Micromethod for the investigation of the interactions between DNA and redox-active molecules

A novel microscale and surface-based method for the study of the interactions of DNA with other redox-active molecules using DNA-modified electrodes is described. The method is simple, convenient, reliable, reagent-saving, and applicable for DNA studies, especially those involving microsamples. Information such as binding site size (s, in base pairs), binding constant (K), ratio (K0x/KRed) of the binding constants for the oxidized and reduced forms of a bound species, binding free energy (delta Gb), and interaction mode, including changes in the mode of interaction, and "limiting" ratio K0x0/KRed0 at zero ionic strength can be obtained using only 3-15 micrograms of DNA samples. The method was developed using [Co(Phen)3]3+/2+ (Phen = 1,10-phenanthroline)/double-stranded DNA (dsDNA)-modified gold electrodes and [Co(bpy)3]3+/2+ (2,2'-bipyridyl)/dsDNA-modified gold electrodes as model systems. For the [Co(Phen)3]3+/2+/dsDNA-modified gold electrode system, a K2+ of (2.5 +/- 0.3) x 10(5) M-1 and an s of 5 bp were obtained in 5 mM pH 7.1 Tris-HCl buffer solution containing 50 mM NaCl. For [Co(bpy)3]3+/2+/dsDNA-modified gold electrodes, K3+ and s values of (1.3 +/- 0.3) x 10(5) M-1 and 3 bp, respectively, were obtained. While the s values are consistent with those reported in the literature obtained by solution methods, the K values are almost an order of magnitude larger. A transition in the nature of the interaction between dsDNA and [Co(Phen)3]3+/2+, from electrostatic to intercalative with increasing ionic strength, was found in our studies. Negative values of delta E0' for [Co(bpy)3]3+/2+ bound to dsDNA suggest that its interaction with dsDNA is predominantly electrostatic over the ionic strength range of 5-105 mM. The "limiting" ratio K3+0/K2+0 of 22 obtained for [Co(Phen)3]3+/2+ bound to dsDNA at zero ionic strength suggests that electrostatic interactions are predominant over intercalative ones under these limiting conditions. The ratio for [Co(bpy)3]3+/2+ of 16 also indicates that the 3+ form binds to dsDNA more strongly than the 2+ form at zero ionic strength. For [Co(Phen)3]3+/2+/single-stranded DNA (ssDNA)-modified gold electrodes, the nonuniformity of the surface structure of ssDNA-modified gold electrodes greatly complicates the analysis. A system consisting of a dsDNA-modified gold electrode and [Co(tppz)2]3+/2+ (tppz = tetra-2-pyridyl-1,4-pyrazine) was studied by this method, with a K2+ value of (5 +/- 1) x 10(5) M-1 and an 8 value of 7 bp being obtained.     

DNA sensor with a dipyridophenazine complex of osmium(lI) as an electrochemical probe

Application of a dipyrido[3,2-a:2',3'-c]phenazine (DPPZ)-type metal complex as an DNA electrochemical probe was studied. The introduction of electron-donating groups (NH2) was effective for controlling the redox potential and binding affinities of the DPPZ-type osmium complex. The [Os(DA-bpy)2DPPZ]2+ complex (DA-bpy; 4,4'-diamino-2,2'-bipyridine) had a lower half-wave potential (E 1/2) of 147 mV (vs Ag/AgCl) and higher binding affinity with DNA (binding constant, K = 3.1 x 10(7) M(-1)) than those of other complexes. With a single-stranded DNA immobilized gold electrode, the hybridization signal (deltaI) of the [Os(DA-bpy)2DPPZ]2+ complex was linear in the concentration range of 1.0 pg mL(-1) - 0.12 microg mL(-1) for the targeted DNA with a regression coefficient of 0.999. The detection limit was 0. 1 pg mL(-1). The 400-bp yAL3 gene was also detected with good sensitivity and selectivity using the [Os(DA-bpy)2DPPZ]2+ complex.     

Voltammetric sensor for oxidized DNA using ultrathin films of osmium and ruthenium metallopolymers

Films containing [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ metallopolymers were assembled layer by layer on pyrolytic graphite electrodes to make sensors that selectively detect oxidized DNA. These films showed reversible, independent electrochemistry for electroactive Os3+/Os2+ and Ru3+/Ru2+ centers, with formal potentials of 0.34 and 0.76 V vs SCE, respectively. The combination of ruthenium and osmium metallopolymers in the films provided a catalytic Os square wave voltammetry (SWV) peak that is mainly selective for 8-oxoguanine and the detection of other oxidized nucleobases from the Ru peak. The method is applicable to measurements on DNA in solution or DNA incorporated into films. Using the Os SWV peak, 1 oxidized nucleobase in 6000 was detected. The sensor is simple and inexpensive, and the approach may be useful for the detection of oxidized DNA as a clinical biomarker for oxidative stress.     

DNA biosensor for detection of Helicobacter pylori using phen-dione as the electrochemically active ligand in osmium complexes

A surface-based method for the study of the interactions of DNA with redox-active 1,10-phenantroline-5,6-dione (phen-dione) osmium complexes is described. The study was carried out using gold electrodes modified with DNA via adsorption and [Os(bpy)(2)(phe-dione)](3+/2+) (bpy = 2,2'-bipyridyl) or [Os(phen)(2)(phen-dione)](3+/2+) (phen = 1,10-phenantroline) as electrochemical reported molecules. The method, which is simple and reagent-saving, allows the accumulation of osmium complexes within the DNA layer. The amount of osmium complex bound by the adsorbed layer of DNA was determined from the voltammetric charge associated with the osmium redox process of the immobilized metal complex. The quinone moiety of the phen-dione ligand was useful as an indicator for electrochemical DNA sensing because of its redox response at low potentials. A thiol-linked single-stranded Helicobacter pylori DNA probe was immobilized, through S-Au bonds on to a gold electrode (density of modification 86 pmol/cm(2)). Following hybridization with the complementary DNA sequence, the osmium complex was electrochemically accumulated within the double-stranded DNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range where the quinone moiety was redox active (i.e., at very low potentials, -0.020 V vs SSCE); with this approach, a sequence of the H. pylori could be quantified over the range from 5 to 20 pmol with a linear correlation of r = 0.9888 and a detection limit of approximately 6 pmol.     

Electrochemiluminescence from Os(phen)2(dppene)2+ (phen = 1,10-phenanthroline and dppene = bis(diphenylphosphino)ethene)

The electrochemiluminescence (ECL) of Os(phen)2(dppene)2+ (phen = 1,10-phenanthroline and dppene = bis(diphenylphosphino)ethene) is reported in mixed CH3CN/H2O (50:50 v/v) and aqueous (0.1 M KH2PO4) solutions with tri-n-propylamine (TPrA) as an oxidative-reductive coreactant. ECL efficiencies (phi(ecl) = photons emitted/redox event) of 2.0 in aqueous, and 0.95 in mixed for Os(phen)2(dppene)2+ were obtained using Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) as a relative standard (phi(ecl) = 1). Photoluminescence (PL) efficiencies of 0.094 and 0.053 were obtained in aqueous and mixed solutions, respectively, as compared to Ru(bpy)3(2+) (phi(em) = 0.042). The ECL spectra were identical to photoluminescence spectra (lambda(max) approximately 584 nm), indicating formation of the same metal-to-ligand (MLCT) excited states in both ECL and PL. The ECL is linear over several orders of magnitude in aqueous and mixed solution, with theoretical detection limits (blank plus three times the standard deviation of the noise) of 16.9 nM in H2O and 0.29 nM in CH3CN/H2O (50:50 v/v).     

Electrogenerated chemiluminescence. 77. DNA hybridization detection at high amplification with [Ru(bpy)3]2+-containing microspheres

An ultrasensitive DNA hybridization detection method based on electrogenerated chemiluminescence (ECL) using polystyrene microspheres/beads (PSB) as the carrier of the ECL labels, namely, tris(2,2'-bipyridyl)ruthenium(II) tetrakis(pentafluorophenyl)borate (Ru(bpy)3[B(C6F5)4]2), is reported. Probe single-stranded DNA (p-ssDNA) was attached to the surface of magnetic beads (MB) and hybridized with target-ssDNA (t-ssDNA) with immobilized PSB containing a large number of water insoluble Ru(bpy)3[B(C6F5)4]2 species (approximately 7.5 x 10(9) molecules/bead). With this approach a large amplification factor of Ru(bpy)3[B(C6F5)4]2 molecules for each t-ssDNA can be achieved, when each PSB is attached to a limited number of t-ssDNA. The p-ssDNA-MB <--> t-ssDNA-PSB/Ru(bpy)3(2+) conjugates formed were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine (TPrA) as an ECL coreactant. ECL was produced with a potential scan from 0 to 3.0 V versus Ag/Ag+, and the integrated ECL intensity was found to be linearly proportional to the t-ssDNA concentration in a range of 1.0 fM to 10 nM under optimized conditions. ECL signals associated with two base pair mismatched ssDNA and noncomplementary ssDNA can be distinguished well from the ECL signal related to the complementary DNA hybridization. A Poisson distribution is followed when a large number of MB reacts with PSB, and the minimum number of 1.0- and 2.8-microm diameter MB required to bind and magnetically separate a single 10-microm diameter PSB from the reaction solution was estimated to be three and one, respectively. The principle described in this paper could be also applied to many other ECL analyses, such as immunoassays.     

Real-Time Electrochemical PCR with a DNA Intercalating Redox Probe

The proof-of-principle of a nonoptical real-time PCR method based on the electrochemical monitoring of a DNA intercalating redox probe that becomes considerably less easily electrochemically detectable once intercalated to the amplified double-stranded DNA is demonstrated. This has been made possible thanks to the finding of a redox intercalator that (i) strongly and specifically binds to the amplified double-stranded DNA, (ii) does not significantly inhibit PCR, (iii) is chemically stable under PCR cycling, and (iv) is sensitively detected by square wave voltammetry during PCR cycling. Among the different DNA intercalating redox probes that we have investigated, namely, methylene blue, Os[(bpy)(2)phen](2+), Os[(bpy)(2)DPPZ](2+), Os[(4,4'-dimethyl-bpy)(2)DPPZ](2+) and Os[(4,4'-diamino-bpy)(2)DPPZ](2+) (with bpy = 2,2'-bipyridine, phen = phenanthroline, and DPPZ = dipyrido[3,2-a:2',3'-c]phenazine), the one and only compound with which it has been possible to demonstrate the proof-of-concept is the Os[(bpy)(2)DPPZ](2+). In terms of analytical performances, the methodology described here compares well with optical-based real-time PCRs, offering finally the same advantages than the popular and routinely used SYBR Green-based real-time fluorescent PCR, but with the additional incomes of being potentially much cheaper and easier to integrate in a hand-held miniaturized device.     

Use of polymeric indicator for electrochemical DNA sensors: poly(4-vinylpyridine) derivative bearing [Os(5,6-dimethyl-1,10-phenanthroline)2Cl]2+

A poly(4-vinylpyridine) (PVP) derivative bearing redox-active osmium complexes, PVP-[Os(5,6-dmphen)(2)Cl](2+) (5,6-dmphen = 5,6-dimethyl-1,10-phenanthroline), was employed as a hybridization indicator for electrochemical DNA sensors. PVP-[Os(5,6-dmphen)(2)Cl](2+) exhibited approximately 1000 times higher sensitivity than the corresponding monomeric analogue, [Os(5,6-dmphen)(3)](2+), in DNA determination due to polymeric effects. The detection limit of the present sensor was approximately 0.5 amol. Another merit of the polymeric indicator is that the redox potential was found to be +360 mV (vs Ag/AgCl), which is significantly lower than that reported for the monomeric analogue (+672 mV). The polymeric indicator was applicable to the discrimination of single- and double-base-mismatched DNAs from fully matched target DNA. The polymeric indicator can be removed from the electrode surface by rinsing the electrode in a high-temperature buffer for 6 min, and thus, the polymeric indicator-based DNA sensor can be used repeatedly.     

A single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by electrogenerated luminescence based on Ru(bpy)(3)2+ in aqueous solution

Ascorbic (H2A) and dehydroascorbic (DA) acids were for the first time directly determined in a single chromatographic run by means of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)2+) based electrogenerated chemiluminescence (ECL) detection. For the first time, it was demonstrated that DA, a nonelectroactive compound, is ECL active and is responsible for the ECL behavior of H2A. This fact, together with the lack of a DA standard, suggested the use of a calibration graph obtained for H2A, for determining both analytes. The proven ECL activity of DA, together with literature data relative to the standard redox potentials of the different species coming from H2A, led to a reconsideration of the proposed ECL reaction mechanism for H2A. The role of the OH- ion in the reaction mechanism of the two analytes appeared to be crucial. H2A and DA could be separated by a suitable C18-reversed-phase HPLC column using an aqueous 30 mM H3PO4 solution as the mobile phase. The optimal ECL response was achieved by polarizing the working electrode at 1.150 Vvs SCE (standard calomel electrode) (oxidation diffusion limiting potential for both H2A and Ru(bpy)(3)2+). The Ru(bpy)(3)2+ solution, at pH 10 for carbonate buffer, was mixed to the eluent solution in a postcolumn system, obtaining, still at pH 10, the final 0.25 mM Ru(bpy)(3)2+ concentration. The detection limit found for the two analytes was 1 x 10(-7) M. The method was successfully applied to the determination of the analytes in a commercially available orange fruit juice.     

Effect of surface immobilization on the electrochemiluminescence of ruthenium-containing metallopolymers

The effect of surface confinement on the electrochemiluminescence (ECL) properties of metallopolymer [Ru(bpy)2(PVP)10]2+, where bpy is 2,2'-bipyridyl and PVP is poly(4-vinylpyridine), is reported. Immobilizing a luminescent material on an electrode surface can substantially modulate its photophysical properties. Significantly, our study revealed that the overall efficiency of the ECL reaction for the metallopolymer film is almost four times higher, at 0.15%, than the highest value obtained for [Ru(bpy)2(PVP)10]2+ dissolved in solution, (phi(ECL) = 0.04%). Electrochemistry has been used to create well-defined concentrations of the quencher Ru3+ within the film. Analysis of both the steady-state luminescence and lifetimes of the film reveals that static quenching by electron transfer between the photoexcited Ru2+ and the Ru3+ centers is the dominant quenching mechanism. The bimolecular rate of electron transfer is (2.5 +/- 0.4) x 10(6) M(-1) s(-1). The implications of these findings for ECL-based sensors, in terms of optimum luminophore loading, is considered.     

L-alpha-glycerophosphate and L-lactate electrodes based on the electrochemical "wiring" of oxidases.

The title electrodes were constructed by coimmobilizing the respective FAD oxidases on solid electrode surfaces with a poly(vinyl pyridine) polymer which was N-derivatized with bromoethylamine and Os(bpy)2Cl2. The redox-polymer-enzyme hydrogels were cross-linked on the electrode surface using poly(ethylene glycol) diglycidyl ether. As in the case of glucose oxidase, the redox polymer acts as an electron relaying "wire" transferring electrons directly from the enzymes' FADH2 centers to the electrode. This transfer competes with the natural process of reoxidation of FADH2 by molecular oxygen. The variation of the response of these electrodes with the atmosphere (N2 or air), pH, and substrate concentration was determined. The pH profile of the electrocatalytic current differs from that of the activity of the free enzymes, exhibiting a broader maximum, shifted to higher pH values. The observed sensitivities and linear ranges are respectively 2 x 10(-2) A M-1 cm-2 and 2.7 mM for L-alpha-glycerophosphate, and 0.3 A M-1 cm-2 and 0.2 mM for L-lactate that may be compared to 2 x 10(-2) A M-1 cm-2 and 10 mM for glucose. The 0-90% response time for all electrodes is 1 s or less.     

Interactions of benzyl viologen with surface-bound single- and double-stranded DNA

The interactions of benzyl viologen (BV) with single- and double-stranded calf-thymus DNA immobilized onto gold electrodes have been studied by electrochemical methods. Benzyl viologen interacts electrostatically with both double-stranded (ds) and single-stranded (ss) DNA, and the strength of the interactions is dependent on ionic strength (mu). The dicationic form (BV2+) binds to dsDNA 9 times more strongly than the singly reduced form, BV*+, in a pH 7.4 Tris-HCl buffer solution at mu = 8.4 mM. BV2+ binds to ssDNA 5 times more strongly than the BV*+ form. From measurements at mu = 8.4 mM, a binding constant (K2+) of 2.0 (+/-0.2) x 10(4) M(-1) and a binding site size (s) of 1 base pair were obtained, respectively, for dsDNA. For ssDNA, at the same ionic strength, the values obtained for K and s were 3.6 (+/-0.4) x10(4) M(-1) and 2 nucleotides, respectively. The amount of BV bound, whether to dsDNA or ssDNA, decreased with increasing ionic strength. Whereas the binding rate of BV to both dsDNA and ssDNA immobilized onto gold electrodes is relatively low, once immobilized, it dissociates rapidly away from the electrode surface. The electron-transfer rate constant for BV is moderately fast at both dsDNA- and ssDNA-modified gold electrodes. The application of benzyl viologen as an electroactive indicator capable of differentiating between surface-immobilized single- and double-stranded DNA in denaturation/regeneration cycles has been explored.     

Electrochemiluminescent detection of metal cations using a ruthenium(II) bipyridyl complex containing a crown ether moiety

The effects of metal ions on the electrochemiluminescence (ECL) properties of (bpy)2Ru(AZA-bpy) (bpy = 2,2'-bipyridine; AZA-bpy = 4-(N-aza-18-crown-6-methyl-2,2'-bipyridine) have been investigated. The electrochemistry, photophysics and ECL of Ru(bpy)3(2+) in the presence of Pb2+, Hg2+, Cu2+, and K+ are reported. The anodic oxidation of Ru(bpy)3(2+) produces ECL in the presence of tri-n-propylamine (TPrA) in 50:50 (v/v) CH3CN:H2O solution. Increases in ECL efficiency (photons generated per redox event) up to 20-fold that depend on both the concentration and nature of the metal ion have been observed, making this an interesting system for electrochemiluminescence metal ion sensing.     

Electrogenerated chemiluminescence of tris (2,2'-bipyridyl)ruthenium(II) ion-exchanged in nafion-silica composite films

The voltammetry and electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3 2+) ion-exchanged in Nafion and Nafion-silica composite materials have been investigated. The major goal of this work was to investigate and develop new materials and immobilization approaches for the fabrication of ECL-based sensors with improved reactivity and long-term stability. Nation-silica composite materials with varying contents of Nation (53-100 wt% relative to silica) were prepared via the two-step acid/base hydrolysis and condensation of tetramethoxysilane. The Nafion doped sols were spin cast on glassy carbon electrodes, dried, and then ion-exchanged with Ru(bpy)3 2+. The shapes of the cyclic voltammetric curves and the amount of Ru(bpy)3 2+ exchanged into the films strongly depends on the amount of Nafion incorporated into the hybrid sol. Nafion-silica films with a low content of Nafion ion-exchanged less Ru(bpy)3 2+ and exhibited tail-shaped voltammetry at 100 mV/s. The ECL of immobilized Ru(bpy)3 2+ in the presence of either tripropylamine or sodium oxalate in pH 5 acetate buffer was also strongly dependent on the amount of Nafion introduced into the composite with greater ECL observed for the Nafion-silica films relative to pure Nafion.     

The effects of nonionic surfactants on the tris(2,2'-bipyridyl)ruthenium(II)--tripropylamine electrochemiluminescence system

The electrochemistry and electrogenerated chemiluminescence (ECL) of Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl) were studied in the presence of the nonionic surfactants Triton X-100, Thesit, and Nonidet P40. The anodic oxidation of Ru(bpy)3(2+) produces ECL in the presence of tri-n-propylamine in both aqueous and surfactant solutions. Increases in both ECL efficiency (> or =8-fold) and duration of the ECL signal were observed in surfactant media. A shift to lower energies of the Ru(bpy)3(2+) ECL emission by approximately 8 nm was also observed. The one-electron oxidation of Ru(bpy)3(2+) to Ru(bpy)3(3t) occurs at + 1.03 V vs Ag/AgCl in aqueous buffered (0.2 M potassium phosphate) solution as found by square wave voltammetry. This potential did not shift in surfactant systems, indicating that the redshifts in ECL emission are due to stabilization of ligand pi* orbitals in the metal-to-ligand charge-transfer excited state. These results are consistent with hydrophobic interactions between Ru(bpy)3(2+) and the nonionic surfactants.     

Electrochemical displacement method for the investigation of the binding interaction of polycyclic organic compounds with DNA

The binding interaction of many organic carcinogens such as polycyclic aromatic hydrocarbons with DNA is the key step in their genotoxic effect. In this work, an electrochemical displacement method was developed to study such interaction. In the method, a DNA film is deposited on an indium tin oxide electrode surface by layer-by-layer assembly, and a redox-active DNA intercalator Ru(bpy) 2(dppz)(BF 4) 2 (bpy = 2,2'-bipyridine, dppz = dipyrido [3,2-a:2',3'-c] phenazine) is employed as an electrochemical indicator. If an organic compound competes with the indicator for the same binding site on DNA in the film, it would displace the ruthenium complex from DNA, resulting in a reduction in the measured electrochemical signal. From the titration curve, the binding constant of the organic compound with DNA can be calculated. With the use of oxalate as an electron donor to chemically amplify the oxidation current of the indicator, chemicals can be tested at low micromolar concentrations. Five well-known DNA binding polycyclic organic compounds, thiazole orange, 4,6-diamidine-2-phenylindole, H33258, ethidium bromide, and quinacrine, were investigated by the displacement method. The binding constants obtained in our experiments fall in the range of (4.3 x 10 (5)) to (1.2 x 10 (7)) M (-1), which are generally consistent with those reported in the literature by some established methods. The electrochemical method provides a general tool that complements the commonly used spectroscopic methods for the study of DNA/small molecule interactions.     

Electrochemiluminescent detection based on solid-phase extraction at tris(2,2'-bipyridyl)ruthenium(II)-modified ceramic carbon electrode

A sensitive electrochemiluminescent detection scheme by solid-phase extraction at Ru(bpy)3(2+)-modified ceramic carbon electrodes (CCEs) was developed. The as-prepared Ru(bpy)3(2+)-modified CCEs show much better long-term stability than other Nafion-based Ru(bpy)3(2+)-modified electrodes and enjoy the inherent advantages of CCEs. The log-log calibration plot for dioxopromethazine is linear from 1.0 x 10(-9) to 1.0 x 10(-4) mol L(-1) using the new detection scheme. The detection limit is 6.6 x 10(-10) mol L(-1) at a signal-to-noise ratio of 3. The new scheme improves the sensitivity by approximately 3 orders of magnitude, which is the most sensitive Ru(bpy)3(2+) ECL method. The scheme allows the detection of dioxopromethazine in a urine sample within 3 min. Since Ru(bpy)3(2+) ECL is a powerful technique for determination of numerous amine-containing substances, the new detection scheme holds great promise in measurement of free concentrations, investigation of protein-drug interactions and DNA-drug interactions, pharmaceutical analysis, and so on.     

Electrochemiluminescence of ruthenium (II) tris(bipyridine) encapsulated in sol-gel glasses

The electrogenerated chemiluminescence (ECL) of Ru(bpy)3 2+ and tripropylamine, tributylamine, triethylamine, trimethylamine, or sodium oxalate encapsulated within sol-gel-derived silica monoliths have been investigated using an immobilized ultramicroelectrode assembly. The major purpose of this study was to investigate the role of the reductant on the magnitude and stability of the ECL in this solid host matrix. For gel-entrapped Ru(bpy)3 2-/tertiary amines, the shape and intensity of the ECL-potential curves were highly dependent on scan rate. At 10 mV/s, the ECL intensity was ca. 6-fold higher relative to that observed at 500 mV/s. When the ECL acquired at low scan rates was normalized by that obtained in solution under similar conditions, a value of 0.03-0.06 was obtained. In direct contrast, the ECL of the Ru(bpy)3 2+-oxalate system showed little dependence on scan rate, and the ECL was ca. 65-75% of that measured in solution. These differences can be attributed to differences in rotational and translational mobility between the reductants (amines vs oxalate) trapped in this porous solid host For both systems, the ECL was found to be stable upon continuous oxidation or upon drying the gels in a high-humidity environment for over 10 days.     

Direct detection of DNA with an electrocatalytic threading intercalator

Herein we report the synthesis, intercalating properties, and analytical applications of an imidazole-substituted naphthalene diimide, N,N'-bis(3-propylimidazole)-1,4,5,8-naphthalene diimide (PIND), functionalized with electrocatalytic redox moieties. PIND was prepared in a single-step reaction from the corresponding dianhydride. Attachment of the redox moieties to PIND relied upon ligand exchange with one of the liable chloride ligands of an Os(bpy)2Cl2 (bpy = 2,2'-bipyridine) complex. The Os(bpy)2Cl2 complex was grafted onto PIND through coordinative bonds with the two imidazole groups at its termini, forming a PIND-[Os(bpy)2Cl]+ compound (PIND-Os). Gel electrophoretic studies revealed that PIND-Os binds more strongly to double-stranded DNA (ds-DNA) than its parent compound 1,4,5,8-naphthalene diimide. The naphthalene diimide group binds to ds-DNA in a "classical" threading intercalation mode, while the two Os(bpy)2Cl+ pendants interact with DNA via electrostatic interaction, reinforcing the intercalation by "locking up" the naphthalene diimide group in place. An electrochemical biosensor was fabricated using the redox-active and catalytic PIND-Os intercalator. An increase in sensitivity of 2500-fold over direct voltammetry was obtained in electrocatalytic amperometry, making this an interesting system for amperometric DNA sensing. Under optimized experimental conditions, the biosensor allowed the detection of a 50-mer target DNA in the range of 1.0-300 pM with a detection limit of 600 fM (1.5 amol, 23 fg).