12796 J. Phys. Chem. 1994,98, 12796-12804

SERRS Study of Acridine Orange and Its Binding to DNA Strands

F. Zimmermann, B. Hossenfelder, J.-C. Panitz, and A. Wokaun' Physical Chemistry II, University of Bayreuth 0-95440 Bayreuth, Germany

Received: June 23, 1994; In Final Form: September 13, 1994@

Surface-enhanced resonance Raman spectra of acridine orange are recorded both on silver island films and on silver colloids, for solution concentrations as low as M. A detailed assignment is achieved by a comparison of the observed band positions with frequencies calculated by normal coordinate analysis. Band specific signal enhancement factors are used to obtain information on the adsorption geometry at the silver surface. Qualitatively similar surface-enhanced spectra are obtained when the acridine orange dye is intercalated into calf thymus DNA. The spectra are compatible with an adsorption geometry in which the axis of the double helix is oriented parallel to the silver surface. The structure and electronic properties of acridine orange are modeled by semiempirical quantum chemical calculations. Excellent agreement with respect to geometric parameters and the long-wavelength absorption maximum is obtained. Analysis of bond order changes upon excitation is used to discuss the band specific dependence of signal intensities on the excitation wavelength.

Introduction

Molecules with planar aromatic ring systems may interact with DNA in a process known as intercalation, which involves insertion of the planar molecules between DNA base pairs in a position approximately perpendicular to the axis of the double

This interaction gives rise to a variety of biological consequences. For the class of acridine dyes, mutagenic, carcinogenic, antibacterial, and antiviral properties have been demon~trated.~ Several acridines, such as the compound acridine orange (AO) investigated in this study, are also used as nucleic acid stains.5

Techniques that have been used to data in investigations of intercalation phenomena include fluorescence,6-8 UV-visible absorp t i~n ,~*~ NMR,'O and resonance Raman spectro~copy.~~' 1-13

Surface-enhanced resonance Raman scattering (SERRS) is a particularly attractive tool for studies of intercalating dyes: The high sensitivity of this method implies the potential of examining fluorescent chromophores at very low concentration^.'^-^^ The efficiency of SERRS for obtaining information on the interaction of dyes with DNA has already been demonstrated in recent studies. 17-21

From the SERRS spectra, a wealth of information on adsorption geometry, molecular structure, and the mode of binding of adsorbates at a metal surface may be d e r i ~ e d * ~ - ~ ~ Le. by analyzing the observed changes in relative band intensities in terms of the well-known selection rules for the electromag- netic enhancement mechanism.22-28 In the present study, the SERRS spectra of acridine orange adsorbed on Ag colloids and silver island film (AgIF) are investigated, and the complex of A 0 with calf thymus DNA on silver colloids is studied. From a comparison of the resonance Raman and SERRS spectra with the results of a normal coordinate analysis, an extensive assignment of the A 0 modes is achieved, and approximate models for the adsorption geometry on both silver substrates are developed.

*Author to whom correspondence should be addressed. Present address: Swiss Federal Institute of Technology, Zurich, and Paul Scherrer Institute, Department F5, CH-5232 Villigen, Switzerland.

@ Abstract published in Advance ACS Abstracts, November 1, 1994.

Experimental Section Solutions and Substrates. All solutions were prepared with

triply distilled water. Acridine orange, as obtained from Aldrich as the zinc chloride double salt, was used without further purification; a stock solution of 1 x M A 0 in water was prepared and employed for all investigations. Calf thymus DNA was purchased from SERVA; a stock solution of 8.6 x mol of nucleotide per liter in phosphate buffer (pH = 7, klerck) was used. The DNA concentration (as referred to as moles of nucleotide per liter) was determined by measuring the UV absorbance and using literature values34 for the extinction coefficient (€260 = 6600 L mol-' cm-').

The AO-DNA complex was prepared by mixing the dye solution with DNA at a dye-to-nucleotide ratio of 1.22 x

Colloidal silver sols were prepared according to the method of C r e i g h t ~ n . ~ ~ After aging for one day, the dye or its complex with DNA was added, for a final A 0 concentration of about 1 x lo-' M. Finally, small amounts of KI or HN03 solution were added in order to suppress the fluorescence of AO. At the same time, a partial coagulation of the silver sol was observed to occur.

Silver island films of 5 nm mass thickness were prepared by slow thermal evaporation (0.5 nm/min) onto glass slides in a bell jar chamber (Balzers, model BA 360). The average mass thickness of the film was measured with a 6 MHz quartz crystal microbalance (film thickness monitor, Balzers, model QSG 301). After aging the films for a week under atmospheric conditions, two drops of the 1 x M A 0 stock solution were deposited onto the island films by spin coating.

Spectroscopic Measurements. UV-visible spectra were recorded on a double-beam instrument (Perkin Elmer, model Lambda 19). For IR spectroscopy, a solid aliquot of A 0 was diluted by the silicon carbide technique. A small amount was dispersed on a silicon carbide substrate (LOT) and placed into a diffuse reflectance cell (Spectra-Tech). The spectrum was recorded on an FTIR instrument (Mattson Polaris); 50 scans were coadded at a resolution of 2 cm-'.

Raman spectra were excited using the 530.9 and 476.2 nm lines of a krypton ion laser (Coherent, model INNOVA 301). The laser beam was focused onto an 8 mm x 0.2 mm line on a quartz cuvette for the liquid samples, with the laser power

0022-365419412098-12796$04.5010 0 1994 American Chemical Society

Acridine Orange and Its Binding to DNA Strands J. Phys. Chem., Vol. 98, No. 48, 1994 12797

Figure 1. Molecular geometry calculated using the PM3 method. Atomic labels were chosen according to the IUPAC nomenclature of the acridine nucleus and conform to those used in the tables.

adjusted between 20 and 30 mW. A backscattering geometry was used for detection.

The silver island films were illuminated at an angle of 45O, by focusing 1.5 mW of power onto a circular spot (diameter 100 pm). The signals were detected at an angle of 90" with respect to the incident laser beam. Raman signals were dispersed in a triple spectrograph (SPEX, model 1877A) and recorded on an optical multichannel analyzer based on a cooled, intensified photodiode array.36 The resolution of the triple spectrograph was set at 6 cm-'. Signal accumulation times on the diode array were adjusted between 10 and 80 s, depending on signal strength; 10-20 exposures were accumulated for each spectrum. Band positions of the Raman signals have been carefully calibrated by means of a neon spectral lamp. Intensi- ties have been normalized with respect to laser power and integration time. The detector sensitivity corresponds to ~ 0 . 2 least significant bit (LSB) per incident photon.

Quantum Chemical Calculations. Semiempirical molecular orbital calculations were carried out on a DECstation (Digital Equipment Corp., model 5000/120) using the program package SCAMP 4.30.37 The latter is based on the MOPAC38 and

programs but offers some additional features. The PM3 parametrizationm was used throughout all calculations. Generation of input files and processing of output files were conveniently handled with the aid of the graphical editor program SHOWMOLE.41

Computations were performed without using any assumptions regarding the molecular geometry and without imposing sym- metry restrictions; all structural parameters were optimized in the calculations. After the molecular geometry had been optimized in a two-step process, it was verified that the resulting structure did represent a stationary point on the energy surface, by calculating the eigenvalues of vibration, rotation, and t ran~la t ion .~~ All eigenvalues had a positive sign, as required. For comparison with the results of FTIR and Raman spectros- copy, the calculated frequencies were multiplied by a constant factor of 0.9.43

Electronic transitions were calculated in a configuration interaction approach. The maximum number of excited elec- trons available in the present implementation of the program was used for this purpose (keyword PECI = 8). In addition, properties of the first excited singlet state (SI) have been calculated: After modeling a vertical transition, the geometry of the excited state was allowed to relax toward the final structure under the SCAMP 4.30 routine SLOW. As before, the maximum number of eight excited electrons was used in

the confguration interaction. We are aware that this represents a limitation, as more than eight electrons would be appropriate for an adequate description of the x-electron system modeled in this study, which contains (14 + 4) n-electrons.

Results and Discussion

Molecular Geometry and Normal Vibrations. The mo- lecular geometry of the bis(dimethy1amino)acridinium cation (Le., of A 0 without its counterion) in its electronic ground state SO is depicted in Figure 1. (A conventional structural formula, as displayed in the inset of Figure 3, shows the CzV symmetry of the most symmetric conformation.) As expected, the result of the calculations shows the acridine ring system to be planar in the ground state. The dimethylamino groups are slightly tilted out of the plane of the acridine moiety.

Calculated geometrical parameters may be compared with the results of a recent X-ray crystal structure determination of acridine orange hydrochloride monohydrate,u which crystallizes in the monoclinic space group P21/a with four molecules per unit cell. In the crystal structure, the acridine ring system is found to have the full "2 (CzJ symmetry; the dimethylamino substituents are displaced from the plane of the acridine nucleus. The latter result is explainedu in terms of intermolecular interactions in the crystal.

The electron density map suggests& that the n-electron density may be located in three different subsystems. A polymethine chain including the centers N15-C3-C4-N10- C5-C6-N16 comprises 10 rc-electrons (atom designations refer to Figure 1). Another subsystem is spread over the centers Cll-C9-C14 and contains 4 delocalized n-electrons. Finally, there are two approximately localized double bonds between centers C1 -C2 and C7-C8, respectively, accommodating 4 more n-electrons. The sum of 18 electrons comprises the acridine ring system plus the two lone pairs from the dimethyl- amino substituents.

Calculated and experimental bond lengths, bond angles, and dihedral angles are juxtaposed in Table 1; an overall good agreement is noted. The standard deviation between experi- mental and calculated bond lengths amounts to 2.2 pm; for the bond angles, the corresponding deviations amount to 1.6'. Dihedral angles relevant for the conformation of the dimethy- lamino groups are less well defined, with a standard deviation of 14". The conformation of these substituents, with large- amplitude degrees of freedom of internal motion, is expected to depend strongly on packing effects in the crystal structure.

12798 J. Phys. Chem., Vol. 98, No. 48, 1994

TABLE 1: Calculated vs Experimental" Bond Lengths (A), Bond Angles (deg), and Dihedral Angles (deg) of Acridine Orange

Zimmermann et al.

PM3 data X-ray data

Cl-C2 C2-C3 c3-c4 C4-Cl2 Cl l -c12 c1-c11 C9-Cll C9-Cl4 C13-Cl4 C5-Cl3 C5-C6 C6-C7 C7-C8 C8-Cl4 C12-Nl0 C13-Nl0 C3-Nl5 C6-Nl6 N15-Cl7 N15-Cl8 N16-Cl9 N16-C20

Cl-C2-C3 c2-c3 -c4 c3-c4-c12 C4-Cl2-Cll C12-Cll-Cl c11 -c1 -c2 c9-c11 -c12 C1 l-Cl2-Nl0 C12-NlO-Cl3 NlO-Cl3-Cl4 c 13 -c 14-c9 C14-C9-Cll C8-Cl4-Cl3 c14-c13-c5 C13-C5-C6 C5-C6-C7 C6-C7-C8 C7-C8-C14 C2-C3-N15 C7-C6-N16 C3-Nl5-Cl7 C3-Nl5-Cl8 C6-N 16-C 19 C6-N16-C20 C17-Nl5-Cl8 C19-N16-C20

C17-N15-C3-C2 C17-N15-C3-C4 C18-N15-C3-C2 C 18 -N15 -C3 -C4 C19-N16-C6-C5 C19-N16-C6-C7 C20-N16-C6-C5 C20-N16-C6-C7

Bond Lengths 135.9 143.8 140.1 140.3 142.4 142.9 139.7 139.7 142.4 140.3 140.1 143.8 135.9 142.9 139.5 139.5 139.8 139.8 148.0 148.0 148.0 148.0

121.2 118.8 120.1 121.0 117.9 121.0 120.1 118.8 121.8 118.8 120.2 120.3 117.9 121.0 120.1 118.8 121.2 121.0 120.1 120.1 120.5 120.8 120.5 120.8 113.7 113.5

Dihedral Angles -9.4 165.0 196.3

12.1 -11.0

-190.9 -163.6

16.4

Bond Angles

133.9 145.2 139.7 138.2 143.6 141.9 138.7 138.5 142.6 139.1 138.9 144.3 134.6 142.1 136.7 136.5 134.8 136.7 146.6 143.9 145.0 146.6

120.4 117.8 121.0 121.6 116.0 123.2 118.9 117.6 124.6 118.0 119.0 121.9 116.3 122.0 119.9 118.9 120.3 122.6 121.0 119.4 122.3 121.4 120.4 122.0 116.5 117.2

0.0 180.0 177.4 -2.9

2.7 -176.0 -170.2

11.1 A further test of the calculated structure may be performed

by comparing calculated bond orders with the experimental electron density maps. In the semiempirical model used, bond orders and bond lengths are linearly dependent. Inspection of Table 2 reveals that bond orders in the ground state conform to the above-mentioned polymethine chain. Also, the decomposi- tion of the n-electron system in three subsets is reproduced very well by our calculation. For example, the isolated double bond Cl=C2 (order 1.69) is surrounded by the single bonds C2-C3 and C 1 -C 1 1 (order 1.15). In the allyl-type four-electron system Cll-C9-C14, as well as in the polymethine chain, the bond orders are intermediate.

TABLE 2: Bond Orders in the Acridine Orange Species

bis(dimethy1- amino)acridinium amino)acridinium bis(dimethy1- bis(dimethy1-

amino)acridine, So cation, So cation, S1

Cl-C2 1.694 1.673 1.614 C2-C3 1.148 1.155 1.177 c3-c4 1.596 1.371 1.298 C4-Cl2 1.155 1.319 1.378 Cl l -c12 1.228 1.218 1.167 c1-c11 1.149 1.159 1.164 C9-C11 1.378 1.360 1.373 C9-Cl4 1.378 1.359 1.230 C13-Cl4 1.228 1.218 1.211 C5-Cl3 1.155 1.319 1.407 C5-C6 1.596 1.371 1.241 C6-C7 1.148 1.155 1.197 C7-C8 1.694 1.673 1.555 C8-Cl4 1.149 1.159 1.229 C12-Nl0 1.382 1.212 1.147 C13-Nl0 1.382 1.212 1.116 C3-Nl5 1.049 1.232 1.197 C6-Nl6 1.049 1.232 1.276 N15-Cl7 0.987 0.974 0.976 N15-Cl8 0.985 0.973 0.975 N16-Cl9 0.987 0.974 0.984 N16-C20 0.985 0.973 0.983

The protonated form of acridine orange consists of 40 atoms; hence, the molecule has 114 normal vibrations. Frequencies of a large number of these normal modes in the optimized ground state, as obtained by the SCAMP program, are included in Table 3. As it is well-known that the frequencies predicted by semiempirical calculations are consistently higher than the experimental ones, the output values have been multiplied by the accepted scaling factor of 0.9, as mentioned in the Experimental Section. For later use, the symmetry species of selected normal vibrations was derived from an inspection of the relevant eigenvectors.

Assignment of Raman and IR Spectra. In order to approach a detailed assignment of the vibrations of such a large molecule, all accessible spectroscopic information must be used and compared with the results of the normal coordinate analysis. As a starting point the infrared and resonance Raman and IR spectra of A 0 have been recorded and are shown in Figure 2. These data are supplemented by frequencies from the SERRS spectra of A 0 on a silver sol (Figure 3, upper trace) and on a silver island film (lower trace). In the SERRS spectra, a large number of well resolved Raman bands is recorded with remarkable intensities; the determined frequencies are listed in Table 3.

A detailed assignment of nearly all Raman bands was achieved on the basis of the normal coordinate analysis and by comparison with literature data for a ~ r i d i n e ~ ~ - ~ * and for anthra~ene!~.~~ Results are given in Table 3. In particular, we note the excellent agreement between the band positions of the detected SERRS signals and the frequencies from normal coordinate analysis.

Only a few important bands with prominent intensities shall be discussed in more detail. The ten bands in the region from 1640 to 1370 cm-' (cf. Table 3 and Figures 2 and 3) have been assigned to C-C and C=N ring stretching vibrations. The intensity of these modes on the silver island film is remarkably weak, as compared to the strength of the same bands on the silver sol. This effect may be due to a different orientation of the ring system relative to the respective silver substrate, as will be discussed below. The detection of vibrations involving the CH3 group (e.g., at 1356, 1324, 1229, and 920 cm-') in the SERRS spectra is important for determining the orientation of adsorbed AO. In view of the pronounced distance dependence

Acridine Orange and Its Binding to DNA Strands

TABLE 3: Vibrational Frequencies of Acridine Orange from SERRS, Resonance Raman, and IR Measurements

J. Phys. Chem., Vol. 98, No. 48, 1994 12799

enhancemenr SERRS on Ag sol SERRS on AgIF resonance Raman IR calc" assignmentb (Ag soVAgIF)

1638 s 1637 m 1643 m 1596 vw 1570 m 1556 sh 1530 1484 m 1452 m 1431 sh 1407 w 1372 s 1356 s 1324 vs 1324 vs

1272 m 1262 sh

1141 vw 1129 vw 963 w 920 sh 906 m 906 m

866 vw 858 w

817 w 804 w 748 m 713 w 698 vw 677 sh 657 s 630 vs 604 m 578 m 559 sh

535 vw 515 sh 496 m

1575 m 1552 sh 1530 vw 1487 w 1449 w 1426 vw 1386 sh 1372 s 1352 sh 1325 m 1325 m 1296 w 1274 vw 1265 vw 1229 w 1142 vw

972 vw 923 vw 909 m 909 m 879 vw 868 w 859 vw 840 vw 812 sh 807 m 752 vs 720 vw 697 vw 680 vw 654 sh 632 vvs 610 vw 586 s 563 sh

536 vw 509 sh 499 s

1580 vw 1544 vw

1437 vw

1404 vw 1376 m

1324 w 1324 w 1292 vw

1266 vw

975 vw 926 vw 901 vw 901 vw

862 vw

808 w 753 m

634 m

587 vw

497 m

1603 m

1502 m

1444 w

1409 w 1383 m 1359 m

1295 w

1250 m 1231 m 1137 m

965 s 922 vs

824 vs 790 s

699 vs

643 m

588 m 570 w

536 vw 506 s

1621, AI 1595, BI 1569, BI 1544, AI 1519, BI 1484, AI 1437, A1 1426, B1 1410, BI 1372, A1 1357 1322, B(i) 1319, A1 1292, B1 1269 1250, AI 1231, 1230, 1227, 1222, 1220 1138, BI 1128, B1 936, B1 926 913, Bz, 899, A2 910, BI 897,895,893,890 866, BZ 851, AI 847, BI 811, A1 809, Bz, 808, Az 766, Az, 754, B2 720, AI 669, BI

661, Bz

591, A1 550, Bz

543, B1 535, A1 507, Bz

642, Az

544, Az

490, Bi

ring st ring st ring st, def ring st ring St ring st CN st + ring st ring st + CN st ring st ring st CH + CH3 def CH3 def + CN st CN st + ring st ring st, def NH def + CN st ring def, st CC, CN CH3 def CH def NH def + CN st, ring ring def, st CH3 def CH def CH def + NH def + NC st CH3 def CH def ring def

CH def CH def ring def, CN, CC ring def

ring def + CH def ring def + CH def ring def, CN, CC ring def ring def + CN def ring def, CC, CN

NH def ring def, CC, CN

+IO +IO +I+ +I+ ++I+ ++I+ ++I+ +I+ +IO +I+ ++I+ ++I+ ++I+ -I+ ++I+ +IO 0/+ +I+ +I+ +IO 010 +I+ +I+ o/+ o/+ ++I+ o/+ ++I+ o/+ o/+ + ++I+ +I+

++I+ ++I+++ ++I+ +I++ +I+ +I+

+I+ o/+

The calculated wavenumbers have been multiplied by an empirical factor of 0.9 to fit the experimental values. * For assignment, the main contributions from the normal coordinate analysis have been used. The relative signal enhancement was determined by subjective comparing of the band intensities in the SERRS spectra with the respective intensities in the resonance Raman spectrum.

of SERRS, the observation of methyl signals implies that at least one of the four CH3 groups must be near the silver substrate.

Most of the strong SERRS bands in the region from 750 to 490 cm-I were assigned to out ofplane ring and CH deforma- tional modes, including the vibrations at 748,657, and 578 cm-' and especially the strongest signal at 630 cm-'. In contrast, the in plane ring vibrations in the same spectral region only give rise to weak signals.

Relative Band Intensities in the SERRS Spectra. Informa- tion on the orientation of the adsorbed A 0 molecule at the silver surface may be derived from analyzing the selective enhance- ment of particular bands and relating them to the symmetry types of the relevant vibrational modes derived from normal coordi- nate analysis. A qualitative measure for the relative enhance- ment was obtained by a subjective comparison of the band intensities in the SERRS spectra with the corresponding intensities of the resonance Raman spectrum; this information is included in the last column of Table 3.

In view of the distance-dependent decrease of both the electromagneticz2-z4 and the c h e m i ~ a l ~ ~ ~ ~ ~ contributions to the surface enhancement effect, vibrational dipoles in the molecule that are close to the surface and strongly coupled to the metal are experiencing strong enhancement. The SERRS enhance-

ment of some methyl deformational bands indicates a molecular orientation in which at least one CH3 group is near the silver surface.

In order to derive information on the adsorption geometry, selection for the electromagnetic enhancement con- tribution are i n v ~ k e d . ~ ~ - ~ ~ The most prominent propensity implies that vibrations modulating the a z z ' element of the polarizability tensor experience the strongest enhancement, whereby Z' denotes the normal relative to the silver surface. Information on the orientation of the acridine ring can thus be derived from comparing the relative enhancements of molecular vibrations corresponding to the AI, BI, A2, and Bz symmetry species. The fact that both A2 and BZ (out ofplane) type modes are observed with significantly enhanced relative intensities (cf. Table 3) can be explained only if the A 0 ring system is not oriented perpendicular to the plane of the surface. The simultaneously observed enhancement of B1 and A1 type modes, on the other hand, excludes the possibility that the ring system be oriented parallel to the surface. The resulting adsorption geometry should therefore lie in between the two extremes, as suggested in Figure 4. The adsorption of AO, as a cationic species, is likely to be associated with the surface binding of a chloride ion, in a manner similar to the one reported for acridine and other positively charged species.31*45,51,5z

12800 J. Phys. Chem., Vol. 98, No. 48, 1994 Zimmermann et al.

1665 634

l " ~ l ~ " l ~ " l " ' l " ~ l ~ ' ~ l ' ~ '

1600 1400 1200 1000 800 600 400

Raman shift / cm-'

IO

Figure 2. IR spectrum (upper trace) of crystalline acridine orange recorded in diffuse reflectance and displayed in the Kubelka-Munk mode. The data have been smoothed using a Gaussian line width of 1.3 cm-l. Resonance Raman spectrum (lower trace) of an A 0 solution (8 x M in HzO, saturated with KI), excited with 25 mW of power at 476.2 nm. Ten exposures of the multichannel detector, of 10 s accumulation time each, have been coadded. A fluorescent background was subtracted, and the spectrum was smoothed with a Gaussian line width of 2.6 cm-l.

, ~ ~ ~ 1 ' ~ ~ 1 ~ ~ ~ , 1 1 1 , ( , , , ~ ' 1 , ( , 1 1

1600 1400 1200 1000 aoo 600 400 2(

Raman shift / cm-' Figure 3. SERRS spectra of acridine orange (1 x lo-' M in Hz0) adsorbed on a silver colloid (upper trace) and of A 0 spin coated onto a silver island film (lower trace). The spectra were excited at a wavelength of 530.9 nm, using 24 and 1.5 mW of power, respectively, and were smoothed with a Gaussian line width of 1.5 cm-l.

The relative SERRS enhancement on the colloid and on the silver island film, respectively, shows remarkable differences with regard to the symmetry species (cf. Table 3). Vibrations of types AZ and BZ experience stronger enhancement on the

Figure 4. Model for the adsorption geometry of A 0 on a silver sol (left) and on a silver island f i (right). The short in-plane axis (C9- N10) of the molecule is denoted by Z, and the surface normal by Z. In defining the adsorption geometry, we start from an orientation in which the ring system is in a plane perpendicular to the surface, with the line through the three nitrogen atoms parallel to the surface. In that plane, the molecule is first rotated around the Y axis (which is perpendicular to the molecular plane) by an angle 6. Subsequently, the molecule is rotated about the Z axis by an angle q. On the colloid (left), 6 = 70" and p = 20"; at the silver island film (right), 6 = 65" and p = 55".

island film, whereas the enhancment of B1 modes is comparable and the one of A, vibrations is weaker, as compared to the relative intensities observed on the silver sol. This result implies that the plane of the acridine ring is oriented closer to parallel to the plane of the silver island surface, as compared to the surface of a colloidal particle. The described trend appears reasonable, as, on the colloid, the adsorbate is solvated by water molecules; hence, an orientation of the ring system away from the substrate and into the surrounding medium appears to be energetically favored.

In passing we note that the differences in relative enhance- ment, as observed on silver island films and sols, are consistent with an hence support the assignments of the bands made in Table 3: Vibrations assigned to a given symmetry species are experiencing similar intensity alterations when changing from one substrate to the other.

Concentration Dependence. In a series of concentration- dependent experiments (Figure 3, the detection limit for A 0

Acridine Orange and Its Binding to DNA Strands J. Phys. Chem., Vol. 98, No. 48, 1994 12801

1600 1400 1200 1000 800 600 4

Raman shift / cm-' I

Figure 5. Concentration dependence of the SERRS spectra of A 0 on a silver sol: (a) 1 x M; (b) 1 x lo-' M; (c) 1 x lo-* M. The spectra have been excited with 30 mW of power at 476.2 nm. Fluorescence was quenched by adding small amounts of solid potassium iodide. All spectra have been smoothed using a Gaussian line width of 1.9 cm-'.

by SERRS on silver colloids was estimated to be less than 1 x M. A spectrum taken at this concentration (Figure 5 , trace

c) exhibits strong bands due to the stabilizer used in the colloid preparation. When the A 0 concentration was further lowered to 1 x M, only signals of the colloid stabilizer could be detected.

In view of these results, the concentration of A 0 is all further experiments was held fixed at a value of 1 x M. At this concentration, the stabilizer bands are weak as compared to the signals of the molecule of interest (Figure 5, trace b). On the other hand, it was considered important to keep the total A 0 concentration as low as possible in the DNA association experiments, to ensure complete binding of the dye to the double helices. In this manner, the signals of any unbound A 0 molecules should be too weak to be detected in the SERRS spectrum, even in the case that partial dissociation of the AO- DNA should be induced upon addition of the colloidal solution.

SERRS of an AO-DNA Complex. The complex of A 0 with calf thymus DNA was prepared as described in the Experimental Section. The SERRS spectrum of the complex is compared with the spectrum of free A 0 in Figure 6. For a valid comparison, care has been taken to use the same experimental conditions: The same colloidal stock solution and A 0 concentration were used, and the spectra were recorded subsequently on the same day. Only weak relative intensity changes are noted when comparing the two spectra. In particular, the difference spectrum included in Figure 6 does not reveal any new bands. For some of the C-C and C-N stretching vibrations, small frequency shifts (12.5 cm-') are noted.

As a reason for the small differences, one has to discuss the possibility of a dissociation of the complex, such that free A 0 would be adsorbed to the silver colloid surface in both cases. However, in view of the experimental conditions (drug-to- nucleotide ratio = 1.22 x and of the strong affinity of A 0 for DNA (association constant =- 1 x 105 L mol-' 5) , it seems unlikely that the dye is released from the DNA- intercalated form. Experimentally, the persistence of the AO- DNA complex was established by UV-visible spectroscopy. The bottom part of Figure 7 illustrates the well-known red shift53 of the A 0 absorption due to complexation with DNA. In the upper part of the figure, we recognize that the same red shift is

1000 1400 1200 1000 800 800 400

Raman shift / cm-' Figure 6. SERRS spectrum of free A 0 (trace a) and of the AO- DNA complex (trace b) recorded under the same experimental conditions. Spectra were excited using 25 mW of power at 476.2 nm and smoothed with a Gaussian line width of 1.9 cm-'; fluorescence was quenched by addition of KI. The top trace (c) displays the difference between the spectra of the AO-DNA complex and of free AO. In order to eliminate spurious signals due to absolute intensity changes in the difference spectrum, the individual spectra have been normalized with respect to the intensity of the ring defomational mode at 630 cm-l. No smoothing has been applied to the difference spectrum.

0.75 4 \

wavelength / n m Figure 7. Absorbance spectra of A 0 (2.5 x M): (a) solution in H20; (b) complex with calf thymus DNA; (c) free A 0 adsorbed on a silver sol; (d) complex with calf thymus DNA adsorbed on a silver sol. The data have been fitted with a Gaussian profie in order to determine the positions of absorbance maxima. The excitation wavelengths of 476.2 and 530.9 nm used in the SERRS experiments are indicated by dotted lines.

maintained after the complex is adsorbed on the silver colloid. In Figure 6, the similar absolute SERRS intensities in traces a and b show that both spectra must be due to a comparable

12802 J. Phys. Chem., Vol. 98, No. 48, 1994 Zimmermann et al.

50 I n

1600 1400 1200 1000 800 600 400 I

Raman shift / cm-' Figure 8. SERRS spectra of A 0 adsorbed on a silver colloid, as excited at a wavelength of 476.2 nm (upper trace, a) and of 530.9 nm (lower trace, b). Excitation power was set at 20 mW in both cases. The spectra have been smoothed with Gaussian line widths of 1.9 and 1.5 cm-l, respectively.

concentration of dye molecules, Le. a majority species. (We are recalling the pronounced concentration dependence evi- denced in Figure 5.) Even if a minority of A 0 probe molecules was released from the complex upon binding to silver, it could not give rise to a spectrum as intense as the one of Figure 6b.

The question why no Raman bands of DNA are observed in the intercalation complex is addressed next. For the used dye concentration of 1 x lo-' M and a dye-to-nucleotide ratio of 1.22 x M. No SERS signals are observed from intact DNA double helices even at considerably higher concentration^;^^ hence, it is not astonishing that DNA Raman bands are absent in the spectrum of Figure 6 b.

A small relative intensity enhancement (15%) due to complexation is visible in Figure 6 for specific bands, Le. mostly vibrations of symmetry type AI. Again applying electromag- netic selection rules, this result suggests that, for A 0 molecules intercalated between DNA base pairs, the plane of the acridine ring system is oriented closer to perpendicular with respect to the silver surface. Such an orientation of intercalated A 0 would be expected if the axis of the DNA double helix itself would be running parallel to the silver surface. This interpretation is in agreement with the SERS results of Koglin and Sequaris, who investigated the binding of native calf thymus DNA on silver electrode surfaces.54

The absence of new bands in the SERRS spectrum of the AO-DNA complex shows that complexation does not lead to a conformational change of AO. In an effort to provoke any changes, SERFS spectra of the dye-DNA complex have been recorded at various pH values (ranging from 2 to 12) and with the addition of different electrolytes (KI, KSCN, KBr, and NaC1). Again, the spectra of the AO-DNA complex and of free A 0 were measured under the same experimental conditions. In all cases, the difference between the relevant pairs of SERRS spectra was no more pronounced than the ones discussed above in connection with Figure 6.

Wavelength Dependence. An interesting effect can be noticed on changing the excitation wavelength from the green to the blue spectral region. If the SERRS spectrum of A 0 is excited at 476.2 nm, the signal intensity of ring stretching vibrations (1200-1600 cm-') is enhanced relative to the one for the deformational vibrations (1000-200 cm-l), as seen by comparing the top and bottom traces of Figure 8. The electronic

the nucleotide concentration amounts to 8 x

absorption spectrum of A 0 (see below) features an experimental maximum at 491 nm, and the absorption at 476.2 nm is higher as compared to the one at 530.9 nm. Resonant excitation should give rise to a stronger resonant enhancement of the ring stretching vibrations which are coupled to the electronic excitation by the relevant Franck-Condon factors.

For a clear attribution of this interesting wavelength-depend- ent effect to a resonant phenomenon, one has to account for a residual reabsorption of the Raman signals. In the case of 476.2 nm excitation, the deformational modes (1000-200 cm-') fall within the long-wavelength tail of the silver colloid absorption band. However, a quantitative evaluation of the absorbance at the relevant Raman wavelengths shows that this effect would not alter the observed intensity ratios by more than 5%. From a more fundamental point of view, a A4 correction must be applied to the experimental Raman intensities prior to a comparison of spectra excited at different wavelengths. A straightforward calculation shows that this correction amounts to a factor of 1.4 for the relative intensity of a Raman band at 1600 cm-', as compared to the frequency origin of the Raman spectrum. In the following, discussion will be restricted to relative intensity changes larger than 25%, in order to exclude any spurious effects due to wavelength-dependent parameters of optics or detectors.

After absorbance and A4 corrections have been taken into account, it emerges that the stretching vibrations at 1638 and 1570 cm-' experience a significant relative intensity enhance- ment as the excitation energy is increased (A = 476.2 nm). On the other hand, the ring deformational modes are stronger when an excitation wavelength of 530.9 nm is employed.

From an inspection of the normal coordinates obtained with the SCAMP calculations, the vibration at 1638 cm-l is found to be strongly localized on centers Cl-C2 and C7-C8, respectively. As mentioned above, chemical bonding between theses centers resembles isolated n-type double bonds, both according to X-ray data and molecular orbital calculations. The mode at 1570 cm-' involves large displacements at the atomic centers of the allyl-type n-electron subsystem. On the other hand, the stretching vibration at 1530 cm-' is attributed to a vibration which primarily involves centers of the polymethine- type electronic n-subsystem.

In order to use these assignments for an interpretation of the SERRS signal intensities, one might argue that the 1530 cm-' mode of the delocalized polymethine chain experiences resonant enhancement for both excitation energies used. In contrast, the excitation of the allyl-type n-electron system and of the isolated double bonds requires higher energies, such that resonant enhancement becomes more significant as the excitation wave- length is shifted toward the blue spectral region. This inter- pretation is supported by the fact that the relative intensity increase upon 476.2 nm excitation is highest for the stretching mode involving the isolated double bonds.

Resonant enhancement for a given vibration is expected to be strong if the electronic transition is associated with a geometry change related to the relevant normal coordinate. In other words, for such a vibration several Franck-Condon factors would be different from zero. Unfortunately, a full analysis of the relevant modes in terms of Franck-Condon factors could not be carried out, as the calculation of force constants for the first excited state of the bis(dimethy1amino)acridinium cation turned out to be too demanding with respect to computation time.

We shall only address the 1638 cm-' mode and calculate approximate Franck-Condon factors under the assumption that the force constants are comparable for both electronic states. From our calculation of the molecular geometry of the relaxed

Acridine Orange and Its Binding to DNA Strands

TABLE 4: Electronic Properties of Investigated Species

J. Phys. Chem., Vol. 98, No. 48, 1994 12803

bis(dimethylamino)acridine, So

total energy/eV -2793.184 ionization potentiallev 8.120 electronic transition wavelengthshun, calc (exp) 370 (435) composition of transition 74% HOMO - LUMO

14% HOMO-1 - LUMO+l 5% HOMO-3 - LUMO+l

electronic transition wavelengthshm, calc 356 composition of transition 51% HOMO-1 - LUMO

32% HOMO - LUMO+l 9% HOMO-3 - LUMO

bis(dimethy1amino)acridinium bis(dimethy1amino)acridinium cation, So cation, S1

-2802.892 -2800.069 11.285 11.185 425 (490) 89% HOMO - LUMO 6% HOMO-2 - LUMO+l 5% HOMO-I - LUM0+2 391 63% HOMO- 1 - LUMO 16% HOMO - LUMOS1 13% HOMO-2 - LUMO

SI state, it follows that the bond length is increased only by 1.6 pm. We model the normal coordinate as a harmonic oscillator, with a reduced mass of 9.86 x kg from the SCAMP output and an experimental frequency of 3.09 x 1014 s-l. From basic quantum mechanics one calculates the relevant Franck- Condon factors, Sm2 = 0.96, Slo2 = 0.035, and S202 = 0.005. The result that S1o2 is small whereas Sm2 remains close to unity implies that the SO - S1 transition does not involve a length change of the bonds involved in the 1638 cm-’ stretching vibration, in agreement with the qualitative argument given above.

Electronic Spectra of Acridine Orange in Its Neutral and Protonated Form. Electronic transitions in the W-visible spectral range were calculated both for the neutral and the protonated forms of acridine orange. The nitrogen center N10 was chosen as the site of protonation, as established in a detailed investigation by Za~ker,’~ in which the spectral properties of acridine orange were studied as a function of dye concentration and of solution pH. The results of our calculation are listed in Table 4, where the total energies and ionization potentials of three relevant states are given. Calculated wavelengths for the lowest energy transitions are compared with the experimental data;55 in addition, the single electron excitations contributing to the respective electronic transitions are given.

In agreement with experimental evidence, our calculations show that the energy of the transition with the dominant HOMO-LUMO contribution is lowered upon protonation of the A 0 dye base, with a concomitant increase in the HOMO- LUMO character of the transition. The difference between the numerical values of the calculated wavelengths and the experi- mental data amounts to -65 nm for both species investigated. This result is interpreted as follows. First, the semiempirical model used is perfectly capable of reproducing the spectral shift which occurs upon protonation. Second, the difference between the absolute values of calculated and experimental wavelengths is attributed to the neglect of interactions with solvent molecules. In the coarsest approximation, both the bis(dimethy1amino)- acridinium cation and the neutral bis(dimethy1amino)acridine may be expected to interact with the sovent in a similar fashion.

Excited State Properties of the Bis(dimethy1amino)acri- dinium Cation. The bis(dimethy1amino)acridinium cation is the dominant species in the range of pH values corresponding to the intracellular medium, and it may probably be identified with the species involved in DNA intercalation. The signals observed by the SERRS technique exhibit a preresonant enhancement as the laser energies used for excitation (wave- lengths around 500 nm) are slightly below the photon energy required to excite the first singlet state of the dye molecule. Therefore, some properties of the electronically excited state S1 of the bis(dimethy1amino)acridiniw-n cation will be discussed in this section. According to our calculation, the S1 state lies at an energy of 2.8 eV above the electronic ground state. This energy, which is equivalent to a wavelength of 439 nm, is indeed

close to the incident laser energy. Therefore, the assumption appears valid that the f i s t excited singlet state takes part in the resonant scattering process.

In order to describe the changes occurring upon So-Sl excitation, changes in bond orders will be analyzed. It was mentioned above that the PM3 model adequately describes the changes in electron density induced by protonation of the bis- (dimethy1amino)acridine molecule. The development of the polymethine chain N15-C3-C4-C12-NlO-C13-C5-C6- N16, with its conconiitant equalization of bond orders between the mentioned centers (cf. Table 2), and the shift in the HOMO- LUMO transition are in close agreement with the experiment results.

We wish to reemphasize that bond orders are a linear function of calculated bond lengths and, therefore, in a strict sense, reflect only changes in molecular geometry. From a close inspection of Table 2 it emerges that structural relaxation in the SI state leads to a reduction in symmetry, such that the molecular point group of the minimum energy conformation of the excited molecule is only C1. (Of course, any of the two enantiomers would be found with equal probability.) As a result, the separation in energy between the molecular orbitals describing the three subsystems of n-electrons is diminished. It is worthwhile to note a shift of electronic bond order between the bonds C3-Nl5 and C6-Nl6: the bond order C3-Nl5 decreases, whereas the bond order C6-Nl6 is increased by about the same amount. A similar observation holds for the pair of bonds C12-Nl0 and C13-Nl0 and for corresponding other pairs.

Conclusions

Surface-enhanced resonance Raman spectra of acridine orange have been recorded with high sensitivity both on silver island films and silver colloids. From a comparison with solid state IR and solution Raman spectra, a complete assignment of the vibrational spectrum has been achieved. An analysis of the relative signal intensities in the SERRS spectra reveals that the bis(dimethy1amino)acridinium cation is bound to the silver surface in an unsymmetrical way, with one auxochromic group in closer proximity to the surface.

The vibrational frequencies of A 0 remain largely unchanged when the dye is intercalated into calf thymus DNA. Small alterations in relative signal enhancements indicate that the angle between the planes of the silver surface and of the intercalated acridine ring system is larger, as compared to that for free A 0 bound directly to the silver surface. This is consistent with an adsorption geometry in which the axis of the DNA double helix is oriented parallel to the surface.

The semiempirical PM3 parametrization provides an accurate description of the electronic ground state of the bis(dimethy- 1amino)acridiniw-n cation. The geometric parameters agree well with the experimental structure derived from X-ray crystal-

12804 J. Phys. Chem., Vol. 98, No. 48, 1994

lography. The x-electron system is found to be divided into a polymethine chain, an allyl-type subsystem, and two double bonds with comparatively little bond delocalization.

Calculated transition energies match the longest wavelength absorption maximum from W-visible spectroscopy; the shift of the latter observed upon protonation is well reproduced by the calculation.

The calculations show that the mini" energy conformation of the f i s t excited singlet state of the free cation exhibits a reduced symmetry. Franck-Condon factors have been esti- mated in order to interpret the observed band specific wave- length dependence of the SERRS enhancement. The vibrations of the 'isolated' double bonds, which do not couple to the SO - S1 excitation, experience the strongest relative enhancement as the energy of laser excitation is increased.

Zimmermann et al.

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Acknowledgment. Sincere thanks are due to M. Sprinzl for stimulating discussions and to T. Clark for making the SCAMP program available. Financial support of this work by grants of the Deutsche Forschungsgemeinschaft (SFB 213) and by the Verband der Chemischen Industrie is gratefully acknowledged.

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