pubs.acs.org/Langmuir © 2011 American Chemical Society
Aldehyde-Functionalized Benzenediazonium Cation for Multiprobe Immobilization on Microelectrode Array Surfaces Al-Monsur Jiaul Haque and Kyuwon Kim* Department of Chemistry, University of Incheon, Incheon 406-772, Korea Received October 25, 2010. Revised Manuscript Received December 12, 2010 We report in situ generation of aldehyde-functionalized benzenediazonium cation (ABD) and its use as a suitable linker molecule for fast and selective immobilization of biomolecules on indium-tin-oxide (ITO) electrode surfaces. We prepared ABD through a new reaction procedure, a simultaneous diazotation of the amine group and deprotection of the aldehyde group from an aniline derivative, 2-(4-aminophenyl)-1,3-dithiane, which was revealed on the ITO electrode surfaces through the electrodeposition of the reaction product and the characterization of the resulting surfaces with cyclic voltammetry, X-ray photoelectron spectroscopy, and protein immobilization. We also showed that successive electrodeposition of ABD and probe molecules on individually addressable microarray electrode surfaces can provide a useful platform for efficient detection of multianalyte. The usage of ABD has been demonstrated by the patterning of three different probe molecules on a single substrate and the simultaneous detection of two target molecules.
Functionalization of electrode surfaces using linker molecules bearing two suitable organic functional groups, one of which is used for reacting with the surfaces and the other for attaching biomolecules, is a very frequently followed primary step in biosensor and bioelectronics research to immobilize biomolecules on the surfaces for sensing applications. Among various linkers studied in the functionalization, an electrochemically active *To whom correspondence should be addressed. Telephone: (þ82) 32-8358243. Fax: (þ82) 32-835-0762. E-mail:
[email protected]. (1) Delamar, M.; Hitmi, R.; Pinson, J.; Saveant, J. M. J. Am. Chem. Soc. 1992, 114, 5883–5884. (2) Allongue, P.; Delamar, M.; Desbat, B.; Fagebaume, O.; Hitmi, R.; Pinson, J.; Saveant, J. M. J. Am. Chem. Soc. 1997, 119, 201–207. (3) Saby, C.; Ortiz, B.; Champagne, G. Y.; Belanger, D. Langmuir 1997, 13, 6805–6813. (4) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038–5045. (5) Allongue, P.; Henry de Villeneuve, C.; Cherouvrier, G.; Cortes, R.; Bernard, M. C. J. Electroanal. Chem. 2003, 161, 550–551. (6) Adenier, A.; Combellas, C.; Kanoufi, F.; Pinson, J.; Podvorica, F. I. Chem. Mater. 2006, 18, 2021–2029. (7) Maldonado, S.; Smith, T. J.; Williams, R. D.; Morin, S.; Barton, E.; Stevenson, K. J. Langmuir 2006, 22, 2884–2891. (8) Laforgue, A.; Addou, T.; Belanger, D. Langmuir 2005, 21, 6855. (9) Liu, G. Z.; Bocking, T.; Gooding, J. J. J. Electroanal. Chem. 2007, 600, 335– 344. (10) Shewchuk, D. M.; McDermott, M. T. Langmuir 2009, 25, 4556–4563. (11) Bahr, J. L.; Tour, J. M. Chem. Mater. 2001, 13, 3823. (12) Adenier, A.; Cabet-Deliry, E.; Chausse, A.; Griveau, S.; Mercier, F.; Pinson, J.; VautrinUl, C. Chem. Mater. 2005, 17, 491. (13) Liu, G.; Gooding, J. J. Langmuir 2006, 22, 7421–7430. (14) Radi, A. E.; Berbel, X. M.; Lates, V.; Marty, J. L. Biosens. Bioelectron. 2009, 24, 1888–1892. (15) Harper, J. C.; Polsky, R.; dirk, S. M.; Wheeler, D. R.; Brozik, S. M. Electroanalysis 2007, 12, 1268–1274. (16) Griveau, S.; Mercier, D.; Vautrin-UI, C.; Chausse, A. Electrochem. Commun. 2007, 9, 2768–2773. (17) Shabani, A.; Mak, A. W. H.; Gerges, I.; Cuccia, L. A.; Lawrence, M. F. Talanta 2006, 70, 615–623. (18) Flavel, B. S.; Gross, A. J.; Garrett, D. J.; Nock, V.; Downard, A. J. ACS Appl. Mater. Interfaces 2010, 2, 1184–1190. (19) Corgier, B. P.; Marquette, C. A.; Blum, L. J. J. Am. Chem. Soc. 2005, 127, 18328–18332. (20) Corgier, B. P.; Laurent, A.; Perriat, P.; Blum, L. J.; Marquette, C. A. Angew. Chem., Int. Ed. 2007, 119, 4186–4188. (21) Marquette, C. A.; Bouteille, F.; Corgier, B. P.; Degiuli, A.; Blum, L. J. Anal. Bioanal. Chem. 2009, 393, 1191–1198. (22) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Dirk, S. M.; Brozik, S. M. Langmuir 2007, 23, 8285–8287. (23) Polsky, R.; Harper, J. C.; Wheeler, D. R.; Dirk, S. M.; Arango, D. C.; Brozik, S. M. Biosens. Bioelectron. 2008, 23, 757–764.
882 DOI: 10.1021/la104270b
benzenediazonium salt has attracted much attention,1-24 because it has several advantages, for example, ease of synthesis with a wide range of functional groups, fair and fast reactivity with various substrates such as as carbon,2-4 silicon,5 metals,6 and even indium-tin-oxide (ITO),7 good stable film forming property on the surfaces through chemical or electrochemical deposition, and moreover individually functionalizable ability of closely spaced electrodes by electrochemical addressing.8,9 Since the first report on electrochemical reduction of diazonium salts,1 various functional groups such as -NO2, -NH2, and -COOH have been introduced onto different surfaces using this method, but further modification steps or additional linkers are required for the immobilization of biomolecules.9,13-18 Recently, a more elegant immobilization method using diazonium salts has been reported.19-23 The method is based on one by one modification of probe biomolecules with diazonium cation prior to the immobilization on the sensor surfaces. However, because it might suffer from the low immobilization yield resulting in the low density of biomolecules as probes, an amplified detection scheme via an enzymatically generated species has been employed to enhance detection signal.19-23 Therefore, it is beneficial to develop a new method that enables us to provide an increased probe density without further modification steps or additional linkers. One potential alternative can be the use of aldehyde-groupfunctionalized benzenediazonium salt (ABD) because an aldehyde group can be directly coupled to any primary amine-groupterminated molecule including biomolecules without the need for additional linkers or further activation steps. In spite of this convenient feature of the aldehyde group, the use of ABD has never been reported for the immobilization of biomolecules on surfaces. This could be because of the lack of stability of the aldehyde group during synthesis and long-time storage. In view of the advantages of using diazonium and aldehyde functionalities, it is highly desirable to find any relatively stable precursors that conceal two functionalities and then find the shortest possible way to obtain ABD. (24) Haque, A. M. J.; Kwon, S. R.; Park, H.; Kim, T. H.; Oh, Y. S.; Choi, S. Y.; Hong, J. D.; Kim, K. Chem. Commun. 2009, 4865–4867.
Published on Web 01/06/2011
Langmuir 2011, 27(3), 882–886
Haque and Kim
Letter Scheme 1 a
a (A) In situ generation of ABD from the precursor aniline derivative through simultaneous diazotation of amine group and deprotection of aldehyde group. (B) Electrodeposition of ABD and subsequent immobilization of biomolecules on ABD-modified surface.
Figure 1. Cyclic voltammograms of (A) electrodeposition of ABD (1.5 mM in ACN containing 0.1 M Bu4NBF4) on ITO surface and (B) anodic oxidation of ABD-modified ITO surface (in ACN/H2O, 95/5 solution containing 0.1 M Bu4NBF4). Three cycles were performed at a scan rate of 50 mV/s.
Here, we report the in situ generation of ABD and its use as a linker molecule for immobilization of biomolecules on ITO electrode surfaces. We have prepared ABD through a new reaction procedure, that is, the simultaneous diazotation of amine group and deprotection of aldehyde group from a precursor, 2-(4aminophenyl)-1,3-dithiane, synthesized in our previous work.24 The present approach employing ABD has superior advantages over the previous work where the highly oxidative potential being essential for deprotecting aldehyde group on the surfaces should cause partial desorption of diazonium-grafted layers as shown in X-ray photoelectron spectroscopy (XPS) study, which can make it difficult to control surface properties such as immobilization density of adsorbents for further processes. This drawback is a problem regardless of electrode type. The oxidative potential also affects the performance of the electrode for further use. For example, Si or C surfaces that have been widely used as a substrate for the diazonium modification will be easily and irreversibly oxidized during the oxidative deprotection, resulting in insulating oxide even after the modification with the diazonium molecule. This drawback limits the type of electrode for further observation through electrical and electrochemical signal. If one uses ABD, the drawbacks due to the oxidation will not be a matter of concern because the process employing ABD needs only an electrochemical reduction step for the attachment of ABD onto the surfaces. We have used ITO as the electrode surface because it has desirable characteristics for biosensors based on electrochemical and optical principles such as high electrical conductivity, wide (25) Manifacier, J. Thin Solid Films 1982, 90, 297.
Langmuir 2011, 27(3), 882–886
potential window, and high optical transparency.25,26 Three different electrodes of interest of the microarray were functionalized with amino-PEG-biotin and two antibodies by successive electrodeposition of ABD and subsequent immobilization of the biomolecules. The resulting platform was used for selective detection of avidin-biotin interaction and the target antigen. Scheme 1A shows in situ generation of ABD as a plausible main product from 2-(4-aminophenyl)-1,3-dithiane. Originally, the reaction condition is one of common procedures for diazotation reaction of aniline derivatives as previously reported.27,28 We also intended to obtain the corresponding diazonium salt of the aniline derivative, 2-(4-aminophenyl)-1,3-dithiane. Interestingly, we found that the aldehyde group was also deprotected during the diazonium synthesis, which was revealed on the ITO electrode surfaces after the electrodeposition of the reaction product. The product mixture was used for further work without purification. The electrodeposition of the product on ITO surfaces as shown in Scheme1B was carried out using cyclic voltammetry (CV); the resulting voltammogram showed a sharp irreversible peak at approximately -0.1 V vs Ag/AgCl during the first cycle followed by greatly diminished current in the subsequent cycles (Figure 1A). The voltammetric response was closely similar in nature to that observed in the case of other diazonium salts,24 which (26) Kim, E. J.; Shin, H. U.; Park, S.; Sung, D.; Jon, S.; Sampathkumar, S. G.; Yarema, K. J.; Choi, S. Y.; Kim, K. Chem. Commun. 2008, 3543–3545. (27) Harper, J. C.; Polsky, R.; Wheeler, D. R.; Brozik, S. M. Langmuir 2008, 24, 2206–2211. (28) Bhar, J. L.; Yang, J.; Kosynkin, D. V.; Bronikowski, M. J.; Smalley, R. E.; Tour, J. M. J. Am. Chem. Soc. 2001, 123, 6536–6542.
DOI: 10.1021/la104270b
883
Letter
Haque and Kim
Figure 2. (A) C1s XP spectrum of ABD modified ITO surface; (B) ITO microelectrode array patterned on glass (left) and fluorescence microscopic image obtained from the selective immobilization of anti-rabbit IgG labeled with TRITC (right). The width of each microelectrode is 150 μm.
indicates that the diazonium cation was successfully obtained by the diazotation reaction. The electrodeposited surface was examined by CV, XPS, and fluorescence microscopy to confirm the presence of the aldehyde group. The electrochemical oxidation of 1,3-dithiane as an aliphatic electroactive group can afford carbonyl functionality in high yield.29 In our previous report, the electrochemical oxidation of the 4-(1,3-dithiane-2-yl)benzenediazonium (DTD)modified ITO surface showed a sharp irreversible anodic oxidation peak at approximately 1.4 V, attributed to the electrochemical deprotection of the aldehyde group in presence of water.24 However, no anodic oxidation peak was observed in the cyclic voltammogram for the electrodeposited surface in this study (Figure 1B). This result implies that the electrochemically oxidizable species is negligible on the modified surface. As shown in Figure 2A, the C1s XP spectrum consists of four chemical components centered at binding energies of 285.0, 286.4, 288.0, and 289.3 eV, corresponding to C-C/C-H, C-O, CdO, and O-CdO groups, respectively. The peak at 288.0 eV corresponds to the CdO group, which most likely originates from the formation of aldehyde groups along with other (probably carboxylic acid) groups during the diazotation reaction. For the XP spectrum after the oxidative scans, there was some decrease in the intensity at the binding energy corresponding to the C-C/C-H group which might be due to the oxidative desorption of the film, and a slight increase in the intensity corresponding to the O-CdO group rather than the CdO group (see the Supporting Information). This result indicates that there was a little amount of protected groups to be disclosed by oxidation and some aldehyde groups already existing on the surface might have been converted to carboxylic acid during the oxidative scans. It is notable that the intensity of the CdO group after oxidation is not much different from that from before oxidation, which implies that additional treatment of the electrochemical oxidation is not necessary to increase aldehyde density on the surfaces. The presence of aldehyde groups was also evidenced by direct immobilization of fluorophore-labeled antibody on the electrodeposited surface that reacts with the primary amine groups of the protein (Scheme 1B). After electrodeposition, on only “electrode 2” of the four electrodes of the microarray, the resulting surface was exposed to phosphate-buffered saline with 0.05% Tween (PBST) solution of 20 μg/mL anti-rabbit IgG labeled with tetramethylrhodamine isothiocyanate (TRITC) for 1 h. Figure 2B shows the fluorescence microscopy image obtained from the immobilization without any activation step. Highly (29) Martre, M.; Mousset, G.; Rhlid, R. B.; Veschambre, H. Tetrahedron Lett. 1990, 31, 2599–2602.
884 DOI: 10.1021/la104270b
bright red fluorescent color was observed only on the electrodeposited electrode, indicating very efficient coupling between ABD and protein. Because the XPS data indicate that probably carboxyl groups are also present besides the aldehyde groups on the electrodeposited ITO surface, we carried out the abovementioned experiment using carboxybenzenediazonium salt (CBD). However, no fluorescence was observed on the resulting surface; this rules out the possibility of any unexpected binding of the protein through the carboxylic groups (see the Supporting Information). CV, XPS, and fluorescence microscopic image results clearly support the formation of an aldehyde group during the diazotation process, which is consequently demonstrating the generation of ABD. We propose that the “-F” group in NOBF4 plays a Lewis acid role to oxidize the dithiane group, resulting in disclosure of the aldehyde group, although another possibility cannot be ruled out. This finding of the simultaneous reaction, diazotation and deprotection, should be noted because the usual chemical deprotection often requires harsh conditions, accordingly performed in the late synthetic stage, while diazonium salts are relatively unstable during following procedures. The ABD-modified surfaces have been applied to a micropatterning of probe molecules based on electrochemical addressing and a sandwich immunoassay based on the observation of fluorescence without signal amplification. We used an ITO microarray consisting of four individually addressable microelectrodes. Two out of the four electrodes of the array were selectively functionalized with two different kinds of antibodies as probes, as described below (see Scheme S1 of the Supporting Information). Following the electrodeposition of ABD only on “electrode 2”, the resulting array surface was exposed to a 20 μL drop of antirabbit IgG solution (10 μg/mL in PBST) for 2 h. After washing the exposed surface with PBST and water, the same procedure was repeated for “electrode 4” with anti-mouse IgG. For the sandwich immunoassay, the antibody-immobilized platform array was incubated with a 20 μL drop of rabbit IgG (5 μg/mL in PBST) solution for 1 h. After washing, the platform surface was exposed to a 20 μL drop of a mixture solution consisting of anti-mouse IgG labeled with fluorescein isothiocyanate (FITC) and antirabbit IgG labeled with TRITC (20 μg/mL in PBST) for 1 h. Figure 3A shows a fluorescence image obtained from the immunoassay; the image shows that only “electrode 2” gives bright red fluorescence due to TRITC, indicating that the rabbit IgG antigen can be detected with a high specificity and negligible nonspecific binding of proteins using our method. To examine multianalyte detection, the platform used in rabbit IgG detection was reused for the detection of another antigen, mouse IgG. Langmuir 2011, 27(3), 882–886
Haque and Kim
Letter
Figure 3. Fluorescence microscopy images on the same surface obtained from sandwich immunoassay for the detection of (a) rabbit IgG and (b) mouse IgG. The width of each microelectrode is 150 μm.
Figure 4. Fluorescence microscopic images obtained as a result of the simultaneous detection of (A) avidin and (B) rabbit IgG. The width of each microelectrode is 150 μm.
The second immunoassay led to a highly contrasted green fluorescence because FITC was observed only on “electrode 4” (Figure 3B). Two differently colored detection images with high signal-to-noise ratio indicate that not only the cross reaction of the two antigens is negligible but also multianalyte detection can be achieved. The usage of ABD was further examined by the patterning of three different probe molecules on a single substrate and the simultaneous detection of two target molecules, which was also based on the fluorescence observation. Successive immobilization of amino-PEG-biotin and two kinds of antibodies as probes was conducted on the microelectrodes array with a manner as described below (see Scheme S2 of the Supporting Information). Following the electrodeposition of ABD only on “electrode 2”, the resulting array surface was exposed to a 20 μL drop of amino-PEG-biotin solution (2 mM in PBS) for 1 h and washed with PBS and water. Then the “electrode 3” was electrodeposited with ABD, and the array was exposed to a 20 μL drop of anti-mouse IgG solution (10 μg/mL in PBST) for 1 h. After washing the exposed surface with PBST and water, the same procedure was repeated for “electrode 4” with anti-rabbit IgG. The resulting platform with amino-PEG-biotin and two antibodies was used to detect target proteins based on avidin-biotin interaction and antibody-antigen interaction. The former is one of the strongest known protein-ligand interactions with a dissociation constant Kd in the order of 10-15 mol/L. The resulting platform was disclosed to a 20 μL drop of rabbit IgG (5 μg/mL in PBST) solution for 1 h. After washing with Langmuir 2011, 27(3), 882–886
PBST and water, the resulting array surface was exposed to a 20 μL drop of a mixture solution of avidin labeled with FITC, anti-mouse IgG labeled with FITC, and anti-rabbit IgG labeled with TRITC (20 μg/mL in PBST) for 1 h. Figure 4 shows the fluorescence microscopic images obtained; the images show that only “electrode 2” and “electrode 4” give bright fluorescence due to FITC (A) and TRITC (B), respectively, indicating the ability of our method to carry out specific detection of target proteins avidin and rabbit IgG with minimum cross-talk between the electrodes and with high specificity. This result supports that the use of ABD in the patterning of multiprobe molecules based on the electrochemical addressing is very efficient, which might be due to the synergistic effect of the fast electrodeposition of ABD and direct coupling ability of the aldehyde functionality. In conclusion, we have shown an unexpected finding of the chemical condition for the simultaneous deprotection and diazotation reaction of the precursor for producing ABD. It has been for the first time demonstrated that ABD can be a good candidate as a linker molecule in the preparation of a multiprobe platform. Multianalyte has been successfully detected at a time on the single substrate, which was conducted by the observation of fluorescence without signal amplification. The good performance of our method might be attributed to the unique property of ABD to ensure the fast and selective biofunctionalization of electrode surfaces without the need for additional linker molecules or further activation steps. We believe that the ABD-based method would be a simple and promising tool for the DOI: 10.1021/la104270b
885
Letter
immobilization of various types of biomolecules, including DNA, peptides, and proteins, on various surfaces such as silicon, carbon, gold, and ITO, which can facilitate the construction of biodevices for multianalyte detection. Acknowledgment. This research was supported by Basic Science Research Program through the National Research
886 DOI: 10.1021/la104270b
Haque and Kim
Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0077502). Supporting Information Available: Experimental details and results from the use of CBD. Schemes for micropatterning and detection of biomolecules. This material is available free of charge via the Internet at http://pubs.acs.org.
Langmuir 2011, 27(3), 882–886
