Articles Anal. Chem. 1995, 67, 787-792
Detection of Aromatic Compounds Based on DNA intercalation Using an Evanescent Wave Biosensor P. C. Pandeyt and H. H. Weetall* Biosensor Technology Group, Biotechnology Division, National Institute of Standards and Technology, Gaithersbug, Maryland 20899
A flow injection analysis system coupled with an evanescent wave biosensor employing total internal reflection of fluorescence radiationfor the detection of the compounds that intercalate within DNA is reported. A highly fluorescent intercalator, ethidium bromide, has been used as the reference compound for the detection. The evanescent wave biosensorwas developed using immobilized doublestrand DNA (dsDNA) over the surface of a cylindrical wave guide. The response of the DNA-modifiedfiber is siguificantly higher than the response obtained with an unmodified fiber. The response of the biosensor at a constant concentration of ethidium bromide increases on increasing the concentration of immobilized dsDNA. At the steady-state response of the biosensor, obtained at a constant concentration of ethidium bromide, there is a decrease in the response to the injection of another DNA intercalator that competes for the intercalation sites on the dsDNA, displacing the ethidium bromide. This is immediately followed by recovery of the steady-state response. The decrease in the sensor response is a linear function of the concentrations of injected interahtor. Response curves for 9,1O-anthraquinoneS,6-disulfonic acid, remazol brillant blue, decacyclene, and 4,6-diamidino-2-phenylindoledihydrochloride are reported. There has been increasing interest in studying DNA-ligand interactions in recent years.’-16 Such studies have application in the development of a technology for the detection and quantitation
’On leave from the Department of Chemistry, Bamaras Hindu University, Varamasi-221005,India. (1) Pandey, P. C.; Weetall, H. H. Anal. Chem. 1994, 66, 1236-1241. (2) Plunno, P. A E.; Liu, F.; Krull, U. J.; Hudson, R H. E.; Damha, H. C. anal. Chim. Acta 1994,288, 205-214. (3) Ye, X.; Klmura, K; Patel, D. J. J. Am. Chem. SOC.1993, 115, 9325-9326. (4) Bally, C.; Waring, M. J. Biochemistry 1993, 32, 5985-5993. (5) Meyaralmes, F. J.; Porschke, D. Biochemistry 1993, 32, 4246-4253. (6) Vanhoute, L. P. A; Vangarderen, C. J.; Patel, D. J. Biochemistry 1993,32, 1667- 1674. (7)Mattes, W. B.; Kapeghian, J. C.; Lasinski, E. R; Olone, S. D.; Puri,E. C.; Matheson, D. W. Environ. Mol. Mutagen. 1993,22,46-53. (8) Lin,M. F.; Lee, M. H.; Yue, K T.; Marzill, L. G. Inorg. Chem. 1993,32, 3217-3226. (9) Iiu, F.;Meadows, K A; Mcmillin, D. R J.Am. Chem. Soc. 1993,115,66996704. (10) Pindur, U.; Haber, M.; Satller, K. J. Chem. Educ. 1993, 70, 263-272. (11)Tang, P.; Juang, C. L.; Harbison, G. S. Science 1990,249, 70-72. (12) Glazer, A N.; Rye, H. S. Nature 1992, 359, 859-861. This article not subject to U S . Copyright. Published 1995 by the American Chemical Society
of several varieties of organic carcinogens and toxins found in the environment, as well as for some antitumor drugs. The DNAethidium complex has been shown to be highly ~tab1e.l~ The stability of this complex and the noncovalent nature of the intercalation mechanism suggest this complex for use in the detection of other intercalators having differential binding afthities with doublestrand DNA (dsDNA). In this direction, Richardson and Schulman18 have developed a technique for the quantitative detection of fluorescent drugs based on the phenomenon of DNA intercalation and detection by fluorescence polarization. These authors used the classic intercalators acridine orange, ethidium bromide, and proflavin with doublestrand calf thymus DNA to measure low (-5 pg/mL) amounts of the drug actinomycin. Ethidium bromide, 7-hydropyridocarbazoles,3,Mamine&phenylphenanthridinium, and acridine orange have high affinities for intercalation into dsDN11l4 There are several reports describing the mechanism of i n t e r c a l a t i ~ n . ~ JThe ~ - ~“intercalation ~ binding strength” appears to be an essential parameter of the test molecule for displacement of the fluorescence dye from the dsDNA. The evanescent wave (EW) sensor^^-^^ are based on the production of an evanescent field when light propagates in an (13) Fomari, F. A; Gewirtz, D. A; Bauer, G. B.; Abraham, D. J.; Kellogg, G. E. Biophys. J. 1993, 64, A284. (14)Rye, H. S.Yua, S.; Quesada, M. A; Haugland, R P.; Mathies, R A; Glazer, A N. Methods Enymol. 1993,217,414-431. (15) Rye, H. S.; Dabora, J. M.; Quesada, M. A; Mathies, R A; Glazer, A N. Anal. Biochem. 1993,208, 144-150. (16) Quesada, M. A; Rye, H. S.; Gingrich, J. C.; Glazer, A N.; Mathies, R A BioTechniques 1991, 10, 616. (17) Rye, H. S.; Quesada, M. A; Peck, R A; Mathies, R A; Glazer, A N. Nucleic Acid Res. 1991, 19, 327. (18) Richardson, C. L; Schulman, G. E. Intercalation Inhibition assay for compounds that interact with DNA or RNA US.Patent 4,257,774, March 24, 1981. (19) Nordmeler, E. J. Bys. Chem. 1992, 96, 6045-6055. (20) Wilson, W. D.; Krishnamoorthy, C. R; Wang, Y.-H.; Smith, J. C. Biopolymers 1 9 8 5 , 2 4 , 1941-1961. (21) Kapuscinski, J.; Darzynkiewicz, 2.J. Biomol. Struct. Dyn. 1987, 5, 127143. (22) Wilson, W. D.; Lopp, I. G. Biopolymers 1979, 18, 3025-3041. (23) Lohman, T. M.; DeHaseth, P. L.; Record, M. T. Biophys. Chem. 1978, 8, 281-294. (24) Yoshida, M.; Shigemori, K; Sugimura, M.; Matano, M. Meas. Sn‘. Technol. 1 9 9 3 , 4 , 1077-1079. (25) Maccraith, B. D.; Mcdongh, C. M.; Okeefe, G.; Keyes, E. T.;Vos, J. G.; Okelly, B.; Mcgilp, J. F. Analyst 1993, 118,385-388. (26) Ge, Z. F.; Brown, C. W.; Sun, L. F.;Yang, S. C. Anal. Chem. 1993, 65, 2335-2338. (27) Xia, Z. M.; Vandeven, T.G. M. Longmuir 1992,8,2938-2946.
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optical fiber or wave guide. The EW sensors have advantage over the conventional distal-face optrodes in that they offer greater control over the interaction parameters (i.e., interaction length, sensing volume, and response time) by the sensor designer. These parameters may be manip~lated~~ (i) by using a optical fiber of special con6guration sensitive to EW interaction all along its length, (ii) by coating the fiber with a suitable polymer with a range of refractive index values, and (iii) by controlling the thickness of the coating. Introduction of a reagent that responds optically (absorption or fluorescence radiation) and can be retained withii the evanescent field may offer significantlyhigh sensitivity with the possibility of real-time measurements. Application of a fluorophore together with an evanescent field produced over the cylindrical wave guide has resulted in highly sensitive "evanescent fluorosensors".34-39These sensors are based on the principle of total internal reflection fluorescence 0 , 4 0 where the excitation is accomplished through the evanescent wave associated with propagation of trapped modes in the wave guide. A method based on this approach for the detection of nucleic acid using ethidium bromide has been developed by Hirschfeld et al.3s39 at ORD Inc. (North Salem, NH). Such sensors are well suited for probing binding of fluorescent intercalator within dsDNA bonded to the surface of a cylindrical wave guide. In the present research investigation,we describe an evanescent wave fluorobiosensor, based on the TIRF of the excitation radiation for the chosen fluorescent intercalator ethidium bromide, for monitoring the intercalaction of the dye into dsDNA The analyte is detected by competition and displacement of the intercalated ethidium bromide by the analyte of intererest. Doublestrand DNA was immobilized over the surface of an optical fiber using acrylamide-methacrylamide-hydrazides prepolymer. The device has been used to detect other DNA intercalators on the basis of their different affinities for dsDNA at a constant concentration of the ethidium bromide. As examples, the detection of 9,l(lanthraquinone2,6disulfonic acid, remazol brillant blue, decacyclene, and 4',&diamidino-2-phenylindole dihydrochloride (DAPI) are reported. Data are also reported on the effects of DNA concentration and flow rate on the response of the EW fluorobiosensor. EXPERIMENTAL SECTION
Materials. The materials used in this investigation and their sources were as follows: calf thymus dsDNA (sodium salt, preparation containing ~ 3 protein % readily soluble in water, av (28) Radhakrishnan, P.; Nampoori, V. P. N.; Vallabhan, C. P. G. Opt. Eng. 1993, 32, 692-694. (29) Carome, E. F.; Coghlan, G. A; Sukenik, C. N.; Zull, J. E. Sens. Actuators 1 9 9 3 , 14,732-733. (30) MacCraith, B. D. Sens. Actuaton 1993, 11, 29-34. (31) Shriverlake, L. C.;Ogert, R A; Ligler, F.S. Sens.Actuators 1 9 9 3 , 1 1 , 2 3 9 243. (32) Pollardknight, D.; Davie, R J.; Buckle, P. E.; Maule, C. H.; Edwards, P. R; Kinning, T.; Watts, H.; Yenung, D. Faseb]. 1993, 7, A1126. (33) Crystall, B.; Rumbles, G.; Smith, T.A; Phillips, D. J. Colloid.Intetfnce Sci. 1993, 247-250. (34) Glass, T. R; Lackle, S.; Hirschfeld, T. B. Appl. Opt. 1987,26, 2181-2187. (35) Wolfbels, 0. S. In Molecular Luminescence SpectroscopyPart I t Schulman, S. J., Ed.; Wiley: New York, 1988; pp 129-281. (36) Lackle, S.; Glass, T. R; Block, M. J. In Biosensors with Fiberoptics; Wise, Wingard, Eds.; Humana: Clifton, NJ, 1991. (37) Rogers, K R; Valdes, J. J.; Eldefrawl, M. E. Anal. Biochem. 1989, 182, 353-359. (38) Hirschfeld, T. B. Canadian Patent 1,299,074, 1992. (39) Hirschfeld, T. B. British Patent 2,190.189, 1990. (40) Hirschfeld, T. B. ]. Can. Spectrosc. 1965, 126.
788 Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
Mw > 100 MDa) and ethidium bromide from Sigma Chemical Co. (St. Louis, MO); anthraquinone2,&disulfonic acid (disodium salt), remazol brillant blue, decacyclene, and 4',&diamidino-2phenylindole dihydrochloride from Aldrich Chemical Co., Inc. (Milwaukee, WI). All other chemicals used were of analytical reagent grade. Immobilizationof Double-StrandDNA Over Optical Fiber. The ORD Inc. lensed optical fiber (injection-moldedpolystyrene) was obtained from Sapidyne Inc. (Wobum, MA). The acrylamide-methacrylamide-hydrazides prepolymer was synthesized as reported in an earlier publication.4l The modified fiber was prepared by aqueous silanization followed by treatment of the silanized fiber with the DNA-copolymer solution as follows. (a) Silanization of Optical Fiber. The optical fiber was washed with methanol and dried in a stream of nitrogen. The washed fiber was dipped into a solution of (3aminopropyl)triethoxysilanefor 20 s. The fiber was then washed with distilled water and dried at room temperature for 1 h. (b) DNA-Copolymer Solution. A 35% (w/v) solution of acrylamide-methacrylamide-hydrazides was prepared in distilled water. Calf thymus (5 mg) ws dissolved in 5 mL of distilled water. DNA-copolymer solution was prepared by adding the desired amount of DNA solution to the polymer solution, followed by the addition of sterilized 0.5 M phosphate buffer (PH 7.2) so that the final concentration of the prepolymer was 25.5% (w/v). The silanized fiber was dipped vertically into the DNAcopolymer solution for 30 s. The excess DNA-copolymer solution was removed by gentle rotation of the fiber. The fiber was dried at room temperature for 3 h. The polymer-modified fiber was then dipped in 1%glyoxal solution at 4 "C overnight. Finally the fiber was dipped in stirred phosphate buffer for 10 min. Flow Injection System. A diagram of the flow injection system is shown in Figure 1. The phosphate buffer (0.5 M, pH 7.2) was pumped by a Waters 501 HPLC pump (Waters, Milford, MA) to the evanescent fluorometer, designed and built at ORD Inc., through an SP 8880 autosampler (Spectra-Physics, Freemont, CA) equipped with a 20 pL sample loop. A sample in the full loop injection mode was introduced, unless othewise stated. The evanescent fluorometer was connected to a Linear Model 1200 x-t recorder (Liinear Instrument Corp., Reno, NV). The flow rate was 24 mWh. The temperature of the carrier buffer stream was maintained at 25 "C with a Brook6eld thermostat (Stoughton,MA). The phosphate buffer was sterilized prior to use by autoclaving and subsequently filtered through a 0.45 pm filter. A schematic diagram of the fiber optic evanescent fluorometer is shown in Figure 2. Ethidium bromide was excited just outside the waveguide boundary of an optical fiber at the excitation wavelength of 499/82 nm (center wavelength, 499 nm; fwhm, 82 nm) . A part of the emitted fluoroescence reentered the waveguide and was transmitted back up the fiber for detection with a Hamamatsu S2387/66R silicon detector after transmission through 563 nm long pass filters at 45" and 592/19 nm (center wavelength, 592 nm; fwhm, 19 nm) . The dead volume of the flow cell equipped with the optical fiber was 40 pL. Measurement of Ethidium Bromide. Ethidium bromide concentrations were measured using (i) unmodified optical fiber, (ii) polymer-modified fiber without DNA, and (ii) DNA-modified (41) Tran-Mmh,C.; Pandey, P. C.; Kumaran, S. Biosens. Bioelectr. 1 9 9 0 , 5 , 4 6 1 471.
0.SM PHOSPHATE BUFFER pH 7.2
il 5 6 1 LP
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Figure 2. Schematic diagram of the optical system for the fluorescence measurement.
4 FIA RESPONSE Flgure 1. Schematic diagram of the FIA fluorobiosensor.
optical fiber in a flow injection system as described above. The mobile phase phosphate buffer was pumped at a flow rate of 24 mL/h. Increasing concentrations of ethidium bromide were injected through the autosampler. The interaction of the fluorophore (ethidium bromide) with EW was recorded by collecting the resulting fluorescence to the detector. Measurement of Competitive Binding between Ethidium Bromide and Other DNA Intercalators. For the measurement of competitive binding between DNA intercalators and ethidium bromide, a constant concentration of ethidium bromide was pumped at a flow rate of 24 mWh along with the mobile phase until a steady-state response of the sensor was observed. While the system was maintained at steady state, varying concentrations of the competitive intercalators (anthraquinone-2,6disulfonicacid (disodium salt), remazol brillant blue, decacyclene, and 4',6 diamidino-2-phenylindoledihydrochloride containing the concentration of ethidium bromide present in the mobile phase were injected. There was a decrease in fluorescence,followed by the recovery of the steady state. RESULTS AND DISCUSSION
Ethidium Bromide-MediatedEvanescent Wave Sensor. The response of the unmodified fiber on the injection of varying concentrations of ethidium bromide was first examined in order to observe EW interaction with ethidium bromide flowing across the surface of the wave guide. F i e 3 shows the typical response of the EW sensor on the injection of varying concentrations of ethidium bromide at the flow rate of 24 mWh. Each assay is
1
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Figure 3. Typical FIA response on the injection of ethidium bromide. (1) 0.60pdmL, (2) 0.68p(9/mL, (3) l.lOpg/mL, (4) 3.6pgmL. Mobile phase used was 0.5 M phosphate buffer, pH 7.2.
reasonably fast, on the order of 3 min, which includes the buildup and decay of the flow injection analysis (FIA) signal back to the baseline. The mechanical thickness of the polymeric membrane is on the order of 5-15 pm, within which dsDNA is entrapped followed by cross-linking with glyoxal through the terminal amino group of DNA Figure 4 shows the response of the fluorosensor on the injection of increasing concentrationsof ethidium bromide using (1) unmodified fiber, (2) polymer-modified fiber without DNA, and (3) polymer-modified fiber with DNA The response of the evanescent fluorobiosensor depends on the magnitude of the fluorescence collected from the site of excitation of the fluorophore to the detector. It has been shown%that the complete collection efficiency includes several additional non-angle-dependentterms, including the exponential decrease in efficiencywith distance from the fiber surface. Hence, the response of the polymer-modiiied fiber without DNA is less where the excitation of the fluorophore Analytical Chemistry, Vol. 67, No. 5, March 1, 1995
789
loo0
s 360
800 h
> E v
1
600
400
200 90
0 0.001
0.01
0.1
1
I
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Ethidium bromide (pg / m L)
Figure 4. Calibration curve for the analysis of ethidium bromide, using (1) unmodified fiber, (2) polymer-modified fiber without DNA, and (3) polymer-modified fiber with DNA (0.4 mg/mL) in phosphate buffer at 25 "C. 0
takes place outside the polymer film. On the other hand, the response of the DNA-modified fiber is signi6cantly higher than the responses observed with the unmodified and polymer-modfied fibers without DNA The data recorded in Figure 4 show that although the response of the polymer-modifiedfiber without DNA is less than that of the ummodiiied fiber, its detection sensitivity is greater than that of the unmodfied fiber. This may be related to the nonspeiiic interaction of fluorophore with the polymer surface. The detection limit for ethidium bromide observed with unmodfied fiber was approximately 200 ng/mL. The DNAmodified fiber detected less than 2 ng/mL. The greater response of the DNA-moditied fiber is due to the intercalation of ethidium bromide within dsDNA bonded to the surface of the fiber, resulting in the enhanced interaction of evanescent energy to excite the ethidium bromide. The effect of DNA concentration immobilized over the surface of the fiber has also been studied. The response increases on increasing the DNA concentration (Figure 5). A DNA concentration (0.2 mg/mL) was used for the modifcation of the fiber for competitive measurement of DNA intercalators in order to obtain better sensitivity. The effect of flow rate on the response of the EW biosensor was determined by the injection of a constant concentration of ethidium bromide at increasing flow rates. The response decreases with increasing the flow rate as expected for a FIA system (Figure 6).
Competitive Binding between Ethidium Bromide and Other Intermlatorswith DNA. The dsDNA has af6nity to bind various aromatic compounds through different binding (42) King, C.-Y.; Weiss, M. A Proc. Natl. Acad. Sci. U S A . 1994, 90, 1199011994. (43) Li, T. H.; Zeng, Z. J. V.; Estevez, V. A; Baldenius, K. U.; Nicolaou, K. C.; Joyce, G. F. J. Am. Chem. SOC.1994, 116,3709-15. (44) Paloma, L. G.; Smith, J. A; Chazin, W. J.; Nicolaou, K. C. J. Am. Chem. SOC. 1994, 116, 3697-3708. (45) Mehrotra, K; Constantin, D.; Wallin, A,; Moldeus, P.; Jemstrom, B. Cancer Lett. 1994, 78, 49-56. (46) Cullinane, C.; Vanrosmalen, A; Phillips, D. R Biochemisty 1994,33,46324638.
790 Analytical Chemistry, Vol. 67, No. 5, March 1, 7995
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Hence, the selective quantitative determination of these compounds in real-world samples is a great problem. The analysis of the aromatic species is based on the reduction in the observed fluorescence, obtained by the intercalation of a constant concentration ethidium bromide into dsDNA by the binding of aromatic residues. This results in the depletion of ethidium bromide concentration in the polymer film bonded to the surface of the fiber. An attempt has been made to analyze several aromatic compounds using the optical fiber made from polystyrene; however, their detection is limited due to the problem of solubility in aqueous solution. Another problem is the quenching of the fluorescence of ethidium bromide by some of the aromatic compounds. Several similar compounds are fluorescent under the conditions used for the excitation of ethidium bromide. (47) Koshlap, K. M.; Gillespie, P.; Dervan, P. B.; Felgon, J. J. Am. Chem. SOC. 1993, 115, 7908-7909.
1.6
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Figure 8. Typical response of the evanescent fluorobiosensor on the injection of 9,lO-anthraquinone(1, 80,ug/mL; 2, 18,uglmL)at the steady-state response of ethidium bromide (4 ng/mL) in phosphate buffer at 25 "C. DNA concentration was 0.2 mg/mL.
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DNA Intercalators ( W L ) Figure 7. Effect of competitive intercalators on the fluorescence of ethidium bromide. t represents the ratio of the fluorescence of a constant ethidium bromide concentration (10 ng/mL) in the presence of varying concentrations of competitive intercalators to the fluorescence of the same concentration of ethidium bromide in the absence
of the competitive intercalators. (1) 9,10-Anthraquinone-2,6-disulfonic acid, (2)remazol brillant blue, (3)decacyclene,(4) DAPI, (5)chrysene, (6) benz[,lfluoroanthene, and (7)pentacene. Hence, the detection of the aromatic residue based on this approach is limited to those which are not fluorescent under this condition and have affinity to bind with dsDNA The analysis of such compounds are reported in this paper. The fluorescence of ethidium bromide was measured in the presence of seven aromatic compounds (chrysene, benz[ilfluoroanthene, pentacene, decacyclene, DAPI, 9,lCLanthraquinone-2,6 disulfonic acid, and remazol brillant blue) in solution by a fluorescence spectrophotometer (SLM 8000, SLh4 Instrument Inc., Urbana, E) in order to select the competitive intercalator, the presence of which does not affect the fluorescent behavior of ethidium bromide. The data recorded in Figure 7 show the fluorescence of a constant concentration of ethidium bromide (excitation wavelength, 499 nm; emission wavelength, 592 nm) in the presence of varying concentrations of the aromatic compounds. The data are plotted in terms of the ratio of the fluorescence of ethidium bromide measured at the various concentrations of aromatic compounds to the fluorescence measured only in the presence of the same concentration of the ethidium bromide. The fluorescence of the ethidium bromide is considerably affected in the presence of chrysene, benz[ilfluoroanthene, and pentacene ( F i r e 7). However, in the presence of 9,1O-anthraquinone-2,6disulfonicacid, remazol brillant blue, decacyclene, and DAPI, the fluorescence is only slightly affected. These four compounds were selected for the measurement of competitive dsDNA intercalation. For the measurement of the competitive intercalation, a steadystate response of the EW biosensor was obtained at a constant concentration of ethidium bromide (4 ng/mL) and a flow rate of 24 mWh. At the steady-state response of the sensor, varying concentrations of competitive intercalators were injected. Figure 8 shows a typical response of the sensor obtained on the repeated injections of 9,lCLanthraquinone-2,6disulfonicacid (80 pg/mL; 18
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Figure 9. Calibration curves for the analysis of (1) anthraquinone2,6-disulfonicacid, (2) remazol brillant blue, (3) decacyclene, and (4) DAPI based on the displacement of intercalated ethidium bromide.
The competitive intercalators were injected at the steady-state response of the evanescent fluorobiosensor obtained after a constant concentration of ethidium bromide (4 ng/mL) was circulated along with the mobile phase. pg/mL). There is a decrease in the steady-state response, followed by the recovery of the steady state. The total time required for recovery of the steady-statevalue is 6.5-12 min. The decrease in the response is associated with the depletion of ethidium bromide concentration from the polymer film,which is due to the competitive binding of the 9,lCLanthraquinone and displacement of the ethidium bromide from the dsDNA However, the competitive displacement of ethidium bromide from the dsDNA depends on the mode of binding of the small aromatic ligands under investigation. Recently, the binding of DAPI to DNA has been reported by Norden et al.48349These authors have demonstrated a heterogeneous nature of the intercalation of a small ligand with DNA Figure 9 shows the calibration curves for anthraquinone, remazol brillant blue, decacyclene, and 4',6 diamindino-2-phenylindoledihydrochloride @APD. The data shown in Figure 9 show the decrease in the fluorescence of the ethidium bromide on the injection of the (48)Jansen, IC;Norden, B.; Kubista, M. J. Am. Chem. SOC.1993,115,1052710529. (49)Kim, S. IC;Eriksson, S.;Kubista, M.; Norden, B. 1.Am. Chem. SOC.1993, 115,3441-3445.
Analytical Chemistry, Vol. 67, No, 5,March 1, 1995
791
competitive intercalator. This approach can be used to detect other DNA intercalators which are not fluorescent within the excitation wavelength of ethidium bromide. It is also possible to substitute other fluorescent intercalators (Le., acridine orange) which bind with dsDNA in the same fashion under the similar condition. The relative binding ratio Q) of the detected intercalator with dsDNA has been calculated using ethidium bromide as the reference and using the following empirical expression:
k = [6E/C~,,l/[k'/Cethidium bromide where 6E is the decrease in the steady-state response of the evanescent fluorobiosensor on the injection of a competitive intercalator of concentration C, and k' is the response of the sensor at the concentration of 4 ng/mL ethidium bromide (Cethidium bromide). The values of the relative binding ratio for different intercalators at the constant concentration of dsDNA are shown in Table 1. The relative ability of the intercalator to displace ethidium bromide based on the relative binding ratio is as follows: decacyclene > DAPI > remazol brillant blue > 9,10anthraquinone-2,&disulfonicacid. Experiments are in progress to study the impact of kinetic limitation on the association constants of the intercalators with dsDNA. The stability of the evanescent fluorobiosensor was investigated by operating the sensor for 13 h each day. A reproducible FIA response (296%)for 100 injections was recorded. The dsDNA within the polymer film is quite stable (> 1month) when the film is stored in 0.1 M phosphate buffer, pH 7.2. However, the frequent
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Table 1. Relatlve Binding Ratio of DNA Intercalators with Respect to Ethidium Bromide
intercalator
re1 binding ratio
anthraquinone-2,Bdisulfonicacid remazol brilliant blue decacyclene 4',Bdiamidmo-2-phenylindole dihydrochloride @MI)
6.6 x 4.7 10-5 3.9 10-3 8.0 10-4
assembling of the dsDNA-modified fiber into the flow cell causes damage to the polymer film. ACKNOWLEWMENT This work was supported by the US. Environmental Protection Agency. We are thankful to Dr. John Horvath for helpful discussions. We also thank the ORD for the loan of the evanescent fluorosensor. Registry numbers suppliedby the authors: Anthraquinone2,6disulfonic acid, 8550-4; ethidium bromide, 1234458,and DNA, 73049-39-5; remazol brillant blue, 2580-78-1; decacyclene, 191-48 0; DAPI, 28718-90-3. Received for review July 20, 1994. Accepted December 13,1994.@
AC9407328 Abstract published in Advance ACS Abstructs, January 15, 1995.
