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Novel multilayered porous silicon-based immunosensor for determining Hydroxysafflor yellow A
Applied Surface Science 257 (2011) 1906–1910
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Novel multilayered porous silicon-based immunosensor for determining Hydroxysafflor yellow A Xiaoyi Lv a , Jiaqing Mo a , Tao Jiang b , Furu Zhong b , Zhenhong Jia b,∗ , Jiangwei Li c , Fuchun Zhang c School of Electronic and Information Engineering, Xi an Jiaotong University, Xi an 710049, PR China College of Information Science and Engineering, Xinjiang University, Urumqi 830046, PR China c Key Laboratory of Xinjiang Biological Resources and Gene Engineering, College of Life Sciences and Technology, Xinjiang University, Urumqi 830046, PR China a
b
a r t i c l e
i n f o
Article history: Received 2 April 2010 Received in revised form 12 September 2010 Accepted 12 September 2010 Available online 25 September 2010 Keywords: Porous silicon Immunosensor Polybasic structure Hydroxysafflor yellow A
a b s t r a c t External random factors have a great influence on the fabrication of accurate photonic crystal, especially porous silicon-based photonic crystals. Compared with the binary photonic crystal, polybasic structure photonic crystal shows more stability and smaller effect of the random fluctuation. In this paper, we have fabricated a novel simple porous silicon polybasic Bragg’s mirror combined with excellent specific antigen–antibody inmunoreaction as an immunosensor for determining Hydroxysafflor yellow A (HSYA), which is the main chemical component of Carthamus tinctorius L. The binding of HSYA and the polyclonal anti-HSYA antibodies causes red shifts in the reflection spectrum of the sensor, and the red shift was proportional to the HSYA concentration with linear relationship ranging from 1 to 3 g mL−1 with a detection limit of 0.78 ng mL−1 . Importantly, this research offers hope for development of a commercial porous silicon-based immunosensor for component determination of C. tinctorius L. or other antigens. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hydroxysafflor yellow A (HSYA), i.e., the main chemical component of Carthamus tinctorius L. (molecular formula: C27 H32 O16 ), has been used extensively for treatment of cerebrovascular and cardiovascular diseases [1]. The level of HSYA serves as an important index of the quality of C. tinctorius L. and corresponding medications. Therefore, methods of rapid determination of HSYA are desired. The traditional method for determining HSYA, high-performance liquid chromatography (HPLC), is a sensitive but inconvenient technique which requires expensive equipment. By contrast, immunoassay techniques based on specific antigen–antibody reactions are sensitive yet inexpensive, such as enzyme-linked immunosorbent assay (ELISA), and fluorescence-labeled antibody assay, and thus can serve as alternative methods for determining HSYA. However, these common immunoassay methods are not label-free and require much time for HSYA detection. In recent years, multilayered porous silicon (PSi) (so-called PSi photonic crystal) has been employed as an immunosensor platform [2,3]. The fabrication of PSi photonic crystal including Bragg’s mirrors, rugate filters and microcavities is an easy way to periodically alter the etching current. Multilayered PSi-based biosensor offers many of the advantages both of PSi and photonic crystal sensors, such as its high surface area, its ease of
∗ Corresponding author. Tel.: +86 991 8582410; fax: +86 991 8580288. E-mail addresses: [email protected] (X. Lv), [email protected] (Z. Jia). 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.09.024
preparation, label-free procedures and compatibility with standard microelectronics processing. Furthermore, multilayered PSi has realized several polytype quasiregular structures with many useful applications, especially as biochemical sensors, such as Thue-morse, Rudin-Shapiro and Fibonacci multilayer [4,5]. PSi polytype quasiregular structures can be easily obtained and exhibit many interesting optical properties. However, many external random factors produced from the electrochemical process in the fabrication of PSi photonic crystal have a great influence on the fabrication of accurate PSi photonic crystal. This problem limits the commercial application of PSi photonic crystal. Wang et al. demonstrated that a simple polybasic photonic crystal made of three kinds of dielectric mediums shows more stability and smaller influence of the disorder compared to the binary photonic crystals [6]. This research inspired us to make superior PSi photonic crystal. In our lab, we have already synthesized the artificial immunogen of HSYA [7] and in this paper, we report a novel simple PSi polybasic Bragg’s mirror following the (n1 n2 n3 )m sequence with more stability as an immunosensor platform, where n1 , n2 and n3 represent different refractive index layers, respectively. We observed the changes of the reflectivity spectrum before and after the antigen–antibody reaction, and as a hapten, HSYA was for the first time directly determined as an antigen by multilayered PSi-based optical biosensor. This research lays the foundation for judging the quality of medicament and developing sensitive labelfree optical immunosensors for determining any other antigen or antibody.
X. Lv et al. / Applied Surface Science 257 (2011) 1906–1910
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tive index of n1 , n2 , and n3 is 2.1, 1.6 and 1.2, respectively, by using Bruggemann’s effective medium theory [7,11]. Layer thickness in the experiment was controlled by etching time, where L10 , L20 , and L30 are 300 nm, 733 nm, and 1300 nm. In Fig. 2 we see a reflectivity band centered around 7 m. For simplicity, L1i is chosen to be L10 , L2i is L20 , and L3i can be chosen as an random series which is produced by MatLab with Gaussian distribution around L30 = 1300 nm. Fig. 2 shows the reflection spectra of PSi polybasic multilayer with D = 0.0%, 0.5%, 1.0% and 2.8% respectively using the transfer matrix method [12]. We can see clearly from Fig. 2 that there is little change in band structure with different degrees of disorder, in accordance with the conclusion in Ref. [6], while the reflective spectra of disordered one-dimensional binary photonic crystals shows a remarkable change in Ref. [8]. Consequently, PSi photonic crystal with polybasic structure effectively lessens the impact of the disturbance from electrochemical etching. 2.2. Fabrication The PSi immunosensor samples were prepared from highly p-type silicon ( = 0.01 cm) by electrochemical etching in an electrolyte solution (49%HF: ethanol = 1:1, in volume) and PSi polybasic multilayer structure was fabricated following the (n1 n2 n3 )6 sequence. The different refractive index layers were obtained by using Labview to alternate the anodization current for different etching times. The n1 layers were formed at a current density of 20.0 mA/cm2 for 30 s, n2 at 40.0 mA/cm2 for 10 s, and n3 at 80.0 mA/cm2 for 15 s with a 10 s pause after each layer formation [13]. After that, 10 mL of a 0.5 mM KOH solution was applied to the PSi chip for 20 min to increase the pore diameter for accommodating the antibody [2]. 2.3. Functionalization and optical data acquisition
Fig. 1. Schematic cross-section of PSi polybasic multilayer.
2. Materials and methods 2.1. PSi polybasic Bragg’s mirror structure Fig. 1 shows a schematic cross-section of PSi polybasic multilayer consisting of three porosity PSi layers following the (n1 n2 n3 )m sequence, where n1 , n2 and n3 represent different refractive index layers, respectively; L1 , L2 , and L3 represent layer thickness; and m is the total number of periods. Disorder produced by outside random factors is an important factor in the fabrication of photonic crystals, especially PSi-based photonic crystals, because disorder greatly influences accuracy. According to Refs. [6,8], disorder D is defined as follows:
D=
m n2 (L i=1 1 1i
− L10 )2 + n22 (L2i − L20 )2 + n23 (L3i − L30 )2 m(n1 L10 + n2 L20 + n3 L30 )
,
(1)
where i varies from 1 to m; L1i , L2i and L3i are the real thickness of three PSi layers; L10 , L20 and L30 are the average thickness of three PSi layers; and m is 6 in our experiment. By choosing the thickness parameter of Eq. (1) randomly in a Gaussian distribution, in Ref. [6] the authors demonstrated that one-dimensional polybasic photonic crystal made of three kinds of dielectric mediums shows more stability and smaller influence of the disorder compared to binary photonic crystals. Due to the low values of absorption coefficients in the infrared region of PSi, especially at wavelengths longer than 1 m [9,10], herein, we determine from the experiment that the actual refrac-
HSYA is a hapten but does not possess immunogenicity, so it must attach to a large carrier such as a BSA as artificial immunogen to obtain the polyclonal anti-HSYA antibody. In the previous work, HSYA-BSA, i.e. the HSYA artificial antigen, was synthesized successfully by the immediate coupling method and the polyclonal anti-HSYA antibody was produced [7]. All the freshly etched sensors were exposed to H2 O2 for 2 h to stabilize the PSi by oxidizing the surface, and also to form a silica-like internal surface as a necessary step for the subsequent biosensing functionalization. After this treatment, the samples were treated with standard coupling chemistry by using aminopropyltriethoxysilane (APTES) and glutaraldehyde as described in detail elsewhere [3,7,14]. Then, some PSi samples were immersed in HSYA antibody solution with a dilution of 1:400, while others were immersed in negative serum for comparison. Next, all chips were incubated at 37 ◦ C for 2 h for the immobilisation of HSYA antibody. To prevent nonspecific adsorption, the chips were exposed to 3% OVA for 2 h. Finally, each sensor was immersed in a series of concentrations of target HSYA for 2 h at 37 ◦ C respectively and rinsing with PBST buffer. After each step, the reflectivity spectra of the sample were taken by Fourier transformed infrared spectroscopic microscopy (FTIR, Brucker VERTEX70) with a resolution of 0.01 nm, which is similar to that used in Ref. [9]. 3. Results and discussion The pore size of P+ wafers usually is about 5–20 nm in diameter, which is too small to accommodate large molecules such as antibodies. DeLouise et al. have demonstrated that dilute KOH postetching can successfully increase the pore size without destroying the PSi multilayer structure [2]. Fig. 3 shows pores larger than 60 nm in diameter, wide enough for HSYA antibodies to infiltrate easily.
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Fig. 2. Reflection spectra for PSi photonic crystal with polybasic structure (D = 0.0%, 0.5%, 1.0% and 2.8%).
The chemical reaction was analyzed by FTIR with a middle infrared (MIR) source. In Fig. 4(A), the initial sample shows Si–H bonds around 2088 cm−1 and 2115 cm−1 [15] (scissors at 918 cm−1 , wag at 632 and 613 cm−1 ) [16,17]. After oxidation, the sample displayed a broad band around 3432 cm−1 because of the Si–OH bonds on the surface [15,18], as well as a band around 1086 cm−1 due to Si–O bond formation [15]. In Fig. 4(B), after the silanization process, the sample showed small bands at 3357 cm−1 and 3285 cm−1 , which are attributed to –NH bonds. The –CH (at 2939 cm−1 )
Fig. 3. Top view SEM image of multilayered PSi-based immunosensor.
groups are also well evident [15]. After the GA treatment, the –NH bonds disappeared [15] while the imine-C N bonds at 1647 cm−1 , characteristic of the reaction with APTES, are easily recognized [19,20]. Control experiments were performed by using negative serum instead of polyclonal anti-HSYA antibodies (positive serum). Fig. 5 shows that there is no detectable shift after exposure to HSYA of 5 g/mL with negative serum. This indicates that the red-shift of the reflectance spectrum is due to selective antigen–antibody binding. The experimental stop band is narrower than the theoretical one, which is responsible for the lower ratio of refractive indices caused by oxidation. Fig. 6 shows that the red-shift of the reflectance spectrum as a function of the HSYA concentration owing to the thickness of PSi pore wall was increased by the coupling of biological molecules, followed by an increase of the refractive index of the PSi layer [21]. The concentration of HSYA solution between 0.01 and 5 g mL−1 in Fig. 6 and the sensor displays a good linear relationship in the range 1–3 g mL−1 with a correlation coefficient of 0.995 approximating to real concentration ranges of vascular effect [22]. In addition, the sensitivity of 12.78 nm g−1 mL−1 was calculated by estimating the slope. As the resolution of device is 0.01 nm, the lowest detection limit of the immunosensor is 0.01 nm/12.78 nm g−1 mL−1 = 0.78 ng mL−1 [23]. The reproducibility of the immunosensor was also good, with a relative standard deviation of 0.01 for 6 experiments with 1 g mL−1 HSYA
X. Lv et al. / Applied Surface Science 257 (2011) 1906–1910
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Fig. 4. FTIR of PSi immunosensor at various stages of immobilization. (A) (a) Before any treatment; (b) after oxidation. (B) (a) Silanized sample; (b) GA activated sample.
Fig. 5. Reflection spectra for multilayered PSi-based immunosensor with antigen (5 g/mL), (a) A 150.4 nm shift was detected after the chip was exposed to the positive serum; (b) no shift upon exposure of multilayered PSi-based immunosensor to negative serum.
solution, and a red-shift of 101.1 nm, 102.3 nm, 100.4 nm, 103.3 nm, 99.8 nm, and 101.7 nm at HSYA concentrations of 1 g mL−1 . In our previous work, ELISA technique experiments for the detection of HSYA showed a linear relationship range from 0.25 to 2 g/mL with theoretical lowest detection limit of 0.25 g/mL [24]. Yang et al. determined that the calibration curve was linear (R2 = 1) in the range of 2.0–100 g/mL for HSYA by HPLC [25]. Each of the three techniques has advantages. Our sensor is labelfree, has a relatively lower detection limit and shorter detection time compared to conventional ELISA techniques, and is more convenient and simpler than HPLC. Experiments are in progress to enhance the effectiveness of PSi-based immunosensor for detecting HSYA. In conclusion, we have successfully experimentally fabricated and demonstrated a label-free immunosensor based on a novel superior PSi multilayer structure for the detection of HSYA. As an immunosensor platform, our sensor is simple and label-free, has large surface area and requires a shorter detection time compared to conventional ELISA techniques. This platform can not only be used for the detection of HSYA, but can also be extended to other biological materials. Thus, the door can be opened for the development of label-free immunosensors and applications in the rapid and sensitive determination of small molecules as well.
Fig. 6. Dose–response curve. Optical variation as a function of the HSYA concentration.
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Acknowledgements This work was supported by the financial support by the National Natural Science Foundation of China (No. 60968002) and Program for New Century Excellent Talents in University of the PR China (Grant number: NCET-05-0897)and by Scientific Research Project for Universities in Xinjiang (Grant number: XJEDU2006I10). References [1] X. Wei, H. Liu, X. Sun, F. Fu, X. Zhang, J. Wang, J. An, H. Ding, Neurosci. Lett. 386 (2005) 58–62. [2] L.A. DeLouise, P.M. Kou, B.L. Miller, Anal. Chem. 77 (2005) 3222–3230. [3] H. Ouyang, LA. DeLouise, B.L. Miller, P.M. Fauchet, Anal. Chem. 79 (2007) 1502–1506. [4] L. Moretti, L. Rea, L. De Stefano, I. Rendina, Appl. Phys. Lett. 90 (2007), 1911121–1191112-3. [5] V. Agarwal, J. Escorcia-Garcia, E. Miguel, Mora-Ramos, Phys. Stat. Sol.(a) 5 (2007) 1367–1371. [6] X. Wang, L. Fang, Y. Ke-zhu, J. Optoelectron. Laser 15 (2004) 104–107. [7] X. Lü, G. Qing, M. Xiang, Z. Jia, R. Li, F. Zhong, J. Li, F. Zhang, Chem. J. Chin. U 30 (2009) 2381–2386. [8] H. Li, H. Chen, X. Qiu, Physica B 279 (2000) 164–167. [9] E. Xifré Pérez, L.F. Marsal, J. Ferré-Borrull, T. Trifonov, J. Pallarès, Physica E 38 (2007) 172–175.
[10] L. De Stefano, L. Moretti, I. Rendina, A.M. Rossi, Sens. Actuators A 104 (2003) 179–182. [11] M. Qian, X.Q. Bao, L.W. Wang, X. Lu, J. Shao, X.S. Chen, J. Cryst. Growth 292 (2006) 347–350. [12] E. Xifré Pérez, T. Trifonov, J. Pallarès, L.F. Marsal, Phys. Stat. Sol. (c) 9 (2005) 3466–3470. [13] Z. Wang, J. Zhang, S.H. Xu, L. Wang, Z. Cao, P. Zhan, Z.L. Wang, J. Phys. D: Appl. Phys. 40 (2007) 4482–4484. [14] G. Rong, N. Ali, J.E. Sipe, S.M. Weiss, Biosens. Bioelectron. 23 (2008) 1572–1576. [15] R.E. Fernandez, E. Bhattacharya, A. Chadha, Appl. Surf. Sci. 254 (2008) 4512–4519. [16] C.A. Canaria, I.N. Lees, A.W. Wun, G.M. Miskelly, M.J. Sailor, Inorg. Chem. Commun. 5 (2002) 560–564. [17] J. Chapron, S.A. Alekseev, V. Lysenko, V.N. Zaitsev, D. Barbier, Sens. Actuators B 120 (2007) 706–711. [18] H.L. Li, A.P. Fu, D.S. Xu, G.L. Guo, L.L. Gui, Y.Q. Tang, Langmuir 18 (2002) 3198–3202. [19] L. De Stefano, A. Lamberti, L. Rotiroti, M. De Stefano, Acta Biomater. 4 (2008) 126–130. [20] E. De Tommasi, L. De Stefano, I. Rea, V. Di Sarno, L. Rotiroti, P. Arcari, A. Lamberti, C. Sanges, I. Rendina, Sensors 8 (2008) 6549–6556. [21] O. Huimin, C. Christopher, Striemer, M. Philippe, Fauchet, Appl. Phys. Lett. 88 (2006), 163108-1–163108-3. [22] L. Zhang, G. Shen, J. Zhang, P. Nie, J. Zhi, J. Shanghai Jiaotong University (Medical Science) 29 (2009) 431–434. [23] G. Rong, J.D. Ryckman, R.L. Mernaugh, S.M. Weiss, Appl. Phys. Lett. 93 (2008), 161109-1–161109-3. [24] S. Hou, L.v. Fang, S. You-hong, F. Zhang, J. Li, Food Sci. 30 (2009) 150–154. [25] R. Yang, J. Liu, Guangdong Chem. Indus. 34 (2007) 112–114.
Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Novel multilayered porous silicon-based immunosensor for determining Hydroxysafflor yellow A Xiaoyi Lv a , Jiaqing Mo a , Tao Jiang b , Furu Zhong b , Zhenhong Jia b,∗ , Jiangwei Li c , Fuchun Zhang c School of Electronic and Information Engineering, Xi an Jiaotong University, Xi an 710049, PR China College of Information Science and Engineering, Xinjiang University, Urumqi 830046, PR China c Key Laboratory of Xinjiang Biological Resources and Gene Engineering, College of Life Sciences and Technology, Xinjiang University, Urumqi 830046, PR China a
b
a r t i c l e
i n f o
Article history: Received 2 April 2010 Received in revised form 12 September 2010 Accepted 12 September 2010 Available online 25 September 2010 Keywords: Porous silicon Immunosensor Polybasic structure Hydroxysafflor yellow A
a b s t r a c t External random factors have a great influence on the fabrication of accurate photonic crystal, especially porous silicon-based photonic crystals. Compared with the binary photonic crystal, polybasic structure photonic crystal shows more stability and smaller effect of the random fluctuation. In this paper, we have fabricated a novel simple porous silicon polybasic Bragg’s mirror combined with excellent specific antigen–antibody inmunoreaction as an immunosensor for determining Hydroxysafflor yellow A (HSYA), which is the main chemical component of Carthamus tinctorius L. The binding of HSYA and the polyclonal anti-HSYA antibodies causes red shifts in the reflection spectrum of the sensor, and the red shift was proportional to the HSYA concentration with linear relationship ranging from 1 to 3 g mL−1 with a detection limit of 0.78 ng mL−1 . Importantly, this research offers hope for development of a commercial porous silicon-based immunosensor for component determination of C. tinctorius L. or other antigens. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Hydroxysafflor yellow A (HSYA), i.e., the main chemical component of Carthamus tinctorius L. (molecular formula: C27 H32 O16 ), has been used extensively for treatment of cerebrovascular and cardiovascular diseases [1]. The level of HSYA serves as an important index of the quality of C. tinctorius L. and corresponding medications. Therefore, methods of rapid determination of HSYA are desired. The traditional method for determining HSYA, high-performance liquid chromatography (HPLC), is a sensitive but inconvenient technique which requires expensive equipment. By contrast, immunoassay techniques based on specific antigen–antibody reactions are sensitive yet inexpensive, such as enzyme-linked immunosorbent assay (ELISA), and fluorescence-labeled antibody assay, and thus can serve as alternative methods for determining HSYA. However, these common immunoassay methods are not label-free and require much time for HSYA detection. In recent years, multilayered porous silicon (PSi) (so-called PSi photonic crystal) has been employed as an immunosensor platform [2,3]. The fabrication of PSi photonic crystal including Bragg’s mirrors, rugate filters and microcavities is an easy way to periodically alter the etching current. Multilayered PSi-based biosensor offers many of the advantages both of PSi and photonic crystal sensors, such as its high surface area, its ease of
∗ Corresponding author. Tel.: +86 991 8582410; fax: +86 991 8580288. E-mail addresses: [email protected] (X. Lv), [email protected] (Z. Jia). 0169-4332/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2010.09.024
preparation, label-free procedures and compatibility with standard microelectronics processing. Furthermore, multilayered PSi has realized several polytype quasiregular structures with many useful applications, especially as biochemical sensors, such as Thue-morse, Rudin-Shapiro and Fibonacci multilayer [4,5]. PSi polytype quasiregular structures can be easily obtained and exhibit many interesting optical properties. However, many external random factors produced from the electrochemical process in the fabrication of PSi photonic crystal have a great influence on the fabrication of accurate PSi photonic crystal. This problem limits the commercial application of PSi photonic crystal. Wang et al. demonstrated that a simple polybasic photonic crystal made of three kinds of dielectric mediums shows more stability and smaller influence of the disorder compared to the binary photonic crystals [6]. This research inspired us to make superior PSi photonic crystal. In our lab, we have already synthesized the artificial immunogen of HSYA [7] and in this paper, we report a novel simple PSi polybasic Bragg’s mirror following the (n1 n2 n3 )m sequence with more stability as an immunosensor platform, where n1 , n2 and n3 represent different refractive index layers, respectively. We observed the changes of the reflectivity spectrum before and after the antigen–antibody reaction, and as a hapten, HSYA was for the first time directly determined as an antigen by multilayered PSi-based optical biosensor. This research lays the foundation for judging the quality of medicament and developing sensitive labelfree optical immunosensors for determining any other antigen or antibody.
X. Lv et al. / Applied Surface Science 257 (2011) 1906–1910
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tive index of n1 , n2 , and n3 is 2.1, 1.6 and 1.2, respectively, by using Bruggemann’s effective medium theory [7,11]. Layer thickness in the experiment was controlled by etching time, where L10 , L20 , and L30 are 300 nm, 733 nm, and 1300 nm. In Fig. 2 we see a reflectivity band centered around 7 m. For simplicity, L1i is chosen to be L10 , L2i is L20 , and L3i can be chosen as an random series which is produced by MatLab with Gaussian distribution around L30 = 1300 nm. Fig. 2 shows the reflection spectra of PSi polybasic multilayer with D = 0.0%, 0.5%, 1.0% and 2.8% respectively using the transfer matrix method [12]. We can see clearly from Fig. 2 that there is little change in band structure with different degrees of disorder, in accordance with the conclusion in Ref. [6], while the reflective spectra of disordered one-dimensional binary photonic crystals shows a remarkable change in Ref. [8]. Consequently, PSi photonic crystal with polybasic structure effectively lessens the impact of the disturbance from electrochemical etching. 2.2. Fabrication The PSi immunosensor samples were prepared from highly p-type silicon ( = 0.01 cm) by electrochemical etching in an electrolyte solution (49%HF: ethanol = 1:1, in volume) and PSi polybasic multilayer structure was fabricated following the (n1 n2 n3 )6 sequence. The different refractive index layers were obtained by using Labview to alternate the anodization current for different etching times. The n1 layers were formed at a current density of 20.0 mA/cm2 for 30 s, n2 at 40.0 mA/cm2 for 10 s, and n3 at 80.0 mA/cm2 for 15 s with a 10 s pause after each layer formation [13]. After that, 10 mL of a 0.5 mM KOH solution was applied to the PSi chip for 20 min to increase the pore diameter for accommodating the antibody [2]. 2.3. Functionalization and optical data acquisition
Fig. 1. Schematic cross-section of PSi polybasic multilayer.
2. Materials and methods 2.1. PSi polybasic Bragg’s mirror structure Fig. 1 shows a schematic cross-section of PSi polybasic multilayer consisting of three porosity PSi layers following the (n1 n2 n3 )m sequence, where n1 , n2 and n3 represent different refractive index layers, respectively; L1 , L2 , and L3 represent layer thickness; and m is the total number of periods. Disorder produced by outside random factors is an important factor in the fabrication of photonic crystals, especially PSi-based photonic crystals, because disorder greatly influences accuracy. According to Refs. [6,8], disorder D is defined as follows:
D=
m n2 (L i=1 1 1i
− L10 )2 + n22 (L2i − L20 )2 + n23 (L3i − L30 )2 m(n1 L10 + n2 L20 + n3 L30 )
,
(1)
where i varies from 1 to m; L1i , L2i and L3i are the real thickness of three PSi layers; L10 , L20 and L30 are the average thickness of three PSi layers; and m is 6 in our experiment. By choosing the thickness parameter of Eq. (1) randomly in a Gaussian distribution, in Ref. [6] the authors demonstrated that one-dimensional polybasic photonic crystal made of three kinds of dielectric mediums shows more stability and smaller influence of the disorder compared to binary photonic crystals. Due to the low values of absorption coefficients in the infrared region of PSi, especially at wavelengths longer than 1 m [9,10], herein, we determine from the experiment that the actual refrac-
HSYA is a hapten but does not possess immunogenicity, so it must attach to a large carrier such as a BSA as artificial immunogen to obtain the polyclonal anti-HSYA antibody. In the previous work, HSYA-BSA, i.e. the HSYA artificial antigen, was synthesized successfully by the immediate coupling method and the polyclonal anti-HSYA antibody was produced [7]. All the freshly etched sensors were exposed to H2 O2 for 2 h to stabilize the PSi by oxidizing the surface, and also to form a silica-like internal surface as a necessary step for the subsequent biosensing functionalization. After this treatment, the samples were treated with standard coupling chemistry by using aminopropyltriethoxysilane (APTES) and glutaraldehyde as described in detail elsewhere [3,7,14]. Then, some PSi samples were immersed in HSYA antibody solution with a dilution of 1:400, while others were immersed in negative serum for comparison. Next, all chips were incubated at 37 ◦ C for 2 h for the immobilisation of HSYA antibody. To prevent nonspecific adsorption, the chips were exposed to 3% OVA for 2 h. Finally, each sensor was immersed in a series of concentrations of target HSYA for 2 h at 37 ◦ C respectively and rinsing with PBST buffer. After each step, the reflectivity spectra of the sample were taken by Fourier transformed infrared spectroscopic microscopy (FTIR, Brucker VERTEX70) with a resolution of 0.01 nm, which is similar to that used in Ref. [9]. 3. Results and discussion The pore size of P+ wafers usually is about 5–20 nm in diameter, which is too small to accommodate large molecules such as antibodies. DeLouise et al. have demonstrated that dilute KOH postetching can successfully increase the pore size without destroying the PSi multilayer structure [2]. Fig. 3 shows pores larger than 60 nm in diameter, wide enough for HSYA antibodies to infiltrate easily.
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Fig. 2. Reflection spectra for PSi photonic crystal with polybasic structure (D = 0.0%, 0.5%, 1.0% and 2.8%).
The chemical reaction was analyzed by FTIR with a middle infrared (MIR) source. In Fig. 4(A), the initial sample shows Si–H bonds around 2088 cm−1 and 2115 cm−1 [15] (scissors at 918 cm−1 , wag at 632 and 613 cm−1 ) [16,17]. After oxidation, the sample displayed a broad band around 3432 cm−1 because of the Si–OH bonds on the surface [15,18], as well as a band around 1086 cm−1 due to Si–O bond formation [15]. In Fig. 4(B), after the silanization process, the sample showed small bands at 3357 cm−1 and 3285 cm−1 , which are attributed to –NH bonds. The –CH (at 2939 cm−1 )
Fig. 3. Top view SEM image of multilayered PSi-based immunosensor.
groups are also well evident [15]. After the GA treatment, the –NH bonds disappeared [15] while the imine-C N bonds at 1647 cm−1 , characteristic of the reaction with APTES, are easily recognized [19,20]. Control experiments were performed by using negative serum instead of polyclonal anti-HSYA antibodies (positive serum). Fig. 5 shows that there is no detectable shift after exposure to HSYA of 5 g/mL with negative serum. This indicates that the red-shift of the reflectance spectrum is due to selective antigen–antibody binding. The experimental stop band is narrower than the theoretical one, which is responsible for the lower ratio of refractive indices caused by oxidation. Fig. 6 shows that the red-shift of the reflectance spectrum as a function of the HSYA concentration owing to the thickness of PSi pore wall was increased by the coupling of biological molecules, followed by an increase of the refractive index of the PSi layer [21]. The concentration of HSYA solution between 0.01 and 5 g mL−1 in Fig. 6 and the sensor displays a good linear relationship in the range 1–3 g mL−1 with a correlation coefficient of 0.995 approximating to real concentration ranges of vascular effect [22]. In addition, the sensitivity of 12.78 nm g−1 mL−1 was calculated by estimating the slope. As the resolution of device is 0.01 nm, the lowest detection limit of the immunosensor is 0.01 nm/12.78 nm g−1 mL−1 = 0.78 ng mL−1 [23]. The reproducibility of the immunosensor was also good, with a relative standard deviation of 0.01 for 6 experiments with 1 g mL−1 HSYA
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Fig. 4. FTIR of PSi immunosensor at various stages of immobilization. (A) (a) Before any treatment; (b) after oxidation. (B) (a) Silanized sample; (b) GA activated sample.
Fig. 5. Reflection spectra for multilayered PSi-based immunosensor with antigen (5 g/mL), (a) A 150.4 nm shift was detected after the chip was exposed to the positive serum; (b) no shift upon exposure of multilayered PSi-based immunosensor to negative serum.
solution, and a red-shift of 101.1 nm, 102.3 nm, 100.4 nm, 103.3 nm, 99.8 nm, and 101.7 nm at HSYA concentrations of 1 g mL−1 . In our previous work, ELISA technique experiments for the detection of HSYA showed a linear relationship range from 0.25 to 2 g/mL with theoretical lowest detection limit of 0.25 g/mL [24]. Yang et al. determined that the calibration curve was linear (R2 = 1) in the range of 2.0–100 g/mL for HSYA by HPLC [25]. Each of the three techniques has advantages. Our sensor is labelfree, has a relatively lower detection limit and shorter detection time compared to conventional ELISA techniques, and is more convenient and simpler than HPLC. Experiments are in progress to enhance the effectiveness of PSi-based immunosensor for detecting HSYA. In conclusion, we have successfully experimentally fabricated and demonstrated a label-free immunosensor based on a novel superior PSi multilayer structure for the detection of HSYA. As an immunosensor platform, our sensor is simple and label-free, has large surface area and requires a shorter detection time compared to conventional ELISA techniques. This platform can not only be used for the detection of HSYA, but can also be extended to other biological materials. Thus, the door can be opened for the development of label-free immunosensors and applications in the rapid and sensitive determination of small molecules as well.
Fig. 6. Dose–response curve. Optical variation as a function of the HSYA concentration.
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Acknowledgements This work was supported by the financial support by the National Natural Science Foundation of China (No. 60968002) and Program for New Century Excellent Talents in University of the PR China (Grant number: NCET-05-0897)and by Scientific Research Project for Universities in Xinjiang (Grant number: XJEDU2006I10). References [1] X. Wei, H. Liu, X. Sun, F. Fu, X. Zhang, J. Wang, J. An, H. Ding, Neurosci. Lett. 386 (2005) 58–62. [2] L.A. DeLouise, P.M. Kou, B.L. Miller, Anal. Chem. 77 (2005) 3222–3230. [3] H. Ouyang, LA. DeLouise, B.L. Miller, P.M. Fauchet, Anal. Chem. 79 (2007) 1502–1506. [4] L. Moretti, L. Rea, L. De Stefano, I. Rendina, Appl. Phys. Lett. 90 (2007), 1911121–1191112-3. [5] V. Agarwal, J. Escorcia-Garcia, E. Miguel, Mora-Ramos, Phys. Stat. Sol.(a) 5 (2007) 1367–1371. [6] X. Wang, L. Fang, Y. Ke-zhu, J. Optoelectron. Laser 15 (2004) 104–107. [7] X. Lü, G. Qing, M. Xiang, Z. Jia, R. Li, F. Zhong, J. Li, F. Zhang, Chem. J. Chin. U 30 (2009) 2381–2386. [8] H. Li, H. Chen, X. Qiu, Physica B 279 (2000) 164–167. [9] E. Xifré Pérez, L.F. Marsal, J. Ferré-Borrull, T. Trifonov, J. Pallarès, Physica E 38 (2007) 172–175.
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