Potential-Dependent Characterization of Bombesin Adsorbed States

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Potential-Dependent Characterization of Bombesin Adsorbed States on Roughened Ag, Au, and Cu Electrode Surfaces at Physiological pH Edyta Podstawka*,† and Gediminas Niaura‡ Regional Laboratory of Physicochemical Analysis and Structural Research, Faculty of Chemistry, Jagiellonian UniVersity, ul. Ingardena 3, 30-060 Krakow, Poland, Department of Bioelectrochemistry and Biospectroscopy, Institute of Biochemistry, Mokslininku¸ 12, LT-08662 Vilnius, Lithuania ReceiVed: April 27, 2009; ReVised Manuscript ReceiVed: June 10, 2009

This paper reports the direct surface-enhanced Raman spectroscopic (SERS) and generalized two-dimensional correlation analysis observations of the different orientations of the neurotransmitter bombesin (BN) chemisorbed on electrochemically roughened Ag, Au, and Cu electrode surfaces at different applied electrode potentials and at physiological pH. The presence of the indole ring of Trp8 and the amide bond between Gln7 and Trp8 of BN on these surfaces generates a specific SERS profile of BN adsorbed on the roughened Ag and Au electrodes that is affected by the electrode potential. Furthermore, for BN on Au, slight changes are observed in the band enhancement in comparison to what is observed for this neurotransmitter immobilized on Ag. In addition, there are larger changes in the spectra triggered by the substitution of Ag with Au electrodes and Ag with Cu electrodes than by substitution of Au with Cu electrodes. Introduction Cancer is a class of progressive diseases in which a group of cells display uncontrolled growth, invasion, and sometimes migration to other locations in the body via the lymph or blood.1,2 These three properties of malignant cancers differentiate between benign, nonoffensive growths, and malignant tumors. Most cancers are caused by abnormalities in the genetic material of the transformed cells, which can be due to exposure to disruptive substances called carcinogens.3,4 Other cancerpromoting genetic irregularities may be related to mutations in DNA or may be present in all cells from birth.5,6 The variation among individual cancers is usually affected by complex interactions between carcinogens and the host genome. One of the host genome agents is bombesin (BN, pGlu-Gln-Arg-LeuGly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2), an endogenous neurotransmitter that affects a broad range of physiological responses.7-9 BN has been shown to stimulate the growth of androgen-independent cancer cells, including small cell lung, glioblastoma, gastric, pancreatic, prostate, breast, and colon cancers,10-13 by expressing four classes of functional receptors belonging to the G protein-coupled superfamily with seven transmembrane-spanning regions.14 This makes these receptors potential receptor-positive cancer markers in early diagnosis and molecular targets for the detection of lesions in which they are expressed. These receptors include the bombesin/GRP-preferring subtype receptor (rGRP-R), the neuromedin B-preferring subtype receptor (rNMB-R), the orphan subtype-3 receptor (hBRS-3R), and the subtype-4 receptor (fBB4). These receptors exhibit close structural homology,15,16 but they require vastly different binding conformations for receptor occupation and activation and differ in the minimal peptide fragment necessary for detectable changes in biological activity and for maximal potency.17,18 Therefore, it is difficult to evaluate the contributions of each * Corresponding author. Phone: +48-12-663-2077. Fax: +48-12-6340515. E-mail: [email protected]. † Jagiellonian University. ‡ Institute of Biochemistry.

receptor subtype in affecting distinct physiological responses to BN. Moreover, the mechanism of BN tumor growth-stimulating effects for each of the four receptors remains unclear. One way to resolve this problem is to use specific antagonists to these receptors. This is also an efficient approach to the design of chemotherapeutic drugs and diagnostic agents. In this regard, several series of the G protein-coupled receptor antagonists have been developed and tested,19-23 including analogs of BN resulting from side-chain modification strategies (amino acid deletions or the retro-inverso modification), those with modified peptide bonds, those with a modified or deleted C-terminal region, and bombesin-related peptides. Attention has been focused on the conformations of these analogues and their possible roles in the interaction with receptors. A complementary approach is to use a surface-enhanced Raman scattering (SERS) technique that makes it possible to obtain further insight into the ways in which BN and its analogs interact with the surrounding medium, such as how they bind to the proper receptors. This is because, due to the nature of the adsorbate geometry, the adsorption phenomenon provides a unique and unprecedented opportunity to probe a protein/surface interface at the molecular level and to obtain specific information about molecular conformational changes occurring at this interface.24 Hence, we have previously determined the adsorbed molecular structures of BN and bombesin-like peptides on an electrochemically roughened silver electrode surface and a silver colloidal sol using SERS.25,26,32,33 Likewise, we have discovered the adsorption mechanism on these surfaces and changes in the adsorption process by substituting natural amino acids with synthetic amino acids for six modified BN analogs, including [D-Phe12]BN, [Tyr4]BN, [Tyr4, D-Phe12]BN, [D-Phe12, Leu14]BN, [Leu13-R-Leu14]BN, and [Lys3]BN.25,27 Very recently, we have also conducted SERS characterization of seven 6-14 fragments of the bombesin amino acid sequence, cyclo[D-Phe6, His7, Leu14]BN6-14, [D-Phe6, Leu-NHEt13, desMet14]BN6-14, [D-Phe6, Leu13, p-chloro-Phe14]BN6-14, [D-Phe6, β-Ala11, Phe13, Nle14]BN6-14, [D-Tyr6, β-Ala11, Phe13, Nle14]-

10.1021/jp903847c CCC: $40.75  2009 American Chemical Society Published on Web 07/14/2009

Characterization of Bombesin Adsorbed States

J. Phys. Chem. B, Vol. 113, No. 31, 2009 10975

BN6-14, [D-Tyr6, β-Phe11, Phe13, Nle14 OH]BN6-14, and [D-Cys6, Asn7, D-Ala11, Cys14]BN6-14.27 For these fragments, we have correlated the relative potency of inhibition of 125I-[Tyr4]BN binding to rat pancreas acini cells with the behavior of the amide bond on the silver surface, whereas the contribution of the structural components to the ability to interact with the rGRP-R was correlated with the SERS patterns. For example, we showed that the first five amino acids of the BN N-terminus do not influence the adsorption mechanism on the silver roughened electrode; they are also not essential for interaction with rGRPR.28 In addition, on the basis of the almost exclusive enhancement of the Trp8 bands in the SERS spectra of BN, its modified analogs, related peptides, and BN 6-14 fragments, we reached the conclusion that Trp8 is responsible for binding to the silver substrates as well as for receptor recognition.29 The strong enhancement of the CdO vibrations for BN, [Tyr4]BN, and [D-Phe12, Leu14]BN and the lack of enhancement for [Leu13-R-Leu14]BN confirmed the key role of the CdO fragment in recognizing the receptor pathway in pancreatic acinar cells.30 Substitution of Met14 with Leu14 ([D-Phe12, Leu14]BN) does not change the general adsorption mechanism of these peptides (except [Leu13-R-Leu14]BN) through the Trp8 residue, CdO fragment, or amide bond. This is in agreement with the biological activity studies showing that such modifications are not particularly important in the expression of biological activity at rGRP-R.31 We have compared these results with those of species adsorbed on a colloidal silver surface.32-34 Further, the observed SERS signals for carboxy terminal fragments of various lengths (X-14 of the amino acid sequence) of bombesin (BN13-14, BN12-14, BN11-14, BN10-14, BN9-14, and BN8-14) in silver colloidal solutions were compared with the contribution of the structural components of these fragments to their interactions with the receptors.35

Experimental Section

These investigations, together with biological activity studies, have shown that the electrochemically roughened Ag electrode surface is a more selective substrate than the colloidal silver surface. Further changes in selectivity might be triggered by the electrode potential and by the nature of the electrode itself. Because biological recognition usually takes place at a charged interface, potential-controlled experiments make it possible to elucidate specific electric field-induced effects. Therefore, our interest in the present work focuses on roughened electrode surfaces of Ag, Au, and Cu. To further facilitate the understanding of the physiological function of BN and its binding affinity for G protein-coupled receptors, as well as to explore the opportunity for therapeutic intervention with BN, we aimed to investigate the effects on bombesin-receptor interaction of potential-dependent BN · · · Ag, BN · · · Au, and BN · · · Cu electrode interactions. To better understand these issues, we characterized changes in the band enhancement, broadness, and wavenumber arising from the constituents’ amino acids/fragments of the adsorbed species depending on the type of metal substrate and the applied electrode potential. In this way, we can provide the missing structural information concerning the chemisorption of BN on the roughened metal electrode surface. Generalized two-dimensional correlation spectroscopy, which emphasizes spectral features not readily observed in the conventional one-dimensional spectra, was additionally applied for a detailed analysis of the SERS spectral signals, which change as a function of the electrode potential due to slight alterations in the molecular geometry of the substrate.

The Au electrode for SERS was electrochemically roughened by potential scanning (50 cycles) in a 0.1 M aqueous KCl solution between -0.30 and 1.31 V at a scan rate of 300 mV, as previously reported.38,39 Electrochemical roughening procedures for the Ag and Cu electrodes were performed by established methods as described in refs 40 and 41, respectively. Spectral Analysis. Second derivatives of the SERS spectra were calculated in the range of the amide I and II bands using the Savitzky-Golay algorithm42 (of the Grams/AI (7.0) program from Galactic Industries Co., Salem, NH) that produces the leastsquares best fit of the data to the selected polynomial. The number of convolution points can range from 5 to 10 000, although values greater than the number of points across a peak are not normally used. Only odd numbers are used for the number of convolution points, and even values are rounded up. The number of convolution points and degree were set at 10 and 5, respectively. These parameters did not distort the spectra, but a larger number of convolution points provided a smoother result.

Neurotransmitter. Bombesin (BN, pGlu-Gln-Arg-Leu-GlyAsn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) was purchased from Bachem Co., Switzerland. Its purity and chemical structure were proven by means of 1H and 13C NMR spectra (Bruker Avance DRX 300 MHz spectrometer) and by electrospray mass spectrometry (Finnigan Mat TSQ 700). SERS Measurements. Near-infrared SERS spectra were recorded using an Echelle type spectrometer RamanFlex 400 (PerkinElmer, Inc.) equipped with a thermoelectrically cooled (-50 °C) CCD camera and fiber-optic cable for excitation and collection of the Raman spectra. The 785 nm beam of the diode laser was used as the excitation source, and a 180° scattering geometry was employed. The laser power at the sample was restricted to 50 mW, and the beam was focused to a 200 µm diameter spot on the electrode, with an integration time of 10 s. Each spectrum was recorded with an accumulation of 30 scans. Spectro-electrochemical measurements were carried out in a cylindrical, three-electrode, moving cell, arranged with a flat circular electrode of ∼5 mm in diameter press-fitted into a Teflon rod; with gold, silver, or copper as the working electrode; platinum wire as the counter electrode; and a KCl saturated Ag/ AgCl reference electrode. All potential values reported below refer to the potential recorded by this reference electrode. During the experiment, the solution was continuously bubbling with ultra pure Ar gas to remove dissolved oxygen. The working electrode was placed approximately 3 mm from the cell window. To reduce photo and thermal effects, the cell and the electrodes were moved linearly with respect to the laser beam at a rate of about 15-25 mm/s.36,37 The Raman frequencies were calibrated using the polystyrene standard (ASTM E 1840) spectrum. Intensities were calibrated by a NIST intensity standard (SRM 2241). Experiments were conducted at 20 °C.

Generalized Two-Dimensional Correlation Analysis. G2DC analysis of the SERS spectra of BN adsorbed on the roughened electrode surfaces of Ag, Au, and Cu was performed using software 2Dshige version 1.3, which was composed by Shigeaki Morita, Kwansei-Gakuin University, 2004-2005. The four potential-dependent SERS spectra of BN were normalized. In the 2D correlation maps, red regions indicate positive correlation intensities, and blue regions indicate negative correlation intensities.

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Podstawka and Niaura

Figure 1. Raman spectrum of BN in the solid state in the wavenumber regions of 500-1800 and 2700-3400 cm-1. Measurement conditions: excitation wavelength, 785 nm; laser power at the sample, 50 mW; total integration time, 5000 s.

Results and Discussion Given the previously discussed band allocations to the normal mode motions for BN adsorbed on colloidal and electrode surfaces of silver,25-27,32-35 the assignments for nonadsorbed BN (Raman spectrum presented in Figure 1) and BN deposited onto the electrochemically roughened Ag, Au, and Cu electrodes are summarized in Table 1. The SERS spectra of BN adsorbed on the roughened Ag and Au electrode surfaces at physiological pH in the potential range of the electrodes from -1.200 to 0.000 V are presented in Figures 2 and 3, respectively. Figure 4 shows the SERS spectra of BN immobilized on the Cu electrode in a potential range between -1.200 and -0.200 V. Note that the intense and broad band near 940-960 cm-1 results from phosphate anions adsorbed at the Au and Cu electrodes (Figures 3 and 4).43 In general, the SERS spectra on the Ag, Au, and Cu electrodes (Figures 2-4) are very similar to each other, considering both the spectral pattern and the relative band intensities. They also closely resemble the SERS spectra of BN, its modified analogs, and fragments in the silver sol acquired at the same excitation wavelength (785 nm).32,34,35 This supports our earlier results that demonstrated that (i) the first five amino acids of the BN N-terminus do not influence the adsorption mechanism on the Ag roughened electrode or Ag nanoparticle surfaces, and they are not essential for the interaction with the BN receptors;28 (ii) the indole ring of the L-tryptophan residue at position 8 of the amino acid sequence (Trp8) of BN is almost exclusively responsible for the adsorption to the silver substrates and receptor recognition (Figure 5 contains additional support of Trp8 importance for the binding of BN, its fragments, and related peptides to the metal substrate and depicts characteristic wavenumber shifts of the enhanced indole ring modes upon H2O/D2O solvent substitution);29 and (iii) depending on the peptide structure, the CdO moiety of the amide bond between the Gln7 and Trp8 residues alters the BN potency as well as the silver surface affinity.30 However, some small, specific differences between the potential-dependent and substrate-dependent spectra can be clearly observed. For instance, for BN deposited onto the roughened Ag electrode (Figure 2), the bands at 758 and 1004 cm-1, characteristic of the in-plane indole ring (W18) and the

out-of-phase breathing vibrations of the phenyl/pyrrole rings (W16), respectively, considerably decrease in relative intensity when the potential of the Ag electrode becomes more positive (-0.400 and 0.000 V). This phenomenon is probably caused by the variations in the strength of indole · · · Ag interactions when the electrode potential is changed. One important property of the interface affecting the adsorption of molecules and ions is the potential of zero charge (pzc).44 For the Ag electrode, the pzc value in surface-inactive electrolytes was found to be near -0.9 V, considerably more negative as compared with the Au electrode (0.0-0.1 V).44-46 Thus, the Ag electrode surface charge is negligible near -0.800 V, whereas it becomes highly positively charged at -0.400 and 0.000 V. Hence, it seems that the indole ring of Trp8 directly and rather strongly interacts with Ag when the electrode potential is more negative (-1.200 and -0.800 V), whereas when the electrode potential is 0.000 V (Figure 2, trace d), the indole · · · Ag contact is no longer direct. The 7-9 cm-1 down-shift in wavenumber and 4 cm-1 band broadening of the ∼1004 cm-1 SERS band in the electrode potential range of -1.200 and -0.800 V (Figure 1, traces a and b, respectively) in comparison to the position (1011 cm-1) and width (fwhm ) 8 cm-1, fwhm: full width at half maximum) of this band in the normal Raman spectrum of neat, solid state BN (Table 1) supports this conclusion. On the other hand, the observed splitting of the W16 mode (1002 cm-1, fwhm ) 12 cm-1; and 1010 cm-1, fwhm ) 8 cm-1) in the BN SERS spectrum at -0.400 V (Figure 1, trace c) may imply that at this electrode potential, reorientation of the indole ring takes place in a monolayer formed by BN. Additionally, from the relative intensity ratio of bands I1002/I1010 equal to 2.3, it can be deduced that at this electrode potential, there are still more indole rings directly interacting with the roughened Ag electrode surface than those merely close to the substrate. The above-described differences can be successfully detected for isolated bands using simple spectral analysis because the relative intensity variations are sufficiently pronounced. If the relative intensity changes are too small, Generalized twodimensional correlation analysis (G2DCA) can be applied. This novel 2D correlation method is useful in the analysis of spectral signals, which change as a function of many kinds of reasonable



TABLE 1: Wavenumbers and Proposed Band Assignments for the RS and SERS Spectra of BN Adsorbed on Roughened Ag, Au, and Cu Electrode Surfaces wavenumbers/cm-1 SERS assignment

Ag(-1.200 V)

Au(-0.400 V)

Cu(-1.200 V)

νas(CH2/CH3)

2925

2924

νs(CH2/CH3) AI unordered/ν(CdO) in Gln7 AI R-helix AI β-turn/antiparallel β-sheet/unordered δas(NH2) W1 [phenyl + pyrrole ν(N1-C8)] W2 [phenyl] W3 [pyrrole ν(C2dC3)] AII and/or W4 [phenyl ν(CdC)] W5 [phenyl] and Fs(CH2) W6 [pyrrole (νs(N1C2C3) + δ(N1-H)) + phenyl δ(CH)], δas(CH3), and/or δ(CH2)

2874

2873

2921 2907 2875

ν(CdO) in Gln7 W7 [pyrrole ring ν(N1-C8); Fermi resonance] and/or Fw(CH2)

Ag colloidal surface ref. 32

Ag electrode surface ref. 25

RS 2973 2930 2873

1681 1653 1663

1671

1607 1565 1530 1439

1662

1670 1622

1626

1591 1547

1618 1594 1552 1486

1579 1552 1493

1436

1435

1438

1592 1562 1508 1454 1435

1621 1579 552

1424 1394 1360

1398 1355

1358

1315

1348 1326

1459 1433

1356

1357

1362

δi.p.(CH), Ft(CH2), and/or W8 [ν(C3-C9) + δ(N1-H)] Ft(CH2) AIII and/or δ(CCRH) W10 [ν(C3-Cβ) + ν(C-H)] and/or Ft(CH2)Trp

1313

1341 1320

1344

1303 1259

1252 1239

1265 1237

δ(N1-H) and/or Ft(NH2) in Asn/Gln Ft(NH2) in Asn ν(C-C)T alkyl chain and/or W13 [phenyl] ν(C-N) and/or Ft(CH2) ν(C-C)T alkyl chain, guanido group of Arg, and/or Ft(CH2)

1275 1253 1234

1188

1290 1274 1254 1225 1150

1119

1126 1107

1125

1136

1071

1077

1084

1007

1063 1049 1033 1011

W16 [phenyl and pyrrole ring out-of-phase breathing] ν(C-N), Fb(NH2), and/or phenyl Fb(CH) ν(C-C) ν(C-C)O) ν(C-C) ν(C-C) W17 [indole + Gb(N1-H) and Fermi resonance between phenyl ring breathing and o.o.p. ring bend overtone] W [o.o.p. CH phenyl deformation] and/or Fr(CH2) ν(C-C) and/or νs(CNC) secondary amide ν(C-C) W18 [sym phenyl/pyrrole ring breathing] W19 and/or ν(C-S) W and/or ν(C-S)

1111

1004

1011

1011

1011 987

875

880

878

1250 1223 1146 1126

966 957 909

958 931 902 890

1284

894

878

856

834

880 836

829

831

758 745

758 733

758 733 681

physical variables affecting the spectra, such as time, temperature, concentration, potential, pressure, and even chemical reaction.47-49 Therefore, to determine the small dissimilarities in the profile of the BN SERS spectra (Figure 2-4), we performed G2DCA using changes of the electrode potential as a variable. Figure 6 presents (a) synchronous and (b) asynchronous G2D-correlation maps in the frequency range of 1700-650 cm-1 generated from the potential-dependent SERS spectra of BN adsorbed on the roughened Ag electrode. The synchronous SERS spectrum (Figure 6a) contains two very strong ((758, 758) and (1002, 1002) cm-1), four medium ((1659, 1659), (1565, 1565), (1307, 1307), and (836, 836) cm-1), and three lowintensity ((1607, 1607), (1259, 1259), and (978, 978) cm-1) autopeaks. The strong intensity of the (758, 758) and (1002, 1002) cm-1 peaks suggests that the enhancement of these bands changes most significantly with the changes of the electrode potential. This supports the above observations. However, there are no peaks located at the diagonal positions that represent the ∼1439 (W6), ∼1356 (W7), and 1235 cm-1 (W10 +

835 814 760 691

760 722 700

Ft(CH2)Trp) SERS signals (Figure 6a). This may lead to the conclusion that there is no relative intensity alternation of these bands for electrode potentials between -1.200 and 0.000 V. Hence, there is no difference between C8-N1-C2dC3 and C3-Cβ(H2) (see Figure 7 for indole ring atom labeling) in the strength of interaction with the Ag surface when the electrode potential is changed. In addition to the autopeaks, several positive cross-peaks at (1659, 758), (1607, 758), (1565, 758), (1307, 758), (1259, 758), (1002, 758), and (836, 758) cm-1 are present in the synchronous spectrum. The positive sign of these cross-peaks indicates that all these SERS signals undergo potential-dependent enhancement changes in the same direction. In addition, the asynchronous SERS map develops several crosspeaks (Figure 6b). The appearance of these peaks in the asynchronous spectrum suggests that the directions of the transition moments of these modes are different. The positive sign of the (1565, 785), (1565, 1002), and (1565, 1307) cm-1 peaks indicates that potential-induced spectral changes take place earlier at 1565 cm-1 than those at 785, 1002, and 1307 cm-1.

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Figure 2. SERS spectra of BN adsorbed on a roughened Ag electrode at -1.200 (a), -0.800 (b), -0.400 (c), and 0.000 V (d) potentials over the 500-1800 (left) and 2700-3200 cm-1 (right) spectral regions. Measurement conditions: 0.1 M Na2SO4 solution containing 0.01 M phosphate buffer (pH ) 7.0) and 10-5 M BN; excitation wavelength, 785 nm; laser power at the sample, 50 mW; integration time, 1500 s.

Figure 3. SERS spectra of BN adsorbed on a roughened Au electrode at -1.200 (a), -0.800 (b), -0.400 (c), and 0.000 V (d) potentials over the 500-1800 (left) and 2700-3200 cm-1 (right) spectral regions. Measurement conditions: 0.1 M Na2SO4 solution containing 0.01 M phosphate buffer (pH ) 7.0) and 10-5 M BN; excitation wavelength, 785 nm; laser power at the sample, 50 mW; integration time, 300 s.

On the other hand, the negative sign of the two example peaks at (1307, 785) and (1002, 785) cm-1 suggests that spectral changes take place earlier at 785 cm-1 than those at 1307 and 1002 cm-1. Careful comparison of the SERS spectra in Figure 2 also shows a slight decrease in the enhancement of the two aforementioned bands due to pyrrole coring vibrations (i.e., ∼1565 (W3) and ∼1607 cm-1 (W1)) and no relative intensity change for ∼1439 (W6) and ∼1356 cm-1 (W7) (see Table 1 for detailed band assignments) when the electrode potential becomes more positive. The prominent wavenumber shift in

comparison with the position in the normal Raman spectrum of BN (Table 1) is observed for W1 (by ∼17 cm-1) only in the spectra at electrode potentials of -0.400 and 0.000 V (Figure 2, traces c and d, respectively) and for W3 (by 11-13 cm-1, respectively) in the spectra at electrode potentials of -1.200 and -0.800 V (Figure 2, traces a and b, respectively). On the other hand, for W7 and W1, a small wavenumber shift (up to 6 cm-1) is observed at all electrode potentials. These results point out that at the more negative electrode potential, the indole ring of Trp8 adsorbs on the roughened Ag electrode mainly via the C2dC3 fragment of the pyrrole coring, and when the



Figure 4. SERS spectra of BN adsorbed on a roughened Cu electrode at -1.200 (a), -0.800 (b), -0.600 (c), and -0.200 V (d) potentials over the 600-1800 (left) and 2700-3200 cm-1 (right) spectral regions. Measurement conditions: 0.1 M Na2SO4 solution containing 0.01 M phosphate buffer (pH ) 7.0) and 10-5 M BN; excitation wavelength, 785 nm; laser power at the sample, 50 mW; integration time, 300 s.

Figure 5. SERS spectra of BN adsorbed on roughened Cu electrode at -0.400 V in solutions prepared with H2O or D2O solvent. Measurement conditions: 0.1 M Na2SO4 solution containing 0.01 M phosphate buffer (pH 7.0) and 10-5 M of BN; excitation wavelength, 785 nm; laser power at the sample, 50 mW; integration time, 300 s.

electrode potential becomes less negative, the strength of this interaction weakens. Another small difference that distinguishes the SERS spectra of BN at different Ag electrode potentials is the relative intensity

of the W8 (ν(C3-C9) + δ(N1-H)) mode. The SERS signal due to this mode is observed around 1307 cm-1, and it only slightly loses its enhancement when the electrode potential becomes more positive (Figures 2 and 6). Hence, it is reasonable to conclude that when the electrode potential becomes more positive, the strength of the C3-C9 · · · Ag interactions weakens. This is probably because of some reorientation of the C2dC3 bond on the Ag surface with the assumption that this rearrangement does not disturb the C3-Cβ(H2) · · · Ag interactions. Hence, the enhancement of the 1235 cm-1 spectral feature stays invariant at different electrode potentials (Figure 6a). Finally, enhancement of the Trp8 phenyl-ring C-H stretching vibration near 3064 cm-1 at less negative electrode potentials (Figure 2) supports the suggestion of reorientation of the indole ring moiety from the near parallel ring plane orientation with respect to the electrode surface at -1200 mV to a more perpendicular geometry at -0.400 and 0.000 V.50 The last notable differences in the SERS spectra of BN adsorbed on the roughened Ag electrode at different applied potentials (Figure 2) are related to the amide I (1663 cm-1), II (1530 cm-1), and III (1259 cm-1) bands (see Table 1 for detailed band positions). As is evident, the width of the contributing component bands within the amide band region of the SERS spectra, 1700-1500 and 1300-1200 cm-1, is greater than the separation between the maxima of adjacent bands (Figure 2). As a consequence, the individual component bands are difficult to resolve in these experimental spectra. A deconvolution procedure usually allows for increasing the separation of the overlapping components present within the broadband envelope.25,51,52 This procedure demonstrates that the calculated second-derivative SERS spectra of BN deposited on this substrate at the different electrode potentials show several components listed in Table 2. Three of these components are observed at 1677-1683, 1659-1662, and ∼1530 cm-1 (Table 2). However, the synchronous G2D-

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Figure 6. Generalized synchronous (on left) and asynchronous (on right) 2D-correlation maps of the SERS spectra of BN adsorbed on a roughened Ag electrode as a function of electrode potential; spectral range of 1700-650 cm-1.

Figure 7. Structure and atom numbering scheme for tryptophan derivatives.

correlation map (Figure 6a) possesses only two autopeaks around these wavenumbers, i.e., (1659, 1659) and (1259, 1259) cm-1. Therefore, the higher-wavenumber component in the secondderivative SERS spectra is due to the ν(CdO) vibrations rather than to the turn structure of the BN polypeptide chain, whereas the lower-wavenumber band may have β-sheet character. In fact, it has been proposed that the active conformation of BN for interaction with rGRP-R is an antiparallel β-sheet structure at the C-terminus with a turn at position 10-13, and hydrogen bonds between the amide NH2 of Met14 and CdO of Trp8, CdO of Leu13 and N-H of Val10, and between N-H of Leu13 and CdO Val10.53-55 The amide I band observed in the experimental SERS spectra loses enhancement and shifts in wavenumber (1663 f 1668 cm-1) toward its position in the normal BN Raman spectrum when the electrode potential becomes more positive. At the same time, the amide III band intensifies and moves toward its position in the normal Raman spectrum (by 9 cm-1 at -1.200 and -0.800 V, 22 cm-1 at -0.400 V, and 27 cm-1 at 0.000 V). Only the amide II band is constant and seems to be almost independent of the applied electrode potential (Figures 2 and 6, Table 2). These observations are likely due to the rearrangement in the orientation of the amide bond with respect to the roughened Ag electrode surface when the electrode potential changes. We propose that the following changes take place when the electrode potential becomes less negative: For all electrode potentials, the -CONH- bond between Gln7 and Trp8 adopts a tilted orientation. At more negative electrode potentials, the CdO fragment of this bond interacts more closely with the roughened Ag electrode. With increasing positive potential, the CdO fragment moves slightly away from the electrode surface, while the N-H unit approaches this surface more closely. On going from the SERS spectra (Figure 2) and G2Dcorrelation maps (Figure 6) of BN adsorbed on the roughened Ag electrode to the SERS spectra (Figure 3) and G2Dcorrelation maps (Figure 8) of this neurotransmitter immobilized

on the Au electrode surface, several important differences in the relative intensity of the previously discussed spectral features are observed. For example, the most intense autopeaks of the synchronous G2D-correlation map of BN on Au (Figure 8a) are (878, 878) and (1008, 1008) cm-1. This means that instead of the 758 cm-1 band, the 878 cm-1 SERS signal on the roughened Au electrode prominently changes the enhancement. Note also that the autopeaks of the synchronous G2D-correlation map on Ag assignable to W1, W19, amide I, and ν(C-C) (see Tables 1 and 2 for band positions) disappear in the synchronous G2D-correlation map of BN deposited on the Au electrode, whereas (1683, 1683), (1357, 1357), (1239, 1239), and (1118, 1118) cm-1 appear. These facts show that a slightly different structure occurs on the Au electrode when its potential becomes more positive than that on Ag. Several positive cross peaks are also observed (i.e., (1008, 878), (1236, 878), (1357, 878), (1239, 1008), (1357, 1008), (1547, 1008), (1547, 878), (1547, 1239), and (1357, 1239) cm-1) (Figure 8a)), suggesting that the relative intensities of these spectral features alter in the same direction. On the other hand, from the asynchronous G2D-correlation map of BN on Au (Figure 8b), it is evident that changes at 1547 cm-1 take place after those at 1008, 878, and 733 cm-1, whereas the relative intensity change at 1008 cm-1 takes place before that at 1357, 1239, and 1118 cm-1. These results highlight the following possible change in the adsorption mechanism of BN on the roughened Au electrode. The indole ring is tilted with respect to the Au surface. When the Au electrode potential becomes more positive, the tilt angle formed between the indole ring and the substrate slightly increases. This is observed as an intensification of the 1011 cm-1 SERS signal (I1011 v) and a lack of shift in the wavenumber (by 3-6 cm-1 at -1.200 and -0.800 V; and 0 cm-1 at -0.400 and 0.000 V), which is contrary to the results on the roughened Ag surface. In this orientation, the lone pair of electrons on the nitrogen has easier access to the Au surface (I878 v and I1356 v). Thus, the C2dC3-Cβ(H2) fragment may interact more closely with Au (I1547 v and I1235 v). The behavior of the amide signals in the SERS spectra of BN adsorbed on the roughened Au electrode (Figures 3 and 8) is dramatically different from that observed on the Ag substrate. First, the relative intensity of the amide I, II, and III bands (see Table 1 for positions) does not change when the electrode potential becomes more positive (Figures 3 and 8), indicating a lack of changes in the -CONH- bond orientation that is also tilted with respect to the Au surface. Second, the shift in wavenumber of these SERS spectral features in comparison to those in the normal BN Raman spectrum (amide I by 7-12 cm-1, amide III by 23-27 cm-1 (Table 2)) and those in the



TABLE 2: Calculated Wavenumbers in Second-Derivative SERS Spectra of BN Adsorbed on Roughened Ag, Au, and Cu Electrode Surfaces at Different Electrode Potentials wavenumber/cm-1 (1700-1500 cm-1) AI

AI

-1.200 V -0.800 V -0.400 V 0.000 V

1677 1680 1683 1682

-1.200 V -0.800 V -0.400 V 0.000 V -1.200 -0.800 -0.600 -0.200

(1350-1200 cm-1)

W1, W2

W3

1659 1662 1661 1659

1607 1607 1607 1607

1565 1562 1558 1550

1682 1679 1681 1684

1663 1662 1659 1658

1599 1593 1593 1592

1554 1556 1553 1551

1681 1683 1681 1678

1660 1661 1661

1619, 1583 1593 1594 1598

1550 1549 1549 1549

AII

AIII

AIII

AIII

1531 1531 1530

1307 1307 1313 1313

1259 1258 1275 1276

1231 1231 1231 1235

1527 1529 1531 1527

1315 1315 1315 1315

1276 1273

1233 1233 1235 1232

1524 1524 1524 1524

1318 1317 1315 1315

1265 1265 1267 1272

1232 1232 1232 1232

Ag

Au

Cu V V V V

SERS spectra on Ag (amide I by 8-11 cm-1, amide III by 8-9 and ∼25 cm-1 at -1.200 to -0.800 V and -0.400-0.000 V, respectively (Table 2)), are indicative of a conformational heterogeneity of -CONH- on Ag and homogeneity on Au. The positions and relative intensities of the SERS signals of BN adsorbed on the roughened Cu electrode (Figure 4) at the different electrode potentials seems to be identical. The same conclusion can be drawn when analyzing the synchronous G2Dcorrelation map (Figure 9a) generated from these spectra, which shows only one peak (at (943, 943) cm-1) belonging to the SERS signal of adsorbed phosphate anions.43 This phenomenon is in obvious contrast with the results obtained for BN adsorbed on the roughened Ag and Au electrodes and points out that the adsorption characteristics of BN on Cu at the different electrode potentials were found to be analogous. Moreover, on the basis of a comparison of the enhancement and position of the bands observed in the BN SERS spectra on Cu (Figure 4) with those on Au at -1.200 V (Figure 3, trace a), it can also be clearly seen that only the phosphate, amide III, and W16 bands (see Table 1 for band allocations) change their relative intensities among the Cu and Au substrates. The two former SERS signals also exhibit slightly lower and higher wavenumbers, respectively, on the roughened Cu electrode than those on Au. The presence of the W7 doublet and W4 modes at roughly 1340/ 1360 and 1490 cm-1 in the BN SERS spectra on Cu,

respectively, also demonstrated that although the Cu and Au substrates have almost the same adsorption mechanism, the geometry of the indole ring on these surfaces and the strength of the indole ring interactions are significantly different. Regarding these matters, W18, W17, W16, W7, and W1 exhibit a more or less comparable wavenumber shift on both the roughened Cu and Au electrodes, whereas W6 and W3 exhibit a more pronounced wavenumber shift on the Au electrode (by 6-17 and 4-6 cm-1, respectively) than on the Cu electrode (by 3-7 and ∼1 cm-1, respectively). Therefore, it can be proposed that the N1-C2dC3 fragment of the indole ring interacts more strongly with the Au surface than with the Cu surface. Due to the weaker N1-C2dC3 · · · Cu complex formation, the CdC8-N1 unit can approach this surface more easily. The contribution of the -CH2- unit of Trp8 to the SERS spectra of BN deposited onto the roughened Ag, Au, and Cu electrodes (Figures 2-4) is manifested by the strong intensity bands at around 2925 and 2874 cm-1. These are due to the asymmetric and symmetric stretching vibrations of the C-H bond, respectively. The relative intensity of the latter spectral feature decreases for BN adsorbed on the Ag electrode when the electrode potential becomes more positive (Figure 2), whereas for BN immobilized onto the roughened Au electrode surface, the former one decreases in the relative intensity (Figure 3). In the case of BN deposited onto the Cu electrode, the

Figure 8. Generalized synchronous (on left) and asynchronous (on right) 2D-correlation maps of the SERS spectra of BN adsorbed on a roughened Au electrode as a function of electrode potential; spectral range of 1700-650 cm-1.

10982



Figure 9. Generalized synchronous (left) and asynchronous (right) 2D-correlation maps of the SERS spectra of BN adsorbed on a roughened Cu electrode as a function of electrode potential; spectral range of 1700-650 cm-1.

relatively strong 2921 cm-1 band splits into two bands (2907 and 2930 cm-1, based on the deconvolution procedure of a complex band) when the electrode potential becomes more positive (Figure 4). From those, the 2907 cm-1 loses enhancement when the electrode potential becomes more positive, whereas the enhancement of the 2875 and 2930 cm-1 spectral features is independent of the applied electrode potential. Applying a method introduced by Pemberton et al.,56 based on the relative intensity behavior of the symmetric and asymmetric C-H stretching vibrations of the methyl groups contained within the adsorbed molecule, we calculated a tilt angle (θ) of the C2 axis versus the metal surface. The tilt angle can be easily calculated from the surface-induced changes in the intensities ratio of the ν asym(C-H) and νsym (C-H) bands (x) for the adsorbed (surf) and non adsorbed (sol) species:

x)

[I(νasym(C - H)/I(νsym(C - H)]surf [I(νasym(C - H)/I(νsym(C - H)]sol

When the C2 axis of the methylene group is perpendicular to the metal surface (θ ) 0°), the x value should be equal to 0.1. As the tilt angle of the C2 axis, relative to the surface normal, increases, the value of x also increases. Hence, for the next limiting cases θ < 45°, θ ) 45°, θ > 45°, x yields values of ∼0.6, 1, and 5, respectively. The calculated tilt angle for BN adsorbed on the Ag and Au electrodes increases from 0.55 to 0.62 to 0.90 to 1.19 and from 0.68 to 0.80 to 1.04 to 1.21, respectively, when the electrode potential becomes more positive. These values indicate that the tilt angle of the C2 axis of the -CH2- group of BN on the Ag and Au electrodes at the electrode potential of -1.200 V is less than 45° and only slightly increases when the electrode potential becomes more positive. In the case of BN on the Cu electrode, θ is equal to 45° (0.68) for all the applied electrode potential. This suggests no changes of the C2 axis tilt angle of the Trp8 -CH2- group with respect to the Cu surface. In addition, this is in agreement with the chance of the observed changes, due to Trp8, in the wavenumber range of 1800-600 cm-1 of the SERS spectra of BN immobilized onto the Cu electrode.

electrode potential on the structure of this neurotransmitter. We showed that only imperceptible dissimilarities in the SERS spectra pattern are observed when the Ag and Au electrode potentials become more positive. To correctly define the extent of these alternations, we applied the G2DCA method. We demonstrated that the interaction of the indole ring of Trp8 with the Ag, Au, and Cu substrates adopts a tilted orientation, with the pyrrole coring being closer to these surfaces than the phenyl coring. However, the mechanism of these interactions is not considerably different, so the SERS spectra profile variations are small. For example, the indole ring on the roughened Ag electrode adsorbs mainly via the C2dC3 fragment of the pyrrole coring, and when the electrode potential becomes less negative, the strength of this interaction decreases. At the same time, the indole ring neither moves away from the Ag surface nor lies down on the surface. On the other hand, when the Au electrode potential becomes more positive, the tilt angle formed between the indole ring and the substrate slightly increases. Hence, the nitrogen lone pair of electrons can more easily approach the Au substrate, allowing the C2dC3-Cβ(H2) fragment to interact more closely with this substrate. In the last case, the Cu substrate, the indole ring orientation at all applied potentials is the same as that on the Au electrode at -1.200 V. In addition, the effects of the metal substrate substitution and electrode potential have an influence on the behavior of the -CONH- bond between Gln7 and Trp8. In the case of Ag, the amide bond adopts a tilted orientation with respect to the Ag electrode surface. At more negative Ag electrode potentials, its CdO fragment interacts more closely with this substrate, whereas at more positive potentials, the N-H unit approaches this surface more closely. In contrast, on the roughened Au and Cu electrodes, the -CONH- bond orientation stays invariant when the electrode potential becomes less negative. Hence, -CONH- shows conformational homogeneity on the roughened Au and Cu electrodes. Acknowledgment. This work was supported by the Polish State Department for Scientific Research (Grant No. N N204 159136 to E.P.). G.N. gratefully acknowledges the Department of Organic Chemistry at the Institute of Chemistry (Vilnius) for use of their Raman spectrometer.

Conclusions This study combined voltammetry and SERS spectroscopy to study the structural properties of BN adsorbed on electrochemically roughened Ag, Au, and Cu electrodes at physiological pH. We investigated the effects of the metal substrate and

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JP903847C

Potential-Dependent Characterization of Bombesin Adsorbed States

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