- Email: [email protected]
Micro-array detection system for gene expression products based on surface plasmon resonance imaging
Sensors and Actuators B 91 (2003) 133–137
Micro-array detection system for gene expression products based on surface plasmon resonance imaging Yu Xinglonga,*, Wang Dongshenga, Wang Dingxina, Ou yang Jian Huab, Yan Ziboa, Dong Yongguia, Liao Weib, Zhao Xin shengb a
b
Department of Precision Instrument, Tsinghua University, Beijing 100084, PR China State Key Laboratory of Molecular Dynamic and Stable Structures, Department of Chemical Biology, CCME, Peking University, Beijing 100871, PR China
Abstract A kind of micro-array detecting system based on SPR imaging has been developed. The light source of this system is a semiconductor laser. The light emitted from the source becomes round and parallel after going through a pinhole and being collimated. This parallel light beam arrives at a polarizer, its s-light component is shielded and p-light component passes through. The p-light component which is of a rectangular shape after going through a rectangular slip is cast onto the glass surface of a sensing chip, and generates an evanescent wave which excites surface plasmon resonance (SPR). The SPR images are accepted by a 2D CCD camera and processed in real time by a computer. The chips are made into bare gold film array by photoetching and cause the linker layer to cover the gold thin film by self-assembling. Then, rabbit IgG is immobilized onto the liker layer, which finished the preparation of array sensing chip. Once the sensing chip is installed, the detecting system begins the investigation of the interaction between rabbit IgG and its antibody. The experimental results indicate that the 2D SPR biosensor is able to detect a number of units in real time for gene expression products. The improvement of technology and the increase of array number can realize high throughput detection, which is significant to proteomics study and new drug discovery and development. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Biosensor; Biomolecular interaction; Surface plasmon resonance (SPR); Micro-array
1. Introduction Surface plasmon resonance (SPR) imaging is an optical technique for measuring the surface sensitivity of affinity coupling of label-free biomolecules. Based on the principle of evanescent wave, it can measure the change of both the refractivity and the thickness of the absorbed layer up to nanometer level, and has found applications in both chemistry and biology [1]. At present, the most popularly used detecting method is single-channel measuring method, which has high precision and can be used for real-time monitoring but is not efficient enough. With the completion of the Human Genome Project and the development of biotechnology, it is required to improve the efficiency and achieve high throughput detection particularly for the drug screening and the forthcoming research on proteomics. Therefore, the SPR-based high throughput measuring technique has become a hot research *
Corresponding author. Tel.: þ86-10-62782454; fax: þ86-10-62784691. E-mail address: [email protected] (Y. Xinglong).
topic. In 1991, ESA Stenberg et al. developed a four-channel measuring method [2], which was later commercialized by Biacore Company. In 1998, based on Stenberg’s studies, Berger et al. realized a much more efficient 4 4 array measurement by processing the sensing chips in four files and making the sensing layer perpendicular to the sample channel when used for testing [3]. In 2000, when studying surface plasma interferometry, Nikitin et al. found that this technique opened up a new channel for micro-array biosensing with a potential of achieving high throughput analyses [4]. In the same year, by employing a polypyrrole-based surface functionalization based on an electrospoting process on gold surface and surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time, Philippe et al. realized real-time monitoring of parallel label-free hybridization experiments, which first introduced the real-time protein interaction studies [5]. In 2001, Hye et al. generated micro-jet channels on gold surface to form a 2D array and monitored DNA–DNA, RNA–DNA, and protein–DNA interaction up to nanomolar concentration by using SPR imaging measurements of DNA array fabricated on gold surface [6]. In the same year, based on spectroscopic
0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0925-4005(03)00077-7
134
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
imaging, O’Brien et al. developed a new SPR array sensor platform which can compensate for changes in bulk index of refraction of the solution containing the analysts due to effects of the change of temperature or solute concentration on sensor chips [7]. Also in that year, based on the combination of parallel sensing channel architecture and spectral discrimination of sensing channels, Homola et al. reported that their measuring method can more accurately quantify biomolecular interaction [8]. In addition, HTS Biosystems Co. has used the grating-coupled SPR technology to make a 2D array [9]. These researches have been undertaken in various areas to enhance the development of the SPR-based micro-array. In general, they can be categorized into three main methods including: (1) multi-channel method based on SPR imaging and a polypyrrole-based surface functionalization based on an electrospoting process on gold surface; (2) optical interferometry method, including optical heterodyne and spectroscopic imaging; (3) grating-coupled method, including optical waveguides. All these methods have their own advantages, among which the imaging approach attracts more attention due to its simple structure. We describe a micro-array measuring system based on SPR imaging. With a uniform parallel light source and micro-array chips, it acquires information with a CCD camera for real-time signal processing. With this system, an interaction study on rabbit IgG and its antibody has been conducted. Experimental results show that the above system can acquire real-time information in high efficiency with a simple structure. After further improvements, it has the potential in application in diagnosis, discovering and developing new drugs.
2. Experiment set-up and micro-array chip preparation 2.1. Experiment set-up The experiment set-up is as shown in Fig. 1. It consists of a semi-conductor laser, 1; a pinhole, 2; an outer cylinder, 3; a polarizer, 4; an attenuater, 5; a rectangular slip, 6; a triangular prism, 7; a micro-array chip, 8; a flow channel, 9; a 2D CCD camera, 10 (MINTRON ENTERPRISE CO. LTD, 12V1E); and a computer, 11. The visual A/D con-
Fig. 1. Micro-array detection system based on SPR imaging.
verter of the image card (Beijing Join Hope Image Technology LTD, OK_M10F) in the computer 11 is 8 bit. The light from semi-conductor laser, 1, becomes parallel after going through the light pinhole, 2, and the outer cylinder, 3, with a circular facular. The polarizer, 4, is adjusted so that the p-light component can pass through while the s-light component cannot. The attenuater, 5, acts to attenuate the light output from polarizer, 4, to a proper intensity to obtain a clear CCD image and a strong gray scale contract for image processing. A diaphragm, 6, serves to change the aligned image spot into a rectangular one to ensure that it is fully projected onto the CCD, 10, target plane. The light beam from the diaphragm, 6, passes through the prism, 7, and is projected onto the interface of the prism, 7, and the micro-array chip, 8. In order to offset the effect of the air gap between the prism, 7, and the micro-array chip, 8, a kind of oil of the same refractivity as the prism and the substrate of the micro-array chip, 8, is filled in the interface. The incoming light is reflected from the interface, passes through the prism, and is projected on the CCD, 10, target plane. When the substance under analysis flows through the micro-channel, 9, the antigen in the sample combines with the antibody immobilized on the micro-array chip, 8, resulting in the change in the reflected light, i.e. the change of the gray scale and the position of the SPR image. The computer, 11, is used for real-time data acquisition and processing. 2.1.1. Fabrication of bare gold film array on the glass substrate Grids are created by using the photoetching technology in the positive photoresist, which is spin-coated onto the glass substrate. Consequently, the glass slide surface isolation caused by the photoetched grids forms the array on the glass slide. Then, a thin (about 2 nm) chromium film followed by a 45 nm gold film is vapor deposited onto the array surface. After finishing the positive photoresist, we accomplish the fabrication of bare gold film array. 2.1.2. Linker layer self-assemble on bare gold film array surface The wafer is cleaned by ultrasonically rinsing with acetone for 5 min twice, and with methanol for 5 min twice. Then, the wafer is dipped into the ultra-pure water and dried naturally. After that, the wafer is oxidized in a 7:3 concentrated H2SO4:30%H2O2 for 15 min at 90 8C, followed by thorough rinsing with ultra-pure water. The wafer is immersed in absolute alcohol before the assembly of MUA monolayer in 1 mM MUA of absolute alcohol solution for about 72 h. After the assembly, the wafer is rinsed with absolute alcohol three times and dried at 120 8C for 15 min. 2.1.3. Immobilizing antibody on linker layer The MUA assembled surface is activated in 10 mM EDC, 1 mM NHS and 25 mM MES solution for 1 h at 4 8C. The
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
activated MUA surface is dipped into PBS for a while to get rid of the extra activators. As quickly as possible, we transfer them into a 0.1 mg/ml rabbit IgG antibody solution, and keep them in an incubator at 37 8C for 1 h. After the incubation, the samples are softly shaked in PBS at 37 8C twice, and each time for 10 min. Then a 1% BSA is used to block the possibly adsorbing site of the surface for 30 min at 37 8C, the samples are softly shaked in PBS at 37 8C twice, and each time for 10 min again. Surfaces treated by antibodies are kept from drying in a PBS at room temperature for further tests. Fig. 2 is a schematic diagram of micro-array sensing chip architecture.
3. Experiment and results 3.1. Process of experiment First of all, the angle of incidence should be adjusted to approach the resonance angle. Therefore, we performed the
Fig. 2. Schematic diagram of micro-array sensing chip architecture.
Fig. 4. The 2D SPR images: (a) before, and (b) after associating.
Fig. 3. SPR images of single channel: (a) before, and (b) after associating.
135
Fig. 5. Detection result from single channel.
136
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
single channel experiment to find the resonance angle, which is the same as that of the 2D array experiment, so that we can adjust the angle of incidence. After the incident light beam was oriented and the sensing chip was assembled into the detection system, the process of the experiment was recorded as follows. At the beginning of the experiment, the wash buffer PBS was injected to clean the sensing surface; at the same time, the intensity distribution of the reflected light was recorded by the computer as the reference point. Then, the 1:30 goat– anti-rabbit IgG sample solution was slowly injected after the injection of 1 ml PBS. The reaction was monitored in real time. When the reaction reached saturation, 1 ml PBS was injected to substitute the sample solution. Successively, the
elution buffer was injected, until the result curve returned to the state before reaction. 3.2. Image signal process 3.2.1. Single channel image processing The SPR images before and after association are shown, respectively, in Fig. 3(a) and (b). By comparing them, we can note that the shapes of the images are just the same, except that the position of the black stripe has changed and moves a small distance to the left. The method for processing is: read the gray-scale values of every pixel of the same column and calculate the weighted average of them. Then, compare all the weighted averages of every column in the
Fig. 6. The 2D detection results during the: (a) associating; and (b) dissociating periods.
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
frame to get the minimum of all the weighted averages. After that, convert the X-coordinate of the point whose gray-scale value is minimum to the corresponding SPR angle and draw the point in the coordinate of ySPR-times in real time. 3.2.2. 2D image signal process Fig. 4(a) and (b), respectively, show the SPR images before and after association. The shapes of the images are just the same, but their gray scale has changed—the gray scale after association is lower than the one before association. The method of processing the image signal is: process every unit in the array in real time. Read the gray-scale values of every pixel of the same section and calculate the weighted average of them; then, take the weighted average as the Y-coordinate of a point and draw the point in the coordinate of ySPR-times in real time. Based on a great deal of experiments, a transformation relationship between grayscale and resonance angle and the ySPR-times curve can be drawn. 3.3. Results Fig. 5 shows the curve of single channel detection for interaction between rabbit IgG and its antibody. From the plot of Fig. 5, we can see that the association time is about 3 s and the deflection of the resonance angle is 18, but the dissociation time is about 2 s. From the result of the 2D array detection for interaction between rabbit IgG and its antibody as shown in Fig. 6, it can be seen that the association time is about 4 s and the gray level difference reaches 2 levels. In addition, it can be also seen that different curve changes at different time due to different arrival time of the sample. For example, a4 lags behind a1. From Fig. 6(b), we can see that dissociation time is about 3 s and dissociations in various regions are still asynchronous with the same influence of the sample arrival order.
4. Conclusion We have developed a micro-array detection system based on SPR imaging. The system has two characteristics: (1) The light beam is generated by the semi-conductor laser and becomes round and parallel through pinholefiltering and collimating. When it passes through the polarizer, s-light component is disabled and p-light component is maintained. After being shaped into a rectangular one through a rectangle-raster, the beam
137
arrives at the surface of the micro-array chip and generates evanescent wave, which causes surface plasmon resonance. (2) The manufacturing process of the micro-array sensing chip: (2.1) First generate bare gold film array on glass substrate by photoetching and vapor deposition. (2.2) Then create the linker layer on bare gold film array surface with self-assemble. (2.3) Finally, immobilize the rabbit IgG on the liker layer. It is concluded from experiments that the sensitivity of angle scanning of single channel or many parallel channels is greater than that of gray scale detection at the array. In order to improve the sensitivity of the latter, high-precision A/D may be applied such as a 10- or 12-bit A/D and new calculating methods should be developed.
Acknowledgements This study is supported by the National Natural Science Foundation of China and with the Tsinghua University Funds for Basic Researches.
References [1] R.S. Sethi, Transducer aspects of biosensors, Biosens. Bioelectron. 9 (3) (1994) 243–264. [2] E. Stenberg, B. Persson, H. Roos, C. Urbaniczky, Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proceins, J. Colloid Interface Sci. 143 (2) (1991) 513–526. [3] C.E.H. Berger, T.A.M. Beumer, R.P.H. Kooyman, Surface plasmon resonance multisensing, Anal. Chem. 70 (4) (1998) 703–706. [4] P.I. Nikitin, A.N. Grrigorenko, A.A. Beloglazov et al., Surface plasmon resonance interferometry for micro-array biosensing, Sens. Actuators B 85 (2000) 189–193. [5] P. Guedon, T. Livache, F. Martiu et al., Characterization and optimization of a real-time, parallel, lable-free, polypyrrole-based DNA sensor by surface plasmon resonance imaging, Anal. Chem. 72 (24) (2000) 6003–6009. [6] L.J. Hye, G.T. Terry, C.M. Robert, SPR imaging measurements of 1D and 2D DNA microarray created from macrofluidic channels on gold thin films, Anal. Chem. 73 (22) (2001) 5525–5531. [7] M.J. O’Brien II, V.H. Perez-Luna, S.R.J. Brueck et al., Surface plasmon resonance array biosensor-based on spectroscopic imaging, Biosens. Bioelectron. 16 (2001) 97–108. [8] J. Homola, H.B. Lu, G.G. Nenninger et al., A novel multichannel surface plasmon resonance biosensor, Sens. Actuators B 76 (2001) 403–410. [9] www.htsbiosystems.com.
Micro-array detection system for gene expression products based on surface plasmon resonance imaging Yu Xinglonga,*, Wang Dongshenga, Wang Dingxina, Ou yang Jian Huab, Yan Ziboa, Dong Yongguia, Liao Weib, Zhao Xin shengb a
b
Department of Precision Instrument, Tsinghua University, Beijing 100084, PR China State Key Laboratory of Molecular Dynamic and Stable Structures, Department of Chemical Biology, CCME, Peking University, Beijing 100871, PR China
Abstract A kind of micro-array detecting system based on SPR imaging has been developed. The light source of this system is a semiconductor laser. The light emitted from the source becomes round and parallel after going through a pinhole and being collimated. This parallel light beam arrives at a polarizer, its s-light component is shielded and p-light component passes through. The p-light component which is of a rectangular shape after going through a rectangular slip is cast onto the glass surface of a sensing chip, and generates an evanescent wave which excites surface plasmon resonance (SPR). The SPR images are accepted by a 2D CCD camera and processed in real time by a computer. The chips are made into bare gold film array by photoetching and cause the linker layer to cover the gold thin film by self-assembling. Then, rabbit IgG is immobilized onto the liker layer, which finished the preparation of array sensing chip. Once the sensing chip is installed, the detecting system begins the investigation of the interaction between rabbit IgG and its antibody. The experimental results indicate that the 2D SPR biosensor is able to detect a number of units in real time for gene expression products. The improvement of technology and the increase of array number can realize high throughput detection, which is significant to proteomics study and new drug discovery and development. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Biosensor; Biomolecular interaction; Surface plasmon resonance (SPR); Micro-array
1. Introduction Surface plasmon resonance (SPR) imaging is an optical technique for measuring the surface sensitivity of affinity coupling of label-free biomolecules. Based on the principle of evanescent wave, it can measure the change of both the refractivity and the thickness of the absorbed layer up to nanometer level, and has found applications in both chemistry and biology [1]. At present, the most popularly used detecting method is single-channel measuring method, which has high precision and can be used for real-time monitoring but is not efficient enough. With the completion of the Human Genome Project and the development of biotechnology, it is required to improve the efficiency and achieve high throughput detection particularly for the drug screening and the forthcoming research on proteomics. Therefore, the SPR-based high throughput measuring technique has become a hot research *
Corresponding author. Tel.: þ86-10-62782454; fax: þ86-10-62784691. E-mail address: [email protected] (Y. Xinglong).
topic. In 1991, ESA Stenberg et al. developed a four-channel measuring method [2], which was later commercialized by Biacore Company. In 1998, based on Stenberg’s studies, Berger et al. realized a much more efficient 4 4 array measurement by processing the sensing chips in four files and making the sensing layer perpendicular to the sample channel when used for testing [3]. In 2000, when studying surface plasma interferometry, Nikitin et al. found that this technique opened up a new channel for micro-array biosensing with a potential of achieving high throughput analyses [4]. In the same year, by employing a polypyrrole-based surface functionalization based on an electrospoting process on gold surface and surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time, Philippe et al. realized real-time monitoring of parallel label-free hybridization experiments, which first introduced the real-time protein interaction studies [5]. In 2001, Hye et al. generated micro-jet channels on gold surface to form a 2D array and monitored DNA–DNA, RNA–DNA, and protein–DNA interaction up to nanomolar concentration by using SPR imaging measurements of DNA array fabricated on gold surface [6]. In the same year, based on spectroscopic
0925-4005/03/$ – see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0925-4005(03)00077-7
134
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
imaging, O’Brien et al. developed a new SPR array sensor platform which can compensate for changes in bulk index of refraction of the solution containing the analysts due to effects of the change of temperature or solute concentration on sensor chips [7]. Also in that year, based on the combination of parallel sensing channel architecture and spectral discrimination of sensing channels, Homola et al. reported that their measuring method can more accurately quantify biomolecular interaction [8]. In addition, HTS Biosystems Co. has used the grating-coupled SPR technology to make a 2D array [9]. These researches have been undertaken in various areas to enhance the development of the SPR-based micro-array. In general, they can be categorized into three main methods including: (1) multi-channel method based on SPR imaging and a polypyrrole-based surface functionalization based on an electrospoting process on gold surface; (2) optical interferometry method, including optical heterodyne and spectroscopic imaging; (3) grating-coupled method, including optical waveguides. All these methods have their own advantages, among which the imaging approach attracts more attention due to its simple structure. We describe a micro-array measuring system based on SPR imaging. With a uniform parallel light source and micro-array chips, it acquires information with a CCD camera for real-time signal processing. With this system, an interaction study on rabbit IgG and its antibody has been conducted. Experimental results show that the above system can acquire real-time information in high efficiency with a simple structure. After further improvements, it has the potential in application in diagnosis, discovering and developing new drugs.
2. Experiment set-up and micro-array chip preparation 2.1. Experiment set-up The experiment set-up is as shown in Fig. 1. It consists of a semi-conductor laser, 1; a pinhole, 2; an outer cylinder, 3; a polarizer, 4; an attenuater, 5; a rectangular slip, 6; a triangular prism, 7; a micro-array chip, 8; a flow channel, 9; a 2D CCD camera, 10 (MINTRON ENTERPRISE CO. LTD, 12V1E); and a computer, 11. The visual A/D con-
Fig. 1. Micro-array detection system based on SPR imaging.
verter of the image card (Beijing Join Hope Image Technology LTD, OK_M10F) in the computer 11 is 8 bit. The light from semi-conductor laser, 1, becomes parallel after going through the light pinhole, 2, and the outer cylinder, 3, with a circular facular. The polarizer, 4, is adjusted so that the p-light component can pass through while the s-light component cannot. The attenuater, 5, acts to attenuate the light output from polarizer, 4, to a proper intensity to obtain a clear CCD image and a strong gray scale contract for image processing. A diaphragm, 6, serves to change the aligned image spot into a rectangular one to ensure that it is fully projected onto the CCD, 10, target plane. The light beam from the diaphragm, 6, passes through the prism, 7, and is projected onto the interface of the prism, 7, and the micro-array chip, 8. In order to offset the effect of the air gap between the prism, 7, and the micro-array chip, 8, a kind of oil of the same refractivity as the prism and the substrate of the micro-array chip, 8, is filled in the interface. The incoming light is reflected from the interface, passes through the prism, and is projected on the CCD, 10, target plane. When the substance under analysis flows through the micro-channel, 9, the antigen in the sample combines with the antibody immobilized on the micro-array chip, 8, resulting in the change in the reflected light, i.e. the change of the gray scale and the position of the SPR image. The computer, 11, is used for real-time data acquisition and processing. 2.1.1. Fabrication of bare gold film array on the glass substrate Grids are created by using the photoetching technology in the positive photoresist, which is spin-coated onto the glass substrate. Consequently, the glass slide surface isolation caused by the photoetched grids forms the array on the glass slide. Then, a thin (about 2 nm) chromium film followed by a 45 nm gold film is vapor deposited onto the array surface. After finishing the positive photoresist, we accomplish the fabrication of bare gold film array. 2.1.2. Linker layer self-assemble on bare gold film array surface The wafer is cleaned by ultrasonically rinsing with acetone for 5 min twice, and with methanol for 5 min twice. Then, the wafer is dipped into the ultra-pure water and dried naturally. After that, the wafer is oxidized in a 7:3 concentrated H2SO4:30%H2O2 for 15 min at 90 8C, followed by thorough rinsing with ultra-pure water. The wafer is immersed in absolute alcohol before the assembly of MUA monolayer in 1 mM MUA of absolute alcohol solution for about 72 h. After the assembly, the wafer is rinsed with absolute alcohol three times and dried at 120 8C for 15 min. 2.1.3. Immobilizing antibody on linker layer The MUA assembled surface is activated in 10 mM EDC, 1 mM NHS and 25 mM MES solution for 1 h at 4 8C. The
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
activated MUA surface is dipped into PBS for a while to get rid of the extra activators. As quickly as possible, we transfer them into a 0.1 mg/ml rabbit IgG antibody solution, and keep them in an incubator at 37 8C for 1 h. After the incubation, the samples are softly shaked in PBS at 37 8C twice, and each time for 10 min. Then a 1% BSA is used to block the possibly adsorbing site of the surface for 30 min at 37 8C, the samples are softly shaked in PBS at 37 8C twice, and each time for 10 min again. Surfaces treated by antibodies are kept from drying in a PBS at room temperature for further tests. Fig. 2 is a schematic diagram of micro-array sensing chip architecture.
3. Experiment and results 3.1. Process of experiment First of all, the angle of incidence should be adjusted to approach the resonance angle. Therefore, we performed the
Fig. 2. Schematic diagram of micro-array sensing chip architecture.
Fig. 4. The 2D SPR images: (a) before, and (b) after associating.
Fig. 3. SPR images of single channel: (a) before, and (b) after associating.
135
Fig. 5. Detection result from single channel.
136
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
single channel experiment to find the resonance angle, which is the same as that of the 2D array experiment, so that we can adjust the angle of incidence. After the incident light beam was oriented and the sensing chip was assembled into the detection system, the process of the experiment was recorded as follows. At the beginning of the experiment, the wash buffer PBS was injected to clean the sensing surface; at the same time, the intensity distribution of the reflected light was recorded by the computer as the reference point. Then, the 1:30 goat– anti-rabbit IgG sample solution was slowly injected after the injection of 1 ml PBS. The reaction was monitored in real time. When the reaction reached saturation, 1 ml PBS was injected to substitute the sample solution. Successively, the
elution buffer was injected, until the result curve returned to the state before reaction. 3.2. Image signal process 3.2.1. Single channel image processing The SPR images before and after association are shown, respectively, in Fig. 3(a) and (b). By comparing them, we can note that the shapes of the images are just the same, except that the position of the black stripe has changed and moves a small distance to the left. The method for processing is: read the gray-scale values of every pixel of the same column and calculate the weighted average of them. Then, compare all the weighted averages of every column in the
Fig. 6. The 2D detection results during the: (a) associating; and (b) dissociating periods.
Y. Xinglong et al. / Sensors and Actuators B 91 (2003) 133–137
frame to get the minimum of all the weighted averages. After that, convert the X-coordinate of the point whose gray-scale value is minimum to the corresponding SPR angle and draw the point in the coordinate of ySPR-times in real time. 3.2.2. 2D image signal process Fig. 4(a) and (b), respectively, show the SPR images before and after association. The shapes of the images are just the same, but their gray scale has changed—the gray scale after association is lower than the one before association. The method of processing the image signal is: process every unit in the array in real time. Read the gray-scale values of every pixel of the same section and calculate the weighted average of them; then, take the weighted average as the Y-coordinate of a point and draw the point in the coordinate of ySPR-times in real time. Based on a great deal of experiments, a transformation relationship between grayscale and resonance angle and the ySPR-times curve can be drawn. 3.3. Results Fig. 5 shows the curve of single channel detection for interaction between rabbit IgG and its antibody. From the plot of Fig. 5, we can see that the association time is about 3 s and the deflection of the resonance angle is 18, but the dissociation time is about 2 s. From the result of the 2D array detection for interaction between rabbit IgG and its antibody as shown in Fig. 6, it can be seen that the association time is about 4 s and the gray level difference reaches 2 levels. In addition, it can be also seen that different curve changes at different time due to different arrival time of the sample. For example, a4 lags behind a1. From Fig. 6(b), we can see that dissociation time is about 3 s and dissociations in various regions are still asynchronous with the same influence of the sample arrival order.
4. Conclusion We have developed a micro-array detection system based on SPR imaging. The system has two characteristics: (1) The light beam is generated by the semi-conductor laser and becomes round and parallel through pinholefiltering and collimating. When it passes through the polarizer, s-light component is disabled and p-light component is maintained. After being shaped into a rectangular one through a rectangle-raster, the beam
137
arrives at the surface of the micro-array chip and generates evanescent wave, which causes surface plasmon resonance. (2) The manufacturing process of the micro-array sensing chip: (2.1) First generate bare gold film array on glass substrate by photoetching and vapor deposition. (2.2) Then create the linker layer on bare gold film array surface with self-assemble. (2.3) Finally, immobilize the rabbit IgG on the liker layer. It is concluded from experiments that the sensitivity of angle scanning of single channel or many parallel channels is greater than that of gray scale detection at the array. In order to improve the sensitivity of the latter, high-precision A/D may be applied such as a 10- or 12-bit A/D and new calculating methods should be developed.
Acknowledgements This study is supported by the National Natural Science Foundation of China and with the Tsinghua University Funds for Basic Researches.
References [1] R.S. Sethi, Transducer aspects of biosensors, Biosens. Bioelectron. 9 (3) (1994) 243–264. [2] E. Stenberg, B. Persson, H. Roos, C. Urbaniczky, Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proceins, J. Colloid Interface Sci. 143 (2) (1991) 513–526. [3] C.E.H. Berger, T.A.M. Beumer, R.P.H. Kooyman, Surface plasmon resonance multisensing, Anal. Chem. 70 (4) (1998) 703–706. [4] P.I. Nikitin, A.N. Grrigorenko, A.A. Beloglazov et al., Surface plasmon resonance interferometry for micro-array biosensing, Sens. Actuators B 85 (2000) 189–193. [5] P. Guedon, T. Livache, F. Martiu et al., Characterization and optimization of a real-time, parallel, lable-free, polypyrrole-based DNA sensor by surface plasmon resonance imaging, Anal. Chem. 72 (24) (2000) 6003–6009. [6] L.J. Hye, G.T. Terry, C.M. Robert, SPR imaging measurements of 1D and 2D DNA microarray created from macrofluidic channels on gold thin films, Anal. Chem. 73 (22) (2001) 5525–5531. [7] M.J. O’Brien II, V.H. Perez-Luna, S.R.J. Brueck et al., Surface plasmon resonance array biosensor-based on spectroscopic imaging, Biosens. Bioelectron. 16 (2001) 97–108. [8] J. Homola, H.B. Lu, G.G. Nenninger et al., A novel multichannel surface plasmon resonance biosensor, Sens. Actuators B 76 (2001) 403–410. [9] www.htsbiosystems.com.