Materials Science and Engineering C 37 (2014) 369–373
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Influence of material properties upon immobilization of histidine-tagged protein on Ni–Co coated chip Yaw-Jen Chang a,b,⁎, Ching-Yuan Ho a, Cheng-Hao Chang a a b
Department of Mechanical Engineering, Chung Yuan Christian University, Chung Li 32023, Taiwan Institute of Biomedical Technology (IBT), Chung Yuan Christian University, Chung Li 32023, Taiwan
a r t i c l e
i n f o
Article history: Received 7 April 2013 Received in revised form 29 June 2013 Accepted 18 January 2014 Available online 24 January 2014 Keywords: Protein chip Histidine Electrodeposition
a b s t r a c t In protein research, protein microarray facilitates high-throughput study of protein abundance and function. An appropriate microarray surface that can be used to immobilize protein samples is a prerequisite for the investigation of molecular interactions. Ni–Co alloy coated protein microarray chip has been found to adsorb histidinetagged proteins effectively based on the method of immobilized metal affinity chromatography. Due to the ingredient of bi-metallic elements, different electroplating conditions resulted in distinct binding affinities. Therefore, the influence of Ni–Co material properties on the immobilization of histidine-tagged protein was systematically investigated in this study. In the experiments, the contact angle measurement suggested that no strong relationship can be established between the wettability of chip surface and its corresponding protein immobilization. ESCA test demonstrated that the major ingredients of the Ni–Co alloy coated protein microarray chip were Ni and Co. In addition, the XRD test concluded that a Ni–Co protein chip that consists mostly of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni–Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Protein microarray chips are measurement devices used in biomedical applications to study protein abundance and function. By detecting the physiological messages generated from protein–protein interactions, protein microarrays have become powerful tools for disease diagnosis, genetic research, and drug discovery [1–5]. However, an appropriate microarray surface that can immobilize protein samples is a prerequisite for the investigation of molecular interactions. Up to the present time, a variety of surface treatments for protein attachment have been developed, and most binding mechanisms can be classified into physical adsorption and covalent immobilization [6,7]. Histidine (His) is one of the most common natural amino acids present in proteins and is usually used in the method of immobilized metal affinity chromatography (IMAC) for protein purification. Most protein microarrays based on the theory of IMAC were fabricated using the chelator or compound of mono-metallic ion, such as Ni+ 2, to capture the histidine-tagged protein [8–11]. In our previous work, a protein chip with Ni–Co alloy layer fabricated on the printed circuit board by electrodeposition was developed [12]. It is a microarray surface with bi-metallic elements. Cobalt is added as one of the two
⁎ Corresponding author at: Department of Mechanical Engineering, Chung Yuan Christian University, Chung Li 32023, Taiwan. Tel.: +886 3 2654307. E-mail address: [email protected] (Y.-J. Chang). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.028
metallic elements to enhance the specific binding capability in order to immobilize functional proteins with His-tag attached. Electrodeposition is a surface treatment technique that uses electrical current to coat a surface with a layer of metal containing some desired properties. Due to the electrochemical principle, nickel and cobalt can be co-deposited on the surface of substrate and it is an innovative microarray surface. Our experimental results showed that the alloy coating provided specific binding due to the affinity adsorption between the intermediate metals and the histidine-tagged protein. Compared with Ni-coated chip and nitrocellulose (NC) chip, this Ni–Co alloy chip was a highly sensitive protein microarray because of its highly detectable fluorescence intensity with a low fluorescent intensity in the background. Although electroplating is a well-developed technique and the electrodeposition of Ni–Co alloys has been widely studied [13,14], the influence of material properties of Ni–Co alloy coating upon the adsorption of His-tagged protein deserves to be studied. In this paper, the surface morphology and phase structure of Ni–Co film had been systematically investigated to acquire more knowledge of its affinity binding to His-tagged protein. 2. Materials and methods A total of eighteen sets of Ni–Co protein chips were fabricated using different electroplating conditions to investigate and draw out the influence factors of Ni–Co layer upon the immobilization of histidine-tagged protein. Printed circuit board (PCB) was used as the substrate of this
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Table 1 Experimental design of L18 orthogonal array. Exp no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Variables (factors) A
B
C
D
E
F
G
H1
H2
10 10 10 10 10 10 10 10 10 15 15 15 15 15 15 15 15 15
2 2 2 4 4 4 6 6 6 2 2 2 4 4 4 6 6 6
40 45 50 40 45 50 40 45 50 40 45 50 40 45 50 40 45 50
4.0 3.5 4.5 4.0 3.5 4.5 3.5 4.5 4.0 4.5 4.0 3.5 3.5 4.5 4.0 4.5 4.0 3.5
0.5 0 1 0 1 0.5 0.5 0 1 1 0.5 0 1 0.5 0 0 1 0.5
4 0 7 0 7 4 7 4 0 0 7 4 4 0 7 7 4 0
5 0 10 10 5 0 0 10 5 0 10 5 10 5 0 5 0 10
3.64 10.93 18.23 18.23 3.64 10.93 18.23 3.64 10.93 3.64 10.93 18.23 10.93 18.23 3.64 10.93 18.23 3.64
20 60 100 100 20 60 100 20 60 20 60 100 60 100 20 60 100 20
proposed Ni–Co protein microarray since its conducting layer, typically made of thin copper foil, can serve as plating seed layer directly. Ni–Co electrodeposition was performed in a W18 cm × L25 cm × H18 cm electroplating tank. Eight plating factors were considered, including (A) Plating distance (cm); (B) Plating current (A/dm2); (C) Plating temperature (°C); (D) Plating mixture pH; (E) Surface smoothing additive/Low-pinhole additive (mL/L); (F) Nickel chloride additive (g/L); (G) Low-stress additive (mL/L); and (H) Co 2 + (g/L) and Co(NH2SO3)·4H2O (mL/L). The electrodeposition was conducted by using the L18 orthogonal array, as listed in Table 1, for comparison. For any electroplating factor, each level has an equal number of occurrences in the experimental matrix so that uniformly distributed coverage of the test domain can be provided. Each of the vectors conveys information different from that of any other vector in the sequence. That is, the effects of factors can be separated from each other and therefore the experimental design can avoid redundancy. Moreover, due to this property of orthogonal arrays, a full factorial experiment to change one variable at one time is not necessary. The numerous experimental runs can be reduced without losing any vital information. Thus, the use of orthogonal arrays in this study fosters meaningful and cost-effective experimental designs.
After the fabrication of Ni–Co coated protein chip, bio-experiments using Uricase protein were performed. Sandwich immunoassay was adopted so that the fluorescence intensities after the immunoassay represent their respective binding capability between protein and Ni–Co membrane. The 6 × His-tagged Uricase antigen was diluted to concentration of 20 μg/mL by coating buffer (0.1 M PB, pH 7.4). The samples were then arrayed on the biochips, followed by a 37 °C/2 h incubation for protein immobilization in a hot circulator oven. After the immobilization step, the biochips were washed with washing buffer (Phosphate Buffer with 0.1% Tween-20) three times for 1 min per wash in order to remove the residual antigen. The first anti-Uricase antibody was prepared by dilute buffer (Phosphate Buffer with 1% BSA) to the final concentration of 2 μg/mL, and was then added onto the top of the antigen layer. One-hour incubation in a 37 °C hot circulator oven was followed by three washes in washing buffer for 1 min per wash. Next, the Cy5-conjugated anti goat IgG was diluted in dilute buffer to 5 μg/mL and added onto the top of the His/anti-His protein complex to report the result. The binding reaction was again incubated in a 37 °C hot circulator oven for 1 h, and then followed by the same washing procedure. Please refer to our previous work for the details [12]. In order to acquire more knowledge of the properties affecting the adhesion characteristics of His-tagged protein on Ni–Co coated surface, four kinds of material tests were performed. • Test of wettability Since the analyte-specific reagents must be spotted on the microarray surface, it is essential to study the wettability of Ni–Co alloy coating to examine whether the adsorption mechanism comes from the attraction of the hydrophilic chip surface. Using the contact angle goniometer (DSA 10, KRÜSS GmbH, Germany), deionized (DI) water was dropped on the chip surface. The droplets were controlled in the same volume of 31.5 μL with 3 droplets per chip for statistical computation. Photos were taken during the experiment and, afterward, analyzed by Drop Shape Analysis (DSA) software to calculate the contact angle. • ESCA test To identify all elements that exist in or on the Ni–Co chip surface responsible for the immobilization of His-tagged proteins, electron spectroscopy for chemical analysis (ESCA) was utilized to analyze the surface chemistry of Ni–Co alloy coating. In this study, ESCA test was performed using Al Kα X-ray radiation source with power of 24.1 W, 187.85 eV and beam size of 100 μm (PHI 5000 VersaProbe, manufactured by ULVAC-PHI). • XRD test
Fig. 1. Wettability property of Ni–Co.
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Fig. 2. ESCA survey spectra of Ni–Co chip (experiment no. 12).
After analyzing the elemental composition of Ni–Co alloy, X-ray diffraction (XRD) was used to examine its crystallographic structure in order to gain the structural or physico-chemical information about the immobilization of His-tagged proteins. In this study, the test was carried out using X'Pert Pro MRD PW3040/60 (PANalytical B.V., Netherlands). A low incident angle of X-ray beam was used to characterize the crystallographic structure of Ni–Co alloy coating. The light source was Cu Kα with a wavelength (λ) of 0.154 nm. Scanning speed was set at 0.02°/min with a scanning angle (2θ) from 40° to 105°. • SEM imaging The high-resolution images of scanning electron microscope (SEM) can provide the information about the surface topography and composition of Ni–Co alloy, allowing us to confirm the inferences drawn from the other tests. In this study, Hitachi S-4800N field emission SEM was used.
3. Results and discussion Among 18 fabrication conditions of the Ni–Co coated protein chips, experiment nos. 1, 4, 5, 8, and 12 had larger signal-to-noise (S/N) ratios in fluorescence intensity. This result explains that these chips had better binding ability to immobilize His-Uricase, because these chips present stronger fluorescence intensities against the fluorescent background. On the other hand, experiment nos. 7, 11, and 13 had lower S/N ratios. The material tests reveal several results regarding the adsorption mechanism of Ni–Co alloy coating to the His-tagged protein. 3.1. Influence of contact angle
Fig. 3. XRD test of Ni–Co alloy coated protein microarray chip: (a) experiment no. 1, (b) experiment no. 4, (c) experiment no. 5, (d) experiment no. 8, and (e) experiment no. 12.
The 6 × His-tagged Uricase antigen was diluted by coating buffer and arrayed on the Ni–Co chip. The coating buffer (0.1 M PB, pH 7.4) is an aqueous solution. Although the measured contact angle of DI water cannot be completely equivalent to that of PB solution, it provides an observation about the wettability of the Ni–Co layer. In this study, eighteen sets of Ni–Co chips were tested. As shown in Fig. 1, the experimental results of contact angle explain that most chips belong to or are close to hydrophobic surface; while only experiment no. 9 is hydrophilic. Furthermore, no strong relationship can be established between the contact angle and the chip's corresponding S/N ratio of fluorescence
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Fig. 4. SEM micrographs (10000×): (a) experiment no. 4, (b) experiment no. 12, (c) experiment no. 6, and (d) experiment no. 11.
intensity of immunoassay. That is, the wettability property of Ni–Co layer is not the dominant factor for the adherence of His-tagged protein. 3.2. Results of ESCA test The characteristic peaks in the ESCA spectra are related to the electron configuration of the photo-emitted electrons within the atoms. We utilized ESCA spectra to identify the elements existing in or on the surface of Ni–Co alloy coating. As shown in Fig. 2, the notations Ni2s and Ni2p explain that photo-emitted electrons escaping from 2s- and 2p-subshell of Ni (orbital energy level L) were detected, respectively. Similarly, electrons from 2s- and 2p-subshell of Co were also detected, as described by Co2s and Co2p, respectively. In addition, electrons from the orbital energy level M of Ni and Co indicated by Ni3s, Ni3p, and Co3s were also discovered. Therefore, the ESCA spectra clearly indicate that the ingredients of the Ni–Co alloy coated protein microarray chip are Ni and Co, without any other metallic compositions. Besides, O1s reveals the existence of oxygen in the chip surface. Since the Ni–Co chips were kept at 45% relative humidity and 23 °C for up to four weeks for the long-term sensibility test, the experimental result shows that the atomic% of oxygen (in the compositions of Ni, Co, and O) raised to 55.7% in the fourth week due to surface oxidization. Moreover, carbon described by C1s mainly comes from the talcum powder on the gloves used during the bio-experiments. 3.3. Results of XRD test The detailed information about the phase composition and structure of Ni–Co alloy coated chips was explored using X-ray diffraction by illuminating the sample with X-rays of a fixed wavelength. A diffraction pattern records the intensity of the reflected radiation as a function of 2-theta angle, in which the vertical axis records X-ray intensity and the horizontal axis indicates angles in degrees 2θ. The peaks on the pattern were compared with the JCPDS card and technical literature [14] in order to point out the crystallographic structure of alloy and to
understand their immobilization mechanism. We discover that the affinity binding between Ni–Co coating and His-tagged protein can be linked to the crystallographic structure of alloy. For the chips with better binding capability (experiment nos. 1, 4, 5, 8, and 12), their Ni–Co deposits exhibit a majority of hexagonal close packed (hcp) lattice. Chip nos. 12 and 4 especially had a strong hcp (0 0 2) texture with a clear (1 0 0) peak, as shown in Fig. 3. On the contrary, for the chips with lower S/N ratios (experiment nos. 7, 11, and 13), their Ni–Co deposits demonstrate a majority of face-centered cubic (fcc) lattice with remarkable (2 0 0) growth orientation. The other chips that lie in between these two cases have crystal structures with mixed hcp and fcc phases. Therefore, the crystallographic structure of Ni–Co alloy determines the binding performance. A Ni–Co protein chip with a majority of hcp lattice has better binding capability.
3.4. Results of SEM SEM image can provide the detailed surface morphology of the material being examined. In this study, the SEM images of all Ni–Co alloys were taken and compared with literatures [13,14]. For the chips with larger S/N ratios (experiment nos. 1, 4, 5, 8 and 12), their SEM micrographs displayed a hcp morphological surface. Fig. 4(a) and (b) shows the SEM images of experiment nos. 4 and 12, respectively. The alloys have a rather regularly branched structure. On the other hand, fcc grain lattice was discovered for the chips with lower S/N ratios (experiment nos. 7, 11, and 13). Fig. 4(c) and (d) shows the SEM images of experiment nos. 6 and 11, respectively. The Ni–Co alloys showed a spherical cluster surface. Unlike the nitrocellulose membrane which provides nonspecific binding to capture the samples by its porous fiber structure and electrostatic force, Ni–Co alloy layer provides specific binding to immobilize His-tagged protein through the crystallographic structure of the alloy. SEM micrographs are direct image evidence.
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4. Conclusion
Acknowledgment
Ni–Co alloy coated protein microarray presented in our previous work is an innovative microarray surface with bi-metallic elements for immobilizing His-tagged proteins. In this paper, the adsorption mechanism of Ni–Co film had been systematically investigated through several material tests. ESCA test demonstrated that the major ingredients of the Ni–Co alloy coated protein microarray chip are Ni and Co. Since these two elements are co-deposited on the surface of the substrate, different crystallographic structures can be formed with different electroplating conditions. The XRD test, by comparing the diffraction pattern, concluded that a Ni–Co protein chip with a majority of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni–Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology, not the wettability property. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein. Understanding the influence factors allows us to fabricate the Ni–Co protein chip with better immobilization ability such that this chip can be used under low concentration of proteins. In addition, the development of microarray surface by electroplating provides a potential mass production approach for producing stable protein chips, because electroplating is a well-developed and inexpensive technique.
We thank for the support of this work by Chung Yuan Christian University, Taiwan. References [1] K. Büssow, Z. Konthur, A. Lueking, H. Lehrach, G. Walter, Am. J. Pharmacogenomics 1 (2001) 1. [2] T. Bacarese-Hamilton, F. Bistoni, A. Crisanti, Biotechniques 33 (2002) 24. [3] V. Espina, A.I. Mehta, M.E. Winters, V. Calvert, J. Wulfkuhle, E.F. Petricoin III, L.A. Liotta, Proteomics 3 (2003) 2091. [4] J. LaBaer, N. Ramachandran, Curr. Opin. Biotechnol. 9 (2005) 14. [5] S.S. Saliterman, Fundamentals of BioMEMS and Medical Microdevices, SPIE, Washington, 2006. [6] P. Angenendt, J. Glökler, J. Sobek, H. Lehrach, D.J. Cahill, J. Chromatogr. A 1009 (2003) 97. [7] M. Schena, Protein Microarrays, Jones and Bartlett Publishers, Massachusetts, 2005. [8] G.B. Sigal, C. Bamdad, A. Barberis, J. Strominger, G.M. Whitesides, Anal. Chem. 68 (1996) 490. [9] E.L. Schmid, T.A. Keller, Z. Dienes, H. Vogel, Anal. Chem. 69 (1997) 1979. [10] J.W. Hyun, M.Y. Yun, S.J. Noh, Y. Ahn, Y.D. Huh, H. Park, J. Pyee, H.G. Ji, Curr. Appl. Phys. 6 (2006) 275. [11] E. Kang, J.W. Park, S.J. McClellan, J.M. Kim, D.P. Holland, G.U. Lee, E.I. Franses, K. Park, D.H. Thompson, Langmuir 23 (2007) 6281. [12] Y.J. Chang, C.H. Chang, Biosens. Bioelectron. 25 (2010) 1748. [13] D. Golodnitsky, Y. Rosenberg, A. Ulus, Electrochim. Acta 47 (2002) 2707. [14] L. Wang, Y. Gao, Q. Xue, H. Liu, T. Xu, Appl. Surf. Sci. 242 (2005) 326.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Influence of material properties upon immobilization of histidine-tagged protein on Ni–Co coated chip Yaw-Jen Chang a,b,⁎, Ching-Yuan Ho a, Cheng-Hao Chang a a b
Department of Mechanical Engineering, Chung Yuan Christian University, Chung Li 32023, Taiwan Institute of Biomedical Technology (IBT), Chung Yuan Christian University, Chung Li 32023, Taiwan
a r t i c l e
i n f o
Article history: Received 7 April 2013 Received in revised form 29 June 2013 Accepted 18 January 2014 Available online 24 January 2014 Keywords: Protein chip Histidine Electrodeposition
a b s t r a c t In protein research, protein microarray facilitates high-throughput study of protein abundance and function. An appropriate microarray surface that can be used to immobilize protein samples is a prerequisite for the investigation of molecular interactions. Ni–Co alloy coated protein microarray chip has been found to adsorb histidinetagged proteins effectively based on the method of immobilized metal affinity chromatography. Due to the ingredient of bi-metallic elements, different electroplating conditions resulted in distinct binding affinities. Therefore, the influence of Ni–Co material properties on the immobilization of histidine-tagged protein was systematically investigated in this study. In the experiments, the contact angle measurement suggested that no strong relationship can be established between the wettability of chip surface and its corresponding protein immobilization. ESCA test demonstrated that the major ingredients of the Ni–Co alloy coated protein microarray chip were Ni and Co. In addition, the XRD test concluded that a Ni–Co protein chip that consists mostly of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni–Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Protein microarray chips are measurement devices used in biomedical applications to study protein abundance and function. By detecting the physiological messages generated from protein–protein interactions, protein microarrays have become powerful tools for disease diagnosis, genetic research, and drug discovery [1–5]. However, an appropriate microarray surface that can immobilize protein samples is a prerequisite for the investigation of molecular interactions. Up to the present time, a variety of surface treatments for protein attachment have been developed, and most binding mechanisms can be classified into physical adsorption and covalent immobilization [6,7]. Histidine (His) is one of the most common natural amino acids present in proteins and is usually used in the method of immobilized metal affinity chromatography (IMAC) for protein purification. Most protein microarrays based on the theory of IMAC were fabricated using the chelator or compound of mono-metallic ion, such as Ni+ 2, to capture the histidine-tagged protein [8–11]. In our previous work, a protein chip with Ni–Co alloy layer fabricated on the printed circuit board by electrodeposition was developed [12]. It is a microarray surface with bi-metallic elements. Cobalt is added as one of the two
⁎ Corresponding author at: Department of Mechanical Engineering, Chung Yuan Christian University, Chung Li 32023, Taiwan. Tel.: +886 3 2654307. E-mail address: [email protected] (Y.-J. Chang). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.028
metallic elements to enhance the specific binding capability in order to immobilize functional proteins with His-tag attached. Electrodeposition is a surface treatment technique that uses electrical current to coat a surface with a layer of metal containing some desired properties. Due to the electrochemical principle, nickel and cobalt can be co-deposited on the surface of substrate and it is an innovative microarray surface. Our experimental results showed that the alloy coating provided specific binding due to the affinity adsorption between the intermediate metals and the histidine-tagged protein. Compared with Ni-coated chip and nitrocellulose (NC) chip, this Ni–Co alloy chip was a highly sensitive protein microarray because of its highly detectable fluorescence intensity with a low fluorescent intensity in the background. Although electroplating is a well-developed technique and the electrodeposition of Ni–Co alloys has been widely studied [13,14], the influence of material properties of Ni–Co alloy coating upon the adsorption of His-tagged protein deserves to be studied. In this paper, the surface morphology and phase structure of Ni–Co film had been systematically investigated to acquire more knowledge of its affinity binding to His-tagged protein. 2. Materials and methods A total of eighteen sets of Ni–Co protein chips were fabricated using different electroplating conditions to investigate and draw out the influence factors of Ni–Co layer upon the immobilization of histidine-tagged protein. Printed circuit board (PCB) was used as the substrate of this
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Table 1 Experimental design of L18 orthogonal array. Exp no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Variables (factors) A
B
C
D
E
F
G
H1
H2
10 10 10 10 10 10 10 10 10 15 15 15 15 15 15 15 15 15
2 2 2 4 4 4 6 6 6 2 2 2 4 4 4 6 6 6
40 45 50 40 45 50 40 45 50 40 45 50 40 45 50 40 45 50
4.0 3.5 4.5 4.0 3.5 4.5 3.5 4.5 4.0 4.5 4.0 3.5 3.5 4.5 4.0 4.5 4.0 3.5
0.5 0 1 0 1 0.5 0.5 0 1 1 0.5 0 1 0.5 0 0 1 0.5
4 0 7 0 7 4 7 4 0 0 7 4 4 0 7 7 4 0
5 0 10 10 5 0 0 10 5 0 10 5 10 5 0 5 0 10
3.64 10.93 18.23 18.23 3.64 10.93 18.23 3.64 10.93 3.64 10.93 18.23 10.93 18.23 3.64 10.93 18.23 3.64
20 60 100 100 20 60 100 20 60 20 60 100 60 100 20 60 100 20
proposed Ni–Co protein microarray since its conducting layer, typically made of thin copper foil, can serve as plating seed layer directly. Ni–Co electrodeposition was performed in a W18 cm × L25 cm × H18 cm electroplating tank. Eight plating factors were considered, including (A) Plating distance (cm); (B) Plating current (A/dm2); (C) Plating temperature (°C); (D) Plating mixture pH; (E) Surface smoothing additive/Low-pinhole additive (mL/L); (F) Nickel chloride additive (g/L); (G) Low-stress additive (mL/L); and (H) Co 2 + (g/L) and Co(NH2SO3)·4H2O (mL/L). The electrodeposition was conducted by using the L18 orthogonal array, as listed in Table 1, for comparison. For any electroplating factor, each level has an equal number of occurrences in the experimental matrix so that uniformly distributed coverage of the test domain can be provided. Each of the vectors conveys information different from that of any other vector in the sequence. That is, the effects of factors can be separated from each other and therefore the experimental design can avoid redundancy. Moreover, due to this property of orthogonal arrays, a full factorial experiment to change one variable at one time is not necessary. The numerous experimental runs can be reduced without losing any vital information. Thus, the use of orthogonal arrays in this study fosters meaningful and cost-effective experimental designs.
After the fabrication of Ni–Co coated protein chip, bio-experiments using Uricase protein were performed. Sandwich immunoassay was adopted so that the fluorescence intensities after the immunoassay represent their respective binding capability between protein and Ni–Co membrane. The 6 × His-tagged Uricase antigen was diluted to concentration of 20 μg/mL by coating buffer (0.1 M PB, pH 7.4). The samples were then arrayed on the biochips, followed by a 37 °C/2 h incubation for protein immobilization in a hot circulator oven. After the immobilization step, the biochips were washed with washing buffer (Phosphate Buffer with 0.1% Tween-20) three times for 1 min per wash in order to remove the residual antigen. The first anti-Uricase antibody was prepared by dilute buffer (Phosphate Buffer with 1% BSA) to the final concentration of 2 μg/mL, and was then added onto the top of the antigen layer. One-hour incubation in a 37 °C hot circulator oven was followed by three washes in washing buffer for 1 min per wash. Next, the Cy5-conjugated anti goat IgG was diluted in dilute buffer to 5 μg/mL and added onto the top of the His/anti-His protein complex to report the result. The binding reaction was again incubated in a 37 °C hot circulator oven for 1 h, and then followed by the same washing procedure. Please refer to our previous work for the details [12]. In order to acquire more knowledge of the properties affecting the adhesion characteristics of His-tagged protein on Ni–Co coated surface, four kinds of material tests were performed. • Test of wettability Since the analyte-specific reagents must be spotted on the microarray surface, it is essential to study the wettability of Ni–Co alloy coating to examine whether the adsorption mechanism comes from the attraction of the hydrophilic chip surface. Using the contact angle goniometer (DSA 10, KRÜSS GmbH, Germany), deionized (DI) water was dropped on the chip surface. The droplets were controlled in the same volume of 31.5 μL with 3 droplets per chip for statistical computation. Photos were taken during the experiment and, afterward, analyzed by Drop Shape Analysis (DSA) software to calculate the contact angle. • ESCA test To identify all elements that exist in or on the Ni–Co chip surface responsible for the immobilization of His-tagged proteins, electron spectroscopy for chemical analysis (ESCA) was utilized to analyze the surface chemistry of Ni–Co alloy coating. In this study, ESCA test was performed using Al Kα X-ray radiation source with power of 24.1 W, 187.85 eV and beam size of 100 μm (PHI 5000 VersaProbe, manufactured by ULVAC-PHI). • XRD test
Fig. 1. Wettability property of Ni–Co.
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Fig. 2. ESCA survey spectra of Ni–Co chip (experiment no. 12).
After analyzing the elemental composition of Ni–Co alloy, X-ray diffraction (XRD) was used to examine its crystallographic structure in order to gain the structural or physico-chemical information about the immobilization of His-tagged proteins. In this study, the test was carried out using X'Pert Pro MRD PW3040/60 (PANalytical B.V., Netherlands). A low incident angle of X-ray beam was used to characterize the crystallographic structure of Ni–Co alloy coating. The light source was Cu Kα with a wavelength (λ) of 0.154 nm. Scanning speed was set at 0.02°/min with a scanning angle (2θ) from 40° to 105°. • SEM imaging The high-resolution images of scanning electron microscope (SEM) can provide the information about the surface topography and composition of Ni–Co alloy, allowing us to confirm the inferences drawn from the other tests. In this study, Hitachi S-4800N field emission SEM was used.
3. Results and discussion Among 18 fabrication conditions of the Ni–Co coated protein chips, experiment nos. 1, 4, 5, 8, and 12 had larger signal-to-noise (S/N) ratios in fluorescence intensity. This result explains that these chips had better binding ability to immobilize His-Uricase, because these chips present stronger fluorescence intensities against the fluorescent background. On the other hand, experiment nos. 7, 11, and 13 had lower S/N ratios. The material tests reveal several results regarding the adsorption mechanism of Ni–Co alloy coating to the His-tagged protein. 3.1. Influence of contact angle
Fig. 3. XRD test of Ni–Co alloy coated protein microarray chip: (a) experiment no. 1, (b) experiment no. 4, (c) experiment no. 5, (d) experiment no. 8, and (e) experiment no. 12.
The 6 × His-tagged Uricase antigen was diluted by coating buffer and arrayed on the Ni–Co chip. The coating buffer (0.1 M PB, pH 7.4) is an aqueous solution. Although the measured contact angle of DI water cannot be completely equivalent to that of PB solution, it provides an observation about the wettability of the Ni–Co layer. In this study, eighteen sets of Ni–Co chips were tested. As shown in Fig. 1, the experimental results of contact angle explain that most chips belong to or are close to hydrophobic surface; while only experiment no. 9 is hydrophilic. Furthermore, no strong relationship can be established between the contact angle and the chip's corresponding S/N ratio of fluorescence
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Fig. 4. SEM micrographs (10000×): (a) experiment no. 4, (b) experiment no. 12, (c) experiment no. 6, and (d) experiment no. 11.
intensity of immunoassay. That is, the wettability property of Ni–Co layer is not the dominant factor for the adherence of His-tagged protein. 3.2. Results of ESCA test The characteristic peaks in the ESCA spectra are related to the electron configuration of the photo-emitted electrons within the atoms. We utilized ESCA spectra to identify the elements existing in or on the surface of Ni–Co alloy coating. As shown in Fig. 2, the notations Ni2s and Ni2p explain that photo-emitted electrons escaping from 2s- and 2p-subshell of Ni (orbital energy level L) were detected, respectively. Similarly, electrons from 2s- and 2p-subshell of Co were also detected, as described by Co2s and Co2p, respectively. In addition, electrons from the orbital energy level M of Ni and Co indicated by Ni3s, Ni3p, and Co3s were also discovered. Therefore, the ESCA spectra clearly indicate that the ingredients of the Ni–Co alloy coated protein microarray chip are Ni and Co, without any other metallic compositions. Besides, O1s reveals the existence of oxygen in the chip surface. Since the Ni–Co chips were kept at 45% relative humidity and 23 °C for up to four weeks for the long-term sensibility test, the experimental result shows that the atomic% of oxygen (in the compositions of Ni, Co, and O) raised to 55.7% in the fourth week due to surface oxidization. Moreover, carbon described by C1s mainly comes from the talcum powder on the gloves used during the bio-experiments. 3.3. Results of XRD test The detailed information about the phase composition and structure of Ni–Co alloy coated chips was explored using X-ray diffraction by illuminating the sample with X-rays of a fixed wavelength. A diffraction pattern records the intensity of the reflected radiation as a function of 2-theta angle, in which the vertical axis records X-ray intensity and the horizontal axis indicates angles in degrees 2θ. The peaks on the pattern were compared with the JCPDS card and technical literature [14] in order to point out the crystallographic structure of alloy and to
understand their immobilization mechanism. We discover that the affinity binding between Ni–Co coating and His-tagged protein can be linked to the crystallographic structure of alloy. For the chips with better binding capability (experiment nos. 1, 4, 5, 8, and 12), their Ni–Co deposits exhibit a majority of hexagonal close packed (hcp) lattice. Chip nos. 12 and 4 especially had a strong hcp (0 0 2) texture with a clear (1 0 0) peak, as shown in Fig. 3. On the contrary, for the chips with lower S/N ratios (experiment nos. 7, 11, and 13), their Ni–Co deposits demonstrate a majority of face-centered cubic (fcc) lattice with remarkable (2 0 0) growth orientation. The other chips that lie in between these two cases have crystal structures with mixed hcp and fcc phases. Therefore, the crystallographic structure of Ni–Co alloy determines the binding performance. A Ni–Co protein chip with a majority of hcp lattice has better binding capability.
3.4. Results of SEM SEM image can provide the detailed surface morphology of the material being examined. In this study, the SEM images of all Ni–Co alloys were taken and compared with literatures [13,14]. For the chips with larger S/N ratios (experiment nos. 1, 4, 5, 8 and 12), their SEM micrographs displayed a hcp morphological surface. Fig. 4(a) and (b) shows the SEM images of experiment nos. 4 and 12, respectively. The alloys have a rather regularly branched structure. On the other hand, fcc grain lattice was discovered for the chips with lower S/N ratios (experiment nos. 7, 11, and 13). Fig. 4(c) and (d) shows the SEM images of experiment nos. 6 and 11, respectively. The Ni–Co alloys showed a spherical cluster surface. Unlike the nitrocellulose membrane which provides nonspecific binding to capture the samples by its porous fiber structure and electrostatic force, Ni–Co alloy layer provides specific binding to immobilize His-tagged protein through the crystallographic structure of the alloy. SEM micrographs are direct image evidence.
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4. Conclusion
Acknowledgment
Ni–Co alloy coated protein microarray presented in our previous work is an innovative microarray surface with bi-metallic elements for immobilizing His-tagged proteins. In this paper, the adsorption mechanism of Ni–Co film had been systematically investigated through several material tests. ESCA test demonstrated that the major ingredients of the Ni–Co alloy coated protein microarray chip are Ni and Co. Since these two elements are co-deposited on the surface of the substrate, different crystallographic structures can be formed with different electroplating conditions. The XRD test, by comparing the diffraction pattern, concluded that a Ni–Co protein chip with a majority of hcp lattice has better binding capability. SEM micrographs provide direct image evidence. These material tests summarize that the Ni–Co alloy coated protein microarray chip adsorbs His-tagged proteins through its surface morphology, not the wettability property. Therefore, it can provide specific binding due to the affinity adsorption between the intermediate metals and the protein. Understanding the influence factors allows us to fabricate the Ni–Co protein chip with better immobilization ability such that this chip can be used under low concentration of proteins. In addition, the development of microarray surface by electroplating provides a potential mass production approach for producing stable protein chips, because electroplating is a well-developed and inexpensive technique.
We thank for the support of this work by Chung Yuan Christian University, Taiwan. References [1] K. Büssow, Z. Konthur, A. Lueking, H. Lehrach, G. Walter, Am. J. Pharmacogenomics 1 (2001) 1. [2] T. Bacarese-Hamilton, F. Bistoni, A. Crisanti, Biotechniques 33 (2002) 24. [3] V. Espina, A.I. Mehta, M.E. Winters, V. Calvert, J. Wulfkuhle, E.F. Petricoin III, L.A. Liotta, Proteomics 3 (2003) 2091. [4] J. LaBaer, N. Ramachandran, Curr. Opin. Biotechnol. 9 (2005) 14. [5] S.S. Saliterman, Fundamentals of BioMEMS and Medical Microdevices, SPIE, Washington, 2006. [6] P. Angenendt, J. Glökler, J. Sobek, H. Lehrach, D.J. Cahill, J. Chromatogr. A 1009 (2003) 97. [7] M. Schena, Protein Microarrays, Jones and Bartlett Publishers, Massachusetts, 2005. [8] G.B. Sigal, C. Bamdad, A. Barberis, J. Strominger, G.M. Whitesides, Anal. Chem. 68 (1996) 490. [9] E.L. Schmid, T.A. Keller, Z. Dienes, H. Vogel, Anal. Chem. 69 (1997) 1979. [10] J.W. Hyun, M.Y. Yun, S.J. Noh, Y. Ahn, Y.D. Huh, H. Park, J. Pyee, H.G. Ji, Curr. Appl. Phys. 6 (2006) 275. [11] E. Kang, J.W. Park, S.J. McClellan, J.M. Kim, D.P. Holland, G.U. Lee, E.I. Franses, K. Park, D.H. Thompson, Langmuir 23 (2007) 6281. [12] Y.J. Chang, C.H. Chang, Biosens. Bioelectron. 25 (2010) 1748. [13] D. Golodnitsky, Y. Rosenberg, A. Ulus, Electrochim. Acta 47 (2002) 2707. [14] L. Wang, Y. Gao, Q. Xue, H. Liu, T. Xu, Appl. Surf. Sci. 242 (2005) 326.
