Monitoring Of DNA Hybridization Research Articles

Luminescent chemosensors based on semiconductor quantum dots

Semiconductor quantum dots are inorganic nanoparticles with unique photophysical properties. In particular, their huge one- and two-photon absorption cross sections, tunable emission bands and excellent photobleaching resistances are stimulating the development of luminescent probes for biomedical imaging and sensing applications. Indeed, electron and energy transfer processes can be designed to switch the luminescence of semiconductor quantum dots in response to molecular recognition events. On the basis of these operating principles, the presence of target analytes can be transduced into detectable luminescence signals. In fact, luminescent chemosensors based on semiconductor quantum dots are starting to be developed to detect small molecules, monitor DNA hybridization, assess protein-ligand complementarities, test enzymatic activity and probe pH distributions. Although fundamental research is still very much needed to understand further the fundamental factors regulating the behavior of these systems and refine their performance, it is becoming apparent that sensitive probes based on semiconductor quantum dots will become invaluable analytical tools for a diversity of applications in biomedical research.

PAMAM Dendrimers‐Based DNA Biosensors for Electrochemical Detection of DNA Hybridization

AbstractGold electrodes were modified with submonolayers of mercaptoacetic acid (RSH) and further reacted with poly(amidoamine) (PAMAM) dendrimers (generation 4.0) to obtain thin films, on which DNA probe was later immobilized to afford a stable recognition layers. The characterization of the PAMAM/RSH‐modified electrode was investigated by cyclic voltammetry (CV) and electrochemical impedance measurement. Differential pulse voltammogram (DPV) measurement was used to monitor DNA hybridization with daunomycin (DNR) as indicator. Experiments carried out with these novel materials not only showed an improved DNA attachment quantity on the dendrimers‐modified electrodes compared to DNA sensors with oligonucleotides directly immobilized on Au electrodes, but also exhibited a high selectivity, sensitivity and stability for the measurement of DNA hybridization.

Monitoring DNA Structures by Dual Fluorescence of Pyrene Derivatives

We have developed a nucleotide modified by a pyrene derivative with dual fluorescence. The dual fluorescence of the fluorophore, which was incorporated into DNA, was effectively controlled at ambient temperature according to DNA structural status. Our nucleoside with dual fluorescence is effective as a conceptually new probe for monitoring DNA hybridization by the color change without multilabeling with fluorescent dyes.

Sensitive, Label-Free DNA Diagnostics Based on Near-Field Microwave Imaging

Biological assays often rely on "reporter labels" to enhance measurement sensitivity, for example, by incorporation of a fluorescent dye or a nanoparticle into a nucleic acid or a protein. Use of labels, however, complicates sample preparation, increases assay costs, and can cause experimental artifacts by interfering with assay thermodynamics or limiting label stability. We evaluate near-field microwave imaging (NFMI) as an alternative, label-free technique for molecular diagnostics. Using DNA monolayers as an experimental model, NFMI is demonstrated to achieve sensitivities comparable to conventional fluorescence bioassays. Moreover, NFMI is shown to be compatible with imaging at resolutions required by microarray applications, as demonstrated by monitoring DNA hybridization in an array format.

Development of a novel DNA detection system for real-time detection of DNA hybridization

Abstract With the recent development in DNA hybridization technology, it is now possible to directly analyze oligonucleotide hybridization events with a variety of DNA detection systems in a rapid and simple way. Hybridization of the immobilized oligonucleotides with target oligonucleotides in solution will lead to changes in the mass, electrical charge, or optical properties of the immobilized oligonucleotide layer, which can be detected by microgravimetric, potentiometric, amperometric or optical transducers. In this study, we propose a new DNA detection system of the probe oligonucleotide hybridization based on evanescent field method. First, we have fabricated a DNA chip microarray. The 5′-biotinylated probe oligonucleotides were immobilized on the surface of DNA chip microarray. The particles with immobilized oligonucleotides were arranged by random fluidic self-assembly on the pattern chips through hydrophobic interaction. Second, we have detected DNA hybridization using evanescent field microscopy. The hybridization with fluorescence conjugated target oligonucleotides was elicited by interaction with the evanescent field microscopy. With the evanescent field excitation and real-time detection method described here, we suggest that a very sharp discrimination of bulk fluorescence against surface excitation in combination with high excitation intensities can be achieved. Moreover, monitoring DNA hybridization in a large analytic concentration range and various mismatching conditions were also performed in a rapid and simple procedure.

A sensitive DNA electrochemical biosensor based on magnetite with a glassy carbon electrode modified by muti-walled carbon nanotubes in polypyrrole

A novel sensitive electrochemical biosensor based on magnetite nanoparticle for monitoring DNA hybridization by using MWNT–COOH/ppy-modified glassy carbon electrode is described. In this new detection system, mercapatoacetic acid (RSH)-coated magnetite nanoparticles, capped with 5′-(NH 2) oligonucleotide, is used as DNA probe to complex 29-base polynucleotide target (a piece of human porphobilinogen deaminase PBGD promoter from 170 to 142). Target sequence hybridized with the probe results in the decrease of the reduction peak current of daunomycin connected with probe. The response of non-complementary sequence was almost the same as the blank, and the response of three-base mismatched sequence within 29-base polynucleotide was obviously distinguished from complementary sequence, which can easily identify point mutation of DNA. The equation of calibration plot is i p (μA) = 0.8255 − 0.0847 c target oligonucleotide × 10 13 in the range of 6.9 × 10 −14 to 8.6 × 10 −13 mol/L, and correlation coefficient is 0.9974. The detective limit is 2.3 × 10 −14 mol/L of target oligonucleotide. This device can be optimized for the detection of complex sequence.

A biosensor monitoring DNA hybridization based on polyaniline intercalated graphite oxide nanocomposite

Polyaniline intercalated graphite oxide nanocomposite (PAI/GO) was synthesized. The microstructure of PAI/GO were investigated using Fourier transform infrared spectrometer (FT-IR), X-ray diffractometer (XRD), transmission electron microscope (TEM) and atomic force microscope (AFM). The results show that polyaniline intercalated between interlayers of GO, changes the structure of GO greatly forming nanograins in the chain and helix-like form. The resulting nanocomposite enveloped in carbon paste electrode displays electrochemical activity and two sharp peak potentials of square wave voltammometry (SWV) are at 668 and 207 mV. Such characteristics of PAI/GO have been employed to investigate the interaction between DNA and PAI/GO. The results indicate that single-strand DNA (ssDNA) and double-strand DNA (dsDNA) can change redox characteristics of PAI/GO on the PAI/GO modified carbon paste electrode (CPE) and the modified electrode has strong electrochemical effect on the redox of ssDNA and dsDNA. The SWV redox potentials in ssDNA and dsDNA solution are 90.99 and 18 mV, respectively. The PAI/GO-modified electrode immobilized with ssDNA can be utilized to monitor hybridization of complementary ssDNA with hybridization peak at −270 mV. The influence of various experimental parameters on PAI/GO-modified CPE in ssDNA solution (i.e. frequency and amplitude of SWV, pH and incubation time) was investigated. Under optimization of experimental variables, a linear relationship between the peak current ( E p is around 90.99 mV) recorded with the electrode not immobilized by ssDNA and the ssDNA concentration in solution was obtained covering range from 34 to 241 μg/mL. And the modified electrode immobilized by ssDNA can be utilized to monitor the hybridization and detect complementary ssDNA, covering linear range from 275 to 551 μg/mL. The PAI/GO-modified CPE exhibited a good stability and reproducibility for detecting ssDNA.

Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers.

The detection of specific DNA sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens. A new assay developed for this purpose is described that harnesses a sensitive electrocatalytic process to monitor DNA hybridization. Two sequences belonging to the pathogenic microbe Helicobacter pylori are used to demonstrate the versatility and specificity of the assay: one that codes for an unique H. pylori protein and one that represents a small portion of the 23S rRNA from this organism. Both sequences can be monitored into the nanomolar concentration range. Target sequences introduced to the electrode surface as synthetic oligonucleotides, PCR products, and RNA transcripts are all detected with high specificity. In addition to reporting the presence of pathogen-related sequences, this assay can accurately resolve single-base changes in target sequences. An A2143C substitution within the H. pylori rRNA that confers antibiotic resistance significantly attenuates hybridization to an immobilized probe corresponding to the WT sequence. The single-base mismatch introduced by this mutation slows the kinetics of hybridization and permits discrimination of the two sequences when short hybridization times are employed. The remarkable sensitivity of this label-free assay to small sequence changes may provide the basis of a new method for the detection and genotyping of infectious bacteria using electrochemical methods.

Electrochemical behavior and detection of hepatitis B virus DNA PCR production at gold electrode

Sequence-known short-stranded hepatitis B virus (HBV) DNA fragment (181 bps) was obtained by PCR method. The strategy for its electrochemical detection was designed by covalently immobilizing single-stranded HBV DNA on gold electrode surface via carboxylate ester as a linkage between 3′-hydroxy end of DNA and carboxyl group of thioglycolic acid (TGA) self-assembled monolayer. The hybridization reaction on surface was evidenced by electrochemical methods using ferrocenium hexafluorophosphate (FcPF 6) as an electroactive indicator. The interactions of Fc + with single-stranded (ss) and double-stranded (ds) HBV DNA immobilized on TGA monolayer were studied. The difference between the responses of Fc + at ss- and ds-DNA/Au electrodes suggested that this hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. AC impedance and XPS techniques have been employed to characterize the immobilization of ss-DNA on the gold surface.

Particle-based detection of DNA hybridization using electrochemical stripping measurements of an iron tracer

Two particle-based procedures for monitoring DNA hybridization based on electrochemical stripping detection of an iron tracer are described. The first protocol involves probes labeled with gold-coated iron core–shell nanoparticles, while the second route relies on detecting the iron content of magnetic-sphere tags. In both cases, the captured iron-containing particles are dissolved following the hybridization, and the released iron is quantified by cathodic-stripping voltammetry in the presence of the 1-nitroso-2-naphthol ligand and a bromate catalyst. Both protocols offer high sensitivity, a well-defined concentration dependence, and minimal contributions from non-complementary nucleic acids. The iron-containing particle signal amplifiers thus represent a very useful addition to the arsenal of metal tracers employed in electrical bioassays.

Monitoring DNA hybridization on alkyl modified silicon surface through capacitance measurement

Single strand oligodeoxynucleotide is attached to the alkyl modified silicon surface through a peptide bond. The oligodeoxynucleotide-modified silicon substrate is used as a working electrode in an electrochemical cell system. After the electrode is treated by a solution containing strands of complementary oligodeoxynucleotide the Mott–Schottky measurements exhibit obvious negative shift in the flat band potential of the electrode, while in a control experiment treated with a solution of non-complementary oligodeoxynucleotide such a shift does not occur. The DNA hybridization is also manifested in a real time capacitance measurement. A DNA sensor based on the capacitance measurement could be more convenient than that based on a fluorescence detection.

Hybridization biosensor using di(2,2 ′-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3 ′-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181 bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy) 2Cl 2] + as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy) 2Cl 2] + in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy) 2Cl 2] + at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.

Surface-enhanced Raman scattering detection of the breast cancer susceptibility gene BRCA1 using a silver-coated microarray platform

Surface-enhanced Raman scattering (SERS) spectroscopy was used to monitor DNA hybridization of a fragment of the BRCA1 breast cancer susceptibility gene on modified silver surfaces. Rhodamine B was covalently attached to a 5′-amino-labeled oligonucleotide sequence (23 mer) through a succinimidyl ester intermediate in methanol. The silver surfaces were prepared by depositing a discontinuous layer (9.0 nm) of silver onto glass slides, which had been etched with HF to form a microwell platform, and subsequently modified with a monolayer of mercaptoundecanoic acid. The complementary probe was covalently attached to the silver surfaces using a succinimidyl ester intermediate in acetonitrile. The silver island substrate allows a very large enhancement of the Raman signal of the DNA-Rhodamine B, and clear distinction between hybridized samples and controls on a microwell array sampling platform.

Novel Silicon Dioxide Sol−Gel Films for Potential Sensor Applications: A Surface Plasmon Resonance Study

Stable silicon dioxide (silica, SiO2) films on noble metals were synthesized using a novel sol−gel technique. Surface plasmon resonance (SPR) spectroscopy was used to compare the stability of silica films generated by this sol−gel technique with those deposited by using thermal evaporation, in the presence of PBS buffer. These films were later chemically functionalized with various reactive groups as a test for their versatility and usefulness in sensoric applications. A surface grating enclosed in a high-pressure and elevated temperature cell was used in the investigation of polymer brushes while SPR coupled with fluorescence detection schemes was used in the Kretschmann prism configuration to monitor DNA hybridization interactions.