in the transmission spectrum. The three troughs are considered as SPP-BW (1, 0) GENA, (1, 1) GENA and (2, 0) GENA order mode. Besides the mentioned three modes, another trough appears at wavelength 830 nm. The trough is due to the SPPs coupling at the water/gold and quartz/metal interfaces. The SPPs coupling of the GENA are decided by the refractive indexes of the medium and substrate. The refractive index difference of the medium and substrate would lead to structural symmetry-broken of the upper and lower interfaces and the SPPs at the both interfaces couple to induce the coupled mode [23, 24]. The resonance peak of SPP-BW (1, 1) GENA order mode is split into two peaks due to the SPPs coupling. The shorter wavelength peak is mainly influenced by the refractive index of medium which is near to the peak of the SPP-BW (1, 1) water/gold order mode, while the longer one is mainly influenced by the refractive index of substrate which is near to the peak of the SPP-BW (1, 1) quartz/gold order mode. Therefore, the shorter wavelength peak (SWP) and the longer wavelength peak are corresponding to the peaks of the SPPs coupling on the medium/GENA and substrate/GENA interfaces, respectively.
Figure 3(b) shows the simulated transmission spectra of the GENA surrounded by the medium of DI water and aqueous NaCl solution. The peak of the SPP-BW (1, 1) medium/gold order mode is red shifted with the nd increasing. The S of this peak is 600 nm/RIU which is a little less than that of the GENA with quartz substrate, in which S is 625 nm/RIU. Some other work demonstrated that the S of the MN was increased with the medium and MN contact area increasing [18, 24]. But for the GENA model, the S will be a little decreasing with the quartz substrate removed. The phenomenon is ascribed to the coupled mode disappearing. The GENA without substrate rarely could be fabricated. Therefore, the coupled mode of the GENA with quartz substrate could ensure the S non-decreasing. Figures 3(c) and 3(d) show the transmission spectra map of the GENA with and without the quartz substrate for nd tuned from 1.33 to 1.39, respectively. In these figures, high and low transmittance are scaled with color in red and blue. The white arrows show each mode. The two figures clearly show the coupled mode is excited in the GENA with substrate, but not excited without substrate. All the peaks of the modes red-shift with the nd increasing. Among these peaks, the shift of the SWP is largest. Besides the high sensitivity, the SWP of the GENA on the quartz substrate is narrower than the GENA without substrate. The narrower peak width at half height leads to the lower detection limit of the GENA biosensor [13, 14].
Besides the quartz substrate, some other substrates are usually adopted to fabricate the MN, such as ITO, Al2O3 and SiN, whose refractive indexes are 1.74, 1.76 and 2, respectively. Figure 4(a) shows the simulation transmission spectra of the GENA on the four substrates in the DI water. It clearly shows that the 794 nm SWPs are weak when the substrate is ITO, Al2O3 or SiN. Figures 4(b) to 4(e) are the calculated electric field intensity distribution of the GENA XZ cross-section with substrate of quartz, Al2O3, SiN and ITO at 794 nm, respectively. Figure 4(b) shows the electric field distribution on the GENA with quartz substrate. The strong and localized electric field intensity manifest the enhanced coupling of the SPPs at both interfaces. And the coupling is larger and more localized than that on other substrates. Therefore, the SWP of the GENA on quartz substrate is higher than that on other substrate material. This phenomenon is attributed to the refractive indexes difference between the medium and the substrate. The intensity of the coupled mode would be enhanced as the refractive index difference become less . Then, the SWP would be stronger. The small weak SWPs of the GENA on ITO, Al2O3 and SiN substrate are more difficult to detect in the experiment. In contrast, the SWP of the GENA on the quartz substrate can be easily measured in the experiment for biosensor. The quartz substrate is the more suitable substrate for the GENA with fine sensing characteristics.
3.1 Fabrication of the GENA biosensor chip
Based on the simulation and analysis, the suitable integrated PDMS microfluidic GENA biosensors were fabricated. Figure 5 is a schematic diagram of the detailed fabrication processes of GENA and PDMS microfluidics. The fabrication of the GENA started with the 5 nm Ti (adhesive layer) and 60 nm Au successively deposited on the quartz glass (2 cm2cm) by electron beam evaporation (EBE). Next, the GENA were patterned by EBL and transferred to gold film by using ion milling. Then, the gold film around the GENA were etched by overlay photolithography.
The PDMS was utilized to fabricate the microfluidics through mold replication technology. The fabrication processes of PDMS microfluidic were described as following: (1) the silicon mold was fabricated by photolithography followed by a deep reactive ion etching; (2) the PDMS prepolymer (Sylgard-184, Dow Corning) was prepared by thoroughly mixing the PDMS curing agent with the PDMS base monomer (mass-ratio 1:10), poured onto the silicon mold, and cured in the furnace at 80°C for 60 min; (3) Fully cured PDMS microfluidic was peeled off from the silicon mold and two holes were punched to be the outlet/inlet; (4) the PDMS microfluidic was cleaned and treated with O2 plasma for PDMS-quartz bonding with the quartz substrate.
3.2 Measurement of the GENA biosensor chip
The optical transmission spectral measurement system is shown in Fig. 6. The test process of the GENA biosensor is briefly described as follow. A light beam from a broadband tungsten halogen source (Ocean Optics HL-2000) was collimated by a collimator and successively goes through a Glan-Taylor polarizer, a silver reflecting mirror and then was focused on the pattern of GENA sample by a long-working-distance 10 × objective. The sample stage was employed to realize precise movement of the GENA sensor chip. The solution was injected to the surface of the GENA sensor chip by the micro pump. The transmission light beam was collected and focused by another long-working-distance 10 × objective and then split into two beams by a splitter, which were detected by a CCD camera and the spectrometer. The location of the light spot on the sample could be observed by the CCD camera. Meanwhile, the transmission light signal from the detection surface was collected by the detection fibers, which were connected with a spectrometer (Ocean QE6500). The transmission spectrum of the GENA biosensor was obtained by commercial signal processing software (Spectra Suits, Ocean Optics).
Figure 7(a) shows the scanning electron microscope (SEM) image of one fabricated GENA with p = 600 nm, t = 60 nm, a = 470 nm and b = 410 nm. The refractive index S of the chip was determined by measuring transmission spectra of the GENA biosensor chip in the DI water and NaCl aqueous solution. Figure 7(b) is the transmission spectra. All the transmission spectra are normalized and smoothed. Obviously, the wavelengths of the SWPs are red shift as nd increase. Figure 7(c) shows a linear shift of the SWPs wavelengths with nd increasing. A linear fit (R2 of 0.994) of the data sets shows a refractive index S of 549 nm/RIU. The experimental S we obtained is about 76 nm/RIU less than that of the simulation, which might be caused by the non-uniformity of the GENA and the rough surface of the gold film induced from the fabrication process, such as Au EBE, EBL and ion milling [16, 19]. The transmission spectra rely on the structural parameters of the GENA. The different transmission spectra of the non-uniformity GENAs are overlapped to be the measured transmission spectra which become broader . The surface roughness of the gold film could cause stronger plasmon coupling to the scattering losses, which leads to shorter length of the plasmon propagation and broader peak width at half height of the transmission spectra .
The GENA has potential application in label-free detecting of the chemical and biomedical molecules. The pre-modified GENA could be used to detect the label-free antigen and antibody in the biomedicine diagnosis. In our experiment, AFP was chosen to demonstrate that the GENA capabilities in detecting antigen-antibody interaction. AFP is a biomarker for hepatocellular carcinoma. If the AFP concentration in the blood is beyond 20 ng/ml, the human would have a high risk of hepatocellular carcinoma . Therefore it is meaningful to detect the low-concentration AFP.
The detection of the low-concentration AFP using GENA chip is a multi-steps process. The detection process is as follows: (1) dithiodiglycolic acid (Sigma) with 2mM aqueous solution was pumped into the chamber and reacting for 30 min; (2) the aqueous solution of mixed 0.4 M EDC (1-ethyl-3 -[3-dimethylaminopropyl] carbodiimide hydrochloride, Sigma) and 0.1 M S-NHS (sulfo-N-hydroxysuccinimide, Sigma) at a 1:1 volume ratio was injected into the chamber and incubated for 30 min, then the carboxyl groups on dithiodiglycolic acid were activated; (3) the solution in the chamber was thoroughly rinsed with 0.01 M PBS (phosphate buffered saline, Sigma) and then dried by N2 blowing; (4) monoclonal human AFP (α-fetoprotein) antibodies (Tianjin Medical University) were bonded to the surface of the GENA biosensor by pumping 0.01 M PBS through chemical reaction between amidogen on the alkaline aminophenol of the AFP antibodies and the active carboxyl; (5) to eliminate the nonspecific binding on the detection surface, redundant active carboxyl groups were enclosed by 1 M ethanolamine (Sigma) aqueous solution; (6) the human AFP (Tianjin Medical University) were bonded to the AFP antibodies by pumping 0.01 M PBS through specific binding.
Figure 7(d) shows the SWP shift from 1.50 nm to 4.48 nm as AFP concentration is raised from 25 ng/ml to 100 ng/ml. The inset figure shows the SWP shift 1.50 nm after 25 ng/ml AFP interacted with the GENA biosensor. These results indicate that the GENA offers a state-of-the-art biosensor chip for label-free immunoreaction testing.
The GENA biosensor chip, which works with the coupled mode, is designed, simulated, fabricated and characterized. The coupled mode SWP of the GENA biosensor chips shows a higher sensitivity than that of the other SPP-BW modes. The SWP sensitivity of the GENA on the quartz substrate is a little higher than that of the GENA without substrate, although the interface area of the GENA on the quartz substrate and medium is smaller. For owning the stronger and more localized electric field distribution, the GENA on the quartz substrate has better biosensing characteristics than that on other common substrate materials. The coupled mode SWP of the fabricated GENA biosensor chip shows a high S which reaches 549 nm/RIU. The liver cancer maker AFP of 25 ng/ml has been detected by the GENA biosensor chip, it is much closed to the clinic concentration in the biomedicine diagnosis. It is believed that the GENA biosensor chip has potential applications in health care, clinical diagnostics, environmental monitoring, especially, cancer biomarker testing.
The authors gratefully acknowledge financial support by fund from the National Key Basic Research Program of China (Nos. 2011CB933102 and 2011CB933203), the Nation Natural Foundation of China (Grant Nos. 61378058 and 61335010), China Postdoctoral Science Foundation (No. 2014M550796) and Science and Technology Research Funding of State Cultural Relics Bureau (No. 20110135).
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