Hybridization by an Electrical Force and Electrochemical Genome Detection Using an Indicator-free DNA on a Microelectrode-array DNA Chip

Detection of disease-related gene expression by DNA hybridization is a useful diagnostic method. In this study a monolayer graphene field effect transistor (GFET) was fabricated for the detection of a particular single-stranded DNA (target DNA). The probe DNA, which is a single-stranded DNA with a complementary nucleotide sequence, was directly immobilized onto the graphene surface without any linker. The VDirac was shifted to the negative direction in the probe DNA immobilization. A further shift of VDirac in the negative direction was observed when the target DNA was applied to GFET, but no shift was observed upon the application of non-complementary mismatched DNA. Direct immobilization of double-stranded DNA onto the graphene surface also shifted the VDirac in the negative direction to the same extent as that of the shift induced by the immobilization of probe DNA and following target DNA application. These results suggest that the further shift of VDirac after application of the target DNA to the GFET was caused by the hybridization between the probe DNA and target DNA.

A direct electrochemical detection procedure for DNA hybridization by using the electrochemical signal changes of conductive poly(m-aminobenzenesulfonic) acid (PABSA)/TiO(2) nanosheet membranes, which were electropolymerized by using the pulse potentiostatic method, is reported. Due to the unique properties of TiO(2) nanoparticles, m-aminobenzenesulfonic acid monomers tend to be adsorbed around the particles, and the electropolymerization efficiency is greatly improved. The combination of TiO(2) nanoparticles and PABSA resulted in a nanocomposite membrane with unique and novel nanosheet morphology that provides more activation sites and enhances the surface electron-transfer rate. These characteristics were propitious for the magnification of PABSA electrochemical signals and the direct detection of DNA hybridization. Owing to the presence of abundant sulfonic acid groups, PABSA could overcome the drawbacks of polyaniline and be used to detect bioanalytes at physiological pH. DNA probes could be covalently attached to the sulfonic groups through the amines of DNA sequences by using an acyl chloride cross-linking reaction. After immobilization of probe DNA, the electrochemical impedance value increased significantly compared to that of PABSA/TiO(2) nanosheet membranes, and then decreased dramatically after the hybridization reaction of the probe DNA with the complementary DNA sequence compared to that of the probe-immobilized electrode. Electrochemical impedance spectroscopy was adopted for indicator-free DNA biosensing, which had an eminent ability for the recognition between double-base mismatched sequences or non-complementary DNA sequences and complementary DNA sequences. A gene fragment, which is related to one of the screening genes for the transgenically modified plants, the cauliflower mosaic virus 35S gene was satisfactorily detected. This is the first report for the indicator-free impedance DNA hybridization detection by using PABSA/TiO(2) membranes under neutral conditions.

This research aims to develop the multiple channel electrochemical DNA chip using microfabrication technology. At first, we fabricated a high integration type DNA chip array by lithography technology. Several probe DNAs consisting of thiol group at their 5-end were immobilized on the gold electrodes. Then target DNAs were hybridized and reacted. Cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. Therefore, it is able to detect a plural genes electrochemically after immobilization of a plural probe DNA and hybridization of non-labeling target DNA on the electrodes simultaneously. It suggested that this DNA chip could recognize the sequence specific genes.

A biocompatible and uniform interface based on silica nanoparticles derivatized with amino groups has been constructed for the effective immobilization and sensitive sequence-specific detection of calf thymus DNA. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) results showed that a monolayer of silica nanoparticles can be formed on a gold electrode under our experimental conditions using cysteine self-assembly monolayer as binder medium. Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy (XPS) verified the successful immobilization of DNA on silica-nanoparticle-modified gold electrodes. Quantitative results demonstrated that enhanced immobilization of single-strand DNA (ss-DNA) up to 1.6 x 10(-8) mol cm(-2) could be achieved owing to the larger surface area and the special properties of silica nanoparticles. In addition, hybridization experiments demonstrated that the immobilized ss-DNA on silica nanoparticles could specifically interact with complementary DNA in solutions.

The visual detection of specific double-stranded DNA sequences possesses great potential for the development of diagnostics. Zinc finger domains provide a powerful scaffold for creating custom DNA-binding proteins that recognize specific DNA sequences. We previously demonstrated sequence-enabled reassembly of TEM-1 β-lactamase (SEER–LAC), a system consisting of two inactive fragments of β-lactamase each linked to engineered zinc finger proteins (ZFPs). Here the SEER–LAC system was applied to develop ZFP arrays that function as simple devices to identify bacterial double-stranded DNA sequences. The ZFP arrays provided a quantitative assay with a detection limit of 50 fmol of target DNA. The method could distinguish target DNA from non-target DNA within 5 min. The ZFP arrays provided sufficient sensitivity and high specificity to recognize specific DNA sequences. These results suggest that ZFP arrays have the potential to be developed into a simple and rapid point-of-care (POC) diagnostic for the multiplexed detection of pathogens.

Sequence-specific detection and quantification of nucleic acids are central steps in many molecular biology procedures which have also been transferred to chip-based procedures. Hybridization-based assays can be used to quantify and discriminate between DNA target sequences down to the level of single base mismatches. Arrays of DNA probes immobilized on a support enable simultaneous testing of multiple sequences of a single sample. DNA arrays can be produced either by in-situ synthesis of oligonucleotides or by immobilization of pre-assembled DNA probes. Covalent and directed immobilization improves the reproducibility and stability of DNA arrays. This is especially interesting with repeated use of transducers or chips. Procedures are described for effective covalent immobilization of pre-assembled amino-linked oligonucleotides, by use of ink-jet techniques, on a modified and heated glass surface, with addressable surface areas ranging from 0.01 mm2 to a few mm2. Almost immediate evaporation of the spotted droplets on the heated surfaces leads to very high surface hybridization capacities. The surfaces are suitable for use with a label-free detection method - reflectometric interference spectroscopy (RIfS). It is shown that hybridization capacity and non-specific interaction at these DNA-surfaces can be characterized by use of RIfS. With a consumption of less than 80 ng mm(-2) oligonucleotide and a specific hybridization capacity of more than 300 fmol mm(-2), the activated aminodextran procedure was usually suitable for setting up a DNA array with label-free detection. Non-specific interactions with random oligomers or protein (ovalbumin) were low. Up to 150 repeated regenerations (stripping) of the surfaces by acid treatment and denaturing agents, and 50 days of storage, have been possible without significant loss of hybridization capacity.

A morphologically complex cellulosic substance (e.g., commercial filter paper) was employed as a substrate for DNA immobilization and successive recognition. A uniform ultrathin zirconia gel film was first deposited on each cellulose nanofiber in bulk filter paper by a facile sol-gel process. Relying on the large surface area of filter paper and the strong affinity of zirconia for the phosphate group, terminal-phosphate probe DNA was abundantly immobilized on the zirconia-modified filter paper so as to convert the composite to a biofunctional material for the sensitive and repetitive recognition of the corresponding complementary target DNA on the nanomolar level. By contrast, in spite of the viability of the immobilization of the probe DNA and the recognition of target DNA on the quartz plate, the amount of captured probe DNA or recognized target DNA on such a flat substrate was much less than that captured or recognized on filter paper, resulting in a relatively insensitive recognition event. Moreover, control experiments on bare filter paper (without a zirconia nanocoating) suggested that the zirconia gel film was essential to probe DNA immobilization and subsequent target DNA recognition.

A label-free electrochemical biosensor for highly sensitive and selective detection of DNA via a dual-amplified strategy

Development of an ultrasensitive electrochemical genosensor for detection of HIV-1 pol gene using a gold nanoparticles coated carbon paste electrode impregnated with lead ion-imprinted polymer nanomaterials as a novel electrochemical probe

DNA microarrays have the potential to revolutionize medical diagnostics and development of individualized medical treatments. However, accurate quantification of scantily expressed genes and precise measurement of small differences between different treatments is not currently feasible. A major challenge remains the understanding of physicochemical processes and rate-limiting steps of hybridization of complex mixtures of DNA targets on immobilized DNA probes. To this end, we developed a mathematical model to describe the effects of molecular orientation and transport on the kinetics and efficiency of hybridization. First, we calculated the hybridization rate constant based on the distance between the complementary nucleotides of the target and probe DNA. The surface reaction rate was then integrated with translational and rotational transport of target DNA to the surface to calculate the kinetics of hybridization. Our model predicts that hybridization of short DNA targets is diffusion limited but long targets are kinetically limited. In addition, for DNA targets with wide size distribution, it may be difficult to distinguish between specific binding of long targets from nonspecific binding of short ones. Our model provides novel insight into the process of DNA hybridization and suggests operating conditions to improve the sensitivity and accuracy of microarray experiments.

Microfluidic system for DNA sequence detection

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated32P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the32P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the32P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.

A proof of concept procedure for the electroaddressable covalent immobilization of DNA and protein on arrayed electrodes along with simultaneous detection of multiple bioagents in the same sample solution is described. Carboxyphenyldiazonium was selectively deposited onto five of nine individually addressable electrodes in an array via bias assisted assembly. Amine functionalized DNA probes were covalently coupled to the carboxyl surface via carbodiimide chemistry. This was followed by the covalent immobilization of diazonium-antibody conjugates into the remaining four electrodes via cyclic voltammetry. Simultaneous electrochemical detection of a DNA sequence related to the breast cancer BRCA1 gene and the human cytokine protein interleukin-12, which is a substantial component in the immune system response and attack of tumor cells, is reported. These results demonstrate the possibility of selective patterning of diverse biomolecules on a single device and may have significant implications for future development of microarrays and biosensors.

The present work describes the fabrication, characterization, and optimization of NH2-derivatized polymer coatings prepared by pulsed-plasma polymerization for applications as adhesion layers in DNA immobilization. Fourier transform infrared spectroscopy and surface plasmon resonance spectroscopy were used to study (i) polymer matrix properties and (ii) oligonucleotide/DNA binding. The successful DNA attachment on amine functionalized surfaces was found to depend on the macromolecular architecture of the plasma films and on the amine group densities. Pulsed and continuous wave plasma polymers deposited at similar equivalent power showed comparable immobilization properties, while low duty cycle plasma-polymerized films showed a higher sensitivity toward DNA binding than high duty cycle plasma-deposited films. The stabilities of the various films in buffer solution and their reactivities as they were affected by variations in pH have been investigated.

Detection of DNA hybridization and extension reactions by an extended-gate field-effect transistor: Characterizations of immobilized DNA–probes and role of applying a superimposed high-frequency voltage onto a reference electrode