Anal. Chem. 1997, 69, 4703-4707
Selective Detection of Deoxyribonucleic Acid at Ultralow Concentrations by SERRS Duncan Graham,† W. Ewen Smith,*,† Adrian M. T. Linacre,† Calum H. Munro,‡ Nigel D. Watson,† and Peter C. White†
Department of Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow G1 1XL, Scotland, and Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Genetic research programs such as the human genome project have demonstrated the need for selective detection of nucleic acids at ultralow concentrations (McKusik, V. A. FASEB J. 1991, 5, 12-20. Cui, X.; et al. Proc. Natl. Acad. Sci. U.S.A. 1989, 89, 9389-9393). Current detection techniques require amplification of the genetic material by the polymerase chain reaction (PCR) and detection of the amplified product by fluorescence. A new reliable method for DNA detection has been developed based on adsorption of DNA on colloidal silver and subsequent signal detection using surface-enhanced resonance Raman scattering (SERRS). It uses specifically designed primers and can be applied directly to genetic analysis. Improved surface and aggregation chemistry, utilizing modified DNA and a novel aggregating agent, has resulted in acceptable reproducibility for biological detection. DNA was detected down to 8 × 10-13 M, which equates to less than one molecule in the interrogation volume at any one time, and an RSD of 10% was obtained. The increased sensitivity compared to fluorescence circumvents the need for an amplification step, and much greater selectivity is obtained due to the sharp vibrational spectra observed, thus reducing the need for separation procedures. The method is potentially sensitive enough to eliminate the need for time-consuming amplification steps and provide a new dimension to labeling chemistry and multiplex analysis of DNA. Recent advances have turned surface-enhanced resonance Raman scattering (SERRS) into an extremely sensitive analytical probe for small quantities of dye.3-5 Adsorption of the analyte to a suitably roughened surface of silver or a related metal produces surface-enhanced Raman scattering (SERS), which gives a signal enhancement of up to 106.6-9 By using a dye and tuning the laser †
University of Strathclyde. University of Pittsburgh. (1) McKusick, V. A. FASEB J. 1991, 5, 12-20. (2) Cui, X.; Li, H.; Goradia, T. M.; Lange, K.; Kazazian, H. H.; Galas, D.; Arnheim, N. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9389-9393. (3) Munro, C. H.; Smith, W. E.; White, P. C. Analyst 1995, 120, 993-1003. (4) Munro, C. H.; Smith, W. E.; Armstrong, D. R.; White, P. C. J. Phys. Chem. 1995, 99, 879-885. (5) Kneipp, K.; Wang, Y.; Dasari, R. R.; Feld, M. S. Appl. Spectrosc. 1995, 49, 780-784. (6) Fleishmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163. (7) Albrecht, M. G.; Creightonb, J. A. J. Am. Chem. Soc. 1977, 99, 5215. (8) Vo-Dinh, T.; Hiromoto, M. Y. K.; Begun, G. M.; Moody, R. L. Anal. Chem. 1984, 56, 1667-1670. (9) Sheng, R. S.; Zhu, L.; Morris, M. D. Anal. Chem. 1986, 58, 1116-1119. ‡
S0003-2700(97)00657-4 CCC: $14.00
© 1997 American Chemical Society
excitation frequency to the maximum of the dye chromophore to obtain SERRS, a significant further enhancement is produced.10,11 A key advantage over SERS is that the greater contribution due to resonance leads to more readily identifiable and stable signals.12 Nonfluorescent as well as fluorescent labels can be used, thus increasing the range of labels available.3 Only vibrations associated with the chromophore are significantly enhanced, and a great degree of selectivity is obtained.3 Sensitivity can surpass fluorescence,12 and the sharp signals provide a “fingerprint” of each molecule, thus facilitating very effective discrimination, even for closely related dyes in mixtures. Recently, single-molecule detection has been reported.13,14 One study obtained a signal from a single colloidal particle immobilized on a surface, and the other analyzed the near-IR SERS obtained from a dye. However, in the study by Kneipp et al.,14 a problem with surface attachment was observed. An estimate of 80% of the molecules involved were attached to the surface. The problem of the equilibrium between surface adsorption and desorption has been encountered previously12 and leads to difficulty in quantifying the amount of analyte present. In previous cases, the surface chemistry involved has not been considered in detail. Although the present study is more concerned with sensitive detection for use in molecular biology than in quantitation, an RSD value was calculated to estimate the precision. The use of SERRS rather than SERS produces a degree of reliability and sensitivity not currently present in SERS labels.4,12 In principle, the method could be used to detect low concentrations of dye-labeled DNA, thus eliminating the need for expensive, time-consuming amplification and providing greater discrimination from a much wider range of chromophores. Other attempts to detect DNA by Raman spectroscopy have focused upon obtaining SERS spectra of the individual bases and of intercalated drugs. Vo-Dinh et al.15,16 reported a system for the detection of DNA sequences based on probe hybridization, which uses a solid substrate. The levels of detection were estimated to be in the micromolar range using detection mainly by SERS, although one label is potentially SERRS active. Kneipp et al.17-20 were able to detect the presence of DNA by allowing the appropriate nucleic acid (DNA or RNA) to adsorb onto the (10) Siiman, O.; Lepp, A.; Kerker, M. J. Phys. Chem. 1983, 87, 5319-5325. (11) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 88, 5935-5944. (12) Rodger, C.; Smith, W. E.; Dent, G.; Edmondson, M. J. Chem. Soc., Dalton Trans. 1996, 791-799. (13) Nie, S.; Emroy, S. R. Science 1997, 275, 1102-1106. (14) Kneipp, K.; Wang, Y.; Kneipp, H.; Perelman, L. T.; Itzkan, I.; Dasari, R. R.; Feld, M. S. Chem. Phys. Lett. 1997, 78, 1667-1670. (15) Vo-Dinh, T.; Houck, K.; Stokes, D. L. Anal. Chem. 1994, 66, 3379-3383. (16) Vo-Dinh, T. U.S. Patent 5,306,403, 1994. (17) Kneipp, K.; Flemming, J. J. Mol. Struct. 1986, 145, 173-179.
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surface of a metal colloid to produce SERS. However, they found that the signals were optimal after 18 h, suggesting a timedependent process. Further, detection of intercalator/DNA complexes by SERRS has been attempted.21-24 This approach suffers from the problem of discriminating between DNA-bound intercalator and intercalator adsorbed on the surface but not complexed by the DNA. This paper describes for the first time detection of dye-labeled DNA by SERRS by utilizing two improvements over existing procedures. The specifically designed oligomer used, which can be directly applied to conventional molecular biology assays, takes SERRS much closer to competing with fluorescence in standard procedures. EXPERIMENTAL SECTION Reagents. All reagents were of analytical grade. Modified DNA was purchased from the Oswel DNA Unit, University of Southampton, U.K. The addition of the substituted fluorescein dye 2,5,1′,3′,7′,9′-hexachloro-6-carboxyfluorescein (HEX) to the 5′terminus of the synthetic oligonucleotide was carried out using standard phosphoramidite chemistry.25 Colloidal Preparation. Colloidal silver suspensions were prepared by reaction of silver nitrate with sodium citrate.26 A sample of silver nitrate (90 mg) was dissolved in distilled water (500 mL) and heated rapidly to boiling under stirring. Immediately when boiling commenced, an aqueous solution of sodium citrate (1.0%, 10 mL) was added rapidly and heating was reduced. After boiling gently for 90 min with stirring, the volume was adjusted to 500 mL with distilled water. Sample Preparation. A solution containing the labeled modified oligomer was prepared in distilled water. An aqueous solution of spermine hydrochloride (8 × 10-4 M, 20 µL) was premixed with the oligomer and this mixture added to an aliquot of the silver colloid suspension. Details of the amounts of oligomer and colloid volumes used are summarized in Table 1. An aliquot of this suspension (400 µL) was then transferred to a microtiter plate for examination.
spermine (20 µL, 8 × 10-4 M) and added to colloid and water (500 µL/500 µL). Once the solution starting with 1 × 10-9 M had been used, an aliquot (100 µL) of the colloidal suspension was added to water (900 µL) to provide a further dilution. Each sample was scanned for 10 s. SERRS Measurements. SERRS was recorded using a 25mW argon ion laser (514.5 nm, 1 mW) as the excitation source and a Renishaw 2000 spectrometer (Renishaw Ltd., Gloucestershire, U.K.) and microscope (10 × objective). The detector was a cooled charge-coupled device (CCD). Control spectra were obtained by examining a suspension of the above components without the DNA present. PCR Procedures. PCR products were generated from the following mixture: 100 ng of genomic DNA, 100 µL reaction mix, 0.2 µM primer, and 2.0 units of Taq polymerase. The forward primer (RS151T) was HEX labeled and contained the propynyl-modified tail. Two reverse primers were used to control the length of PCR product (RS152, RH5A). The following cycle was used 30 times for each PCR mixture to produce the products which were purified by gel electrophoresis: 95 °C 15 s, 55 °C 15 s, and 72 °C 30 s. RESULTS AND DISCUSSION A surface that has proved particularly suitable to produce strong SERRS is a silver colloid aggregated to provide regions of high electric fields in the interstices.27 However, when an attempt was made to use the conventional method of SERRS detection with colloid, of DNA labeled at the 5′-terminus with HEX (Figure 1) to produce a resonant probe, poor scattering was observed. The problem appeared to be that the surface of the silver had a negative charge and the negatively charged DNA did not adsorb to the surface of the silver due to the electrostatic repulsion. This problem will also have adversely affected previous SERS studies using colloid.
Table 1. Summary of Conditions Used for SERRS Dilution Study of the Modified 17-Mer stock concn (M)
vol of DNA used (mL)
colloid/ water (mL)
overall concn (M)
scan time (s)
1 × 10-7 1 × 10-8 1 × 10-9 1 × 10-10 1 × 10-11 blank
20 20 20 20 40
500:500 500:500 500:500 250:250 100:400 500:500
2 × 10-9 2 × 10-10 2 × 10-11 4 × 10-12 8 × 10-13
10 60 3 × 50 3 × 50 3 × 50 60
For the modified 38-mer, 20 µl of the oligomer solution (dilutions went from 1 × 10-7 to 1 × 10-9 M) was premixed with (18) Flemming, J.; Kneipp, K. Stud. Biophys. 1989, 130, 45-50. (19) Kneipp, K.; Pohle, W.; Fabian, H. J. Mol. Struct. 1991, 244, 183-192. (20) Kneipp, K.; Dasari, R. R.; Wang, Y. Appl. Spectrosc. 1994, 48, 951-955. (21) Lecomte, S.; Moreau, N. J.; Aubard, J.; Baron, M. H. Biospectrocopy 1995, 1, 423-426. (22) Nabiev, I.; Chourpa, I.; Manfrait, M. J. Phys. Chem. 1994, 98, 1344-1350. (23) Nabiev, I.; Baranov, A.; Chourpa, I.; Beljebbar, A.; Sockalingum, G. D.; Manfrait, M. J. Phys. Chem. 1995, 99, 1608-1613. (24) Zimmerman, F.; Hossenfelder, B.; Panitz, J. C.; Wokaun, A. J. Phys. Chem. 1994, 98, 12796-12804. (25) Carruthers, M. H. Science 1985, 230, 281-285. (26) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391-3395.
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Figure 1. 2,5,1′,3′,7′,9′,-Hexachloro-6-carboxyfluorescein (HEX), λmax ) 540 nm.
Two new procedures were utilized to overcome the problem. In order to achieve the maximum enhancement from the surface of the colloid, the aggregation procedure is critical. Inorganic ions (Na+, Mg2+, Cl-) or acids are often used as aggregating agents. They appear to act by reducing the surface charge on the colloid.27 These methods are ineffective with DNA because aggregation is achieved without attachment of the DNA to the silver surface. In a new departure, the organic polyamine, spermine, was used. It interacts with DNA to balance the charged phosphate groups, thus acting as a charge modifier of DNA.28 It was found to be an effective agent for controlled aggregation of the colloidal particles. A transmission electron micrograph (TEM) of the aggregated colloid shows how the colloidal particles are (27) Munro, C. H.; Smith, W. E.; Garner, M.; Clarkson, J.; White, P. C. Langmuir 1995, 11, 3712-3720. (28) Basu, H. S.; Marton, L. J. Biochemical J. 1987, 244, 1, 243-246.
Figure 2. Transmission electron microscope photograph of spermine-aggregated citrate-reduced silver colloid. Magnification, ) 11500×.
Figure 3. Absorbance spectrum of citrate-reduced colloid and spermine-aggregated colloid. Key: (1) unaggregated colloid, (2) colloid aggregated with spermine.
Figure 4. 5-(3-Amino-propyn-1-yl)-2′-deoxyuridine.
packed closely together to provide the areas of high electric field necessary for the surface enhancement (Figure 2). Further proof of the aggregation process was obtained by monitoring the absorbance spectrum for the colloid and that of the spermine aggregated colloid (Figure 3). Figure 3 clearly shows a distinct change in the absorbance of the colloid with the peak shape at 400 nm changing and a new peak appearing at 790 nm. This shows that the bulk property of the colloid has changed due to the addition of the spermine. In a further step, the DNA was chemically modified to incorporate a propargylamino-modified nucleoside (Figure 4) which was prepared according to the procedure of Cruickshank and Stockwell.29 By modifying several bases in the oligonucle(29) Cruickshank, K. A.; Stockwell, D. L. Tetrahedron Lett. 1988, 29, 41, 52215224.
Figure 5. Spermine-induced aggregation. Initially propargylaminomodified DNA labeled at the 5′-terminus with HEX is mixed with an excess of spermine hydrochloride. This balances the charges and produces a solution with an excess of positive ions. The mixture is then added to the colloidal suspension. The propargylamino groups attach to the surface of the colloid as the excess spermine aggregates the colloid. The HEX label is then close enough to the surface to experience the surface enhancement from the silver.
otide, positively charged amine groups within the DNA are produced30 that interact strongly with the negative colloidal surface. This improved surface attraction of the DNA and permitted SERRS of the active label to be obtained. The addition of the propargylamino-modified nucleosides will stabilize any duplex formed by hybridization,31 and it is suspected to improve base specificity.32 Hence, the modified oligonucleotides will not only adhere to the colloidal surface but will act as very specific capture probes for target sequences. The target signals were obtained when the DNA was mixed with an excess of spermine prior to addition to the colloidal suspension to neutralize the DNA. After addition of the DNA/ spermine mixture to the colloidal suspension, the propargylamino modification attaches the DNA to the surface of the colloid. The excess spermine aggregates the colloidal particles to produce a mixture that allows SERRS signals to be accumulated (Figure 5). By adopting this procedure, we have been able to detect both single- and double-stranded DNA. The first sequence successfully detected was a 17-mer labeled at the 5′-terminus with the commercially available dye, HEX (Figure 6) Four main bands were observed, 1302, 1341, 1502, and 1629 cm-1, which correspond to symmetrical modes of in-plane C-C stretching vibrations. The (30) Hashimoto, H.; Nelson, M. G.; Switzer, C. J. Am. Chem. Soc. 1993, 11, 7128-7134. (31) Graham, D. Ph. D. Thesis, University of Edinburgh, 1996.
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Figure 7. Dilution study involving modified 38-mer. (1) 1 × 10-7; (2) 1 × 10-8; (3) 1 × 10-9; and (4) 1 × 10-10 M and (5) blank.
Figure 6. SERRS spectra obtained for modified 17-mer. Key: (a) 1× 10_7; (2) 1 × 10-8; (3) 1 × 10-9; (4) 1 × 10-10; and (5) 1 × 10-11 and (6) blank.
17-mer contained six of the propargylamino-modified bases, HEXT*T*C GCC T*T*A GCC AAT* T*C [T*, 5-(3-aminopropyn-1-yl)2′-deoxyuridine]. SERRS was obtained down to an overall concentration of 8 × 10-13 M, 4 × 10-16 mol, by starting with a 1 × 10-11 M solution and adding 40-500 µL of colloid and water (1:4). One inconsistency of the dilution study was that the peak found at 1502 cm-1 appeared to diminish in size as the concentration was reduced and the peak at 1543 cm-1 appeared to increase. Nie and Emroy13 found small changes when examining a sample over a time period of 300 s. They explained this as being due to each molecule being adsorbed at different sites. Our spectra are stable for at least 15 min and the changes found are much smaller, but it seems reasonable to assume that as the DNA is diluted the pattern of surface adsorption will alter, thus producing a change in enhancement of different modes. To provide an estimate on the sensitivity of the system, the actual number of molecules examined at any one time in the interrogation volume was calculated. The interrogation volume was assumed as being a cylinder of dimensions d ) 5 µm and h ) 20 µm. This produced a volume of 4 × 10-16 m3 or 4 × 10-10 cm3. The lowest concentration of oligomer detected was 1 × 10-11 M. A 40-µl sample of this solution was added to 500 µL of colloidal suspension, thus producing a final concentration of 8 × 10-13 M or 4 × 10-16 mol. Of that solution, only 400 µL was used which equates to 3.2 × 10-16 mol or, by multiplying by Avogadro’s constant, 192.7 million molecules. By ratio of interrogation to sample volumes, we can say that there is only 0.08 of a molecule being irradiated at any one time. (32) Wagner, W. R.; Matteucci, M. D.; Lewis, J. G.; Guttierrez, A. J.; Moulds, C.; Froehler, B. C. Science 1993, 260, 1510-1513.
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Various longer chain oligonucleotides have been synthesized to examine the effect length had on the signal. A 26-mer primer was synthesized with a (T*C)6 repeat at the 5′-terminus and also a HEX label, HEX-T*CT* CT*C T*CT* CT*C GTG CTG CAG GTG TAA ACT TGT ACC AG. The resulting modified 38-mer provided strong SERRS signals, thus indicating that the HEX label was close enough to the surface to experience surface enhancement (Figure 7). The relative standard deviation (RSD) from the peak area of the peak at 1629 cm-1 was calculated to be 10% (n ) 6). Normally RSD values for SERRS studies are between 5 and 30%, depending upon the nature of the surface attachment. The value of 10% indictates acceptable reproducibility in this case and strong surface attachment. It should be noted in evaluating this figure that the equipment is not optimized for quantitative analysis and the technique is in the early stages of development. As the modified 5′-tail was sufficient in this case, it was decided to examine extension products primed from this 38-mer and also a reverse primer. The reverse primer enabled the controlled synthesis of two extension products, a 120- and a 254-mer. Both extension products were purified by gel electrophoresis prior to examination and were double stranded containing six propynylmodified groups and one HEX label per duplex. Detection of the 120-mer was achieved at an estimated concentration of 4 × 10-11 M in 1mL, which translates to ∼10 molecules under examination at any one time. A similar result was obtained for the longer extension product. CONCLUSIONS Previous levels of detection, of individually labeled DNA sequences, by colloidal SERRS have been surpassed by several orders of magnitude. This was made possible by examining the surface chemistry in detail and devising chemical modifications to overcome the surface adsorption problem. The experiments described above illustrate that SERRS is capable of detecting DNA without the need for an amplification step currently required for genome analysis. The process itself is reliable, reproducible, and
highly sensitive. It is selective in that the sharp signals provide a “fingerprint” of the specific dye chromophores3 and single components of mixtures can be readily identified, thus diminishing the need for a separation step. In addition, the use of nonfluorescent as well as fluorescent labels will increase the range of labels available for use with DNA. This unique approach, requiring surface binding rather than solution detection, produces opportunities to develop a range of new DNA analytical methods. Work is currently under way to detect specific extension products isolated from the modified primers by means not involving chromatographic separation.
ACKNOWLEDGMENT The authors thank Zeneca Diagnostics for funding D.G. and especially Drs. N. Gibson and D. Whitcombe for valuable assistance. The work described here is protected by patent WO 97/05280. Correspondence and requests for material to (e-mail)
[email protected]. Received for review June 24, 1997. Accepted August 27, 1997. AC970657B X
Abstract published in Advance ACS Abstracts, October 15, 1997.
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