J. Phys. Chem. B 2005, 109, 2739-2744
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Raman Mapping and In Situ SERS Spectroelectrochemical Studies of 6-Mercaptopurine SAMs on the Gold Electrode Haifeng Yang,†,‡ Yanli Liu,† Zhimin Liu,† Yu Yang,† Jianhui Jiang,† Zongrang Zhang,‡ Guoli Shen,† and Ruqin Yu*,† State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan UniVersity, Changsha 410082, People’s Republic of China, and Department of Chemistry, Shanghai Normal UniVersity, 100 Guilin Road, Shanghai 200234, People’s Republic of China ReceiVed: August 31, 2004; In Final Form: December 14, 2004
The self-assembled monolayers (SAMs) of 6-mercaptopurine (6MP) were formed at the roughened polycrystalline gold surfaces in acid and alkaline media. The time-dependent Raman mapping spectral analysis in conjunction with the quantum calculations for the vibrational modes using ab initio BLYP/6-31G method suggested that both of the resulted 6MP SAMs adopted the same adsorption mode through the S atom of pyrimidine moiety and the N7 atom of the imidazole moiety anchoring the gold surface in a vertical way. The in situ surface-enhanced Raman scattering spectroelectrochemical experiment was conducted to examine the stability of the SAMs at various bias potentials. It was found that the detaching process of the 6MP SAMs from the surface involved one electron reduction as the voltage was applied at ca. 0.7 V vs a standard calomel electrode.
I. Introduction 6-Mercaptopurine (6MP) has been modified on the surfaces of various metallic electrodes to promote the charge-transfer procedures of some metalloproteins,1-4 in particular, by the selfassembled monolayers (SAMs) technique as a promising approach to construct controllably the surface in orderly uniform manner. For this purpose, the structural properties of 6MP adsorbed at various metallic surfaces have been investigated by conventional electrochemical methods,5 Raman spectroscopy,6 and scanning tunneling microscopy.7 Of the above-mentioned methods, Raman spectroscopy, especially the advent of the high sensitive surface-enhanced Raman scattering (SERS) technique, provides much more information about the molecular structure and the orientation of the species adsorbed at the surfaces by the molecular monolayers. As an aromatic thiol molecule, 6MP possesses several possible sites of the N1, N3, N7, and S10 atoms to attach to the metallic surface (Scheme 1), the SERS measurement is especially useful for the understanding of the adsorption modes of the 6MP molecule self-assembled at various surfaces. Hosten and co-workers6 reported that 6MP attaches head-on through the N1 atom with respect to the silver surface on the basis of the SERS data obtained in a slightly basic 6MP solution over the surface. By comparison of the differences between the normal Raman spectrum of solution and SERS spectral data acquired from a gold surface, Taniguchi et al.3 drew a conclusion that 6MP in an alkaline form would adsorb onto the gold surface via the S atom in SdC6 and the N7 atom with a vertical orientation under the applied potential at 0 V vs a standard calomel electrode (SCE). In addition, the features of the 6MP SAMs formed at Hg and Au (111) electrodes from the alkaline * Author to whom correspondence may be addressed. Phone/Fax: +860731-882-2782. E-mail:
[email protected]. † Hunan University. ‡ Shanghai Normal University.
SCHEME 1: Structure of 6MP in Thiol Form and Its Tautomer
media have been studied by electrochemical methods, and Blazquez et al.5 suggested that the interaction between 6MP and the gold surface adopted the sites of the N7 and S atoms in SsC6, while in the case of Hg, the interaction involved the N1 and S atoms. Further investigation is desired to shed light on the adsorption mode of 6MP SAMs at the metallic surface. To take account of the existing form of 6MP molecule in the dependence of pH values in the present paper, the 6MP SAMs formed at the roughened gold surface were prepared from dilute 6MP in the acid and basic solutions. The self-assembled procedures were monitored by Raman mapping technique. The desorption procedure of the resulted 6MP SAMs from the gold surface was observed with in situ SERS spectroelectrochemical measurement. II. Experimental Section Chemicals. 6-Mercaptopurine monohydrate (99.5+ %) was obtained from Acros Organics. Two stock solutions of 10-1 mol/L 6MP were prepared with dissolution in 0.01 mol/L HCl and 0.01 mol/L KOH solutions, respectively. Doubly distilled water was freshly prepared and bubbled with nitrogen for ∼15 min to remove oxygen. All other reagents were of analytical grade. Roughening the Surface of the Gold Electrode. The gold electrode was constructed from polycrystalline material (99.99%
10.1021/jp046082l CCC: $30.25 © 2005 American Chemical Society Published on Web 02/02/2005
2740 J. Phys. Chem. B, Vol. 109, No. 7, 2005
Yang et al. SCHEME 2: Possible Forms of 6MP in the Alkaline Solution
Figure 1. Raman spectra of the 0.1 mol/L 6MP recorded in (a) 0.01 mol/L KOH solution and (b) 0.01 mol/L HCl solution.
pure). Before Raman measurements, the gold electrode was polished successively with emery paper, 1, 0.3, and 0.05 µm alumina, and then treated in a 0.1 mol/L KCl solution by an ORC method.9 After washing the gold electrode with doubly distilled water, it was put into a homemade electrochemical cell with a quartz window at the top. During this pretreatment and in situ spectroelectrochemical measurement, the experiments were conducted by a CHI 760B electrochemical workstation. Pt wire and a SCE served as counter and reference electrodes, respectively. SAMs. After the cell was installed onto the XY stage below the sampling objective of Raman spectroscopy, the stock solution of 6MP was freshly diluted into the concentration of 10-4 mol/L, and then the diluted solution was injected into the cell for forming monolayers at the roughened gold surface. The self-assembling procedure was investigated by Raman mapping measurement. Raman Mapping and In Situ SERS Spectroelectrochemical Measurements. Raman spectra were recorded with a Jobin Yvon Micro-Raman spectroscope (RamLab-010). It comprises an integral Olympus BX40 microscope with a 50× objective (8 mm) that can focus the laser on the sample to collect the backscattered radiation, a notch filter to cut the exciting line, a holographic grating (1800 g/mm), which provides a spectral resolution of 2 cm-1, and a semiconductor-cooled 1024 × 256 pixels charge-coupled device detector. A laser of 632.8 nm with a power of ca. 5 mW and a spot of ca. 3 µm in diameter on the electrode surface was used as the excitation line. The slit and pinhole were set at 100 µm and 300 µm, respectively, in confocal configuration to increase the spatial spectral resolution. The collection time was 30 s, and for each spectrum, there were two separate accumulations. An XY stage with the capability of one moving step for 1 µm controlled by PC was utilized to perform Raman mapping. The mapping area of ca. 80 × 60 µm2 was squared randomly on a monochrome image of the surface obtained by an optical axis conjugate camera and a white light source. Calibration was done referring to the 519 cm-1 line of silicon. Theoretical Methods. Full geometry optimization and computation of vibrational modes of 6MP were performed by ab initio BLYP/6-31G method in Gaussian 98 software. III. Results and Discussion Solution Raman Spectra of 6MP. For comparison purposes, the Raman spectra of the 0.1 mol/L 6MP in 0.01 mol/L KOH and 0.01 mol/L HCl solutions were recorded. In Figure 1, one
noticed the clear discrepancies between the spectral features of 6MP obtained from the solutions with pH 1.82 and 12.8, which indicate that the structure of 6MP molecule is dependent on the pH condition. Taniguchi et al.3 and Hosten et al.6 have reported Raman spectra of solution 6MP under alkaline conditions. Although the excitation laser lines used were different and Taniguchi attached a hydrogen atom to the N7 of 6MP and named it 7Hpurine-6-thiol, the two sets of Raman spectra were almost identical.6 As shown in Figure 1a, the most prominenetly observed Raman bands occur near 1596, 1554, 1522, 1432, 1410, 1371, 1325, 1299, 1274, 1244, 1214, 1136, 1006, 876, 685, 605, 440, and 232 cm-1. Taniguchi et al.3 suggested that in a strongly alkaline solution two protons from SH and the N7 of the imidazole ring would release, leading to 6MP existing in alkaline form (Scheme 2a). On the basis of the absence of an S-H near 918 cm-1 and N1-H at 1407 cm-1 assigned by the results of semiempirical quantum PM3 method together with Urey-Bradley force-field calculations for solid 6MP in its tautomer form, Hosten et al.6 concluded that, in alkaline solution, the 6MP molecule ionizes and loses the H on the N1 of pyrimidine ring (Scheme 2b). However, one noticed that the band around 1410 cm-1 can be observed in the spectrum of Figure 1a and can also be found in the spectrum given by Taniguchi et al. Because of the absence of an analysis of the normal modes of 6MP molecule in the paper reported by Taniguchi et al. herein, the calculations for vabritional modes of alkaline forms (Scheme 2a) of 6MP along with 6MP in its thiol form and its tautomer were conducted by ab intio the BLYP/6-31G method that is considered as a more promising approach with less error.10-11 In Table 1, the observed Raman bands of 6MP in an alkaline solution can be found in the two sets of the scaled calculation results of vibrational modes for the ionized 6MP tautomer and the alkaline form suggested by Taniguchi et al.3 It might reveal that the two 6MP forms shown in parts a and b of Scheme 2 coexist in the alkaline solution. To date, no Raman spectrum of 6MP recorded in an acid solution has been reported. The Raman spectrum of solution 6MP under the acid condition was acquired in present work, and the feature bands are peaked at 1519, 1449, 1421, 1377, 1330, 1265, 1023, 675, 615, 588, 441, and 299 cm-1. In Table 2, one noticed that the observed Raman bands in an acid solution mostly matched the data set of quantum calculation results for vibrations modes of the 6MP thiol form; particularly the calculated bands near 1018 and 587 cm-1 due to S-H were found in the recorded Raman spectrum occurring at 1023 and 588 cm-1. It led us to infer that, in solution with such a pH value as 1.82, 6MP molecules tend to exist in a thiol form. Raman Mapping of 6MP SAMs at the Roughened Gold Surface. In some sense, so far, the Raman mapping technique is the only available approach to obtain the information concerning the formation of SAMs at a surface in the aqueous solution at molecular level.8,12 The Raman mapping spectra
Studies of 6-Mercaptopurine SAMs
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TABLE 1: Observed Raman Bands of 6MP in an Alkaline Solution (Wavenumbers Are Given in cm-1) along with the Vibrational Bands from the Quantum Calculations scaled BLYP/ 6-31G
scaled BLYP/ 6-31G
Raman alkaline forma tautomer Raman alkaline formb tautomer 1680 1584 1497 1460
1478
1596 1554 1522
1006 1612 1592 1582 876
1432 1410
1413
1371 1325 1299 1274 1244
1355
1438 1404 1393 1368
1306
1307
1136
1185 1131
972 967 961 930
1236 1188 1087 1083 1040
685
848 820 808 686 662
605 564 508 440
232
950 948 869 859 819 797 687 673 670 606 581 566 494 430
425 407 251
277
232
217
1023 a Alkaline form of 6MP suggested by Taniguchi et al. b The scale factor was 0.994.
TABLE 2: Observed Raman Bands of 6MP in an Acid Solution (Wavenumbers Are Given in cm-1) along with the Vibrational Bands from the Quantum Calculations scaled BLYP/ 6-31G Raman
tautomer
thiol form
scaled BLYP/ 6-31G Raman
tautomer
1631 1612 1592 1582 1519 1449 1421 1377
1478 1438 1404 1393 1368
1588 1494 1460 1422 1403
675
950 948 869 859 819 797 687 673 670
thiol form 969 945 884 836 825 676 667 641
1343 1330 1307 1265 1236 1188 1087 1083
1292 1271 1237 1162 1094
615
606
588
581 566 494 430
441
1023
587 497 391 321 297
299 1040
Figure 2. Raman mapping of the formation of SAMs at the roughened gold surface recorded in 10-4 mol/L 6MP in an acid solution (pH 1.82) after (a) 30 min (b) 6 h.
277 1018
229 217
recorded at the roughened gold surface with the self-assembled species can provide evidences for examining the features of SAMs involving the orderly uniform and, in particular, help to understand the interaction way between the species and the surface on the basis of the mechanism of SERS.13-15 Figure 2 shows the Raman mapping spectra of 6MP SAMs formed at the SERS-active gold surface in a solution with pH 1.82. It can be seen that there are only slight changes of Raman mapping spectra recorded after a queue time of 6 h as compared to those after 30 min. To take account of the impacts on the intensitities of Raman bands from diffierent enhanced factors
due to the varying roughnesses of the surface point by point, one concluded that 6MP molecules should quite quickly selfassemble at the gold surface to form uniform monolayers in an acid medium. From Figure 3a, it is interesting to note that the profiles of Raman spectra obtained at various points of surface in a basic medium are not identical ones. The diversity of SERS spectra proposes that, at the very beginning in the alkaline solution, 6MP molecules in various structures might all attach onto the gold surface, It might be attributed to all of them possessing the S and N atoms served as the possible adsorption sites to bind the surface. This hypothesis could be supported by the careful observation on two spectra presented in Figure 4, which were taken from the Raman mapping profiles of Figure 3a. After comparison of the bands in the spectra of Figure 4 with the results of quantum calculations listed in Tables 1 and 2, for example, the SERS band near 1678 cm-1 should originate from the alkaline form (Scheme 2a) and bands at 1367 and 1620 cm-1 should be due to the ionized tautomer, but the most of bands result from the ionized thiol form. However, taking a queue time of ca. 6 h, one noticed that, in Figure 3b, the recorded profiles of Raman mapping from the 6MP adsorbed layers in the alkaline media were altered to be observed in the same way as in the acid solution. These shifts of the spectral features could be deduced to the modification of the adsorption mode and the adsorbed species due to the interaction between the interface and the bulk solution. Formation of SAMs might naturally tend to select a conformation of molecules with the lowest minimized energy such as herein the 6MP thiol form. In addition, the ionized thiol form of 6MP quickly bound onto the gold surface via the Au-S covalent bond and eventually competitively replaced the relatively unstable adsorption site formed through the interaction of the
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Yang et al.
Figure 3. Raman mapping of the formation of SAMs at the roughened gold surface recorded in 10-4 mol/L 6MP in an alkaline solution (pH 12.8) after (a) 30 min (b) 6 h.
SdC of the alkaline form or the ionized tautomer with the surface after a dynamically self-organized procedure. Adsorption Mode of 6MP SAMs at the Gold Surface. As mentioned above, both in the acid and alkaline media, the 6MP molecules tended to adopt the same adsorption mode to selfassemble monolayers at the gold surface, which was suggested by the profiles of Raman mapping obtained after a due time. It is well-known that the SERS technique can provide not only a high enhancement16 in Raman signals on the order of 106 even up to 1015 to detect Raman scattering signals from a single molecule17 but the special surface selection rules that can be
Figure 4. Two feature Raman spectra selected out of the profiles of Raman mapping recorded from the gold surface in 10-4 mol/L 6MP in an alkaline solution (pH 12.8) after 30 min.
employed to determinate the adsorption mode. Table 3 contains the observed SERS bands of 6MP SAMs together with the possible assignments based on the scaled quantum calculation results for vibrational modes. It can be seen in Table 3 and the SERS spectra of 6MP SAMs in Figure 1b that the band of 1023 cm-1 assigned to SH vibration is absent; additionally, in 6MP, a thiol forms the band at 441 cm-1 associated to C6-S vibration downward shifts about 12 cm-1. These observations indicate that the S atom with the
TABLE 3: Assignments for the Observed SERS Bands of 6MP SAMs at the Roughened Gold Surface (Wavenumbers Are Given in cm-1)a SERS bands 1588(m) 1532(w) 1467(s) 1389(s) 1325(vs) 1287(vs) 1257(vs) 1230(sh) 1199(m) 1135(s)
observed bands in an acid solution 1519 1449 1421 1377 1330 1265
1023 934(w) 852 (s) 679(m) 511(w) 429(w)
675 615 588 441
scaled bands of thiol form from BLYP/6-31G
assignments
1588 1494 1460 1422 1404 1343 1292 1271 1237 1162 1094 1018 945 836 676
νC2 - N1 + νC6C5C4 + δN9 - H νN7C8 + δC8 - H + δN9 - H δC2 - H δN9 - H + δC2 - H + νN9C8 + δC2 - H δC2 - H δC8 - H + br(pyrim + Imid) + δC2 - H δC2 - H + δC8 - H + νN1C2N3 νC6N1C2 + δC2 - H + δC8 - H + δN9 - H νC6 - S + δC2 - H + δC8 - H νC2 - N1 + δC8 - H + δC2 - H + δS - H δC8 - H + δN9 - H + νC8N9 δS - H δC5N7C8 + δS - H br(pyrim) + δS - H δC8 - Hop + δN9 - Hop
587 497 391
δC8 - H + δN9 - H + δC2 - H + δS - H νS - C6 + br(pyrim)
a Key: ν, stretching vibration; δ, deformation vibration; br, ring breathing vibration; op, out of plane; pyrim, pyrimidine; imid, Imidazole; sh, shoulder peak; vs, very strong; s, strong; m, medium; w, weak.
Studies of 6-Mercaptopurine SAMs SCHEME 3: A Suggested Adsorption Mode of 6MP SAMs at the Gold Surface
J. Phys. Chem. B, Vol. 109, No. 7, 2005 2743 SAMs was washed carefully using doubly distilled water and then a 0.1 mol/l KCl solution was injected into the cell. In Figure 5, one noticed that the SERS signals were sharply changed when the potential varied from -0.6 to -0.7 V vs SCE and disappeared as the potential negatively moved to -0.8 V, demonstrating that the 6MP molecules were detached from the gold surface. The similar phenomenon of electrochemical desorption of 6MP molecules from Au(111) in 0.1 mol/L KOH solution has been observed by Blazquez and co-workers5 using the cyclic voltammetry method, and the peak of desorption potential occurred at -0.65 V vs SCE. Their suggested adsorption mode of 6MP at the gold surface by the electrochemical method is in agreement with that proposed in this paper. According to the published papers5,18-29 on the reductive desorption process, the mechanism can be described by
Au - 6MP + 1 e f 6MP- + Au pyrimidine ring might be vertically bound onto the gold surface. It leads to the bands at 1467, 1389, 1287, and 1257 cm-1 due to deformation vibrations of C2-H of the pyrimidine ring to be drastically enhanced. Accordingly, the breathing vibration of the pyrimidine ring at 852 cm-1 unobservable in Raman spectrum of solution 6MP is enhanced to be observed in SERS spectra. Occurrence of the bands at 1532 and 934 cm-1 attributed to vibrations involving the N7 atom along with the observation of the SERS bands of C8-N9, C8-H, and N9-H might suggest that the N7 atom of imidazole ring should be another adsorption site with respect to the gold surface. The possible mode of 6MP adsorbed at the gold surface is presented in Scheme 3. In Situ SERS Spectroelectrochemical Measurements. There has been considerable interest in the examination of the stability of the 6MP SAMs on the gold electrode at the different potentials due to a wide field of its electrochemical applications. The spectroelectrochemical measurements were carried out in situ after the bulk solution containing the 6MP molecules was removed from the cell; the gold electrode surface with the 6MP
IV. Conclusions Formation processes of the 6MP SAMs at gold surfaces in acid and alkaline media were investigated using Raman mapping technique, and the stability of the SAMs was examined by in situ SERS spectroelectrochemical measurements. From these experiments, some conclusions could be reached as follows: (1) Both in acid and in alkaline solutions, 6MP molecules have a natural tendency to form SAMs at the gold surface via the S and N7 atoms so as to make the pyrimidine ring and imidazole ring standing at the surface as a vertical mode. (2) Desorption of 6MP molecules from a polycrystalline gold surface with a mechanism of one electron reduction processes occurring at the applied potential around -0.7 to -0.8 V vs SCE lead to destroying the SAMs. Acknowledgment. The financial support from the National Natural Science Foundation of China (Grant Nos. 20375012, 20075006, and 29975006), the Foundation for Ph.D. Thesis Research (Grants No. 20010532008), the Foundation of Science Commission of Hunan Province, and the Foundation of Shanghai Higher Education (Grant No. 03DZ16), and NSF in Shanghai are greatly appreciated. References and Notes
Figure 5. In situ SERS spectra of the 6MP SAMs on the gold surface recorded in a 0.1 mol/L KCl solution with shift of the applied voltages vs SCE.
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