Anal. Chem. 1997, 69, 4056-4059
Electrochemical Measurements of Oligonucleotides in the Presence of Chromosomal DNA Using Membrane-Covered Carbon Electrodes Joseph Wang,* Xiaohua Cai,† Joa˜o Roberto Fernandes,‡ Douglas H. Grant,§ and Mehmet Ozsoz|
Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003
Substantial improvements in the selectivity of electrochemical measurements of trace nucleic acids are obtained by using membrane-covered carbon disk electrodes. Access to the electrode surface can be manipulated via a judicious choice of the membrane molecular weight cutoff (MWCO). The resulting separation step, performed in situ at the electrode surface, adds a new dimension of selectivity based on molecular size to electroanalysis of nucleic acids. Transport properties are evaluated with respect to the oligonucleotide length and membrane MWCO. A highly selective response is observed for synthetic oligonucleotides in the presence of otherwise interfering chromosomal DNAs. Discrimination among oligonucleotides of different lengths is also possible. Short accumulation periods (1-5 min) are sufficient for convenient measurements of low milligram per liter concentrations. Due to the important biological role of nucleic acids, reliable analytical techniques are required for their quantitation. Electroanalysis has been shown to be very useful for this task.1 The electroreduction of nucleic acid bases has led to the development of advanced pulse polarographic procedures for trace measurements of DNA and RNA.2 Coupling these reduction processes with the strong adsorption of nucleic acids at the hanging mercury drop electrode resulted in highly sensitive adsorptive stripping voltammetric schemes for their quantitation.3,4 We have demonstrated recently that analogous stripping measurements of nucleic acids can be performed at various carbon electrodes by coupling the electrooxidation of the guanine moiety with the surface activity of nucleic acids and constant-current potentiometric stripping analysis (PSA).5-7 Unlike solid electrode voltammetric measurements of nucleic acids, which suffer from †
Present address: Nova Biomedical Co. Waltham, MA 02254. Permanent address: Departamento de Quı´mica, Universidade Estadual Paulista, Bauru-SP, POB 473, Brazil. § Permanent address: Department of Chemistry, Mount Allison University, Sackville, N. B. EOA 3CO, Canada. | Permanent address: Faculty of Pharmacy, Ege University, Izmir 35100, Turkey. (1) Palecek, E. Electroanalysis 1996, 8, 7. (2) Vojtiskova, M.; Lukasova, E.; Palecek, E. Bioelectrochem. Bioenerg. 1981, 8, 487. (3) Palecek, E.; Fojta, M. Anal. Chem. 1994, 66, 1566. (4) Palecek, E. Anal. Biochem. 1988, 170, 421. (5) Wang, J.; Cai, X.; Jonsson, C.; Balakrishnan, M. Electroanalysis 1996, 8, 20. (6) Wang, J.; Cai, X.; Wang, J.; Jonsson, C.; Palecek, E. Anal. Chem. 1995, 67, 4065. (7) Cai, X.; Rivas, G.; Farias, P.; Shiraishi, H.; Wang, J.; Palecek, E. Electroanalysis 1996, 8, 753. ‡
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a large background current around the guanine peak, the computerized PSA operation offers a sophisticated background correction and results in a well-defined response. Such anodic measurements of DNA and RNA represent a major advance in the analysis of nucleic acids, as it opens the door to modern solidstate sensing devices. Yet, while offering remarkable sensitivity, such probes would greatly benefit from improvements in the discrimination among nucleic acids. In view of the similarity in the guanine peak potentials, only differences in the peak height can be exploited for partial differentiation between native and denaturated DNAs or between tRNA and DNA.1,3 The objective of this work is to investigate the utility of membrane-covered carbon paste electrodes for PSA measurements of synthetic oligonucleotides in the presence of chromosomal DNA. Modern molecular biology and biotechnology is shifting to studies and applications of well-defined oligonucleotides of known nucleotide sequence. The widespread use of synthetic oligonucleotides in methodologies such as PCR, in situ hybridization, and in vivo genetic manipulations means that these oligomers require extensive analytical characterization. A drawback of the above advanced electroanalytical protocols is the lack of resolution between small oligonucleotides and large nucleic acid macromolecules. Accordingly, previous electrochemical studies have rarely focused on the detection of synthetic oligonucleotides.8 Coverage of various electrodes with different membranes or polymeric films has been widely used for enhancing the selectivity and stability of electroanalytical measurements.9-11 Stripping measurements of trace metals have been greatly improved (with respect to surfactant interferences and overlapping peaks) upon covering the mercury film electrode with dialysis membranes12 or permselective coatings.13-15 In the following sections, we will similarly demonstrate that placing dialysis membranes (of different molecular weight cutoffs) over the carbon paste electrode imparts a new dimension of selectivity to the electroanalysis of nucleic acids. In particular, such coated electrodes facilitate the detection of short oligonucleotides in the presence of large DNA and RNA molecules and offer discrimination among oligonucleotides of different lengths. The size discrimination of the membranemodified electrode should thus be useful for the detection of (8) Palecek, E.; Kolar, V.; Jelen, F.; Heinemann, U. Bioelectrochem. Bioenerg. 1990, 23, 285. (9) Wang, J. Electroanalysis 1991, 3, 255. (10) Murray, R. C.; Ewing, A. G.; Durst, R. A. Anal. Chem. 1987, 59, 379A. (11) Emr, S.; Yacynych, A. Electroanalysis 1995, 7, 913. (12) Stewart, E.; Smart, R. Anal. Chem. 1984, 56, 1131. (13) Wang, J.; Hutchins-Kumar, L. Anal. Chem. 1986, 58, 402. (14) Hoyer, B.; Florence, T.; Bately, G. Anal. Chem. 1987, 59, 1608. (15) Wang, J.; Taha, Z. Electroanalysis 1990, 2, 383. S0003-2700(97)00431-9 CCC: $14.00
© 1997 American Chemical Society
synthetic oligonucleotides, for evaluating the purity of nucleic acids preparations, or for studying DNA degradation or cleavage. While not offering discrimination at the sequence level, the membrane coverage should be useful for minimizing nonspecific adsorption effects in sequence-specific hybridization detection. Useful information on the base pair composition can also be obtained based on the dependence of the stripping signals upon the guanine and adenine content. The characteristics and advantages of membranecovered nucleic acid-sensing electrodes are discussed below. EXPERIMENTAL SECTION Apparatus. Constant-current chronopotentiometic measurements or PSAs were performed with a TraceLab unit (PSU 20, Radiometer) interfaced with an IBM PS/2 55SX. Potentials were sampled at a frequency of 10 KHz, and the derivative signal (dt/ dE) was recorded against the potential. The peak area, following baseline fitting, was used as the analytical signal. A diode array spectrophotometer (Model 8452A, Hewlett Packard) was employed to measure the concentration of nucleic acids by monitoring the absorbance at 260 nm. The three-electrode system consisted of a carbon paste working electrode (with or without a dialysis membrane), a Ag/ AgCl reference electrode (Model RE-1, BAS Inc.) and a platinum wire auxiliary electrode. The electrodes entered 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 mass 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 on a weighing paper before use. All water and pipet tips were sterilized by autoclaving for 30 min. The electrochemical cells were 4-mL vials (16 × 20 mm, Kimble Glass Inc.); they were cleaned with diluted nitric acid (1: 4), rinsed thoroughly with water, and then dried at room temperature before use. Reagents and Materials. DNA oligomers were obtained (as their ammonium salts) from Life Technologies (Grand Island, NY). The base sequences and molecular weight (MW) of these oligomers are as below: 10-mer 15-mer 21-mer 27-mer 36-mer 38-mer 42-mer
5’-AGT GTT CTA C-3′ (mw 3171) 5’-ACG CCA CAT CTT GCG-3′ (mw 4845) 5’-ACT GCT AGA GAT TTT CCA CAT-3′ (mw 6720) 5’-GTC GTC AGA CCC AAA ACC CCG AGA GGG-3′ (mw 8769) 5’-GTC GTC AGA CCC AAA ACC CCG AGA GGG GAC GGA AAC-3′ (mw 11 702) 5’-GTT AAG ACT ACG ACG GTA TCT GAT CGT CTT CGA TCC CC-3′ (mw 12 381) 5’-ACT GCT AGA GAT TTT CCA CAC TGA CTA AAA GGG TCT GAG GGA-3′ (mw 13 683)
Double-stranded calf thymus DNA (dsDNA, Catalog No. D4522), single-stranded calf thymus DNA (ssDNA, Catalog No. D8899), transfer RNA (tRNA, Catalog No. R8759), diethyl pyrocarbonate (DEPC), and sodium acetate buffer (3 M, pH 5.2 ( 0.1 at 25 °C, certified free of DNase and RNase, Catalog No. S7855) were received from Sigma. All chemicals were used as received. All stock solutions of dsDNA, ssDNA, and DNA oligomers (∼1000 mg/L) were prepared with the TE solution (10 mM Tris-
HCl, 1 mM EDTA, pH 8.0). The tRNA solution was prepared with DEPC-treated water.16 Dilute solutions were prepared with water just before use. The supporting electrolyte was 0.2 M sodium acetate buffer (pH 5). Three Spectra/Por membranes (MWCO: 6-8000; 12-14 000; 50 000) were received from Spectrum Medical Industries, Inc. (Laguna Hills, CA). The first two (MWCO: 6-8000; 12-14 000) were washed thoroughly and then soaked for 30 min at 70 °C in sterilized water, followed by further washing and soaking in sterilized water for 24 h prior to use. The third membrane (MWCO, 50 000) was received wet in 0.1% sodium azide and was thoroughly washed with sterilized water before use. Preparation of Membrane-Covered Carbon Paste Electrode. The carbon paste electrode was prepared as described above. A drop of the sodium acetate buffer was placed on the electrode surface while the latter was held vertically. A piece of wet membrane (∼2 × 2 cm) was positioned on the electrode surface and held in place by two O-rings. Care was taken not to overstretch the membrane and not to introduce an air bubble between the membrane and electrode surface. Procedure. The analysis of nucleic acids consisted of two steps: accumulation and stripping. The electrode was first pretreated by applying a potential of +1.7 V for 1 min in a stirred acetate buffer solution (0.2 M, pH 5) containing analytes. These were subsequently preconcentrated onto the activated electrode surface by adsorptive accumulation at +0.5 V for an appropriate time. The accumulated nucleic acid was measured using chronopotentiometry with a constant stripping current of +6 µA at a starting stripping potential of +0.5 V. All the experiments were conducted at room temperature (22.0 ( 0.5 °C). RESULTS AND DISCUSSION Compared to mercury drop electrodes (commonly used for cathodic detection of nucleic acids), the carbon paste disk electrode can be readily covered with dialysis membranes. Such membrane coverage imparts a new dimension of selectivity, based on molecular size, to electroanalytical measurements of nucleic acids. Figure 1 shows the dependence of the permeability of different membranes for six different oligonucleotides (of different lengths), as well as for denatured (ss) DNA. The ratio of the guanine signal at the membrane-coated electrode to that at the bare one (S/So × 100) is used as a measure of the permeability. Both membranes display a rapid decrease in the permeability at first upon increasing the number of nucleobases from 10 to 21; a slower decrease in the permeability is observed between 21 and 42 bases. The large (15K base) denatured DNA is completely excluded from the surface by both membranes. Such discrimination between small oligonucleotides and large nucleic acid macromolecules provides the basis for the analytical utility of the new membrane-coated carbon paste electrode (see below). Notice also the differentiation among the various oligonucleotides. While both membranes display similar trends, the 50 000 MWCO one displays higher permeability (particularly toward oligonucleotides in the 21-42 range). Such a separation step, performed in situ on the electrode surface, allows discrimination among nucleic acids on the basis of size. For example, short oligonucleotides can thus be readily (16) Maniati, T.; Fritzsch, E.; Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1982.
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Figure 1. Permeability of membranes [MWCO: 50 000 (A); 1214 000 (B)] to synthetic oligonucleotides of different lengths. S/S0, the “permeability”, is the ratio of PSA signals with and without the membrane. The electrode was pretreated at +1.7 V for 1 min, followed by adsorptive accumulation at +0.5 V for 10 min: electrolyte, 0.2 M sodium acetate buffer (pH 5); concentration, 10 mg/L; constant current, +6 µA; initial potential, +0.5 V. The oligonucleotide length is represented by the number of bases; the last point (∼15K) corresponds to ssDNA.
Figure 3. Chronopotentiograms for (a) the 1 mg/L 27-mer oligomer at the bare (A) and membrane (MWCO, 50 000) covered (B) carbon paste electrodes. (b) Same as (a) after addition of 3 mg/L ssDNA; (c) same as (a) after addition of 6 mg/L ssDNA. Conditions, as in Figure 1.
Figure 4. (A) Chronopotentiograms for (a) 10 mg/L 17-mer oligomer and (b) same as (a) after adding 10 mg/L 10-mer oligomer at the membrane (MWCO, 6-8000) covered CPE. (B) Chronopotentiograms for (a) 10 mg/L 38-mer oligomer and (b) same as (a) after adding 10 mg/L 27-mer oligomer at the membrane (MWCO,1214 000) covered CPE. Conditions as in Figure 1. Figure 2. Chronopotentiograms for dsDNA, ssDNA and their mixture with (A, B) 10- and (C, D) 27-mer oligomers at the bare (A, C) and membrane (MWCO: 12-14 000; MWCO 50 000, respectively) covered (B, D) carbon paste electrodes: (a) 10 mg/L dsDNA; (b) same as (a) but after addition of 10 mg/L ssDNA; (c-e) same as (b) but after addition of 5, 10, and 15 mg/L (A, B) 10-mer and (C, D) 27-mer oligomers, respectively. Conditions, as in Figure 1, except for using a 5-min accumulation period.
detected in the presence of a large excess of DNA macromolecules. Such selective PSA measurements are illustrated in Figure 2 for the detection of a 10- (A, B) or 27-mer (C, D) oligomer in the presence of chromosomal DNA. The bare electrode (A, C) exhibits large contributions of dsDNA (a) and ssDNA (b), which do not permit selective detection of the spiked oligomers (c-e). In contrast, the dialysis membrane (B, D) effectively restricts the large chromosomal DNA molecules from reaching the surface, hence allowing highly selective detection of the short oligomers. Similar improvements were obtained for the detection of the 10mer oligomer (over the 5-20 mg/L range) in the presence of 10 mg/L tRNA (not shown; conditions, as in Figure 2A). Figure 3 displays the improved selectivity in the detection of 1 mg/L 27-mer oligonucleotide in the presence of 3-(b) and 6-(c) fold excess of ssDNA. With the bare electrode (A), it is not feasible to selectively detect the short oligomer in the presence of the chromosomal DNA, because of the additivity of the guanine signal. In contrast, the membrane-covered electrode (B) hinders 4058 Analytical Chemistry, Vol. 69, No. 19, October 1, 1997
the transport of ssDNA toward the surface, and its response to the 27-mer oligomer is not affected by the presence of excess concentrations of the DNA macromolecule. Similar improvements were obtained for measurements of 10 mg/L of the 27-mer oligomer in the presence of 100 mg/L ssDNA (not shown; 5-min accumulation). The permeability profiles of Figure 1 indicate also that a judicious choice of the membrane would allow differentiation among individual oligonucleotides. Such possibility is demonstrated in Figure 4. For example, the use of the 6000-8000 MWCO membrane allows selective detection of a 10-mer oligonucleotide in the presence of the larger 17-mer one (A). Similarly, the 27-mer oligomer can be selectively measured in the presence of a 38-mer oligonucleotide using a carbon paste electrode covered with the 12 000-14 000 MWCO membrane. Such fine tuning of the selectivity (i.e., differentiation between oligonucleotides of slightly different molecular size) should further benefit from the design of multiple-electrode arrays, each covered with a membrane of different MWCO. Figure 5 displays calibration plots for the 10-mer oligomer at carbon paste electrodes covered with membranes of different MWCOs [(A) 6000-8000; (B) 12 000-14 000; (C) 50 000]. The three membrane-covered electrodes result in defined and nearly linear calibration plots over the 5-40 mg/L range tested. As expected from the change in the MWCO, the sensitivity increases
Figure 5. Calibration plots for the 10-mer oligomer at the membranecovered carbon paste electrodes. (A) MWCO, 6-8000; (B) MWCO, 12-14 000; (C) MWCO, 50 000. Conditions as in Figure 1, except for using a 5-min preconcentration period. Inset compares calibration plots at the bare (a) and the 50 000 MWCO membrane-covered (b) electrodes.
Figure 6. Effect of accumulation time on the response for 5 mg/L of the 10-mer oligomer at the bare (A) and membrane-coated (B, C) electrodes. MWCO: (B) 50 000; (C) 12 000-14 000. Other conditions as in Figure 1.
upon increasing the membrane porosity. The response of the bare electrode (shown in the inset) is 8-15 times larger and displays a substantial curvature above 20 mg/L. Such surface saturation effects are minimized by the “built-in” dilution action of the membrane. Because of the remarkable sensitivity of the adsorptive PSA operation, the decrease in sensitivity associated with the membrane coverage is not a matter of major concern. It should also be pointed out that the sensitivity of membrane-free adsorptive electrochemical measurements increases upon decreasing the molecular size of the nucleic acid molecule.17 This has been attributed to the more ready penetration of the shorter molecules into the pores and grooves of “rough” carbon surfaces (i.e., higher surface coverage). Hence, higher sensitivity is expected for short synthetic oligonucleotides compared to chromosomal DNA. Hence, despite the membrane coverage, a very low detection limit of 0.5 mg/L was obtained for the 10-mer oligomer following a 20-min accumulation at the 12 000-14 000 MWCO membranecoated electrode (not shown). Lower detection limits are expected for membranes with higher MWCO or with longer accumulation periods. Figure 6 displays the dependence of the peak area for the 10mer oligomer upon accumulation time in the absence (A) and presence (B, C) of membranes. At the bare electrode, the (17) Brabec, V.; Koudelka, J. Bioelectrochem. Bioenerg. 1980, 7, 793.
response rises rapidly with the time at first up to 5 min and then it levels off. The response of the coated electrodes, in contrast, increases linearly with the time over the entire range. The response is also very reproducible. A relative standard deviation of 5.4% was obtained for a series of eight repetitive measurements of 5 mg/L of the 10-mer oligomer at the 12 00014 000 MWCO membrane-coated electrode (5-min accumulation; not shown). As expected for adsorptive stripping measurements, the response of the membrane-coated electrode increases rapidly upon extending the accumulation period. For example, measurements of 5 mg/L of the 10-mer oligomer at the 12 000-14 000membrane MWCO coated electrode resulted in a nearly linear dependence of the signal upon the accumulation time over the 1-30 min range (slope, 2.2 ms/min). The absence of curvature is attributed to the dilution action of the membrane. In conclusion, this study has illustrated that the use of membrane-coated carbon electrodes can dramatically enhance the power of electroanalytical measurements of nucleic acids. The additional separation step, performed at the surface, permits effective exclusion of ssDNA, dsDNA, and tRNA. As a result, substantial improvements in the selectivity are observed for the detection of biotechnologically important synthetic oligonucleotides. Discrimination among oligonucleotides of different lengths is also possible. The new strategy thus offers considerable opportunities for molecular biology research, including great promise for studying of DNA degradation or cleavage (including DNA damage or enzymatic hydrolysis), for assessing the purity of nucleic acid preparations, or for elucidating the interaction of DNA with molecular weight substances. The membrane coverage should also be useful for extending the linear range, and for protecting the surface against passivation (hence reducing the attention necessary for sample cleanup). The use of membranecoated electrodes should also be useful for minimizing nonspecific adsorption effects in sequence-specific hybridization detection. While the concept has been illustrated in the context of carbon paste electrodes and microdialysis membranes, it could be readily extended to other solid electrode materials and membrane coverages. Additional advantages can be achieved by designing multiple-electrode arrays, with each electrode covered with a membrane of different MWCO, thus providing a patterned response based on the nucleic acid size. This and similar developments should bring electroanalysis closer to the needs of contemporary molecular biology research.
ACKNOWLEDGMENT J.W. acknowledges the financial support from the Department of Energy (DOE) and the DOE-WERC program. J.R.F., D.H.G, and M.O. acknowledge fellowships from FAPESP (Brazil), Mount Allison University (Canada), and Scientific & Technical Research Council (Turkey), respectively.
Received for review April 23, 1997. 1997.X
Accepted July 3,
AC9704319 X
Abstract published in Advance ACS Abstracts, August 15, 1997.
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