Anal. Chem. 2003, 75, 4229-4235
Voltammetric Sensor for Chemical Toxicity Using [Ru(bpy)2poly(4-vinylpyridine)10Cl)]+ as Catalyst in Ultrathin Films. DNA Damage from Methylating Agents and an Enzyme-Generated Epoxide Bingquan Wang† and James F. Rusling*,†,‡
Department of Chemistry, University of Connecticut, U-60, 55 North Eagleville Road, Storrs, Connecticut 06269-3060, and Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06032
Films containing presynthesized [Ru(bpy)2poly(4-vinylpyridine)10Cl)]Cl and ds-DNA grown layer by layer by alternate electrostatic assembly were used to detect DNA damage from an epoxide metabolite and methylating agents on a reaction time scale of minutes. The redox polymer [Ru(bpy)2poly(4-vinylpyridine)10Cl)]Cl was used as an inner layer in films 14-25 nm thick to catalyze the voltammetric oxidation of guanine bases of ds-DNA in the outer layers. This film architecture provides a self-contained, reagentless sensor for toxicity screening based on detection of DNA damage. Films were incubated with reactants and washed, and then DNA damage was analyzed by square wave voltammetry (SWV). Bioactivation of styrene to its metabolite styrene oxide was accomplished by incorporating the protein myoglobin into the films to catalyze the conversion. DNA damage caused the catalytic SWV peaks at ∼0.75 V vs SCE to increase nearly linearly over the first 10-20 min of reaction, depending on the damage agent employed. Such prototype toxicity biosensors hold promise for in vitro screening of new agricultural chemicals and drugs for potential genotoxicity. DNA damage can be caused by lipophilic pollutants and drugs or their metabolites bioactivated by liver cytochrome P450 (cyt P450) enzymes, which constitutes a major toxicity pathway.1-4 Detection of DNA damage can serve as the basis for toxicity screening of new chemicals. While in vivo animal testing is considered a reliable method for prediction of chemical carcinogenicity in humans,3 the enormous number of new compounds being synthesized annually exceeds the capacity and cost of applying in vivo tests to all target chemicals. Thus, there is a need for rapid, inexpensive screening at early stages of a chemical’s commercial development. * Corresponding author. E-mail:
[email protected]. † Department of Chemistry, University of Connecticut. ‡ Department of Pharmacology, University of Connecticut Health Center. (1) Schenkman, J. B., Greim, H., Eds. Cytochrome P450; Springer-Verlag: Berlin, 1993. (2) Ortiz de Montellano, P. R., Ed. Cytochrome P450; Plenum: New York, 1995. (3) Pauwels, W.; Vodiceka, P.; Severi, M.; Plna, K.; Veulemans, H.; Hemminki, K. Carcinogenisis 1996, 17, 2673-2680. (4) McConnell, E. E.; Swenberg, J. A. CRC Crit. Rev. Toxicol. 1994, 24, S49S55. 10.1021/ac034097u CCC: $25.00 Published on Web 07/01/2003
© 2003 American Chemical Society
Electrochemistry provides simple, sensitive, inexpensive approaches to detecting DNA.5-8 Thorp et al. showed that Ru(bpy)32+ is an efficient catalyst that specifically oxidizes guanines in DNA,9,10 providing enhanced voltammetric peaks. We showed that this soluble metal complex can be used to detect DNA damage. When double-stranded (ds) DNA in ultrathin films was damaged by formation of styrene oxide-nucleobase adducts, guanines became more available to react rapidly with the catalyst, providing an increased current at ∼1 V versus SCE.11 In our first attempt to develop reagentless DNA damage sensors, we adsorbed poly(vinylpyridine) (PVP) onto pyrolytic graphite electrodes and then attached Ru(bpy)22+ based on coordinating ligation.12 The [Ru(bpy)2-PVP]2+ polymer formed in situ gave reversible voltammetry at 0.75 V versus SCE and was used to catalyze the oxidation of guanines in DNA. We successfully used DNA/polycation films with an internal [Ru(bpy)2-PVP]2+ layer to detect DNA damage from styrene oxide.13 In this paper, we incorporated a presynthesized catalytic metallopolycation, [Ru(bpy)2poly(4-vinylpyridine)10Cl)]Cl, with reversible electroactivity into inner layers of DNA films on pyrolytic graphite using layer-by-layer alternate electrostatic assembly.14 DNA damage in the films caused by styrene oxide and by methylating agents dimethyl sulfate and methyl methanesulfonate was detected using square wave voltammetry (SWV). Finally, styrene was bioactivated to metabolite styrene oxide by (5) Mikkelsen, S. R. Electroanalysis 1996, 8, 15-19. (6) Thorp, H. H. Trends Biotechnol. 1998, 16, 117-121. (7) Wang, J. Chem. Eur. J. 1999, 5, 1681-1685. (8) Palacek E.; Fojta, M. Anal. Chem. 2001, 73, 74A-83A. (9) (a) Johnston, D. H.; Glasgow, K. C.; Thorp, H. H. J. Am. Chem. Soc. 1995, 117, 8933-8938. (b) Napier, M. E.; Thorp, H. H. Langmuir 1997, 13, 63426344. (c) Yang, I. V.; Thorp, H. H. Inorg. Chem. 2000, 39, 4969-4976. (10) . (a) Armistead, P. M.; Thorp, H. H. Anal. Chem. 2000, 72, 3764-3770. (b) Sistare, M. F.; Codden, S. J.; Heimlich, G.; Thorp, H. H. J. Am. Chem. Soc. 2000, 122, 4742-4749. (c) Szalai, V. A.; Thorp, H. H. J. Phys. Chem. B 2000, 104, 6851-6859. (11) Zhou, L.; Rusling, J. F. Anal. Chem. 2001, 73, 4780-4786. (12) Mugweru, A.; Rusling, J. F. Electrochem. Commun. 2001, 3, 406-409. (13) Mugweru, A.; Rusling, J. F. Anal. Chem. 2002, 74, 4044-4049. (14) (a) Lvov, Y. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mhwald, H., Eds.; Marcel Dekker: New York, 2000; pp 125-167. (b) Rusling, J. F. In Protein Architecture: Interfacing Molecular Assemblies and Immobilization Biotechnology; Lvov, Y., Mhwald, H., Eds.; Marcel Dekker: New York, 2000; pp 337-354. (c) Lvov, Y. In Handbook Of Surfaces And Interfaces Of Materials. Vol. 3. Nanostructured Materials, Micelles and Colloids; Nalwa, R. W., Ed.; Academic Press: San Diego, CA, 2001; pp 170-189.
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using myoglobin in the films, and the resulting DNA damage was detected by SWV. EXPERIMENTAL SECTION Chemicals. Calf thymus (CT) ds-DNA (type XV), poly(guanylic acid)′, adenine, and guanine were from Sigma. Horse heart myoglobin (Mb, MW 17 400) from Sigma filtered through an Amicon YM30 filter (30 000 MW cutoff).15 Ruthenium trichloride (99.9%) was from Fluka.. Styrene oxide, styrene, methyl methanesulfonate (MMS), dimethyl sulfate (DMS), poly(diallyldimethylammonium chloride) (PDDA), poly(sodium 4-styrenesulfonate) (PSS), and PVP were from Aldrich. Other chemicals were reagent grade. Water was purified with a Hydro Nanopure system to specific resistance of >18 MΩ cm. [Ru(bpy)2poly(4-vinylpyridine)10Cl)]Cl. The metallopolymer was synthesized according to Clear et al.,16 with minor modifications. cis-Dichlorobis(2,2′-bipyridine)ruthenium (Ru(bpy)2Cl2) was prepared by a reported procedure.17 Typically, 0.4 g of Ru(bpy)2Cl2 and 0.8 g of PVP in 200 mL of ethanol were refluxed in the dark for 100 h. Ethanol was removed using a rotary evaporator, and the residue was dissolved in 80 mL of dichloromethane. After the solvent was evaporated, the metallopolymer was precipitated with hexane. Finally, the dark solid was dried in a vacuum in the dark for 10 h. Elemental analysis was C, 63.32; H, 5.61; N, 11.37. Anal. Calcd for C90H90N14O2Cl2Ru C, 68.70; H, 5.77; N, 12.46. UV-visible spectra in ethanol gave peaks at 354 and 498 nm, consistent with previous reports.16 Electrochemistry. A CHI 660A electrochemical analyzer was used for cyclic voltammetry (CV) and SWV. A three-electrode thermostated cell contained a saturated calomel reference electrode (SCE), a Pt wire counter electrode, and a pyrolytic graphite (PG) disk (Advanced Ceramics, A ) 0.16 cm2) working electrode. SWV conditions were 4-mV step, 25-mV pulse, and 5-Hz frequency. Solutions were purged with pure nitrogen for 15 min prior to each series of experiments. Ohmic drop was compensated >95% by the CHI system. Film Assembly. Basal plane PG electrodes were polished with 400-grit SiC paper and then ultrasonicated for 30 s each in ethanol and then in water. Electrodes were dried in nitrogen and dipped into 6 mg mL-1 PSS containing 0.5 M NaCl for 15 min. After washing with water, these PG electrodes were then dipped into 0.05% (m/V) [Ru(bpy)2(PVP)10Cl)]Cl (denoted hereafter as ClRuPVP) in 5% ethanol/water for 15 min. Adsorption for 15 min provides steady-state adsorption of PSS on oppositely charged surfaces.18,19 After fabricating PSS/ClRu-PVP films, electrodes were dipped into 2 mg mL-1 CT ds-DNA in pH 7.0 Tris buffer containing 0.5 M NaCl for 15 min, rinsed with water, and then dipped into 2 mg mL-1 PDDA. Alternate adsorption cycles were repeated until the desired number of layers were made. Final film structure is denoted as PSS/ClRu-PVP/CT-ds-DNA/PDDA/CT-ds-DNA. (15) Nassar, A.-E. F.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1995, 67, 23862392. (16) Clear, J. M.; Kelly, J. M.; O’Connell, C. M.; Vos, J. G. J. Chem. Res. 1981, 3039-3068. (17) Sprintschnik, G.; Sprintschnik, H. W.; Kirsch, P. P.; Whitten D. G. J. Am. Chem. Soc. 1977, 99, 4947-4953. (18) Lvov, Y. M.; Lu, Z.; Schenkman, J. B.; Zu, X.; Rusling, J. F. J. Am. Chem. Soc. 1998, 120, 4073-4080. (19) Lvov, Y.; Ariga, K.;. Ichinose, I.; Kunitake, T. J. Am. Chem. Soc. 1995, 117, 6117-6123.
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Table 1. Films Employed in This Work film composition
short name used in text
PSS/ClRu-PVP PSS/ClRu-PVP/DNA/PDDA/CT-ds-DNA PSS/ClRu-PVP/DNA/(Mb/CT-ds-DNA)2
ClRu-PVP film Ru/PDDA/DNA film Ru/Mb/DNA film
For films containing Mb, PG electrodes were abraded with 400grit SiC paper, rinsed with water, further roughened using medium Crystal Bay emery paper (PH4 3M 001K), and then ultrasonicated in ethanol and water successively for 30 s. Roughening of the PG surface provides films with more active Mb.20 Additional steps were the same as above except PDDA was replaced with 3 mg mL-1 Mb in pH 5.5 buffer. Final film structure is denoted as PSS/ ClRu-PVP/CT-ds-DNA/(Mb/CT-ds-DNA)2. Electrochemical surface area was estimated at 0.21 cm2 using peak currents of soluble ferricyanide and the Randles-Se´vcı´k equation. Where possible, films used in this work are referred to in the subsequent text as denoted in Table 1. Reactions of DNA Films. Incubations of Ru/PDDA/DNA films in saturated styrene oxide solutions (∼10 mM) were done at 37 °C. A 120-µL aliquot of styrene oxide was added to 10 mL of pH 5.5 acetate buffer + 50 mM NaCl to make a saturated emulsion.21 Toluene was used for controls. DNA films were incubated in stirred emulsions, and then electrodes were rinsed with water and transferred to another cell containing pH 5.5 buffer for SWV analysis. Incubations of films with 2 mM MMS or DMS were done in pH 6.5 phosphate buffer at 37 °C. After washing, SWV analysis was done in pH 6.5 buffer + 20 mM NaCl. Reaction of MMS with DNA under our conditions was confirmed by capillary electrophoresis (CE). A 2-mL sample of 1 mg mL-1 CT ds-DNA in pH 6.5 buffer was reacted with 2 mM MMS. Samples were incubated at 37 °C after which 400-µL aliquots were placed in centrifuge tubes and evaporated to dryness under N2. The residue was dissolved in 300 µL of formic acid for hydrolysis and then reacted in a vacuum at 150 °C for 40 min. Also, 2.5 mL of 1 mg mL-1 guanine or adenine at pH 6.5 was reacted with 2 mM MMS at 37 °C. CE was done on a Beckman P/ACE 5000 with UV detector at 254 nm using a 20 mM bicarbonate + 100 mM SDS buffer, pH 9.6, employing a 50 cm × 75 µm silica capillary at 25 °C and 15 kV. Safety Note: Styrene oxide, MMS, and DMS are suspected human carcinogens and relatively volatile. Gloves were worn, and all weighing and manipulations were done under a closed hood. All reactions were carried out in closed vessels. Mb-Catalyzed Conversion of Styrene to Styrene Oxide. Incubations of Mb-containing DNA films in saturated styrene + H2O2 in pH 5.5 buffer were done at 37 °C. A 200-µL aliquot of styrene was added to 10 mL of buffer. Ru/Mb/DNA films were incubated in the stirred emulsion, rinsed with water, and then transferred to an electrochemical cell containing pH 5.5 buffer for analysis. Quartz Crystal Microbalance. A quartz crystal microbalance (QCM, USI System) was used with Au-coated quartz QCM resonators (9 MHz, AT-cut, International Crystal Mfg. Co.) Clean Au surfaces were first coated by immersion in 0.3 mM 3(20) Ma, H.; Hu, N.; Rusling, J. F. Langmuir 2000, 16, 4969-4975. (21) pH 5.5 gave the largest reaction rate of styrene oxide with DNA. See ref 13.
Table 2. Results from QCM during Film Assembly film
bilayer thickness, nm
total thickness, nm
DNA, µg cm-2
polyion or protein, µg cm-2
Ru/PDDA/DNA Ru/Mb/DNA
PDDA/DNA, 3.1 ( 0.2 Mb/DNA, 7.3 ( 0.5
14.6 ( 0.8 25.5 ( 0.9
1.4 ( 0.3 1.5 ( 0.3
ClRu-PVP, 2.3 ( 0.4 Mb, 3.6 ( 0.7
mercaptopropionic acid (MPA) and 0.7 mM 3-mercapto-1-propanol in ethanol to make a partially negative surface.11,22 Then, PDDA was adsorbed, followed by assembly of films as above for the PG electrodes. Resonators were immersed in a given adsorbate solution for 15 min, washed with water, and dried in a stream of nitrogen. The frequency change for the dry film was measured at ambient temperature. The Sauerbrey equation gives the relation between adsorbed mass and frequency shift ∆F (Hz) in the absence of viscoelasticity changes. For 9-MHz quartz resonators, the film mass per unit area M/A (g cm-2) is19
M/A ) -∆F/(1.83 × 108)
(1)
for our resonators of A ) 0.16 ( 0.01 cm2 on one side. The nominal thickness (d) of dry films was estimated from19
d (nm) ) -(0.016 ( 0.002) ∆F(Hz)
(2)
RESULTS QCM Characterization. QCM was used to monitor growth of films on gold QCM resonators coated with mixed monolayers of MPA and mercaptopropanol to mimic a partly negative carbon surface.11 PDDA was first coated on the MPA-Au surface to give a positive charge and then PSS/ClRu-PVP/CT-ds-DNA/PDDA/ CT-ds-DNA films (Ru/PDDA/DNA films; see Table 1) were assembled on this layer. -∆F varied linearly with layer number (Figure 1), suggesting regular film growth with reproducible layers of DNA, polyions, and proteins. The -∆F for the initial MPA layer on gold was smaller that that of the polyion layers, consistent with the short-chain molecules in the resulting thiol monolayer.11,31 For PSS/ClRu-PVP/ds-DNA/PDDA/ds-DNA films, the PSS layer adsorbed from 0.5 M NaCl caused ∆F to decrease by 190 Hz, giving nominal layer thickness from eq 2 of 3 nm. The estimated ClRu-PVP layer thickness was 6.8 ( 0.6 nm. Table 2 gives (22) Njue, C. K.; Rusling, J. F. J. Am. Chem. Soc. 2000, 122, 6459-6463. (23) Cassidy, J. F.; Vos, J. G. J. Electroanal. Chem. 1987, 218, 341-345. (24) Doherty, A.; Stanley, M. A.; Leech, D.; Vos, J. G. Anal. Chim. Acta 1996, 319, 111-120. (25) Rusling, J. F.; Zhang, Z. In Handbook Of Surfaces And Interfaces Of Materials. Vol. 5. Biomolecules, Biointerfaces, and Applications; Nalwa, R. W., Ed.; Academic Press: New York, 2001; pp 33-71. (26) Yang, J.; Zhang Z.; Rusling, J. F. Electroanalysis 2002, 14, 1494-1500. (27) Lewis, R. J. In Carcinogenically Active Chemicals: A Reference Guide; Lewis, R. J., Ed.; Van Nostrand Reinhold: New York, 1990. (28) Op Het Veld, C. W.; Jansen J.; Zdzienicka, M. Z.; Vrieling, H.; Van Zeeland, A. A. Mutat. Res. 1998, 398, 83-92. (29) Adams, S. P.; Laws, G. M.; Storer, R. D.; Deluca, J. G.; Nichols, W. W. Mutat. Res. 1996, 368, 235-248. (30) (a) Zu, X.; Lu, Z.; Zhang, Z.; Schenkman, J. B.; Rusling, J. F. Langmuir 1999, 15, 7372-7377. (b) Munge, B.; Estavillo, C.; Schenkman, J. B.; Rusling, J. F. ChemBioChem 2003, 4, 82-89. (31) Zhou, L.; Yang, J.; Estavillo, C.; Stuart, J. D.; Schenkman, J. B.; Rusling, J. F. J. Am. Chem. Soc. 2003, 125, 1431-1436.
Figure 1. QCM frequency shifts for cycles of alternate Mb/CT-dsDNA and PDDA/CT-ds-DNA adsorption on gold resonators coated first with mixed monolayers of mercaptoproionic acid/mercaptopropanol (first point) as the first layer and PDDA/PSS as the second and third layers. Finals films are PDDA/PSS/ClRu-PVP/CT-ds-DNA/ (Mb/CT-ds-DNA)2 (b) and PDDA/PSS/ClRu-PVP/CT-ds-DNA/PDDA/ CT-ds-DNA (O). (Average values for five replicate films).
thicknesses and component weights of the films used. For PSS/ ClRu-PVP/DNA/Mb/DNA/Mb/DNA films, each Mb adsorption step brought about a -320-Hz change in frequency, corresponding to a layer thickness of ∼5 nm. (Table 2). Voltammetry of PSS/[Ru(bpy)2(PVP)10Cl]Cl. [Ru(bpy)2(PVP)10Cl]Cl was initially adsorbed directly onto bare PG electrodes from a 5% ethanol/aqueous solution, but these films gave relatively small voltammetric peaks. To increase the amount of adsorbed metallopolymer, we first deposited a layer of PSS on the PG,20 and then the metallopolymer was assembled onto this surface. The resulting film electrodes exhibited good stability. After extensive rinsing with water and storage in the dark at 4 °C for 2 weeks, there was ∼10% decrease in the peak current. Figure 2 shows cyclic voltammograms of a PSS/ClRu-PVP film at pH 5.5. Pairs of oxidation-reduction peaks corresponding to the RuII/RuIII redox couple were observed with midpoint potential ∼0.75 V versus SCE, similar to previous reports.23,24 The separation between anodic and cathodic peaks was 50 mV at 25 mV s-1, and peak current increased linearly with scan rate, with r ) 0.999. For all scan rates, the ratio of anodic to cathodic peak currents was unity, suggesting that the electron transfer is chemically reversible. These results are consistent with nonideal thin-film voltammetry.25 Integration of peak areas at low scan rates gave an average surface concentration of 0.35 nmol cm-2, much larger than the 0.075 nmol cm-2 for the in situ formed ClRuPVP in our previous report.12 Electrodes coated with PSS/ClRu-PVP showed catalytic peak increases by CV in solutions of poly(guanylic acid), ds-DNA, and ss-DNA (see Supporting Information). Peak current was larger with ss-DNA than with ds-DNA because in the ss-DNA case the average distance of closest approach of guanine to Ru sites is probably smaller than in ds-DNA. A small shift in potential to less Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
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Figure 2. Cyclic voltammograms of PSS/ClRu-PVP-coated PG electrode at different scan rates in 10 mM acetate buffer (pH 5.5) + 50 mM NaCl. Scan rates from top to bottom are 150, 125, 100, 75, 50, and 25 mV s-1.
Figure 4. Difference SWV of Ru/PDDA/DNA films after incubations at 37 °C for different times and then washing: (a) incubation in saturated styrene oxide for 0, 5, 10, 20, and 30 min; (b) incubation in toluene for 0, 5, 10, and 30 min.
Figure 3. Difference SWV of various films in pH 5.5 buffer: (a) PSS/ClRu-PVP; (b) PSS/ClRu-PVP/CT-ds-DNA/PDDA/DNA; (c) PSS/(ClRu-PVP/CT-ds-DNA)2; (d) PSS/(ClRu-PVP/PSS)2.
positive values is most likely due to electrostatic stabilization of the RuIII sites by the negatively charged DNA, similar to effects observed for Co(bpy)33+ and DNA.26 These results confirmed the catalytic properties of the films toward oxidation of guanines in DNA and were similar to those found for ClRu-PVP layers formed in situ.12 SWV Detection of Damaged DNA. Figure 3 compares SWV of metallopolymer electrodes in the absence and presence of DNA layers. When ds-DNA was immobilized on the PSS/ClRu-PVP layer in PSS/ClRu-PVP/DNA/PDDA/DNA films, peak current increased consistent with catalytic oxidation of DNA.6,12,13 Figure 3d shows films with two layers of ClRu-PVP and PSS instead of DNA. When the PSS was replaced by DNA, the peak current again increased (Figure 3c) because of catalytic DNA oxidation. However, there is no clear advantage of this architecture, so further studies were done with films having a single layer of ClRu-PVP. Because guanine is oxidized irreversibly, catalytic peaks of the films decreased gradually under continuous scanning due to the decreasing numbers of guanines available. Thus, a series of Ru/ PDDA/DNA electrodes were used to study the time course of DNA damage, one for each incubation period. After incubation with saturated styrene oxide at 37 °C for different times, electrodes were rinsed and analyzed by SWV. Figure 4a shows SWV of Ru/ PDDA/DNA films after incubation with styrene oxide for different times. Peak increases were found with increased incubation time. Control experiments were done with toluene, for which no chemical reactions with DNA have been reported. The peak currents were nearly constant (Figure 4b), suggesting that no detectable DNA damage was caused. 4232 Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
Figure 5. Influence of incubation time on the ratio of final SWV peak current of Ru/PDDA/DNA films after incubation to peak current of ClRu-PVP film (no DNA). Incubations in saturated styrene oxide (b) and toluene (O) in pH 5.5 buffer at 37 °C. Error bars show standard deviations for three trials.
Denoting the initial SWV peak current of ClRu-PVP electrodes as Ip,i, and the peak current of Ru/PDDA/DNA films after incubation as Ip,f, we plotted the ratio Ip,f/Ip,i versus incubation time (Figure 5). This ratio increased during the first 20 min of incubation with styrene oxide and then leveled off after ∼30 min. DNA films incubated with unreactive toluene showed small decreases in catalytic peak currents. We also investigated methylating agents DMS, a confirmed human carcinogen, and MMS, a suspected carcinogen.27 Reaction between MMS and DNA films was studied in phosphate buffers, and pH 6.5 was found to be the optimum pH. Increases in SWV peak current were observed for up to 10-min incubation of Ru/ PDDA/DNA films with MMS or DMS (Figure 6). Peak currents decreased at t > 10 min. Control DNA films incubated in the absence of methylating agents gave nearly constant peak current ratios. To confirm DNA methylation under our incubation conditions, reaction products of MMS with ds-DNA in solution were analyzed
Figure 6. Influence of incubation time on the ratio of final SWV peak current of Ru/PDDA/DNA films after incubation to peak current of ClRu-PVP film (no DNA). Incubations at pH 6.5 in 2 mM DMS (b), in 2 mM MMS (O), and in pure buffer (4) at 37 °C. Error bars show standard deviations for three trials.
Figure 8. Difference square wave voltammograms of Ru/Mb/DNA films on rough PG electrodes in the (b) absence and (c) presence of saturated oxygen and (a) for a ClRu-PVP electrode with no DNA or Mb.
Figure 7. Partial capillary electropherograms for incubated guanine, adenine, and DNA after acid hydrolysis, showing region of retention times of peaks identified with damaged bases. Incubations were done with 2 mM MMS at pH 6.5 and 37 °C. Peaks for the four intact DNA bases (not shown) have retention times of
