Surface-enhanced Raman scattering analysis of etheno adducts of adenine

359

vibrational Spectroscopy, 4 (1993) 359-364

Elsevier Science Publishers B.V., Amsterdam

Surface-enhanced

Raman scattering analysis of etheno adducts of adenine A.M. Helmenstine

University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (USA)

Y.S. Li 1 and T. Vo-Dinh Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (USA)

(Received 26th May 1992)

Abstract We report smfaceenhanced Raman spectroscopy (SERS) detection and analysis of several etheno adducts formed from the metabolism of vinyl chloride: 1,N6-ethenoadenine, 1,N6-ethenoadenosine, and l,N’-etheno-2’-deoxyadenosine. Spectra of the etheno adducts are compared with unmodified adenine and 2’-deoxyadenosine. Chemical group assignments are made for the SERS peaks of these adducts and the limit at which SERS can detect the adducts is assessed. These assignments are compared with SERS analyses of nucleotides and nucleosides. Keywords: Raman spectrometry; Adenine; DNA; Etheno adducts; Vinyl chloride

The development of selective and sensitive analytical tools to measure chemically induced DNA damage in specific tissues is important in risk assessment because alteration of DNA is a key step in the initiation of carcinogenesis. Measurement of DNA adduct levels would give a useful measure of exposure to genotoxic agents. In a previous study, we have used the surface-enhanced Raman spectroscopy (SERS) technique to detect DNA adducts of benzo(a)pyrene [l], a carcinogenic polycychc compound of great environmental interest [2]. In this work we further investigate the usefulness of SERS for the analy-

sis of 1,N6-ethenoadenine, a cyclic base adduct produced from the metabolism of vinyl chloride. Vinyl chloride, which is carcinogenic in man and laboratory animals [3], is biotransformed to reactive intermediates capable of covalently interacting with cellular macromolecules. Alkylation of RNA by vinyl chloride metabolites in vivo and in vitro has been shown to produce 1,N6-ethenoadenosine [4]. SERS was used for the study of this adduct because it has very high sensitivity and because other spectroscopic techniques have proven inadequate in comparing bases with their modified counterparts.

Correspondence to: T. Vo-Dinh, Advanced Monitoring Development Group, Health and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (USA). i On sabbatical leave from Memphis State University, Memphis, TN 38152 (USA).

EXPERIMENTAL

SERS spectra were recorded using a Spex double monochromator (Model 1403) equipped gth

A.M. Hehenstine et al. / Vii. S’ctmsc.

360

a gallium arsenide photomultiplier tube (RCA Model C31034) operated in single-photon counting mode. Raman scattering signals were acquired, processed, and stored using a Spex DM3000 computer. The 647.1-nm line of a krypton ion laser (Coherent Innova 70) filtered with an Oriel 647.1~nm laser filter (narrow bandpass) was used for sample excitation. Scattering signals at 180” were collected with a 600 pm diameter silica fiber (NA = 0.26; General Fiber Optics), which was positioned as close to the sample as possible without damaging the silver film. This distance was approximately one micrometer and was not altered between scanning of subsequent samples. Negligible loss of collected radiation resulted from this distance, since the optical fiber was considerably larger than the laser spot (600 pm fiber diameter versus 10 pm beam diameter). A stepping motor was used to position the collection fiber and caused no noticeable variation in spectral quality. The collected radiation was carried to the other end of the fiber and was focused onto the monochromator slit. The experimental setup is schematically depicted in Fig. 1. Nucleotides and nucleosides were obtained from Sigma and used as received. Unless otherwise noted, solutions were prepared using a 1 + 1 (v/v) mixture of absolute ethanol and water as solvent. Silvered microparticle-coated SERS sub-

COLLECTION FIBER ,

I 1800

I

II

l 1400

I

I

I

I IO00

Raman Shift

I

I

I

I 600

4 (1993) 359-364

I

I, 200

(cm-l)

Fig. 2. SERS spectra of (top) 1,N6-ethenoadenine and (bottom) adenine from 1.0~~1drops of 1 X 10T4 M (100 picomole) solutions in ethanol. Laser excitation was 70 mW (1,N6ethenoadenine) and 130 mW (adenine) at 647.1 nm.

strates were prepared by spin-coating (20 s, 2000 r.p.m.1 5.0% aqueous agglomerate-free alumina (0.1 wrn diameter, Baikowski International) onto a piece of a microscope slide and then thermally evaporating 80.0 to 100.0 mn silver onto the alumina-coated glass surface. Analyte solutions spotted onto the substrates were allowed to dry at room temperature before exposure to the laser beam. The optical setup and sample preparation procedures have been described previously [5,6].

RESULTS AND DISCUSSION rl

.U

SEW3 EMISSION

ANALYTE SILVER COATING ALUMINA GLASS SUPPORT

LASER

Fig. 1. Schematic diagram of the SERS fiber-optic experimental setup.

A filtered laser excitation of 25 to 70 mW proved adequate for signal detection from these samples. Spectra could be recorded repeatedly with samples studied within this excitation power range without sample decomposition or diminished SERS peak intensities. Laser power above 75 mW resulted in photodegradation of the sample and these samples could not be re-analyzed by SERS. The signal-to-noise ratio was fairly low, but most SERS bands could be readily distinguished from the background noise. Sample SERS spectra of 1,N6-ethenoadenine and adenine adsorbed on the silver-coated alu-

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A.M. Helmenstine et aL / Vii. Spectrosc. 4 (1993) 359-364

mina are displayed in Fig. 2. Many reports have appeared in the literature dealing with the adsorption of adenine on various SERS substrates such as silver sols and electrochemically roughened silver surfaces [7-121. To our knowledge, no SERS analysis has been performed on 1,N6ethenoadenine. The SERS spectrum of adenine (Fig. 2) exhibits two prominent bands at 738 and 1372 cm- ‘. The 73&m- ’ band corresponds to the most intense SERS band observed for adenine on silver sols [7-91 and roughened electrodes [lo-121 within 10 cm-‘. This band may be assigned to ring breathing vibration. It is unlikely that this band arises from the coupled NH, deformation and ring vibration as suggested for the metal-adenine complex [ 131 because l,N 6ethenoadenine does not have a primary amino group, but does show a corresponding band at 340 cm-‘. It is noteworthy that the relative intensity of the prominent 738~cm-’ band for adenine (Fig. 2) was weak when high laser power was used for excitation. This may arise from either a change of molecular orientation of the analyte molecule on the silver film or from thermal decomposition of the sample.

The measured SERS frequencies and tentative assignments for the peaks of adenine and 1,N6ethenoadenine are given in Table 1. Note that the bands in Fig. 2 through Fig. 5 do not necessarily correspond to the band assignments given in Table 1. For these samples, the signal-to-noise ratio is low because these chemicals were relatively difficult to detect with SERS, requiring extremely fine optical alignment. Since an SERS peak was defined as the maximum number of photons per wavenumber, it followed that one particular wavenumber had to be defined as the peak, even when two or more wavenumbers had the same photon counts. SERS bands did not shift in subsequent measurements, although there was some uncertainty in identification of the peak wavenumber. Table 1 assignments were made for the average band positions for all spectra studied. Some peaks which appear in the spectra but which are not accounted for in Table 1 are due to very reproducible background noise from the optical setup and silvered alumina-coated substrate. These background peaks, for the main part from the silica collection fiber, are denoted by asterisks on Fig. 2. A background spectrum is included

TABLE 1 Observed SERS frequencies (cm-‘) and assignments for adenine, 1,N6-ethenoadenine (EADENINE), 1,N6-ethenoadenosine (EADENO), 2’-deoxyadenosine (dADENO and 1,N6-etheno-2’-deoxyadenosine (MADENO) a ADENINE

EADENINE

1574 w, b

1582 sh

EADENO

dADEN

EdADENO 1642 w, sh

1650 w, sh 1568 w, b

1454 w

1500 s 1472 m, sh

1536 s, sh 1508 s, sh 1472 s, sh 1460 s, sh

1372 s 1338 m, sh

1338 m, sh

1346 m, b

1468 m, b

1340s 1332 s

Ndouble bond stretching] 1542 s, sh 1510 s, sh 1472 s, sh 1464 s, sh 1338 s, vb

1300 m, sh 1252 w, sh

I[double bond stretching] A[N,-H in-plane bending]

NC,-H in-plane bending] A[in-plane ring vibration] A[in-plane ring vibration] A[Kekule vibration]

109Om

738 s

Assignment

994w 826 w 798 vw 740w

730 m

1109 m, b

748s 694 m

736 s 688m

I vibration I vibration I vibration A[ring breathing]

598 w a Abbreviations: s, strong; m, medium; w, weak; sh, shoulder; b, broad; v, very; a = adenine; I = C,N,,C,,N,

ring.

362

I 1600

A.M. Hehenstine

I

I

I

I 1400

a

I,1

I

1000

I,1

I

600

I,

1 200

Raman Shift (cm-l) Fig. 3. SERS spectra of l,i@-ethenoadenine and corresponding background noise. Sample was 1.0 ~1 of a 2.561 mg ml-’ solution in ethanol. Laser excitation was 50 mW at 647.1 nm.

with a 1,N6-ethenoadenine spectrum from the same SERS substrate in Fig. 3 to clearly illustrate the differences between the sample spectrum and background noise levels. The background was reproducible enough that peaks near the noise level could readily be distinguished. The noise was not substractable in this study because application of a sample to the substrate altered and in some cases reduced background levels. This effect was always reproducible for a given analyte, but subtraction of the background could cause weak peaks to be undetectable. Enough trials were taken for each chemical in this study (lo-20 per chemical, all on fresh substrates) that weak bands not necessarily appearing on every spectra could be documented. While this background may be high enough to inhibit detection of SERS peaks from 200-500 cm-‘, this optical setup did give excellent detection from 500 to 1800 cm-‘. The relative intensities of the SERS bands were also very reproducible. Most vibrational assignments in Table 1 are in good agreement with assignments made for adenine by Hirakawa et al. [14]. In Suh and Moskovits’ [81 study of adenine adsorbed onto silver colloid particles, the adenine molecule was shown to be flat on the metal surface for the weak band at 1317 cm-’ was as expected for the

et al. / Vii. Spectrosc. 4 (1993) 359-364

C,-N,, stretching mode. In another SERS study of adenine on silver sols Kim et al. [9] observed a frequency decrease of ring-breathing vibration from 746 to 735 cm-r when the sample concentration was increased from 7.5 x 10m7 M to 9.0 x lO-‘j M. They suggested that the molecular adsorption was “ring-on” at lower concentrations, but “end-on” at higher concentrations. Several different vibrational analyses have been used to derive the molecular orientations of analytes on the surfaces of SERS-active substrates. In an SERS study of adenine on a silver electrode, Watanabe et al. 1111 assigned the strong band at 1328 cm-’ to N,-C, stretching vibration. From this assignment and from the intensity of the band, it followed that at 5 mM concentration, the molecule was adsorbed “end-on” via the N, atom. The SERS peak observed around 1330 cm-’ when adenine was adsorbed onto silver particles [7-91 and electrodes [lo-121 was generally strong. In our study, however, the corresponding band at 1338 cm-’ appears as a shoulder on an intense band at 1372 cm-‘. This may suggest that the adsorption of adenine on the substrate is not in the “end-on” position via the N, atom. One possible assignment of the strong band at 1372 cm-’ is to the C,-H in-plane bending. If this assignment is correct, the molecule would be expected to “stand” or “tilt” on the surface via the N, and N, atoms. It should, however, be noted that the true orientation of the molecule depends upon correct vibrational assignment of the bands as well as on information about whether the molecules are adsorbed in a monolayer or are stacked upon each other. The concentration of adenine used in this study may have resulted in the stacking of molecules and not in a monolayer. Samples at a 1 x 10m4 M concentration are probably at the threshold between stacked and monolayer molecules; all lower concentrations form monolayers on the substrate. The majority of sample solutions examined in this study were around 1 x lop4 M concentration. Vibrational assignments for 1,N6-ethenoadenine are essentially based on the assumption that force fields around the adenine ring did not appreciably vary upon the formation of adducts. A change in the molcular orientation on the

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surface might affect spectral features. Several characteristic bonds of 1,N6-ethenoadenine may be used to differentiate it from adenine. As shown in Fig. 2, the most prominent band of 1,N6ethenoadenine at 1500 cm-r is not present in adenine. This peak may be assigned to a double band stretching mode. The medium intensity band at 1472 cm-’ may correspond to the weaker band at 1454 cm-’ in adenine. This band could be assigned to N,-H in-plane bending. The 1372 cm-r band CC,-H in-plane bending) was prominent in adenine, but is not observed in the SERS spectrum of 1,N6-ethenoadenine. Also absent from the 1,N6-ethenoadenine spectrum are the 1300 cm-’ (in-plane ring vibration) and 1252 cm-’ (Kekule vibration) bands of adenine. Several weak peaks at 994, 826, and 798 cm-’ appear in 1,N6-ethenoadenine, but not in adenine. As seen in Table 1, results comparable to those for adenine and 1,N6-ethenoadenine were obtained for 1,N6-ethenoadenosine, 2’-deoxyadenosine, and 1,N6-etheno-2’-deoxyadenosine. Sample spectra for these chemicals are shown in Figs. 4 and 5. A background spectrum from the same substrate used to produce these spectra of 1,N6-ethenoadenosine, 2’-deoxyadenosine, and 1,N6-etheno-2’-deoxyadenosine is included for

1’ ”

”

”

Raman

”

”

Shift

(cm-l)

”

’ II

Fig. 4. SERS spectra of (top) 2’-deoxyadenosine and (bottom) corresponding background noise. Sample was 1.0 1.11of a 1.363 mg ml-’ solution in ethanol. Laser excitation was 70 mW at 647.1 nm.

1800

1475

1150

Raman Shift

625

500

(cm-t)

Fig. 5. SERS spectra of (top) l,N’-ethenoadenosine and (bottom) l,N’-etheno-2’-deoxyadenosine. Samples were 1.0 1.11of 1.613 mg ml-’ (1,N6-ethenoadenosine) and 1.415 nig ml-’ (1,N6-etheno-2’-deoxyadenosine) solutions in ethanol. Laser excitation was 70 mW at 647.1 nm. Spectra may be directly compared with those presented in Fig. 4.

comparison purposes in Fig. 4. Bands appearing in the spectra but not listed in Table 1 may be attributed to this optical setup background, primarily from the collection fiber. Each compound has a unique spectrum. SERS may be used to quickly and reliably differentiate between the unaltered bases and their etheno adducts, presumably in both DNA and RNA. An SERS limit of detection study was performed using serial dilutions of 1,N6-ethenoadenine. Sample spectra from this study are shown in Fig. 6. Laser power for the limit of detection study was 50 mW. Under these conditions, SERS could be used to detect 1,N6ethenoadenine from a dried 1.0 /II spot of 1 x lo-’ M (10 picomoles) analyte. Since the laser beam did not impinge on the entire sample spot, but only on one percent of the sample area, the limit of detection was in actuality better than 100 femtomoles. Similar limits of detection for SERS were observed for the other compounds used in this study. Conclusions These experiments demonstrated that SERS spectra of adenine and its etheno adducts can be

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This research was supported by the Office of Health and Environmental Research, US Department of Energy, under contract DE-AC% 840R21400 with Martin-Marietta Energy Systems. YSL acknowledges support of the US Department of Energy Faculty Research Participation program administered by Oak Ridge Associated Universities. AMH acknowledges support from a joint contract between the University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences and the Health and Safety Research Division of Oak Ridge National Laboratory. 1475

1350 Ruan

1225

1100

Shift (cm-l)

Fig. 6. Sample SERS spectra of l,hr’-ethenoadenine. Solutions were (top-to-bottom) 8.9196 X 10v4 M, 8.9196 X 10-4.5 M, 8.9196X lo-’ M, and 8.9196X 10-5.5 M in phosphatebuffered saline (pH = 7.0). Lase.r excitation was 50 mW at 647.1 mn.

obtained and differentiated from each other with relatively simple apparatus and experimental procedures. The results of this study may be of interest to researchers involved in the characterization of DNA adducts from exposure to small molecules such as vinyl choride. While DNA adducts of large compounds, such as the polyaromatic hydrocarbons, can be analyzed by spectroscopic techniques such as fluorescence 1151 and phosphorescence [16], it is difficult to use these techniques to analyze DNA adducts from less luminescent small molecules. While IR spectroscopy could be used to differentiate between specific chemical groups, it is generally not sensitive enough for trace detection and analysis. In addition, the IR technique is limited by strong influences from the water absorption band. The SERS technique, which combines the sensitivity of the surface-enhancement effect with the spectral selectivity of Raman, may be an important biomonitoring tool for the sensitive detection of DNA adducts of small molecules.

REFERENCES

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