Micromethod for the Investigation of the Interactions between DNA and Redox-Active Molecules
ABSTRACT 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.
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- "(B) Cyclic voltammograms of 80 mM His, 90 mM SDS + 80 mM His, 80 mM His + 10 M hematin, and 90 mM SDS + 80 mM His + 10 M hematin. for the three-component system, it can be deduced that the electron transfer process in this system is facilitated and from the positive shift for the peak potential, it can be deduced that hydrophobic attractions are dominant in the system  . "
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- ", LLO hlyA , N-hydroxysulfosuccinimide(NHS ) N-(3-dimethylamion) propyl-N'ethyl carbodiimidehydrochloride (EDC), DNA , [Co(phen) 3 ](ClO 4 ) 3 (Cyclic voltammetry, CV), CV , 1 1.1 材料 Listeria monocytogenes 1.2 方法 1.2.1 工作电极的预处理 , , , , 1 mmol/L K 3 Fe(CN) 6 0.1 mol/L KCl( pH=7.1 Tris-HCl ), , 100 mV/s , , 100 mV/s 1 mmol/L K 3 Fe (CN) 6 + 0.1 mol/L KCl CV 70 mV 1.2.2 常温下电极的修饰 (H 2 SO 4 /30% H 2 O 2 , 7: 3 v/v) 5 min, , min 1 mmol/L AET(2-Aminoethanethiol, Sigma ) 16 h, AET , AET/Au , 1.2.3 工作电极的 DNA 自组装 AET/Au 5 mmol/L EDC(N-(3- dimethylamion) propyl-N'-ethyl carbodiimidehydrochloride , Sigma ) 8 mmol/L NHS(N-hydroxysulfosuccinimide, Acros ) pH 7.0 (Na 2 HPO 4 NaH 2 PO 4 )(Phosphate Buffer, PB) 15 min , 1 mg/mL HlYAP 5 mmol/L NaCl Tris-HCl (pH7.1) 1 h, ssDNA 1.2.4 引物设计、DNA 提取和 PCR 扩增 , : HlYAF : 5 -AA 514 HEREDITAS (Beijing) 2010 32 GCTGTTACTAAAGAGCAGTTGCAAGC-3 , HlYAR: 5 -CTGGCAAATAGATGGACGATGTGAAATG-3 ; : HlYAP 5 -HS-TTTTTTTTTTTTTTTGG TTCCGCAAAA GATGAAGTTC-3 DNA DNA (Bacteria gDNA Isolation Mini Kit, ) Listeria monocytogenes DNA, DNA (Wizard® SV Genomic DNA Purification System, Promega) HlYAF HlYAR hlyA 820~1 419 bp , 600 bp HlYAP hlyA 1 053~10 74 bp HlYAF HlYAR hlyA , PCR 25 L , DNA 35 ng, HlYAF 5 pmol/L, HlYAR 20 pmol/L, dNTP 200 mol/L, PCR (10 mmol/L Tris-HCl, 50 mmol/L HCl, 2.5 mmol/L MgCl 2 , pH8. 3) 0.35 U DNA (TaKaRa) : 95 5 min , 94 30 s, 54 30 s, 72 30 s, 5101520 2530 , 72 8 min 1.2.5 杂交反应 DNA(ssDNA) PB , ( DNA ) ( 20 min), Tris-HCl (pH 7.1) 1.2.6 电化学信号检测 Co (phen) 3 (ClO 4 ) 3 ( 0.12 mol/ L) Tris-HCl (pH 7.1), + 15 V , 1 min , pH7.1 Tris-HCl pH7.1 Tris-HCl , (CV), 0.2~+0.6 V , 0.1 V/s , Au ssDNA/Au dsDNA , 2 2.1 Co(phen) 3 (ClO 4 ) 3 (AET) (SAM)  , DNA DNA (SAM), EDC NHS Na 2 HPO 4 NaH 2 PO 4 pH7.0 (PB), EDC NHS , , Tris-HCl , Co(phen) 3 (ClO 4 ) 3 , ( 1),   "
ABSTRACT: Listeria monocytogenes (LM) is a food-borne pathogen inducing listeriosis, an illness characterized by encephalitis, septicaemia, and meningitis. Listeriolysin O (LLO) is absolutely required for virulence by L. monocytogenes, and is found only in virulent strains of the species. One of the best ways to detect and confirm the pathogen is detection of one of the virulence factors, LLO, produced by the microorganism. This paper focused on the electrical method used to detect the LLO toxin gene in food products and organism without labeling the target DNA. The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto the gold electrode with the mercaptan activated by N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N'-ethyl carbodiimidehydrochloride (EDC). The hy-bridization reaction that occurred on the electrode surface was evidenced by Cyclic Voltammetry (CV) analysis using [Co(phen)3](ClO4)3 as an indicator. The covalently immobilized single-stranded DNA could selectively hybridize to its complementary DNA in solution to form double-stranded DNA on the gold surface. A significant increase of the peak cur-rent of Cyclic Voltammetry (CV) upon hybridization of immobilized ssDNA with PCR amplification products in the solu-tion was observed. This peak current change was used to monitor the amount of PCR amplification products. Factors deter-mining the sensitivity of the electrochemical assay, such as DNA target concentration and hybridization conditions, were investigated. The coupling of DNA to the electrochemical sensors has the potential of the quantitative evaluation of gene.Hereditas (Beijing) 05/2010; 32(5):512-6. DOI:10.3724/SP.J.1005.2010.00512
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- "When the voltammetric signals of [Cu(phendione)(DAP)] 2+ at bare GCE, ssDNA/GCE and dsDNA/GCE reached steady-state, the electrochemical data for the redox peaks were determined and the results showed that, compared with bare GCE, the formal potential (E ° ') at ssDNA/GCE shifted negatively, which indicated that [Cu(phendione)(DAP)] 2+ bound to ssDNA mainly via electrostatic action at electrode surface . While at dsDNA/GCE, it was found that, contrasted with the case at ssDNA/GCE, the formal potential showed a positive shift, suggesting a characteristic of intercalative mode of [Cu(phendione)(DAP)] 2+ with dsDNA . These different electrochemical responses indicated that the copper complex has the ability for the discrimination of ssDNA and dsDNA. "
ABSTRACT: Discrimination of a mix-ligand polypyridyl copper complex, [Cu(phendione)(DAP)]SO(4) (phendione=1,10-phenanthroline-5,6-dione, DAP=2,3-diaminophenazine) to single-stranded (ss-) and double-stranded (ds-) DNA is presented in this paper. UV absorption spectra suggested that the copper complex could be a typical metallointercalator for dsDNA. Surface-based electrochemical experiments showed that [Cu(phendione)(DAP)](2+) underwent different electrochemical behaviors at ssDNA and dsDNA modified GCE because of the different affinities of the copper complex with ssDNA and dsDNA, which suggested that the copper complex could be an effective discriminator for ssDNA and dsDNA in sensing analysis. The different binding parameters such as binding constant (K) and binding site size (s), and the kinetic and thermodynamic binding parameters of [Cu(phendione)(DAP)](2+) with ssDNA and dsDNA were also evaluated, further quantificationally proving the discrimination ability.Bioelectrochemistry (Amsterdam, Netherlands) 02/2009; 75(1):32-6. DOI:10.1016/j.bioelechem.2008.12.006 · 3.87 Impact Factor