- Email: [email protected]
A CMOS active pixel sensor based DNA micro-array with nano-metallic particles detection protocol
Solid-State Electronics 49 (2005) 1933–1936 www.elsevier.com/locate/sse
A CMOS active pixel sensor based DNA micro-array with nano-metallic particles detection protocol Yijin Wang
a,b,*
, Chen Xu b, Jiong Li b, I-Ming Hsing
a,c
, Mansun Chan
a,b
a
b
Bioengineering Graduate Program, HKUST, Clear Water Bay, Hong Kong Department of Electrical and Electronic Engineering, HKUST, Clear Water Bay, Hong Kong c Department of Chemical Engineering, HKUST, Clear Water Bay, Hong Kong
Received 18 March 2005; received in revised form 14 September 2005; accepted 30 September 2005 Available online 14 November 2005
The review of this paper was arranged by Prof. S. Cristoloveanu
Abstract A DNA micro-array (DMA) for DNA detection is reported. The DMA combines a standard CMOS active pixel image sensor with a DNA detection protocol utilizing the binding of DNA targets and probes functionalized with gold nano-particles that can modify the opaqueness at the detection site. The DMA has been fabricated using a 0.5 lm CMOS process together with on-chip timing control and correlated double sampling. Experimental results show that the system can detect DNA samples with extremely low concentration down to 10 pM using ordinary light source. 2005 Elsevier Ltd. All rights reserved. Keywords: CMOS compatible; Adjustable dynamic range; High sensitivity
1. Introduction Biochip fabricated with micro-electronic technology has become a new powerful tool in molecular biology. In particular, high-density deoxyribonucleic acid (DNA) microarray can significantly reduce cost and time to perform complicated clinical diagnosis like DNA mutations responsible for some genetic diseases (such as cancers) and DNA sequence analysis for virus detection [1–5]. Currently, the most well developed DNA detection methodology is based on the modification of DNA sample with fluorescent labels so that DNA matched with a particular probe immobilized at a specific location of a solid support (e.g., glass slides) can be identified by the optical emission from the fluorescence label [6,7]. This method requires some expensive optical instruments to excite the fluorescent labels (such as laser
*
Corresponding author. Tel.: +852 23588842; fax: +852 23581458. E-mail address: [email protected] (Y. Wang).
0038-1101/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.sse.2005.09.015
or UV light sources) and to capture the output of the fluorescence (such as fluorescent microscope or array scanner with CCD camera). In addition, the signal intensity of the fluorescence quenches with time, which makes the experimental result depend on the transient response of the label [8]. Some efforts to combine fluorescence-based DNA detection with CCD camera have been reported [9], but the need of specialized excitation UV light source and color filter results in complication of the surface chemistry between the DNA material and the array. Besides fluorescent label, nano-metallic particle can also be used as labels. Compared with the fluorescence-based detection method, nano-particle based DNA detection method has the advantages of: (1) physical property (such as conductivity and opacity) is easier to be electronically detected; (2) signal is stable with time; (3) no external excitation needed. These three advantages lead to more consistent experimental results. Previous work using the conducting property of the metal particle has been proposed but it requires modifications to the CMOS process
1934
Y. Wang et al. / Solid-State Electronics 49 (2005) 1933–1936
to include inert metal (such as gold or platinum) and different surface passivation techniques, making it more difficult to be fabricated in ordinary IC foundries [10]. In this work, we have developed a DNA micro-array based on CMOS active pixel sensor (APS) together with the nano-metallic particle detection protocol. The DMA is fully compatible with the standard CMOS process and has been fabricated using a standard 0.5 lm CMOS technology. The experimental result is reported in this paper. 2. Chip design and fabrication The fabricated DMA is composed of active pixel sensors (APS). Each pixel consists of a photodiode for sensing and active pixel sensor for readout. Its schematic together with a cross-section of a detection cell is shown in Fig. 1. The active pixel sensor is in a traditional 3-transistor structure. The DNA probes are attached on the surface of the detection cell. The photodiode is formed with an n+ diffusion and p structure. A metal layer is used to create an optical window and guard ring to prevent any crosstalk from the neighboring pixels. The cell size is 15 · 20 lm2 with a fill factor of 50%. Furthermore, due to the elimination of direct contact of the metal layer to the biomaterial, we do not need to introduce the inert metal material as in our previous work [10]. This makes the DMA fully CMOS compatible and can be fabricated in any CMOS foundry. Unlike fluorescent detection, the nano-particle detection protocol does not require exciting light filtering and thus no on-chip filter is required. It allows the use of the chip surface as a direct solid support for the DNA hybridization. 3. Experimental procedures The post-fabrication experiment composed of two parts: (1) surface treatment of the chip to attach DNA probes for
detection; and (2) applying the sample DNA for detection. It is illustrated in Fig. 2 and described below in detail. 3.1. Surface treatment for DNA detection The top SiO2 passivation layer of the chip is modified by mercaptopropyltrimethoxysilane (MPTS) to form covalent bonding with the DNA probes [11]. The MPTS serves as a molecular bridge layer to chemically connect DNA to silicon dioxide layer. The methoxyl groups on one end of the MPTS molecule can react with SiOH groups on the SiO2 surface. On the other end, the thiol group could react with another thiol group, which was chemically labeled on the five-terminal of DNA probes. Different kinds of DNA probes with known sequences and 16mer selected thiol-modified oligonucleotides are attached to the chip surface by a pin spotter machine, micro-printing system (MicroSysTM 5100). Each spot size has a diameter of 100 lm and covers around 20 pixels to provide redundancy and reduce error reading. Some dummy rows/columns are left uncovered for background subtraction and dynamic range adjustment. Fig. 2(b) illustrates the condition of the chip after immobilizing the DNA probes. The chips are ready to be used for DNA detection. 3.2. DNA detection process The application of sample to the DMA is done by two hybridizations and one silver enhancement steps as shown in Fig. 2(c–e). In the first hybridization step, target DNA with special added tails is applied to the DMA in solution form by pipetting. The sample DNA that matches one of the immobilized probes is captured, while those mismatched DNA targets will be washed away. In the second hybridization step, the DMA is treated with a solution of
Fig. 1. The APS structure used for DNA detection.
Y. Wang et al. / Solid-State Electronics 49 (2005) 1933–1936
1935
Fig. 2. Illustration of detection. (a) the surface of the chip is coated with MPTS, (b) DNA probe immobilization, (c) DNA hybridization, (d) gold nanoparticle labeling, (e) silver enhancement. The length of DNA target sequence is 15 bases.
Fig. 3. The SEM pictures of the DNA chip surface after (a) 5 min and (b) 30 min silver enhancement showing that there is an increasing density of gold nano-particles.
140
700 600
800lux
100
500
80
400
525lux
60
300 307lux
40
200
197lux 102lux
20
100
52lux
0 0
1
Dark Current (fA)
120
Photocurrent (nA)
oligonucleotide-modified gold nano-particle that will attach to the special added tails of the DNA targets. The gold particles by themselves, however, are too sparse to be reliably detected. The size of gold nano-particle can be enlarged in silver enhancement step. The silver enhancement only takes place at the location with the presence of gold particles, which serve as a catalyst to initiate the silver deposition. Fig. 3 shows the SEM pictures at the site with matched DNA after 5 min and 30 min silver enhancement, indicating a successful enlargement of the nano-gold particle and increasing the opaqueness at the location with gold particles. For locations with mismatched DNA molecules, the imaging cell surface remains transparent to optical illumination. The transparency difference at location with matched and mismatched DNA molecule can be easily distinguished by the underlying APS array.
0 2
3
Bias Voltage (V) Fig. 4. I–V characteristic and dark current of a photodiode in the detection cell under different illumination.
4. Experimental result and discussion To study the feasibility of the combined nano-particle detection protocol with optical detection, the optical response from the sensor is first studied. The I–V characteristic and dark current of a photodiode before DNA attachment under different illumination was first measured by using a Precision Parameter Analyzer HP4156 and the results are shown in Fig. 4. The dark current is about six orders of magnitude below that of the optical current indi-
cating the sensor can provide sufficient dynamic range for optical detection. When matched and mismatched DNA samples are applied to the chip followed by the detection protocol described in Section 3, the outputs from the photodiode under illumination of ordinary room light are shown in Fig. 5 The difference in optical signal at location with matched and mismatched DNA is about two times, which
1936
Y. Wang et al. / Solid-State Electronics 49 (2005) 1933–1936
about the degree of matching. Nevertheless, the system is useful for many DNA detection applications.
900
Photocurrent (nA)
800 Mismatched DNA
700 600
Matched DNA
500 400 Match with 5min Match with 10min Mismatch with 5 min Mismatch with 10min
300 200 0.0
0.5
1.0
1.5
2.0
2.5
3.0
Bias Voltage (V)
Digitized Output from the CMOS APS
Fig. 5. Photocurrent comparison between match and mismatch DNA samples with two silver enhancement time.
300 250
5. Conclusion In this paper, a DNA micro-array detection compatible with standard CMOS technology is developed. This system is based on integrated APS to detect the change in opacity when an opaque nano-particle is deposited at the location with perfectly matched DNA samples. The protocol has been demonstrated by DNA micro-array fabricated with a standard 0.5 lm CMOS process. The optical property of the deposited nano-particles has been carefully studied and calibrated indicating a two-time difference can be measured under most optical spectrum in the visible light region. With the built-in dynamic range adjustment circuit, the DMA can work with a wide verity of light source with different intensity. The sensitivity of the system can be enhanced by using longer silver deposition time, and a low concentration of 10 pM can be detected.
Matched DNA
Acknowledgement
200 Single base mismatched DNA
This work is supported by an Earmarked Grant from the Research Grant Council of Hong Kong under the contract number HKUST6110/03E.
150 100 50
References
0
0
10
20
30
40
50
60
Address of the image sensor array Fig. 6. The relative intensity for DNA samples and background after 30 min silver enhancement. The length of DNA target sequence is 15 bases.
is sufficient for optical detection. It has been demonstrated that an extremely low DNA concentration down to 10 pM can be detected using the described method. In the fabricated DNA array, the optical current is converted to output voltage by the active pixel sensor (APS). After the A/D converter, the output is digitized into eight-bit digital data ready for digital signal processing. The digitized value after scanning a row of 64 pixels with different degree of DNA matching is given in Fig. 6. As indicated in the figure, the detection method is very sensitive, and the digital output for even a single base mismatch is already half that of the perfectly matched DNA. The noise margin provided in this method is more than sufficient for most digital circuits to give a reliable out to distinguish matched and mismatched DNA samples. One limitation of the system is it can only distinguish matched or mismatched DNA, but cannot give detail information
[1] Hacia JG, Collins FS. Mutational analysis using oligonucleotide microarrays. J Med Genet 1999;36(10):730–6. [2] Foldes-Papp Z, Egerer R, Birch-Hirschfeld E, Striebel HM, Demel U, Tilz GP, et al. Detection of multiple human herpes viruses by DNA microarray technology. Mol Diagn 2004;8(1):1–9. [3] Stremmel C, Wein A, Hohenberger W, Reingruber B. DNA microarrays: a new diagnostic tool and its implications in colorectal cancer. Int J Colorectal Dis 2002;17(3):131–6. [4] Anthony RM, Brown TJ, French GL. DNA array technology and diagnostic microbiology. Expert Rev Mol Diagn 2001;1(1): 30–8. [5] Todd R, Wong DT. DNA hybridization arrays for gene expression analysis of human oral cancer. J Dent Res 2002;81(2):89–97. [6] Glazer AN, Mathies RA. Energy-transfer fluorescent reagents for DNA analyses. Curr Opin Biotechnol 1997;8(1):94–102. [7] Luke S, Shepelsky M. FISH: recent advances and diagnostic aspects. Cell Vis 1998;5(1):49–53. [8] Song L, Hennink EJ, Young IT, Tanke HJ. Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys J 1995;68:2588–600. [9] Xue M, Xu W, Lu G, YIp N, KO PK, Chan M. Optical response of photodiode in integrated DNA detection system. Electron Devices Meeting, 2001, Proceedings, 2001 IEEE Hong Kong. [10] Li J, Xue M, Wang H, Cheng L, Gao L, Lu Z, et al. Amplifying the electrical hybridization signals of DNA array by multilayer assembly of Au nanoparticle probes. The Analyst 2003;128:917–23. [11] Rogers YH, Jiang-Baucom P, Huang ZJ, Bogdanov V, Anderson S, Boyce-Jacino MT. Immobilization of oligonucleotides onto a glass support via disulfide bonds: a method for preparation of DNA microarrays. Anal Biochem 1999;266:23–30.
A CMOS active pixel sensor based DNA micro-array with nano-metallic particles detection protocol Yijin Wang
a,b,*
, Chen Xu b, Jiong Li b, I-Ming Hsing
a,c
, Mansun Chan
a,b
a
b
Bioengineering Graduate Program, HKUST, Clear Water Bay, Hong Kong Department of Electrical and Electronic Engineering, HKUST, Clear Water Bay, Hong Kong c Department of Chemical Engineering, HKUST, Clear Water Bay, Hong Kong
Received 18 March 2005; received in revised form 14 September 2005; accepted 30 September 2005 Available online 14 November 2005
The review of this paper was arranged by Prof. S. Cristoloveanu
Abstract A DNA micro-array (DMA) for DNA detection is reported. The DMA combines a standard CMOS active pixel image sensor with a DNA detection protocol utilizing the binding of DNA targets and probes functionalized with gold nano-particles that can modify the opaqueness at the detection site. The DMA has been fabricated using a 0.5 lm CMOS process together with on-chip timing control and correlated double sampling. Experimental results show that the system can detect DNA samples with extremely low concentration down to 10 pM using ordinary light source. 2005 Elsevier Ltd. All rights reserved. Keywords: CMOS compatible; Adjustable dynamic range; High sensitivity
1. Introduction Biochip fabricated with micro-electronic technology has become a new powerful tool in molecular biology. In particular, high-density deoxyribonucleic acid (DNA) microarray can significantly reduce cost and time to perform complicated clinical diagnosis like DNA mutations responsible for some genetic diseases (such as cancers) and DNA sequence analysis for virus detection [1–5]. Currently, the most well developed DNA detection methodology is based on the modification of DNA sample with fluorescent labels so that DNA matched with a particular probe immobilized at a specific location of a solid support (e.g., glass slides) can be identified by the optical emission from the fluorescence label [6,7]. This method requires some expensive optical instruments to excite the fluorescent labels (such as laser
*
Corresponding author. Tel.: +852 23588842; fax: +852 23581458. E-mail address: [email protected] (Y. Wang).
0038-1101/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.sse.2005.09.015
or UV light sources) and to capture the output of the fluorescence (such as fluorescent microscope or array scanner with CCD camera). In addition, the signal intensity of the fluorescence quenches with time, which makes the experimental result depend on the transient response of the label [8]. Some efforts to combine fluorescence-based DNA detection with CCD camera have been reported [9], but the need of specialized excitation UV light source and color filter results in complication of the surface chemistry between the DNA material and the array. Besides fluorescent label, nano-metallic particle can also be used as labels. Compared with the fluorescence-based detection method, nano-particle based DNA detection method has the advantages of: (1) physical property (such as conductivity and opacity) is easier to be electronically detected; (2) signal is stable with time; (3) no external excitation needed. These three advantages lead to more consistent experimental results. Previous work using the conducting property of the metal particle has been proposed but it requires modifications to the CMOS process
1934
Y. Wang et al. / Solid-State Electronics 49 (2005) 1933–1936
to include inert metal (such as gold or platinum) and different surface passivation techniques, making it more difficult to be fabricated in ordinary IC foundries [10]. In this work, we have developed a DNA micro-array based on CMOS active pixel sensor (APS) together with the nano-metallic particle detection protocol. The DMA is fully compatible with the standard CMOS process and has been fabricated using a standard 0.5 lm CMOS technology. The experimental result is reported in this paper. 2. Chip design and fabrication The fabricated DMA is composed of active pixel sensors (APS). Each pixel consists of a photodiode for sensing and active pixel sensor for readout. Its schematic together with a cross-section of a detection cell is shown in Fig. 1. The active pixel sensor is in a traditional 3-transistor structure. The DNA probes are attached on the surface of the detection cell. The photodiode is formed with an n+ diffusion and p structure. A metal layer is used to create an optical window and guard ring to prevent any crosstalk from the neighboring pixels. The cell size is 15 · 20 lm2 with a fill factor of 50%. Furthermore, due to the elimination of direct contact of the metal layer to the biomaterial, we do not need to introduce the inert metal material as in our previous work [10]. This makes the DMA fully CMOS compatible and can be fabricated in any CMOS foundry. Unlike fluorescent detection, the nano-particle detection protocol does not require exciting light filtering and thus no on-chip filter is required. It allows the use of the chip surface as a direct solid support for the DNA hybridization. 3. Experimental procedures The post-fabrication experiment composed of two parts: (1) surface treatment of the chip to attach DNA probes for
detection; and (2) applying the sample DNA for detection. It is illustrated in Fig. 2 and described below in detail. 3.1. Surface treatment for DNA detection The top SiO2 passivation layer of the chip is modified by mercaptopropyltrimethoxysilane (MPTS) to form covalent bonding with the DNA probes [11]. The MPTS serves as a molecular bridge layer to chemically connect DNA to silicon dioxide layer. The methoxyl groups on one end of the MPTS molecule can react with SiOH groups on the SiO2 surface. On the other end, the thiol group could react with another thiol group, which was chemically labeled on the five-terminal of DNA probes. Different kinds of DNA probes with known sequences and 16mer selected thiol-modified oligonucleotides are attached to the chip surface by a pin spotter machine, micro-printing system (MicroSysTM 5100). Each spot size has a diameter of 100 lm and covers around 20 pixels to provide redundancy and reduce error reading. Some dummy rows/columns are left uncovered for background subtraction and dynamic range adjustment. Fig. 2(b) illustrates the condition of the chip after immobilizing the DNA probes. The chips are ready to be used for DNA detection. 3.2. DNA detection process The application of sample to the DMA is done by two hybridizations and one silver enhancement steps as shown in Fig. 2(c–e). In the first hybridization step, target DNA with special added tails is applied to the DMA in solution form by pipetting. The sample DNA that matches one of the immobilized probes is captured, while those mismatched DNA targets will be washed away. In the second hybridization step, the DMA is treated with a solution of
Fig. 1. The APS structure used for DNA detection.
Y. Wang et al. / Solid-State Electronics 49 (2005) 1933–1936
1935
Fig. 2. Illustration of detection. (a) the surface of the chip is coated with MPTS, (b) DNA probe immobilization, (c) DNA hybridization, (d) gold nanoparticle labeling, (e) silver enhancement. The length of DNA target sequence is 15 bases.
Fig. 3. The SEM pictures of the DNA chip surface after (a) 5 min and (b) 30 min silver enhancement showing that there is an increasing density of gold nano-particles.
140
700 600
800lux
100
500
80
400
525lux
60
300 307lux
40
200
197lux 102lux
20
100
52lux
0 0
1
Dark Current (fA)
120
Photocurrent (nA)
oligonucleotide-modified gold nano-particle that will attach to the special added tails of the DNA targets. The gold particles by themselves, however, are too sparse to be reliably detected. The size of gold nano-particle can be enlarged in silver enhancement step. The silver enhancement only takes place at the location with the presence of gold particles, which serve as a catalyst to initiate the silver deposition. Fig. 3 shows the SEM pictures at the site with matched DNA after 5 min and 30 min silver enhancement, indicating a successful enlargement of the nano-gold particle and increasing the opaqueness at the location with gold particles. For locations with mismatched DNA molecules, the imaging cell surface remains transparent to optical illumination. The transparency difference at location with matched and mismatched DNA molecule can be easily distinguished by the underlying APS array.
0 2
3
Bias Voltage (V) Fig. 4. I–V characteristic and dark current of a photodiode in the detection cell under different illumination.
4. Experimental result and discussion To study the feasibility of the combined nano-particle detection protocol with optical detection, the optical response from the sensor is first studied. The I–V characteristic and dark current of a photodiode before DNA attachment under different illumination was first measured by using a Precision Parameter Analyzer HP4156 and the results are shown in Fig. 4. The dark current is about six orders of magnitude below that of the optical current indi-
cating the sensor can provide sufficient dynamic range for optical detection. When matched and mismatched DNA samples are applied to the chip followed by the detection protocol described in Section 3, the outputs from the photodiode under illumination of ordinary room light are shown in Fig. 5 The difference in optical signal at location with matched and mismatched DNA is about two times, which
1936
Y. Wang et al. / Solid-State Electronics 49 (2005) 1933–1936
about the degree of matching. Nevertheless, the system is useful for many DNA detection applications.
900
Photocurrent (nA)
800 Mismatched DNA
700 600
Matched DNA
500 400 Match with 5min Match with 10min Mismatch with 5 min Mismatch with 10min
300 200 0.0
0.5
1.0
1.5
2.0
2.5
3.0
Bias Voltage (V)
Digitized Output from the CMOS APS
Fig. 5. Photocurrent comparison between match and mismatch DNA samples with two silver enhancement time.
300 250
5. Conclusion In this paper, a DNA micro-array detection compatible with standard CMOS technology is developed. This system is based on integrated APS to detect the change in opacity when an opaque nano-particle is deposited at the location with perfectly matched DNA samples. The protocol has been demonstrated by DNA micro-array fabricated with a standard 0.5 lm CMOS process. The optical property of the deposited nano-particles has been carefully studied and calibrated indicating a two-time difference can be measured under most optical spectrum in the visible light region. With the built-in dynamic range adjustment circuit, the DMA can work with a wide verity of light source with different intensity. The sensitivity of the system can be enhanced by using longer silver deposition time, and a low concentration of 10 pM can be detected.
Matched DNA
Acknowledgement
200 Single base mismatched DNA
This work is supported by an Earmarked Grant from the Research Grant Council of Hong Kong under the contract number HKUST6110/03E.
150 100 50
References
0
0
10
20
30
40
50
60
Address of the image sensor array Fig. 6. The relative intensity for DNA samples and background after 30 min silver enhancement. The length of DNA target sequence is 15 bases.
is sufficient for optical detection. It has been demonstrated that an extremely low DNA concentration down to 10 pM can be detected using the described method. In the fabricated DNA array, the optical current is converted to output voltage by the active pixel sensor (APS). After the A/D converter, the output is digitized into eight-bit digital data ready for digital signal processing. The digitized value after scanning a row of 64 pixels with different degree of DNA matching is given in Fig. 6. As indicated in the figure, the detection method is very sensitive, and the digital output for even a single base mismatch is already half that of the perfectly matched DNA. The noise margin provided in this method is more than sufficient for most digital circuits to give a reliable out to distinguish matched and mismatched DNA samples. One limitation of the system is it can only distinguish matched or mismatched DNA, but cannot give detail information
[1] Hacia JG, Collins FS. Mutational analysis using oligonucleotide microarrays. J Med Genet 1999;36(10):730–6. [2] Foldes-Papp Z, Egerer R, Birch-Hirschfeld E, Striebel HM, Demel U, Tilz GP, et al. Detection of multiple human herpes viruses by DNA microarray technology. Mol Diagn 2004;8(1):1–9. [3] Stremmel C, Wein A, Hohenberger W, Reingruber B. DNA microarrays: a new diagnostic tool and its implications in colorectal cancer. Int J Colorectal Dis 2002;17(3):131–6. [4] Anthony RM, Brown TJ, French GL. DNA array technology and diagnostic microbiology. Expert Rev Mol Diagn 2001;1(1): 30–8. [5] Todd R, Wong DT. DNA hybridization arrays for gene expression analysis of human oral cancer. J Dent Res 2002;81(2):89–97. [6] Glazer AN, Mathies RA. Energy-transfer fluorescent reagents for DNA analyses. Curr Opin Biotechnol 1997;8(1):94–102. [7] Luke S, Shepelsky M. FISH: recent advances and diagnostic aspects. Cell Vis 1998;5(1):49–53. [8] Song L, Hennink EJ, Young IT, Tanke HJ. Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys J 1995;68:2588–600. [9] Xue M, Xu W, Lu G, YIp N, KO PK, Chan M. Optical response of photodiode in integrated DNA detection system. Electron Devices Meeting, 2001, Proceedings, 2001 IEEE Hong Kong. [10] Li J, Xue M, Wang H, Cheng L, Gao L, Lu Z, et al. Amplifying the electrical hybridization signals of DNA array by multilayer assembly of Au nanoparticle probes. The Analyst 2003;128:917–23. [11] Rogers YH, Jiang-Baucom P, Huang ZJ, Bogdanov V, Anderson S, Boyce-Jacino MT. Immobilization of oligonucleotides onto a glass support via disulfide bonds: a method for preparation of DNA microarrays. Anal Biochem 1999;266:23–30.