Anal. Chem. 1996, 68, 4365-4369
DNA-Modified Electrode for the Detection of Aromatic Amines Joseph Wang,* Gustavo Rivas,† Denbai Luo,‡ Xiaohua Cai, Florenda S. Valera,§ and Narasaiah Dontha
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
A new electrochemical biosensing strategy has been developed for trace measurements of toxic aromatic amine compounds. The device relies on the intercalative collection of aromatic amines onto the immobilized dsDNA layer followed by potentiometric stripping quantitation of the accumulated species. The enhanced sensitivity, accrued from the DNA collection process, is coupled to new selectivity dimensions provided by the structural requirements for such intercalative binding. The extent and rate of the accumulation are strongly dependent upon the structure of the aromatic amine species. Having the amino substituent in a slightly different position produces a dramatic effect upon the response. Nanomolar detection limits are obtained after a 10-min accumulation. Applicability to river water and groundwater samples is demonstrated. Such DNA-based devices hold great promise for environmental screening of toxic aromatic amines and for elucidating molecular interactions between intercalating pollutants with DNA. Amine-substituted aromatic compounds constitute a very important class of environmental pollutants. These compounds are widely used as intermediates in the production of azo dyes and pigments. Because of their toxicological significance, aromatic amines are included in the EPA list of priority pollutants.1 Accordingly, there is considerable interest in the determination of aromatic amines in environmental matrices. Such assays are commonly carried out in central laboratories using various separation techniques.2,3 However, in view of the time delays and cost of laboratory-based chromatographic analyses, there are growing needs for innovative devices for environmental screening of aromatic amines. Fast and compact biosensors, relying on the toxic action of pollutants, should be particularly promising for the task of on-site monitoring of aromatic amines. This article describes a new strategy, based on DNA-modified electrodes, for detecting nanomolar levels of toxic aromatic amines. The use of DNA recognition layers represents an exciting area in analytical chemistry.4 Most of the activity in DNA biosensors has focused on the development of sequence-specific hybridization devices for early detection of genetic or infectious † Permanent address: Dept. Fisico Quimica, Universidad Nacional de Cordoba, Cordoba, Argentina. ‡ Permanent address: Dept. of Chemistry, South-Central Institute for Nationalities, Wuhan 430074, P.R. China. § Permanent address: Inst. of Chemistry, University of the Philippines, Philippines. (1) Fed. Regist. 1979, 44, 69464. (2) Lu, C. S.; Huang, S. J. Chromatogr. 1995, 696, 201. (3) Oostdyg, T.; Grab, R.; Snyder, J.; McNally, M. Anal. Chem. 1993, 65, 596. (4) McGown, L.; Joseph, M.; Pitner, J.; Vonk, G.; Linn, C. Anal. Chem. 1995, 67, 663A.
S0003-2700(96)00650-6 CCC: $12.00
© 1996 American Chemical Society
diseases.5 In contrast, only few studies have been devoted for designing environmental DNA biosensors,6-8 despite the fact that the toxic action of many carcinogenic and mutagenic pollutants is related to their interaction with DNA. Pandey and Weetall7 have recently developed an optical DNA biosensor for the detection of aromatic compounds based on the displacement of a fluorescent indicator from the surface-bound DNA duplex. In the following sections we exploit the intercalative binding of aromatic amines to double-stranded (ds) calf thymus DNA, and their inherent electroactivity, for designing new affinity electrochemical biosensors for these pollutants. Besides their sensing utility, the new DNA-coated electrodes hold great promise for exploring the binding interaction of aromatic amines (and other contaminants) with DNA double helix. Structural representations of the compounds examined are given in Figure 1. EXPERIMENTAL SECTION Apparatus. Potentiometric stripping analysis (PSA) was performed with a TraceLab unit (PSU 20, Radiometer) in connection to an IBM PS/2 55SX. Potentials were sampled at a frequency of 30 kHz, and the derivative signal (dt/dE) was recorded against the potential. The peak area following baseline fitting was used as the analytical signal. The three-electrode system consisted of a carbon paste working electrode, a Ag/AgCl reference electrode (Model RE-1, BAS Inc., W. Lafayette, IN), and a platinum wire auxiliary electrode. The electrodes joined the cell through holes in the Teflon cover. The carbon paste was prepared in the usual way by hand-mixing graphite powder (Acheson 38, Fisher Scientific) and mineral oil (Sigma Chemical Co., Catalog No. M5904, free of DNase, RNase, and protease). The ratio of graphite powder to mineral oil was 70:30. The resulting paste was packed tightly into a Teflon sleeve (3.5-mm i.d.) body. Electrical contact was established with a stainless steel screw. The surface was polished to a smooth finish before use. The electrochemical cells were 2-mL vials (19 × 20 mm, Kimble Glass Inc.). All glassware, containers, and pipet tips (with exception of the electrodes) were sterilized by autoclaving for 30 min. The cells were cleaned with diluted nitric acid (1:4), rinsed thoroughly with water, and then dried in the oven (150 °C) before use. The electrodes were rinsed with sterilized water prior to use. A diode array spectrophotometer (Model 8452A, Hewlett Packard) was employed to measure the concentration of nucleic acids by monitoring the absorbance at 260 nm. (5) Nature 1996, 379, 389. (6) Pandey, P. C.; Weetall, H. H. Anal. Chem. 1994, 66, 1236. (7) Pandey, P. C.; Weetall, H. H. Anal. Chem. 1995, 67, 787. (8) Wang, J.; Chicharro, M.; Rivas, G.; Cai, X.; Dontha, N.; Farias, P.; Shiraishi, H. Anal. Chem. 1996, 68, 2251. (9) Arrigan, D. W. Analyst 1994, 119, 1953.
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Figure 1. Structures of polynuclear aromatic compounds examined in the present study.
Reagents. Double-stranded calf thymus DNA (dsDNA, Catalog No. D4522), and 2-anthramine (Catalog No. A1381) were received from Sigma Chemical Co. 1-Anthramine (Catalog No. A3,860-62), aminonaphthalene (Catalog No. A6,640-5), 9,10-diaminophenanthrene (Catalog No. D2,290-8), 1,2-diaminoanthraquinone (Catalog No. D1,158-2), and 1-aminopyrene (Catalog No. A7,790-3) were products of Aldrich. All chemicals were used as received. Caution: In view of their toxicity aromatic amine compounds should be handled with special care. The stock solution of the dsDNA (∼1000 µg/mL) was prepared with TE solution (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), and its concentration was estimated through UV measurement. All analyte stock solutions were prepared with ethanol. Sterile distilled water was used for all aqueous media. The groundwater sample was collected from the Hanford Site (Richland, WA), while the river one was obtained from the Rio Grande (at Las Cruces, NM). Procedure. The biosensing protocol at the DNA-modified electrode consisted of three steps: DNA immobilization, association/accumulation of the aromatic compound onto the dsDNA layer, and chronopotentiometric detection of the accumulated analyte. (I) DNA immobilization. A freshly smoothed carbon paste surface was immersed into a stirred acetate buffer (0.2 M, pH 5.0) containing 5 µg/mL dsDNA. The electrode was first polarized at +1.7 V for 1 min followed by application of a potential of +0.2 V for 2 min to accumulate the DNA onto the electrode surface. (II) Association/accumulation. The DNA-modified electrode was transferred to the stirred sample solution (0.02 M phosphate buffer, pH 7.0) for the desired time, while the electrode was held at a given potential (see figure captions). (III) Signal transduction. Chronopotentiometric measurements were performed in an acetate buffer blank solution with an initial potential of +0.2 V and a constant current of +8 µA. The surface was rinsed carefully with water for a short time (5 s) prior to each medium exchange. Successive measurements were carried out by renewing the surface and repeating the above procedure (using a new 4366 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
Figure 2. Chronopotentiograms for 0 (a) and 0.5 µM (b and c) 2-anthramine (A) and 9,10-diaminophenanthrene (B) after a 3-min accumulation at the DNA-modified (a and b) and bare (c) carbon paste electrodes and transfer of the electrodes to a blank solution (0.2 M acetate buffer, pH 5.0). DNA immobilization: 1 min at +1.7 V followed by 2 min at +0.2 V in acetate buffer medium containing 5 µg/mL dsDNA. Accumulation: 3 min at +0.3 (A) or +0.1 (B) V in 0.02 M phosphate buffer (pH 7.0) containing 0.5 µM 2-anthramine or 9,10-diaminophenanthrene. PSA transduction: in the blank acetate buffer with an initial potential of +0.2 V and a constant current of +8 µA.
“stripping” solution and cell). All data were obtained at room temperature (23.0 ( 0.5 °C). RESULTS AND DISCUSSION The operation of the new affinity DNA biosensor is analogous to that of preconcentrating chemically modified electrodes9 in that the uptake of the target analyte(s) into the preconcentrating layer is followed by electrochemical quantitation of the accumulated species. The high sensitivity, accrued from the preconcentration step, is coupled to the selectivity provided by the nature of the analyte-DNA interaction and by the ability to perform the measurement in a blank/electrolyte solution (i.e., medium exchange). The strong attachment of the dsDNA to the surface of the pretreated carbon paste transducer8 facilitates such operation in blank solutions. The strong intercalative association of aromatic amines with the surface-confined DNA layer is indicated from experiments involving such medium-exchange procedure. Figure 2 compares chronopotentiograms for 2-anthramine (A) and 9,10-diaminophenanthrene (B) following a 3-min accumulation at the DNA-coated (b) and bare (c) carbon paste electrodes and transfer of the electrode to a blank solution. Also shown (a) is the response of the DNA biosensor in the absence of the aromatic amines. Such response is associated with the oxidation of the DNA guanine residue.10 The bare electrode does not permit quantitation of both analytes under these conditions. In contrast, well-defined anodic peaks (II) are observed at the DNA sensor (Ep of +0.72 and +0.54 V, respectively). These peaks are related to the oxidation of the accumulated aromatic amines to their cation radicals.11 Coupled with the flat baseline of the chronopotentiometric transduction mode, the DNA biosensor offers convenient quantitation of submicromolar concentrations following a short accumulation period. Notice also the decrease of the DNA (guanine) oxidation peak (I) following the association with the aromatic compound (10) Palecek, E. Electroanalysis 1996, 8, 7 and references therein. (11) Lines, R. In Organic Electrochemistry; Baizer, M., Lund, H., Eds.; Dekker; New York, 1983; Chapter 15.
TIME (MIN)
Figure 3. Effect of accumulation time on the PSA signals for 10 µM 2-aminonaphthalene (A), 0.5 µM 2-anthramine (B), 0.1 µM 1,2diaminoanthraquinone (C), and 0.5 µM 1-aminopyrene (D) at the bare (a) and the DNA-modified (b) carbon paste electrodes. Accumulation potential: 0 (A and C), +0.3 (B) , and +0.1 (D) V; all other conditions as in Figure 2.
[compare (a) and (b)]. Such lowering of the DNA intrinsic response is attributed to changes in the surface accessibility of the guanine moiety upon binding of the aromatic amines to the DNA duplex. The high sensitivity of the aromatic amines DNA sensor is attributed to its preconcentration step. Figure 3 displays the dependence of the chronopotentiometric response of the bare (a) and DNA-based (b) electrodes on the accumulation time for different aromatic amines. The response of the DNA biosensor toward all four compounds increases rapidly with time at first and then more slowly, indicating appreciable binding. Yet, the exact time-dependent profile differs from compound to compound. For example, 2-aminonaphthalene (A) exhibits a faster signal enhancement at shorter periods, as compared to the slower changes observed for 2-anthramine (B) and 1,2-diaminoanthraquinone (C) and to the sigmoidal change of 1-aminopyrene (D). The different shapes of the temporal profiles reflect differences in the binding kinetics of the analyte-DNA association. As expected, these compounds display some adsorptive stripping response at the plain carbon paste surface (a). However, the corresponding signals at the DNA biosensor are significantly higher (e.g., about 5, 20, 4 and 15-fold larger for 2-aminonaphthalene, 2-anthramine, 1,2diaminoanthraquinone, and 1-aminopyrene, respectively, following a 3-min accumulation). We also examined the effect of the preconcentration potential upon the response of the various aromatic amines (not shown). While the peaks of 2-aminonaphthalene and 1,2-diaminoanthraquinone were nearly independent of the accumulation potential, best results for 2-anthramine and 9,10-diaminophenanthrene were obtained after accumulation at +0.3 and +0.1 V, respectively. The data of Figure 3 indicate also that the DNA biosensor results in different sensitivities toward various aromatic amines. Figure 4 displays calibration data for different aromatic amines. The 3-min accumulation period resulted in well-defined peaks for the submicromolar and micromolar concentration ranges exam-
Figure 4. Chronopotentiometric response of the DNA biosensor for 2-aminonaphthalene (A), 2-anthramine (B), 1,2-diaminoanthraquinone (C), 9,10-diaminophenanthrene (D), and 1-aminopyrene (E) solutions of increasing concentrations (a-f): 0-25 (A, 5 µM increments), 0-1.0 (B, 0.2 µM increments), 0-0.25 (C, 0.05 µM increments), 0-0.5 (D, 0.1 µM increments), and 0-1.0 µM (E, 0.2 µM increments). Also shown are the resulting calibration plots (A, 0-40 µM; B, 0-1.6 µM; C, 0-0.4 µM; D 0-0.8 µM; E, 0-1.6 µM). Threeminute accumulation at 0 (A, C), +0.3 (B), or +0.1 (D, E) V; other conditions as in Figure 2.
ined. The response for 2-aminonaphthalene (A), 1,2-diaminoanthraquinone (C), and 1-aminopyrene (E) increases linearly over the entire ranges tested (5-40, 0.05-0.40, and 0.2-1.6 µM, respectively). In contrast, deviations from linearity are observed for 2-anthramine (B) and 9,10-diaminophenanthrene (D) for concentrations exceeding 1.2 × 10-6 and 5 × 10-7 M, respectively. The slopes of the linear portions correspond to sensitivities of 3.6 (A), 126.8 (B), 306.0 (C), 99.2 (D), and 86.0 (E) ms/µM. Such trend in sensitivity (1,2-diaminoanthraquinone > 2-anthramine > 9,10-diaminophenanthrene > 1-aminopyrene > 2-aminonaphthalene) appears to be related to the strength of the DNA-aromatic compound interaction. It is known that the size and conformation of the molecule play a major role in its intercalative binding.12 Aminonaphthalene, with its two-ring structure, has the lowest sensitivity. The highest sensitivity is observed for the anthracene derivatives (1,2-diaminoanthraquinone, 2-anthramine) possessing three adjacent rings. The different ring system conformation of 9,10-diaminophenanthrene and 1-aminopyrene may account for their lower sensitivity (despite their three or four benzene rings). Substituent position is another factor influencing the intercalative DNA binding13 and hence the sensor response. Having the amino substituent in a slightly different position is sufficient to produce a dramatic effect upon the response. For example, while a sensitive response was observed for trace levels of the wellknown mutagen 2-anthramine (2-aminoanthracene), no response (12) Lesko, S.; Smith, A.; Tso, P.; Umans, R. Biochemistry 1968, 7, 434. (13) Tanius, F.; Jenkins, T.; Neidle, S.; Wilson, W. Biochemistry 1992, 31, 11632.
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Figure 5. Chronopotentiograms for 2-anthramine in the presence of 0.5 µM 1-anthramine after a 3-min accumulation at the bare (A) and DNA-modified (B) carbon paste electrodes and transfer of the electrodes to the acetate buffer blank solution. Concentration of 2-anthramine: 0 (a), 0.5 (b), 1.0 (c), and 1.5 µM (d). Accumulation potential: +0.3 V; other conditions as in Figure 2.
was obtained for its 1-anthramine (1-aminoanthracene) isomer. Such ability of the DNA recognition layer to discriminate between aromatic amine isomers (based on the amine position) adds a new and important dimension of selectivity. Figure 5B demonstrates the quantitation of 2-anthramine in the presence of 1-anthramine. Successive 5 × 10-7 M concentration increments of 2-anthramine to the 1-anthramine solution resulted in well-defined concentrationdependent peaks (b-d); no response is observed for the 1-anthramine solution (a). The response for 2-anthramine in the presence of 1-anthramine is similar to that observed for 2-anthramine alone (not shown). In contrast, an additive response is observed for analogous measurements at the bare carbon paste electrode (A). Note again the significantly lower sensitivity, and inferior signal-to-noise characteristics, in the absence of the DNA layer. It should be pointed out that the medium-exchange protocol employed throughout this study further enhances the selectivity by discriminating against solution-phase (nonaccumulated) electroactive species. The favorable signal-to-noise characteristics of the DNAmodified electrodes result in extremely low detection limits. For example, Figure 6b displays chronopotentiograms for 2 × 10-6 M 2-aminonaphthalene (A), 1 × 10-7 M 2-anthramine (B), 2 × 10-8 M 1,2-diaminoanthraquinone (C), 5 × 10-8 M 9,10-diaminophenanthrene (D), and 1 × 10-7 M 1-aminopyrene (E) following a 10-min accumulation. Also shown (a) are the corresponding potentiograms for the blank solution. Detection limits around 1.8 × 10-6 M 2-aminonaphthalene, 8 × 10-8 M 2-anthramine, 1 × 10-8 M 1,2-diaminoanthraquinone, 2 × 10-8 M 9,10-diaminophenanthrene, and 6 × 10-8 M 1-aminopyrene can be estimated from the signal-to-noise characteristics of these data (S/N ) 3). Such detection limits are low enough for assays of polluted environmental samples. Note that the pyrene molecule displays multiple peaks associated with its oxidation to the cation radical followed by oxidation to the dication.11 The peak at +0.74 V was used for all quantitative work. The reproducibility of the DNA biosensor protocol was evaluated from eight repetitive measurements of 2.5 × 10-7 M 1,2diaminoanthraquinone, 5 × 10-7 M 2-anthramine, and 2 × 10-5 M aminonaphthalene (3-min accumulation at 0.0, +0.3, and 0.0 V, respectively). These series resulted in mean peak areas of 78.6, 70.8, and 68.4 ms and relative standard deviations of 8.4, 9.4, and 4368 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
Figure 6. Chronopotentiograms for (A) 2-aminonaphthalene (a, 0 µM; b, 2 µM), (B) 2-anthramine (a, 0 µM; b, 0.1 µM), (C) 1,2diaminoanthraquinone (a, 0 µM; b, 0.02 µM), (D) 9,10-diaminophenanthrene (a, 0 µM; b, 0.05 µM), and (E) 1-aminopyrene (a, 0 µM; b, 0.1 µM) after a 10-min accumulation at the DNA-modified carbon paste electrodes and transfer of the electrodes to an acetate buffer blank solution. Accumulation potential: 0 (A, C), +0.3 (B), +0.1 V (D, E); other conditions as in Figure 2.
Figure 7. Assay of untreated natural water sample. Chronopotentiograms for the groundwater sample (water sample/phosphate buffer (pH 7.0; 0.2 M) 8:2), spiked with 0 (a) 0.4 (b), 0.8 (c), and 1.2 (d) µM 2-anthramine. Accumulation time: 3 min; other conditions as in Figure 2.
9.0%, respectively. No removal of the intercalated analyte is required at the end of each measurement cycle, since the carbon paste immobilization strategy permits a fast and simple surface renewal. Applicability to relevant environmental samples is illustrated in Figure 7. Three standard additions of 4 × 10-7 M 2-anthramine to the untreated groundwater sample resulted in well-defined concentration-dependent peaks (b-d, II) following a 3-min accumulation. Note again the simultaneous decrease of the DNA guanine peak (I). The natural constituents of the sample did not yield any detectable (interfering) signal (a). Yet, comparison to operation in “synthetic” samples (e.g., Figure 2A) indicates a somewhat lower sensitivity. Such change is attributed to the presence of electroinactive species possessing DNA intercalation properties. Proper calibration in the relevant sample matrix would be required to address this effect. Similar response characteristics were obtained for measurements of 2-anthramine in a river water sample (not shown). In summary, we have presented here a new screening tool for aromatic amine compounds. Such use of DNA intercalation as a basis for electrochemical biosensors results in a rapid, sensitive, and simple detection of aromatic amine compounds. The enhanced sensitivity accrued from the DNA collection process is coupled to a new dimension of selectivity provided by the structural requirements for such intercalative binding. Compact (hand-held) PSA instruments, originally developed for decentral-
ized trace metal testing,14 may be combined with the new DNA preconcentrating devices to offer convenient field monitoring of aromatic amine pollutants. Further development work, aimed at automating the various steps, would be required prior to the realization of such on-site applications. Preliminary experiments, aimed at coupling such instruments with DNA-coated microfabricated carbon electrode strips were very successful. The concept could be extended to other intercalating electroactive species (e.g., nitro-substituted aromatic compounds). Nonelectroactive aromatic pollutants may be detected via the displacement of an electroactive marker, in a manner analogous to the displacement of fluorescent indicators.7 Alternately, one can use changes in the DNA guanine peak for following changes in the concentration of nonelectroactive species. The new DNA biosensors should be useful for in situ (14) Wang, J. Analyst 1994, 119, 763. (15) Meyer-Almes, F.; Porschke, D. Biochemistry 1993, 32, 4246.
probing of the interaction of pollutants with DNA and may thus shed useful insights into the complex intercalation mechanism of aromatic residues.15 Future work will examine the correlation between the sensor response and the analyte carcinogenicity. ACKNOWLEDGMENT J. W. acknowledges the financial support from the U.S. Department of Energy (DOE), and the WERC-DOE funding program. G.R., D.L., and F.S.V. acknowledge fellowships form CONICET (Argentina), the State Education Committee (PR China), and UP and DOST-ESEP (Philippines) R&D, respectively. Received for review July 2, 1996. Accepted October 9, 1996.X AC960650E X
Abstract published in Advance ACS Abstracts, November 15, 1996.
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