Examination of Electron Transfer Through DNA Using

Oct 7, 2008 - Teresa C. Cristarella , Adam J. Chinderle , Jingshu Hui , and Joaquín Rodríguez-López. Langmuir 2015 31 (13), 3999-4007. Abstract | F...
0 downloads 0 Views 679KB Size
Download PDF
J. Phys. Chem. C 2008, 112, 16999–17004

16999

Examination of Electron Transfer Through DNA Using Electrogenerated Chemiluminescence Tommie Lyndon Pittman and Wujian Miao* Department of Chemistry and Biochemistry, The UniVersity of Southern Mississippi, Hattiesburg, Mississippi 39406 ReceiVed: July 1, 2008; ReVised Manuscript ReceiVed: September 5, 2008

Three aminoalkanethiols that have large electron-transfer rate constants, SH-(CH2)n-NH2 (n ) 6, 8, and 11), were individually self-assembled on Au electrodes, followed by covalent attachment of tris(2,2′bipyridyl)ruthenium(II) (Ru(bpy)32+) moieties onto the end of the thiols. Two separate electrogenerated chemiluminescence (ECL) waves were observed upon anodic potential scanning from 0 to 1.40 V vs Ag/ AgCl (3 M KCl) over the electrode placed in 0.10 M tri-n-propylamine (TPrA)/0.10 M phosphate buffer (pH 7.4) solution. The first ECL wave, located at ∼0.88 V vs Ag/AgCl, was associated with the direct oxidation of TPrA at the electrode, and the second ECL wave, located at ∼1.12, 1.22, and 1.35 V vs Ag/AgCl for n ) 6, 8, and 11, respectively, was directly related to the oxidation of the tethered Ru(bpy)32+ species. The electron transfer behavior through DNA was examined at Au electrodes, which were covalently immobilized with 15-mer and 20-mer single-stranded (ss) DNA, respectively, and then hybridized with the relevant complementary ssDNA tagged with Ru(bpy)32+ ECL labels. Under the same experimental conditions described above for Au/aminoalkanethiol-Ru(bpy)32+ studies, both double-stranded (ds) DNA displayed similar ECL responses, with the first ECL peak at ∼0.88 V and the second one at ∼1.22 V vs Ag/AgCl. No peak potential shift for the second ECL wave and no impact of the dsDNA on the entire electron transfer processes were observed, suggesting that complementary dsDNA helical structures can transfer electrons at a very large rate constant and that dsDNA studied were very conductive. In contrast, an electrode attached with 15-mer ssDNARu(bpy)32+ did not show the second ECL wave, implying that ssDNA was not electronically conductive. Introduction The charge transport (electrons and holes) through DNA, or the electronic property of DNA, has been the subject of numerous recent studies.1-5 Many of the efforts have profound biological implications, since understanding charge transport through DNA is essential to characterize and control important life processes, such as aging and radiation damage and repair.6-8 DNA could also have potential applications in nanoscale electronic devices, both as a template for assembling nanocircuits and as an element of such circuits.9-11 Despite its importance, a simple question of whether DNA is an electronic conductor still remains unsettled. DNA has been reported to be metallic,12 semiconducting,13 insulating,14,15 and even a proximity effect induced superconductor.16 Studies based on spectroscopic, biochemical, and biophysical methods have indicated that the DNA stack can provide a medium for charge transport.17-23 Electron transfer through a DNA monolayer immobilized on the surface of Au was also investigated by electrochemistry when an electroactive species was either attached to the end of DNA or dissolved in the solution.24,25 As a result, the fabrication of biosensors to detect DNA sequences and other biological species has become an interest to many scientific workers.26-35 However, questions have been raised with regard to the role played by electrical contacts; length effects; and the manner in which electrostatic damage, residual salt concentrations, traces amount of water effect, and other contaminations may have affected these results,36-38 in additional to the quality of DNA monolayer formed on the * Corresponding author. Phone: +1 601-266-4716. Fax: +1 601-2666075. E-mail: [email protected].

electrode surface. Clearly, more studies, particularly those with new experimental designs, need to be done to obtain a reliable picture of the electronic behavior of DNA. Electrogenerated chemiluminescence (ECL) has been investigated since 1964 as a method of producing light at an electrode.39 In a sense, ECL represents a marriage between electrochemical and spectroscopic methods and possesses significantly high sensitivity and selectivity for its target analyte.40 The most widely used ECL system consists of tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) or its derivatives as the ECL label and tri-n-propylamine (TPrA) as the coreactant because the combination of these two gives the highest ECL efficiency in all coreactant ECL systems known so far. As revealed in recent studies41-45 and demonstrated in Figure 1, two separated ECL waves can be observed at a Au or glassy carbon electrode when a solution containing micro- or nanomolar levels of Ru(bpy)32+ and 0.1 M TPrA is oxidized at the electrode from 0 to ∼1.4 V vs Ag/AgCl. These two ECL waves, one located at ∼0.89 V (1st ECL) and the other at ∼1.13 V vs Ag/AgCl (2nd ECL), are associated with the direct oxidation of TPrA, and Ru(bpy)32+ at the electrode, respectively (Schemes 1 and 2). In other words, without direct oxidation of Ru(bpy)32+ at the electrode, the second ECL wave would not exist. In the present paper, the electron transfer behavior of DNA is studied with ECL technology, in which a gold electrode is attached with DNA sequences tagged with Ru(bpy)32+-type labels and the ECL responses are recorded upon anodic potential scanning using TPrA as a coreactant. It is hoped that findings reported herein could guide other researchers to design and construct some high-performance ECL-based DNA biosensors.

10.1021/jp805791p CCC: $40.75  2008 American Chemical Society Published on Web 10/07/2008

17000 J. Phys. Chem. C, Vol. 112, No. 43, 2008

Figure 1. ECL and CV responses obtained from a 1.0 µM Ru(bpy)32+ 0.10 M TPrA/0.10 M phosphate buffer solution (pH 7.4) at a 2-mmdiameter Au electrode with a scan rate of 50 mV/s. The small anodic/ cathodic redox pair on the CV in the potential range of ∼0.2-0.6 V results from the Au surface oxide formation and its reduction.41

SCHEME 1: ECL Mechanism of the First ECL Wave for the Ru(bpy)32+/TPrA System

SCHEME 2: ECL Mechanism of the Second ECL Wave for the Ru(bpy)32+/TPrA System

Experimental Section Chemicals and Materials. 6-Amino-1-hexanethiol [NH2(CH2)6-SH], 8-amino-1-octanethiol [NH2-(CH2)8-SH], and 11-amino-1-undecanethiol [NH2-(CH2)11-SH] were from Dojindo (Gaithersburg, MD). 1-Methylimidazole (99%), ethanol (200 proof), tri-n-propylamine (TPrA, 99%), hydrogen peroxide (H2O2, 35%), and 3-mercaptopropanoic acid (HS(CH2)2COOH, 3-MPA, 99+%) were from Aldrich (Milwaukee,WI). Sodium phosphate dibasic anhydrous (Na2HPO4, 99%), N-hydroxysuccinimide (NHS), and potassium phosphate monobasic anhydrous (KH2PO4, 99%) were from Sigma (St. Louis, MO). 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and BupH phosphate buffered saline pack (PBS, pH 7.2) were from Pierce (Rockford, IL). Sulfuric acid (H2SO4, 98%), hydrochloric acid (HCl, 37.5%), and phosphoric acid (H3PO4, 85.5%) were from Fisher Scientific (Fairlawn, NJ). Bis(2,2′bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester bis(hexafluorophosphate) [Ru(bpy)2(Me-bpyCOOH-NHS)(PF6)2]wasfromFluka(Milwaukee,WI).Ru(bpy)32+phosphoramidite was from Igen (now Roche Diagnostics, Gaithersburg, MD). All were used without further purification. Synthetic HPLC purified 15- and 20-mer single-stranded DNA (ssDNA) oligonucleotides were obtained from Qiagen Operon (Alameda, CA) and had the following sequences: (a) probe 15mer, 5′-[AmC3]-CTA CGA GCC TCG TCC-3′ (p-15-ssDNA,

Pittman and Miao MW ) 4626.02 g/mol); (b) complementary 15-mer, 3′-GAT GCT CGG AGC AGG-[AmC3] (c-15-ssDNA. MW ) 4795.14 g/mol); (c) probe 20-mer, 5′-[AmC3]-GGA TCC TAC GAG CCT CGT CC-3′ (p-20-ssDNA, MW ) 6191.04 g/mol); and (d) complementary 20-mer, 3′-CCT AGG ATG CTC GGA GCA GG-[AmC3]-5′ (c-20-ssDNA, MW) 6320.13 g/mol). All sequences were modified with a primary amino group at the end of a three-carbon spacer linked to the 5′ position (“5′-[AmC3]-”) for coupling reactions. Gold-coated silicon wafers (Au/Si) with a titanium adhesion layer were purchased from Platypus Technologies (Madison, WI). The thickness of the Au was ∼1000 Å. The compressed nitrogen gas was obtained from Nordan Smith (Hattiesburg, MS). Unless otherwise stated, all solutions were prepared with deionized-distilled water produced from a Barnstead MP-6A Mega-Pure System (Barnstead International, Dubuque, IA). Preparation of Self-Assembled Monolayers (SAMs) of Alkanethiols on Au. Two types of gold electrodes (2-mmdiameter Au disk from CH Instruments, Austin, TX and 1 × 2 cm2 Au/Si wafers) and four alkanethiols [3-MPA and NH2-(CH2)n-SH, n ) 6, 8, and 11] were used for the preparation of SAMs on Au. Au disk electrodes, which were prepolished with 0.3-µm alumina slurry and washed with water, along with Au/Si wafers were cleaned with freshly prepared piranha solution (98% H2SO4/30% H2O2, 70:30, v/v) for ∼10 min, followed by washing with copious amounts of distilled water and then ethanol. The piranha solution was handled carefully because of its ability to react violently with organic materials. The Au electrodes were subsequently dried in a stream of N2 before being transferred into each of the 1 mM thiol ethanol solutions for 24 h. The newly formed Au/SAMs were rinsed with ethanol and water, dried with a stream of N2, and then stored properly to prevent contamination from impurities in laboratory atmosphere. Attachment of Ru(bpy)32+ type ECL Labels to Au/ S-(CH2)n-NH2 (n ) 6, 8, and 11) SAMs. This was achieved by immersing Au/S-(CH2)n-NH2 (n ) 6, 8, and 11) SAMs electrodes into freshly prepared 0.25 mM Ru(bpy)2(Me-bpyCOOH-NHS)(PF6)2 ester, 0.10 M 1-methylimidazole buffer (pH 7) for 1 h and then rinsing thoroughly with ethanol and water before drying the electrodes with a stream of N2. Modification of Complementary ssDNA with ECL Labels. Ru(bpy)32+-type ECL labels were covalently attached to amino ssDNA through the carbodiimide reaction in the presence of freshly prepared EDAC. The added EDAC could ensure efficient cross-linking reactions taking place, since Ru(bpy)32+ NHS ester derivatives tend to hydrolyze during the course of storage. Experimentally, about 60-fold excess of solid Ru(bpy)2(Mebpy-COOH-NHS)(PF6)2 was added to 1.0 mL of 0.10 M 1-methylimidazole buffer (pH 7.0) containing an appropriate amount of 15- or 20-mer complementary ssDNA and 0.10 M EDAC. The mixture was shaken with a Thermolyne Speci-mix shaker (Thermolyne Corp., Dubuque, IA), incubated at room temperature for ∼2 h in the dark, and then transferred to a Sephadex G-25 PD-10 desalting column (Amersham Pharmacia Biotech, Piscataway, NJ) preequilibrated with 0.10 M PBS buffer solution (pH 7.2) for product separation and purification according to the manufacturer’s protocol. The eluted yellowish fraction was collected, identified by UV-visible spectroscopy (Shimadzu UV-2401 PC Spectrometer, Columbia, MD), diluted with 0.10 M PBS (pH 7.2), and stored at 4 °C before use. Immobilization of Probe ssDNA onto the Au/SAMs. The self-assembled 3-MPA monolayer/Au electrodes were placed in a 5 µM amino ssDNA (15- or 20-mer probe DNA) 0.10 M,

Electron Transfer Through DNA 1-methylimidazole buffer solution (pH 7.0) containing freshly prepared 0.10 M EDAC and 0.10 M NHS and then incubated in an oven at a temperature of 37 °C for ∼2 h, followed by rinsing with copious amounts of water and ethanol, and finally dried with a stream of N2. With this treatment, amino-modified probe ssDNA was covalently bound to the Au substrate. Commonly used Tris or phosphate buffers were intentionally avoided because of their possible coupling reactions with amino DNA sequences in the presence of EDAC.46 Modification of Au/3-MPA-p-15-ssDNA with ECL Tag. The above obtained 15-mer probe ssDNA attached onto the Au electrode (Au/3-MPA-p-15-ssDNA) was immersed into 1 mL of 0.10 mM Ru(bpy)32+-phosphoramidite ECL tag solution, which had been freshly prepared with MeCN. The reaction was allowed to take place for ∼2 h in the dark, followed by thorough washing of the electrode with MeCN, ethanol, and water. To eliminate the nonspecific adsorption of the ECL tag, the electrode was incubated in a washing buffer solution (5 mM Tris/HCl-0.5 mM EDTA-1.0 M NaCl, pH 7.5) at 37 °C for ∼1 h.47 Finally, the electrode, designated as Au/3-MPA-p-15ssDNA-Ru(bpy)32+, was washed with copious amounts of water, dried with a stream of N2, and kept at ∼4 °C before use. DNA Hybridization. The DNA hybridization reaction was carried out between Au/3-MPA-p-ssDNA (15- and 20-mer ssDNA) and the Ru(bpy)32+-tagged complementary ssDNA (15and 20-mer ssDNA, respectively) at 37 °C in 0.10 M PBS buffer (pH 7.2) for ∼1 h, where 5 µM of the c-ssDNA-ECL tag was used. The electrode was washed with water and ethanol, respectively, incubated in the washing buffer solution at 37 °C for ∼1 h before further washed with copious amounts of water. The electrode immobilized with complementary double-stranded DNA (dsDNA) was kept in 0.10 M phosphate buffer solution (pH 7.4, containing no chloride ions) at 4 °C before any voltammetric and ECL measurements. Instrumentation. The diamond crystal attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic experiments were performed using a Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Corp., Madison, WI) to verify the formation of aminoalkanethiol SAMs and the thiol SAMs covalently linked to Ru(bpy)32+ on the Au/Si electrode surface. The ECL along with the cyclic voltammetric (CV) signals were measured simultaneously with a 660A electrochemical workstation (CH Instruments, Austin, TX) combined with a photomultiplier tube (PMT, Hamamatsu R928, Japan) installed under the electrochemical cell. This cell had a conventional three-electrode configuration, with a Pt wire as the counter electrode and a Ag/AgCl/Cl-(3.0 M KCl) as the reference electrode. Au-disk electrodes (2-mm diameter) immobilized with various SAMs and DNA were used as the working electrodes. Detailed configuration of this homemade ECL instrument has been described previously.48 To minimize the possible effect of chloride ions leaked from the reference electrode on ECL signals,45 CV and ECL measurements were conducted immediately after the electrode was placed in the electrolyte solution. Unless otherwise stated, all measurements were conducted at a temperature of 20 ( 2 °C. Results and Discussion ATR-FTIR Spectroscopy of SAMs on Au/Si. The formation of aminoalkanethiol SAMs on Au and their interactions with Ru(bpy)32+-NHS ester derivatives were characterized by diamond crystal ATR-FTIR spectroscopy. As shown in the Supporting Information section, densely packed SAMs were

J. Phys. Chem. C, Vol. 112, No. 43, 2008 17001 formed on Au for all three aminoalkanethiols, SH-(CH2)n-NH2 (n ) 6, 8, and 11). However, the Ru(bpy)32+ type ECL labels were attached only to some of the surface -NH2 groups of the thiols, probably due to the large Ru(bpy)32+ molecule’s steric hindrance effect. UV-Visible Absorption Spectra. The formation of the 1:1 mol ratio c-20-ssDNA-Ru(bpy)32+-type NHS ester complex was characterized with UV-visible spectroscopy, as detailed in the Supporting Information section. ECL Behavior of Aminoalkanethiol Monolayers Tethered with Ru(bpy)32+ Moieties on Au. Previous studies have demonstrated that the standard heterogeneous electron transfer rate constants between substrate gold electrodes and selfassembled monolayers formed from various types of alkanethiols (up to 16 methylenes in the alkyl chain) tethered with either ferrocene or pentaamminepyridine ruthenium redox couples are in the range of 107-103 s-1.49,50 These rate constants are very large and suggest that the formed SAMs on Au can act as electron tunneling mediators. Therefore, three aminoalkanethiols (SH-(CH2)n-NH2, n ) 6, 8, 11) were chosen to form SAMs on Au and tethered with carboxylated Ru(bpy)32+ at the amino terminals in an attempt to examine if two ECL waves could be produced after the electrode placed in contact with a phosphatebuffered TPrA solution was scanned anodically. As shown in Figure 2, on the forward scan of the first potential cycling, both 6- (Figure 2A(a)) and 8-carbon chain thiols (Figure 2A(b)) present two ECL waves, whereas the 11-carbon chain thiols only show one ECL wave located in a relative positive potential range (Figure 2B(a)). On the subsequent potential cycling, two ECL waves with lower light intensities were observed for all three thiols (not shown for 6- and 8-carbon thiols but shown in Figure 2B(b) for 11-carbon ones). Table 1 summarizes the ECL peak potential and light intensity values for all three thiols attached with Ru(bpy)32+ moieties along with the corresponding data obtained from free Ru(bpy)32+ at a bare Au electrode. Clearly, the first ECL peak potentials are essentially the same for all three thiols (∼0.88 V vs Ag/AgCl, the second cycle for 11-carbon thiols) but the second ECL peak potentials are shifted to more positive values with the increase in the length of the thiols (Table 1 and Figures 2 and 3). As reviewed in the Introduction section above, the first ECL wave is produced from the direct oxidation of TPrA (Scheme 1), which in the current case can occur at the thiol surface; at the Au through the defects (“pinholes”) of the SAMs that could be present even in wellordered, crystalline alkanethiolate domains;51 or both. The same first ECL wave peak potentials for all three SAMs and for the bare Au electrode strongly suggests that the direct oxidation of TPrA predominantly occurred at pinholes; otherwise, their peak potentials would gradually shift positively with the increase in the carbon chains as the second ECL waves do. This is not surprising, because TPrA is a small molecule, and the fraction of uncovered SAMs on Au could be as high as 1%.52 Lessordered SAMs have been observed after the introduction of terminal groups such as -NH2 in alkanethiols.51,53 The second ECL wave results from the direct oxidation of tethered Ru(bpy)32+ (see Scheme 2), and its shift in peak potential is consistent with the change in electron transfer rate constant. The longer the thiol is, the slower the rate constant would be.49,50 As a result, the Ru(bpy)32+ moieties oxidize at more positive potentials when they are attached to longer thiols, resulting in the shift of the ECL waves to the positive potential direction. Because the quality of SAMs formed on Au increases with the increasing length of the alkyl carbon chain,51,52 much less defect density is expected from 11-aminoalkanethiol SAMs

17002 J. Phys. Chem. C, Vol. 112, No. 43, 2008

Pittman and Miao TABLE 1: Average ECL Peak Potential and Intensity Values Obtained on the Basis of Three to Four Replicated Experiments from Au/S-(CH2)n-NHCO-Ru(bpy)32+ Systems in 0.10 M Phosphate Buffer (pH 7.4) Containing 0.10 M TPrA at a Scan Rate of 50 mV/s Ep,ECL ( 0.010 V (V vs Ag/AgCl)

ip,ECL (nA)

Au/S-(CH2)nNHCO-Ru(bpy)32+

1st wave

2nd wave

1st wave

2nd wave

n ) 0a n)6 n)8 n ) 11

0.89 0.89 0.88 / 0.88

1.13 1.12 1.22 1.35 1.35

450 ( 20 95 ( 10 68 ( 10 / 4.6 ( 1

979 ( 20 75 ( 10 51 ( 10 16 ( 2 2.9 ( 1

1st cycle 2nd cycle

a Data obtained from a 2-mm-diameter bare Au electrode placed in contact with 1.0 µM Ru(bpy)32+ and 0.10 M phosphate buffer (pH 7.4) containing 0.10 M TPrA at a scan rate of 50 mV/s (Figure 1).

Figure 3. Correlation between the number of methylene groups in Au/S-(CH2)n-NH-CO-Ru(bpy)32+ and the peak potential of the 2nd ECL wave.

Figure 2. ECL and CV profiles of Au/SAMs formed from aminoalkanethiols tethered with Ru(bpy)32+ moieties placed in contact with 0.10 M phosphate buffer (pH 7.4) containing 0.10 M TPrA at a scan rate of 50 mV/s. (A) First potential cycle of SAMs formed from (a) 6-amino-1-hexanethiol and (b) 8-amino-1-octanethiol. (B) (a) First and (b) second potential cycle of SAMs formed from 11-amino-1undecanethiol. The size of the Au electrode was 2 mm diameter.

relative to 8- and 6-aminoalkanethiol SAMs. Consequently, the average ECL peak intensity obtained on the basis of three to four replicated experiments decreases with the increase in the number of the alkyl carbons. The 11-aminoalkanethiol SAMs were so well-packed and -ordered that there were almost no defects that could allow the TPrA to be oxidized directly at pinholes, leading to the absence of the first classical ECL wave

during the first potential cycling. The appearance of the first ECL wave for 11-aminoalkanethiol SAMs and the decrease of the ECL wave intensities for other thiol SAMs on the subsequent potential cycles can be attributed to the gradual oxidative deformation of the thiol molecules.54 This is supported by the cyclic voltammograms shown in Figure 2B, where the oxidation of Au in the potential range of 0.2-0.6 V vs Ag/AgCl is absent during the forward scan of the initial potential cycle, but it becomes evident in the following cycle (see also Figure 1). The ECL “bump” appearing around 1.13 V vs Ag/AgCl in Figure 2B(a) was occasionally observed for 11-carbon chain thiols and can be ascribed to the ECL production originating from the direct oxidation of TPrA on the surface of 11-aminoalkanethiol SAMs. Data presented in this section proves that two distinct ECL waves should be observed if Ru(bpy)32+ moieties are tethered to “conductive” SAMs, and the positions of the second ECL waves are directly related to the electron transfer capabilities of the SAMs. ECL Behavior of Au/3-MPA-dsDNA (or -ssDNA) Tethered with Ru(bpy)32+ Moieties. Figure 4 shows the ECL and CV responses of the 15-mer (Figure 4A) and 20-mer dsDNA (Figure 4B) immobilized on the Au/3-MPA SAMs and tethered with Ru(bpy)32+ moieties upon the anodic potential scanning

Electron Transfer Through DNA

J. Phys. Chem. C, Vol. 112, No. 43, 2008 17003 TABLE 2: ECL Peak Potentials Obtained from Systems Involving Electron Transfer through DNA Ep,ECL ( 0.010 (V vs Ag/AgCl) electrode configuration 2+

Au/15-mer dsDNA-Ru(bpy)3 Au/20-mer dsDNA-Ru(bpy)32+ Au/15-mer ssDNA-Ru(bpy)32+ Au/S-(CH2)8-Ru(bpy)32+ Au/S-(CH2)2-COOH a

1st wave

2nd wave

0.88 0.90 0.88 0.88 0.90

1.23 1.21 no wave 1.22 1.20

a Data obtained from a Au/S-(CH2)2-COOH electrode placed in contact with 1.0 µM Ru(bpy)32+-0.10 M phosphate buffer (pH 7.4) containing 0.10 M TPrA at a scan rate of 50 mV/s.

Figure 5. ECL and CV responses obtained from a Au/3-MPA-15p-ssDNA-Ru(bpy)32+ electrode in the presence of 0.10 M TPrA/0.10 M phosphate buffer (pH 7.4) at a scan rate of 50 mV/s. Figure 4. ECL and CV responses obtained from Au/3-MPAdsDNA-Ru(bpy)32+ electrodes in the presence of 0.10 M TPrA/0.10 M phosphate buffer (pH 7.4) at a scan rate of 50 mV/s. (A) 15-mer dsDNA and (B) 20-mer dsDNA.

in the presence of 0.10 M TPrA (0.10 M phosphate buffer, pH 7.4). In each case, two ECL waves, located at ∼0.88 and ∼1.22 V vs Ag/AgCl, are observed. The first ECL wave has a very close peak potential to that obtained from a bare Au electrode as well as from the Au/aminoalkanethoil-Ru(bpy)32+ (Table 1). In addition, no obvious oxidation of Au is seen around 0.35 V vs Ag/AgCl on the forward scan of the CV. These data suggest that the dsDNA-immobilized Au electrode was wellpacked, and the direct oxidation of TPrA occurred probably (1) in the spots of free 3-MPA monolayers that had not linked to probe ssDNA (including insignificant fractions of defects) and (2) through the dsDNA. Unlike in the case of Au/S(CH2)n-NHCO-Ru(bpy)32+, where the peak potential of the second ECL wave was CH2-number-dependent (Figure 3), both 15-mer and 20-mer dsDNA show similar peak potentials for the second wave (Figure 4 and Table 2), indicating that under the present experimental conditions, five extra dsDNA bases have no influence on the electron transfer processes. Furthermore, the second ECL peak potential of ∼1.22 V vs Ag/AgCl is found to be slightly more positive than that obtained from Au/3-MPA placed in contact with free Ru(bpy)32+/TPrA solution (∼1.20 V vs Ag/AgCl, Table 2) but almost the same as that obtained from Au/S-(CH2)8-NHCO-Ru(bpy)32+ (Figures 2 and 4, and Table 1). Given the fact that overall, eight CH2 groups exist within the Au/3-MPA-dsDNA-Ru(bpy)32+ configuration (see the Experimental Section ), namely, two from 3-MPA, three from p-ssDNA and another three from c-ssDNA, the impact of

the dsDNA to the entire ECL peak shift is negligible. In other words, the electron transfer rate constant through dsDNA is too large to be distinguished with the present ECL technology, or the dsDNA sequences studied are very conductive. Note that charge transfer over DNA has been previously interpreted as a single step or multistep charge-hopping with G:C sites acting as stepping stones for short- and long-distance DNA, respectively.8 In addition, solution-phase DNA base oxidation, especially G through electrocatalytic reactions using various metal complexes such as Ru(bpy)32+ as a redox mediator, has been extensively studied by Thorp’s group.55-57 In contrast to the ECL responses from the dsDNA presented in Figure 4, only the first ECL wave is observed from a 15-mer ssDNA (i.e., 15-p-ssDNA) attached to the Au/3-MPA and tethered with Ru(bpy)32+ moieties (Figure 5). The absence of the second ECL wave indicates that no tethered Ru(bpy)32+ species can be oxidized directly through the 3-MPA-ssDNA chain. Thus, the 15-p-ssDNA is incapable of electron transfer and is not electronically conductive. Note that, as described in the Experimental section, proper treatment of the Au/3-MPA-p15-ssDNA-Ru(bpy)32+ electrode, via thorough washing with a series of solvents, incubating in a washing buffer solution, and finally drying with a stream of N2, is crucial to eliminate possible nonspecific adsorption of Ru(bpy)32+ moieties on the surface of Au/3-MPA. In addition, under the present experimental conditions, no ECL was observed from the DNA monolayers in the absence of TPrA. Conclusions Ru(bpy)32+ ECL labels were covalently attached to different lengths of aminoalkanethiol SAMs on Au and showed two ECL

17004 J. Phys. Chem. C, Vol. 112, No. 43, 2008 waves in the presence of the ECL coreactant TPrA in phosphate buffer (pH 7.4) solution. The first ECL which is associated with the direct oxidation of TPrA was located essentially at the same peak potential as that of free Ru(bpy)32+/TPrA solution at a bare Au electrode. The second ECL wave, which is related to the direct oxidation of tethered Ru(bpy)32+ moieties, however, was SAM carbon-length-dependent and could be correlated to the electron transfer rate constant of the SAMs. Electron transfer behavior in dsDNA was studied by using amino-modified ssDNA covalently coupled to a Au electrode precoated with 3-MPA SAMs and then hybridized with the complementary ssDNA tagged with Ru(bpy)32+ ECL labels. Both 15-mer and 20-mer dsDNA displayed two ECL waves with the same ECL peak potentials, implying that dsDNA helical structures can transfer electrons at a very fast rate constant. In contrast, ssDNA did not show the second ECL wave, suggesting that ssDNA is not electronically conductive. Acknowledgment. The abstract was presented at PITTCON 2006, March 12-17, Orlando, FL. Financial support from USM startup (W.J.M.), NSF-MRSEC (NSF-DMR 0213883), and NSF-IGERT (NSF-DGE 0333136) is gratefully acknowledged. Supporting Information Available: The ATR-FTIR spectroscopic studies of SAMs on Au/Si and the UV-visible absorption spectra of c-20-ssDNA, Ru(bpy)32+-type NHS ester as well as the DNA-Ru(bpy)32+ complex are available in the SI. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Endres, R. G.; Cox, D. L.; Singh, R. R. P. ReV. Mod. Phys. 2004, 76, 195–214. (2) Schuster, G. B., Ed. Top. Curr. Chem.; Springer-Verlag: Berlin, Heidelberg, New York; 2004, Vol. 237. (3) Schuster, G. B., Ed. Top. Curr. Chem.; Springer-Verlag: Berlin, Heidelberg, New York; 2004, Vol. 236. (4) Xu, B.; Zhang, P.; Li, X.; Tao, N. Nano Lett. 2004, 4, 1105–1108. (5) Wagenknecht, H.-A., Ed. Charge Transfer in DNA: From Mechanism to Application; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2005. (6) Berlin, Y. A.; Burin, A. L.; Ratner, M. A. Superlattices Microstruct. 2000, 28, 241–252. (7) Berlin, Y. A.; Kurnikov, I. V.; Beratan, D.; Ratner, M. A.; Burin, A. L. Top. Curr. Chem. 2004, 237, 1–36. (8) Boussicault, F.; Robert, M. Chem. ReV. 2008, 108, 2622–2645. (9) Stanca, S. E.; Eritja, R.; Fitzmaurice, D. Faraday Discuss. 2006, 131, 155–165. (10) Koh, S. J. Nanoscale Res. Lett. 2007, 2, 519–545. (11) Tumpane, J.; Kumar, R.; Lundberg, E. P.; Sandin, P.; Gale, N.; Nandhakumar, I. S.; Albinsson, B.; Lincoln, P.; Wilhelmsson, L. M.; Brown, T.; Norden, B. Nano Lett. 2007, 7, 3832–3839. (12) Fink, H. W.; Schonenberger, C. Nature 1999, 398, 407–410. (13) Porath, D.; Bezryadin, A.; De Vries, S.; Dekker, C. Nature 2000, 403, 635–638. (14) de Pablo, P. J.; Moreno-Herrero, F.; Colchero, J.; Gomez Herrero, J.; Herrero, P.; Baro, A. M.; Ordejon, P.; Soler, J. M.; Artacho, E. Phys. ReV. Lett. 2000, 85, 4992–4995. (15) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature 1998, 391, 775–778. (16) Kasumov, A. Y.; Kociak, M.; Gueron, S.; Reulet, B.; Volvkov, V. T.; Klinov, D. V.; Bouchiat, H. Science 2001, 291, 280–282. (17) Delaney, S.; Barton, J. K. J. Org. Chem. 2003, 68, 6475–6483. (18) Giese, B. Curr. Opin. Chem. Biol. 2002, 6, 612–618.

Pittman and Miao (19) Giese, B. Biorg. Med. Chem. 2006, 14, 6139–6143. (20) Giese, B.; Spichty, M. ChemPhysChem 2000, 1, 195–198. (21) Lewis, F. D.; Letsinger, R. L.; Wasielewski, M. R. Acc. Chem. Res. 2001, 34, 159–170. (22) Nunez, M. E.; Barton, J. K. Curr. Opin. Chem. Biol. 2000, 4, 199– 206. (23) Schuster, G. B. Acc. Chem. Res. 2000, 33, 253–260. (24) Drummond, T. G.; Hill, M. G.; Barton, J. K. J. Am. Chem. Soc. 2004, 126, 15010–15011. (25) Liu, T.; Barton, J. K. J. Am. Chem. Soc. 2005, 127, 10160–10161. (26) Yang, V. C.; Ngo, T. T., Eds. Biosensors and Their Applications; Kluwer Academic/Plenum Publishers: New York, 2000. (27) Cunningham, A. J. Introduction to Bioanalytical Sensors; J. Wiley & Sons, Inc.: New York, 1998. (28) Liron, Z.; Bromberg, A.; Fisher, M., Eds. NoVel Approaches in Biosensors and Rapid Diagnostic Assays; Kluwer Academic/Plenum Publishers: New York, 2000. (29) Mikkelsen, S. R. Electroanalysis 1996, 8, 15–19. (30) Wang, J. Anal. Chim. Acta 2002, 469, 63–71. (31) Mannelli, I.; Minunni, M.; Tombelli, S.; Mascini, M. Biosens. Bioelectron. 2003, 18, 129–140. (32) Sutherland, G.; Mulley, J. In Nucleic Acid Probes; Symons, R. H., Ed.; CRC Press: Boca Raton, FL, 1989, pp 159-201. (33) Park, S.-J.; Taton, T. A.; Mirkint, C. A. Science 2002, 295, 1503– 1506. (34) Kenten, J. H.; Gudibande, S.; Link, J.; Willey, J. J.; Curfman, B.; Major, E. O.; Massey, R. J. Clin. Chem. 1992, 38, 873–879. (35) Christodoulides, N.; Tran, M.; Floriano, P. N.; Rodriguez, M.; Goodey, A.; Ali, M.; Neikirk, D.; McDevitt, J. T. Anal. Chem. 2002, 74, 3030–3036. (36) Dekker, C.; Ratner, M. A. Phys. World 2001, 14, 29–33. (37) Endres, R. G.; Cox, D. L.; Singh, R. R. P. Los Alamos National Laboratory, Preprint ArchiVe, Condensed Matter 2002, 1–23, arXiv:condmat/0201404. (38) Briman, M.; Armitage, N. P.; Helgren, E.; Gruener, G. Nano Lett. 2004, 4, 733–736. (39) Bard, A. J.; Debad, J. D.; Leland, J. K.; Sigal, G. B.; Wilbur, J. L.; Wohlsatdter, J. N. In Encyclopedia of Analytical Chemistry: Applications, Theory and Instrumentation; Meyers, R. A., Ed.; John Wiley & Sons: New York, 2000; Vol. 11, pp 9842-9849. (40) Miao, W. Chem. ReV. 2008, 108, 2506–2553. (41) Miao, W.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478–14485. (42) Zu, Y.; Bard, A. J. Anal. Chem. 2000, 72, 3223–3232. (43) Zu, Y.; Li, F. Anal. Chim. Acta 2005, 550, 47–52. (44) Zheng, H.; Zu, Y. J. Phys. Chem. B 2005, 109, 12049–12053. (45) Wang, S.; Neshkova, M. T.; Miao, W. Electrochim. Acta 2008, 53, 7661–7667. (46) Hermanson, G. T. Bioconjugate Techniques; Academic Press: New York, 1996. (47) Miao, W.; Bard, A. J. Anal. Chem. 2003, 75, 5825–5834. (48) Rosado, D. J., Jr.; Miao, W.; Sun, Q.; Deng, Y. J. Phys. Chem. B 2006, 110, 15719–15723. (49) Smalley, J. F.; Finklea, H. O.; Chidsey, C. E. D.; Linford, M. R.; Creager, S. E.; Ferraris, J. P.; Chalfant, K.; Zawodzinsk, T.; Feldberg, S. W.; Newton, M. D. J. Am. Chem. Soc. 2003, 125, 2004–2013. (50) Smalley, J. F.; Feldberg, S. W.; Chidsey, C. E. D.; Linford, M. R.; Newton, M. D.; Liu, Y.-P. J. Phys. Chem. 1995, 99, 13141–13149. (51) Vericat, C.; Vela, M. E.; Salvarezza, R. C. Phys. Chem. Chem. Phys. 2005, 7, 3258–3268. (52) Finklea, H. O. In Electroanalytical Chemistry: A Series of AdVances; Bard, A. J. Rubinstein, I., Eds.; Marcel Dekker, New York, 1996; Vol. 19. (53) Azzaroni, O.; Vela, M. E.; Martin, H.; Hernandez Creus, A.; Reasen, G.; Salvarezza, R. C. Langmuir 2001, 17, 6647–6654. (54) Zu, Y.; Bard, A. J. Anal. Chem. 2001, 73, 3960–3964. (55) Johnston, D. H.; Thorp, H. H. J. Phys. Chem. 1996, 100, 13837– 13843. (56) Sistare, M. F.; Codden, S. J.; Heimlich, G.; Thorp, H. H. J. Am. Chem. Soc. 2000, 122, 4742–4749. (57) Thorp, H. H. In Top. Curr. Chem.; Schuster, G. B., Ed.; SpringerVerlag: Berlin, Heidelberg, New York, 2004; Vol. 237, pp 159-181.

JP805791P