DNA single-base mismatch study with an electrochemical enzymatic genosensor

A thorough selectivity study of DNA hybridization employing an electrochemical enzymatic genosensor is discussed here. After immobilizing on a gold film a 30-mer 3'-thiolated DNA strand, hybridization with a biotinylated complementary one takes place. Then, alkaline phosphatase is incorporated to the duplex through the interaction streptavidin-biotin. Enzymatic generation of indigo blue from 3-indoxyl phosphate and subsequent electrochemical detection was made. The influence of hybridization conditions was studied in order to better discern between fully complementary and mismatched strands. Detection of 3, 2 and 1 mismatch was possible. The type and location of the single-base mismatch, as well as the influence of the length of the strands was studied too. Mutations that suppose displacement of the reading frame were also considered. The effect of the concentration on the selectivity was tested, resulting a highly selective genosensor with an adequate sensitivity and stability.     

DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations.The method is based on the formation and deformation of double-stranded DNA(dsDNA)membranes on a gold surface.It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface.In these systems,Fe(CN)63- was used as the reporter.As the temperature increases,a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears.At a special temperature,the and single base mutation target.Thus,the system provides a simple and sensitive method to detect DNA point mutations without labeling targets.     

DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations. The method is based on the formation and deformation of double-stranded DNA (dsDNA) membranes on a gold surface. It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface. In these systems, Fe(CN)₆ ³⁻ was used as the reporter. As the temperature increases, a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears. At a special temperature, the method gives 100:1 selectivity for the perfect complement and single base mutation target. Thus, the system provides a simple and sensitive method to detect DNA point mutations without labeling targets. __________ Translated from Acta Biophysica Sinica 2005, 21 (2) [译 自: 生物物理学报, 2005,21(2)]     

Detection of single-base DNA mutations by enzyme-amplified electronic transduction

Here we describe a method for the sensitive detection of a single-base mutation in DNA. We assembled a primer thiolated oligonucleotide, complementary to the target DNA as far as one base before the mutation site, on an electrode or a gold-quartz piezoelectric crystal. After hybridizing the target DNA, normal or mutant, with the sensing oligonucleotide, the resulting assembly is reacted with the biotinylated nucleotide, complementary to the mutation site, in the presence of polymerase. The labeled nucleotide is coupled only to the double-stranded assembly that includes the mutant site. Subsequent binding of avidin-alkaline phosphatase to the assembly, and the biocatalyzed precipitation of an insoluble product on the transducer, provides a means to confirm and amplify detection of the mutant. Faradaic impedance spectroscopy and microgravimetric quartz-crystal microbalance analyses were employed for electronic detection of single-base mutants. The lower limit of sensitivity for the detection of the mutant DNA is 1 x 10-14 mol/ml. We applied the method for the analysis of polymorphic blood samples that include the Tay-Sachs genetic disorder. The sensitivity of the method enables the quantitative analysis of the mutant with no PCR pre-amplification.     

Optimization of DNA immobilization on gold electrodes for label-free detection by electrochemical impedance spectroscopy

The ability to immobilize DNA probes onto gold substrates at an optimum surface density is key in the development of a wide range of DNA biosensors. We present a method to accurately control probe DNA surface density by the simultaneous co-immobilization of thiol modified probes and mercaptohexanol. Probe surface density is controlled by the thiol molar ratio in solution, with a linear relationship between thiol molar ratio and probe density spanning (1-9) x10(12)/cm2. The probe surface density per microscopic surface area was determined using chronocoulometry, and a detailed analysis of the method presented. Using this sample preparation method, the effect of probe density and hybridization on the charge transfer resistance with the negatively charged ferri/ferrocyanide redox couple was determined. Above a threshold probe surface density of 2.5 x 10(12)/cm2, electrostatic repulsion from the negatively charged DNA modulates the charge transfer resistance, allowing hybridization to be detected. Below the threshold density no change in charge transfer resistance with probe density or with hybridization occurs. The probe surface density was optimized to obtain the maximum percentage change in charge transfer resistance with hybridization.     

Characterization of antimicrobial peptide activity by electrochemical impedance spectroscopy

Electrochemical impedance spectroscopy performed on surface-supported bilayer membranes allows for the monitoring of changes in membrane properties, such as thickness, ion permeability, and homogeneity, after exposure to antimicrobial peptides (AMPs). We show that two model cationic peptides, very similar in sequence but different in activity, induce dramatically different changes in membrane properties as probed by impedance spectroscopy. Moreover, the impedance results excluded the “barrel-stave” and the “toroidal pore” models of AMP mode of action, and are more consistent with the “carpet” and the “detergent” models. The impedance data provide important new insights about the kinetics and the scale of the peptide action which currently are not addressed by the “carpet” and the “detergent” models. The method presented not only provides additional information about the mode of action of a particular AMP, but offers a means of characterizing AMP activity in reproducible, well-defined quantitative terms.     

Microbial degradation of polymeric coatings measured by electrochemical impedance spectroscopy

This paper reports results of biodegradation studies of polyimide coatings exposed to a mixed fungal culture using electrochemical impedance spectroscopy (EIS). The fungal consortium was originally isolated from degraded polyimides and identified species include Aspergillus versicolor, Cladosporium cladosporioides, and a Chaetomium species. Actively growing fungi on polyimides yield distinctive EIS spectra through time, indicative of failure of the polymer integrity compared to the uninoculated controls. An initial decline in coating resistance was related to the partial ingress of water molecules and ionic species into the polymeric matrices. This was followed by further degradation of the polymers by activity of the fungi. The relationship between the changes in impedance spectra and microbial degradation of the coatings was further supported by scanning electron microscopy, showing extensive colonization of the polyimide surfaces by the fungi. Our data indicate that EIS can be a sensitive and informative technique for evaluating the biosusceptibility of polymers and coatings.     

Digestion of restriction enzyme for the detection of single-base mismatch in DNA

DNA hybridization and enzymatic digestion for the detection of mutation was investigated on the gold nanoparticles-calf thymus DNA (AuNPs-ctDNA) modified glassy carbon electrode (GCE). The thiol modified probe oligonucleotides (SH-ssDNA) were assembled on the surface of AuNPs-ctDNA modified GCE. The electrochemical response of the electrode was measured by differential pulse voltammetry and cyclic voltammetry. Methylene blue (MB) was used as the electroactive indicator. AuNPs were then dispersed effectively on the GCE surface in the presence of ct-DNA. When hybridization occurred, a decrease in the signal of MB current was observed. The modified electrode was used for the detection of mutations during the enzymatic digestion reaction in DNA. During this reaction, an increase in the signal of MB current was observed. So, the modified SH-ssDNA had a higher electrochemical response on the AuNPs-ctDNA/GCE because of the strong affinity of MB for guanine residues in it. The electrochemical detection of restriction enzyme digestion can provide a simple and practical method for observing single-base mismatches that can help in distinguishing mismatch sequences of DNA from the complementary ones.     

Fold-back structures at the distal end influence DNA slippage at the proximal end during mononucleotide repeat expansions.

Polymerase slippage during DNA synthesis by the Klenow fragment of DNA polymerase across A, C, G and T repeats (30 bases) has been studied. Within minutes, duplexes that contain only repeats (30 bp) expand dramatically to several hundred base pairs long. Rate comparisons in a repeat duplex when one strand was expanded as against that when both strands were expanded suggest a model of migrating hairpin loops which in the latter case coalesce into a duplex. Moreover, slippage (at the proximal or 3'-end) is subject to positive and negative effects from the 5'-end (distal) of the same strand. Growing T and G strands generate T.A:T and G-G:C motif fold-back structures at the distal end that hamper slippage at the proximal end. On the other hand, growing tails at the distal end upon annealing with excess complementary template accentuates proximal slippage several-fold.     

Specific oligonucleotide invasion into the end of DNA duplex

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: First, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 μF/cm² for DPhyPC bilayers and 0.75 and 0.77 μF/cm² for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 MΩ cm² were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Specific oligonucleotide invasion into an end of a DNA duplex

A new phenomenon was described: a double-stranded DNA fragment interacted with a single-stranded oligonucleotide complementary to the terminal region of one strand of the duplex to yield a complex with oligonucleotide invasion. Generation of Holliday junctions by homologous linear DNA fragments was less efficient in the presence of single-stranded oligonucleotides complementary to duplex ends. The effect depended on the oligonucleotide concentration, size, and complementarity to a duplex strand. Sequence-specific complexes with single strand invasion were detected in mixtures containing radiolabeled oligonucleotides and duplexes. A single-stranded oligonucleotide invaded a duplex even when its concentration was far lower than the duplex concentration. Complexes with single strand invasion were analyzed by chemical cleavage of noncanonical base pairs. Analysis showed that an oligonucleotide interacts with the complementary region of one strand of the duplex, gradually displacing the other strand. The extent of oligonucleotide invasion into the duplex considerably varied. Oligonucleotide invasion into duplexes became more efficient with increasing oligonucleotide size.     

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch.     

AC impedance spectroscopy of native DNA and M-DNA

Monolayers of thiol-labeled DNA duplexes of 15, 20, and 30 basepairs were assembled on gold electrodes. Electron transfer was investigated by electrochemical impedance spectroscopy with Fe(CN)(6)(3-/4-) as a redox probe. The spectra, in the form of Nyquist plots, were analyzed with a modified Randles circuit which included an additional component in parallel, R(x), for the resistance through the DNA. For native B-DNA R(x) and R(ct), the charge transfer resistance, both increase with increasing length. M-DNA was formed by the addition of Zn(2+) at pH 8.6 and gave rise to characteristic changes in the Nyquist plots which were not observed upon addition of Mg(2+) or at pH 7.0. R(x) and R(ct) also increased with increasing duplex length for M-DNA but both were significantly lower compared to B-DNA. Therefore, electron transfer via the metal DNA film is faster than that of the native DNA film and certain metal ions can modulate the electrochemical properties of DNA monolayers. The results are consistent with an ion-assisted long-range polaron hopping mechanism for electron transfer.     

2'-anthraquinone-conjugated oligonucleotide as an electrochemical probe for DNA mismatch

We previously prepared the oligonucleotides (ODNs) conjugated to an anthraquinone (AQ) group via one carbon linker at the 2'-sugar position. When these modified ODNs bind to cDNA sequences, the AQ moiety can be intercalated into the predetermined base-pair pocket of a duplex DNA. In this paper, 2'-AQ-modified ODNs are shown to be an excellent electrochemical probe to clarify the effect of a mismatch base on the charge transfer (CT) though DNA. Two types of DNA-modified electrodes were constructed by assembly of disulfide-terminated 2'-AQ-ODN duplexes onto gold electrodes. One type of electrodes (system I) contains fully matched base pairs or a single-base mismatch in duplex DNA between the redox center and the electrode. The other (system II) consists of the mismatch but at the outside of the redox center. The modified electrodes were analyzed by cyclic voltammetry to estimate the CT rate through duplex DNA. In system I, the CT rate was found to be approximately 50 s (-1) for the fully matched AQ-ODN duplexes, while the CT rates of the mismatched DNA were considerably slower than that of the fully matched DNA. In system II, the AQ-ODN duplexes showed almost similar CT rates ( approximately 50 s (-1)) for the fully matched DNA and for the mismatched DNAs. The detection of a single-base mismatch was then performed by chronocoulometry (CC). All the DNA duplexes containing a mismatch base in system I gave the reduced electrochemical responses when compared to the fully matched DNA. In particular, the mismatched DNAs including G--A mismatch can be differentiated from fully matched DNA without using any electrochemical catalyst. We further tested the usefulness of single-stranded (ss) AQ-ODN immobilized on a gold electrode in the electrochemical detection of a single-base mismatch through hybridization assay. The ss-AQ-ODN electrodes were immersed in target-containing buffer at room temperature, and the CC measurements were carried out to see the changes in the integrated charge. Within 60 min, the mismatched DNA was clearly distinguishable by the CC differences from the fully matched target. Thus, the electrochemical hybridization assay provides an easy and convenient detection for DNA mutation that does not require any extra reagents, catalyst, target labeling, and washing steps.     

Immobilization of rhodopsin on a self-assembled multilayer and its specific detection by electrochemical impedance spectroscopy

Rhodopsin, the G protein-coupled receptor (GPCR) which mediates the sense of vision, was prepared from calf eyes and used as receptor enriched membrane fraction. In this study it was immobilized onto gold electrode by two different techniques: Langmuir-Blodgett (LB) and a strategy based on a self-assembled multilayer. We demonstrated that Langmuir and LB films of rhodopsin are not stable. Thus, in this study a new protein multilayer was prepared on gold electrode by building up layer-by-layer a self-assembled multilayer. It is composed of a mixed self-assembled monolayer formed by MHDA and biotinyl-PE, followed by a biotin-avidin system which allows binding of biotinylated antibody specific to rhodopsin. The immobilization of rhodopsin in membrane fraction, by the specific antibody bound previously on self-assembled multilayer, was monitored with electrochemical impedance spectroscopy (EIS). In addition, the specificity and sensitivity of this self-assembled multilayer system to the presence of rhodopsin were investigated. No effect was observed when the system was in contact with olfactory receptor I7 in membrane fraction used for control measurements. All these results demonstrate that rhodopsin can be immobilized efficiently, specifically, quantitatively and stably on gold electrode through the self-assembled multilayer.     

Monitoring of cell growth and assessment of cytotoxicity using electrochemical impedance spectroscopy

Electrochemical impedance spectroscopy (EIS) was used to monitor the growth of mammalian cancer cells and evaluate the cytotoxicity of chemicals using Fe(CN)6(3-/4-) as a redox probe. Cancer cells, the human hepatocarcinoma cell line (BEL7404), were grown on optically transparent indium tin oxide (ITO) semiconductor slides, which were used as the working electrodes in electrochemical experiments. Attachment and proliferation of cancer cells on ITO surfaces resulted in increase of electron-transfer resistance (R(et)) between the redox probe of Fe(CN)6(3-/4-) in electrolyte solution and ITO electrode surface. For cytotoxicity assessment, cells grown on ITO substrates were further cultured in the presence of different cytotoxicants and electrochemical impedance measurements were carried out at different time intervals. Gemcitabine, a promising antineoplastic drug showing activity against a wide spectrum of human solid tumors, was selected as a model for long-term cytotoxicity effect study, whereas mercury chloride represented a model for acute toxicants. The inhibitions of gemcitabine and mercury chloride on the viability and proliferation of BEL7404 cells were observed from the electrochemical impedance experiments, and the different action modes were discriminated. Additionally, microscope images were also used to observe the effects of these two chemicals on the morphology of the cells. General consistency has been found between the electrochemical impedance response and the morphological observation. Such an impedance method provides a simple and inexpensive way for in vitro assessment of chemical cytotoxicity.     

S1 nuclease does not cleave DNA at single-base mis-matches

Three assays have been designed to detect the cleavage of duplex phi X174 DNA at single-base mis-matches. Studies with S1 nuclease failed to detect cleavage at mis-matches. S1 nuclease digestion at 37 and 55 degrees C failed to produce a preferential degradation of a multiply mis-matched heteroduplex when compared to a mis-match-free homo-duplex as analyzed by sedimentation on sucrose gradients. Other heteroduplex templates were not cleaved by S1 nuclease at a defined single-base mis-match when assayed by gel electrophoresis or by marker rescue. In all cases, the amount of S1 nuclease employed was at least 10-times more than that required to render a single-stranded phi X174 DNA molecule completely acid soluble. The rate of hydrolysis of single-base mis-matches by S1 nuclease was estimated to be less than 0.016% of the rate at a base in single-strand phi X174 DNA. In no instance did we detect activity by S1 nuclease directed at mis-matched sites in our template molecules. Similarly, the single-strand specific endonuclease from Neurospora crassa does not cleave heteroduplex templates at a defined single-base mis-match when assayed by marker rescue.     

Solution structure of an oncogenic DNA duplex containing a G.A mismatch

The DNA duplex 5'-d(GCCACAAGCTC).d(GAGCTGGTGGC), which contains a central G.A mismatch has been studied by one and two-dimensional NMR techniques. The duplex corresponds to the sequence 29-39 of the K-ras gene. The mismatch position is that of the first base of the Gly12 codon, a hot spot for mutations. The observed NOEs of the nonexchangeable protons show that both of the bases of the mismatched pair are intrahelical over a wide range of pH. However, the structure of the G.A mispair and the conformation of the central part of the duplex change with pH. This structural change shows a pK of 6.0. At low pH, the G.A bases are base paired with hydrogen bonds between the keto group of the G residue and the amino group of the A residue and, secondly, between the N7 of the G and a proton on N1 of A. This causes the G residue to adopt a syn conformation. On raising the pH, the N1-H proton of the protonated A residue is removed, and the base pair rearranges. In the neutral G.A base pair both residues adopt an anti conformation, and the mismatch is stabilized by hydrogen bonds. Our results on the exchangeable and A(H2) protons of the mismatched pair indicate a shift from a classical face-to-face two hydrogen-bonded structure to a slipped structure stabilized by bifurcated hydrogen bonds. This may be a particular characteristics of this oncogenic sequence in which the G.A error is poorly repaired.