共查询到20条相似文献,搜索用时 15 毫秒
1.
DNA sensors and sensor arrays (biochips) have become an important tool in molecular biology and biotechnology in recent years. For low-throughput, easy-to-use devices it is desirable that they be of low cost, reagentless, and label-free. Displacement sensors with electrochemical detection offer these advantages, and therefore the development of such a detection principle is show in this work. An HRP-labeled oligonucleotide was sub-optimally pre-hybridized with a capture probe and was displaced upon introduction of the fully complementary probe target, producing a decrease in signal that was proportional to the sample concentration. This detection scheme has been demonstrated colorimetrically and electrochemically, obtaining a total signal displacement of 55% only 5 min after introduction of the sample. 相似文献
2.
Liangliang Huang Xiaojuan Yang Cui Qi Xiaofang Niu Chunling Zhao Xiaohui Zhao Dihua Shangguan Yunhui Yang 《Analytica chimica acta》2013
In order to develop a sensor for opium alkaloid codeine detection, DNA aptamers against codeine were generated by SELEX (systematic evolution of ligands by exponential enrichment) technique. An aptamer HL7-14, which is a 37-mer sequence with Kd values of 0.91 μM, was optimized by the truncation-mutation assay. The specificity investigation shows that HL7-14 exhibits high specificity to codeine over morphine, and almost cannot bind to other small molecule. With this new selected aptamer, a novel electrochemical label-free codeine aptamer biosensor based on Au-mesoporous silica nanoparticles (Au-MSN) as immobilized substrate has been proposed using [Fe(CN)6]3−/4− as electroactive redox probe. The linear range covered from 10 pM to 100 nM with correlation coefficient of 0.9979 and the detection limit was 3 pM. Our study demonstrates that the biosensor has good specificity, stability and well regeneration. It can be used to detect codeine. 相似文献
3.
Aysegul Uygun 《Talanta》2009,79(2):194-198
A simple and label-free electrochemical sensor for recognition of the DNA sensor event was prepared by electrochemical polymerization of 4-hydroxyphenyl thiophene-3-carboxylate. Poly(4-hydroxyphenyl thiophene-3-carboxylate) (PHPT) was synthesized electrochemically onto glassy carbon electrode and characterized by cyclic voltammetry, FTIR and AFM measurements. An ODN-probe was physisorbed onto PHPT film and tested on hybridization with complementary ODN segments. A biological recognition can be monitored by comparison with electrochemical signal (cyclic voltammogram) of single and double strand state oligonucleotide. The oxidation current of double strand state oligonucleotide is lower than that of single strand, that is corresponding to the decrease of electroactivity of PHPT with the increase of stiffness of polymer structure. Physisorbed ODN-probe and its hybridization were observed morphologically onto ITO electrodes using AFM. The sensitivity of the electrochemical sensor is 0.02 μA/nmol, detection limit is 1.49 nmol and it has good selectivity. 相似文献
4.
Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection 总被引:5,自引:0,他引:5
A novel and sensitive electrochemical DNA biosensor based on multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH) for covalent DNA immobilization and enhanced hybridization detection is described. The MWNTs-COOH-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides with the 5'-amino group were covalently bonded to the carboxyl group of carbon nanotubes. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using an electroactive intercalator daunomycin as an indicator. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity. This is the first application of carbon nanotubes to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization. 相似文献
5.
Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene. 相似文献
6.
One of the most time consuming and complex steps in the detection of DNA target with a biosensor is the previous labeling of the target. In this paper, a novel target label-free, reagentless and easy to use DNA biosensor is reported. Electrochemical transduction (cyclic voltammetry, differential pulse voltammetry and impedance spectroscopy) and optical red out by surface plasmon resonance were chosen for the platform optimization. This target label-free DNA detection method is based on displacement of sub-optimum labeled oligonucleotide. This strategy requires the pre-hybridization of the capture probe immobilized on the electrode surface with a sub-optimum mutated oligonucleotide pre-labeled with an electrochemically active ferrocene moiety. Due to the higher affinity of the target that is fully complementary to the capture probe, the sub-optimum ferrocene-labeled sequence is displaced when the fully complementary target is introduced into the system. The decrease of the electrochemical signal from the ferrocene verifies the presence of the target, which is proportional to the target concentration. A variation of this strategy was employed to enhance the ferrocene signal. A diffusional mediator, ferrocyanide, was introduced in the system to help in this purpose. This platform attains a stable, specific and reproducible response (5-15%), with a detection limit in the range of microM. This electrochemical sensor is the first example of this kind of sensor to detect cystic fibrosis, however, this configuration could be generically applied to any application where the detection of a DNA target is involved. 相似文献
7.
This paper described a novel electrochemical DNA biosensor for rapid specific detection of nucleic acids based on the sulfonated polyaniline (SPAN) nanofibre and cysteamine-capped gold nanoparticle (CA-GNP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-GNP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-GNP were alternately layer-by-layer assembled on the stable substrate by electrostatic force. Cyclic voltammetry was used to monitor the consecutive growth of the multilayer films by utilizing [Fe(CN)6]3−/4− as the redox indicator. The (CA-GNP/SPAN)n films showed satisfactory ability of electron transfer and excellent redox activity in neutral media. Negatively charged probe ssDNA was immobilized on the outer layer of the multilayer film (CA-GNP) through electrostatic affinity. Chronopotentiometry and electrochemical impedance spectroscopy were employed to obtain the direct electrochemical readout for probe ssDNA immobilization and hybridization using [Fe(CN)6]3−/4− in solution as the mediator. While electrochemical impedance spectroscopy led to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provided the total resistance at the interfaces of the modified electrodes. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports was found. Chronopotentiometry was suggested as a rapid transduction means (a few seconds). Based on the (CA-GNP/SPAN)n films, the target DNA with 20-base could be detected up to 2.13 × 10−13 mol/L, and the feasibility for the detection of base-mismatched DNA was also demonstrated. 相似文献
8.
The preparation and attractive performance of carbon-nanotube modified glassy-carbon (CNT/GC) electrodes for improved detection of purines, nucleic acids, and DNA hybridization are described. The surface-confined multiwall carbon-nanotube (MWCNT) facilitates the adsorptive accumulation of the guanine nucleobase and greatly enhances its oxidation signal. The advantages of CNT/GC electrodes are illustrated from comparison to the common unmodified glassy carbon, carbon paste and graphite pencil electrodes. The dramatic amplification of the guanine signal has been combined with a label-free electrical detection of DNA hybridization. Factors influencing the enhancement of the guanine signal are assessed and optimized. The performance characteristics of the amplified label-free electrochemical detection of DNA hybridization are reported in connection to measurements of nucleic-acid segments related to the breast-cancer BRCA1 gene. 相似文献
9.
An electrochemical DNA hybridization biosensor was developed for the detection of DNA hybridization using MDB and proflavine
as electrochemical labels. The biosensor was based on the interaction of 7-dimethyl-amino-1,2-benzophenoxazi-nium Meldola’s
Blue (MDB) and proflavine with double stranded DNA (dsDNA) The electrochemical behaviour of MDB and proflavine as well as
its interaction with double stranded (dsDNA) were investigated by cyclic (CV) and square wave voltammetry (SWV) and screen
printed electrodes (ScPE). Furthermore, DNA-hybridization biosensors were developed for the detection of hybridization between
oligonucleotides, which was detected by studying changes in the voltammetric peaks of MDB (reduction peak at −0.251 V) and
proflavine (reduction peak at 0.075 V). MDB and proflavine were found to intercalate between the base pairs of dsDNA and oligonucleotides.
Several factors affecting the dsDNA or oligonucleotides immobilization, hybridization and indicator preconcentration and interaction
time, were investigated. As a result of the interaction of MDB with dsDNA and hybridized oligonucleotides, the voltammetric
signals of MDB increased. Furthermore, guanine’s oxidation peak (at 0.901 V) was decreased as MDB’s concentration was increased.
As a result of the interaction of proflavine with dsDNA and hybridized oligonucleotides, the voltammetric signals of proflavine
decreased. These results were similar for carbon paste and screen printed electrodes. A comparison of the performance between
CPE and ScPE was done. Our results showed that lower concentrations of MDB and proflavine were detected using screen printed
electrodes. Moreover, reproducibility was better using screen printed electrodes and the detection was faster (regarding the
experimental steps), but they are more cost effective.
相似文献
10.
A label-free electrochemical biosensor for detecting DNA hybridisation was developed by monitoring the change in the voltammetric
activity of ferrocenecarboxylic acid at the biosensor–solution interface. The biosensor was constructed by initially immobilising
on a glassy carbon electrode an anchoring layer consisting of chitosan, carboxyl group functionalised carbon nanofibres and
glutaraldehye. Chitosan acted as an adhering agent and carbon nanofibres were strategically used to provide a large surface
area with binding points for DNA immobilisation, while glutaraldehye was a linker for DNA probes on the electrode surface.
Based on a two-factorial design, cyclic voltammetry of [Fe(CN)6]3−/4− was performed to optimise the composition of the anchoring layer. Next, a 17-base pair DNA probe was attached to the anchoring
layer, followed by its complementary target. Zr(IV) ion, known to exhibit affinity for oxygen-containing electroactive markers,
for example, ferrocenecarboxylic acid, was then coordinated in the DNA duplex. In this way, ferrocenecarboxylic acid was attracted
towards the biosensor for oxidation. A change in the voltammetric oxidation current of ferrocenecarboxylic acid pre- and post-hybridisation
was used to provide an indication of hybridisation. A linear dynamic range between 0.5 and 40 nM and a detection limit of
88 pM of DNA target were then achieved. In addition, the biosensor exhibited good selectivity, repeatability and stability
for the determination of DNA sequences. 相似文献
11.
The in situ hybridization kinetics of label-free DNA on mixed monolayers of peptide nucleic acid (PNA) and 6-mercapto-1-hexanol (MCH) on Au electrodes was investigated by electrochemical impedance spectroscopy (EIS) and used to discriminate the fully complementary DNA from the single-base mismatched hybrids. 相似文献
12.
In this study, SnO2 nanoparticles (SNPs)-poly(vinylferrocenium) (PVF+) modified single-use graphite electrodes were developed for electrochemical monitoring of DNA hybridization. The surfaces of polymer modified and polymer-SNP modified pencil graphite electrodes (PGEs) were firstly characterized by using SEM analysis. The electrochemical behaviours of these electrodes were also investigated using the differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The polymer-SNP modified PGEs were then tested for the electrochemical sensing of DNA based on the changes at the guanine oxidation signals. Experimental parameters, such as; different modifications in DNA oligonucleotides, DNA probe concentrations were examined to obtain more sensitive and selective electrochemical signals for nucleic acid hybridization. After optimization studies, DNA hybridization was investigated in the case of complementary of hepatitis B virus (HBV) probe, mismatch (MM), and noncomplementary (NC) sequences. 相似文献
13.
Mo Wang Huanshun Yin Zhengliang Fu Yunlong Guo Xinxu Wang Yunlei Zhou Shiyun Ai 《Journal of Solid State Electrochemistry》2014,18(10):2829-2835
MicroRNAs (miRNAs), a class of small endogenous nonprotein-coding RNAs, regulate a wide range of biological processes, and their abnormal expressions are related to the growth and development of plants. Thus, a simple, rapid, and highly sensitive assay for miRNA detection is of great significance. In this work, a label-free and ultrasensitive assay for miRNA detection using protein cage nanoparticles has been developed. Apoferritin-encapsulated Cu nanoparticles (Cu-apoferritin) could be immobilized on the electrode through special reaction between amino and carboxyl. Hybridization event between the probe DNA and the target miRNA-159a is confirmed by electrochemical oxidation signal after Cu released into the detection buffer by adjusting the pH. This assay is highly selective and sensitive with a low detection limit of 3.5 fM. Moreover, the developed method can even discriminate single-base mismatched strand between the complementary targets. The effect of abscisic acid on the expression level of miRNA-159a in Arabidopsis thaliana seeds was also investigated. 相似文献
14.
Yangyang Chen Lin Xiao Ying Liu Xiaoxi Li Juan Zhang Yongqian Shu 《Mikrochimica acta》2014,181(5-6):615-621
A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The other end of the probe DNA is linked, via carboxy groups, to magnetic nanoparticles. The binding of target DNA transforms the hairpin structure of the probe DNA and causes the exposure of ester bonds. This results in the release of electro-active ferrocene after hydrolysis of the ester bonds, and in an observable electrochemical response. The quantity of target DNA in the concentration range between 1?×?10?12 mol·L?1 and 1?×?10?8 mol·L?1 can be determined by measuring the electrochemical current. The method can detect target DNA with rapid response (30 min) and low interference. Figure
A lipase-based electrochemical biosensor has been fabricated for the quantitative determination of target DNA. It is based on a stem-loop nucleic acid probe labeled with ferrocene containing a butanoate ester that is hydrolyzed by lipase. The method can detect target DNA with rapid response (30 min) and low interference. 相似文献
15.
A sensitive label-free DNA hybridization biosensing platform was fabricated based on the synergistic effect of polyaniline nanotubes (PANInt) and poly-L-lysine (pLys). The composite of pLys and PANInt was coated onto the carbon paste electrode (CPE) to form a uniform and very stable nanocomposite membrane. The pLys in the composite film not only acts as a membrane to retain good electron transfer capability of PANInt even at physiological pH, but also possesses fine biocompatibility for bio-analytes. DNA probes with negatively charged phosphate groups were readily linked to the positively charged pLys surface due to the strong electrostatic affinity. The synergistic effect of PANInt and pLys could significantly enhance the sensitivity of DNA hybridization recognition. The phosphinothricin acetyltransferase (PAT) gene fragment from transgenic corn and the polymerase chain reaction amplification of the terminator of nopaline synthase gene from the real sample of a kind of transgenic soybean were detected by this DNA electrochemical biosensor via label-free impedance method. This stable composite gives convenient permselectivity properties as a transducer material for the design of modern electrochemical impedance biosensor using [Fe(CN)6]3?/4? as an indicator. 相似文献
16.
Exploring graphene oxide (GO), DNA hybridization detection usually relies on either GO decoration or DNA sequences labeling. The former endows GO with desired chemical, optical, and biological properties. The latter adopts labeled molecules to indicate hybridization. In the present work, we propose a simple, label-free DNA assay using undecorated GO directly as the sensing platform. GO is anchored on diazonium functionalized electrode through electrostatic attraction, hydrogen bonding or epoxy ring-opening. The π–π stacking interaction between hexagonal cells of GO and DNA base rings facilitates DNA immobilization. The adsorbed DNA sequence is specially designed with two parts, including immobilization sequence and probe sequence. In the absence of target, the two sequences lie nearly flat on GO platform. In the presence of target, probe hybridizes with it to form double helix DNA, which ‘stands’ on GO. While the immobilization sequence part remains ‘lying’ on GO surface. Hence, DNA hybridization induces GO interfacial property changes, including negative charge and conformational transition from ‘lying’ ssDNA to ‘standing’ dsDNA. These changes are monitored by electrochemical impedance spectroscopy and adopted as the analytical signal. This strategy eliminates the requirement for GO decoration or DNA labeling, representing a comparatively simple and effective way. Finally, the principle is applied to the detection of conserved sequence of the human immunodeficiency virus 1 pol gene fragment. The dynamic detection range is from 1.0 × 10−12 to 1.0 × 10−6 M with detection limit of 1.1 × 10−13 M with 3σ. And the sequences with double- or four-base mismatched are readily distinguishable. In addition, this strategy may hold great promise for potential applications from DNA biosensing to nanostructure framework construction based on the versatile DNA self-assembly. 相似文献
17.
Dandan Ma Yali Yuan Xilin Xiao Yangyang Gao Yuhui Li Wenhui Xu Wei Long 《Journal of Radioanalytical and Nuclear Chemistry》2014,299(3):1911-1919
A novel and highly sensitive electrochemical DNAzymes biosensor was fabricated using Au nanoparticles (AuNPs) immobilized on the surface of Au electrode that had been previously modified with self-assembled monolayers of 1,6-hexanedithiol. Different modified electrodes were prepared and characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The AuNPs were found to have a large surface area to anchor a large number of negatively charged phosphate backbones of DNAzymes, which further absorbed the electroactive indicator of hexaammineruthenium(III) ([Ru(NH3)6]3+) to amplify the electrochemical signal. In the presence of target molecules, a large amount of DNA partly associated with [Ru(NH3)6]3+ were removed from the electrode surface, leading to a significant decrease in peak current. Differential pulse voltammetry signals of [Ru(NH3)6]3+ provided quantitative measures of the concentrations of uranyl ion (UO2 2+), with linear calibration ranging from 13 pM to 0.15 nM and a detection limit of 5 pM. The presence of other metal ions did not affect the detection of UO2 2+, which indicated the high specificity of UO2 2+. Therefore, a new electrochemical DNAzymes sensor was designed with specific DNAzymes and AuNPs as immobilization platform and signal amplifier. 相似文献
18.
A new protocol is described for amplifying label-free electrochemical measurements of DNA hybridization based on the enhanced accumulation of purine nucleobases in the presence of copper ions . Such electrical DNA assays involve hybridization of the target to inosine-substituted oligonucleotide probes (captured on magnetic beads), acidic dipurinization of the hybrid DNA, and adsorptive chronopotentiometric stripping measurements of the free nucleobases in the presence of copper ions. Both amplified adenine and guanine peaks can be used for detecting the DNA hybridization. The dramatic signal amplification advantage of this type of detection has been combined with efficient magnetic removal of non-complementary DNA, use of microliter sample volumes and disposable transducers. Factors influencing the signal enhancement were assessed and optimized. A detection limit of 40 fmol (250 pg) was obtained with 10 min hybridization and 5 min adsorptive-accumulation times. The advantages of this procedure were demonstrated by its application in the detection of DNA segments related to the BRCA1 breast cancer gene. The copper enhancement holds great promise not only for the detection of DNA hybridization, but also for trace measurement of nucleic acids. 相似文献
19.
A label-free sensing assay for ethanolamine (EA) detection based on G-quadruplex-EA binding interaction is presented by using G-rich aptamer DNA (Ap-DNA) and electrochemical impedance spectroscopy (EIS). The presence of K+ induces the Ap-DNA to form a K+-stabilized G-quadruplex structure which provides binding sites for EA. The sensing mechanism was further confirmed by circular dichroism (CD) spectroscopy and EIS measurement. As a result, the charge transfer resistance (RCT) is strongly increased as demonstrated by using the ferro/ferricyanide ([Fe(CN)6]3−/4−) as a redox probe. Under the optimized conditions, a linear relationship between ΔRCT and EA concentration was obtained over the range of 0.16 nM and 16 nM EA, with a detection limit of 0.08 nM. Interference by other selected chemicals with similar structure was negligible. Analytical results of EA spiked into tap water and serum by the sensor suggested the assay could be successfully applied to real sample analysis. With the advantages of high sensitivity, selectivity and simple sensor construction, this method is potentially suitable for the on-site monitoring of EA contamination. 相似文献
20.
We review the development of reagentless, electrochemical sensors for the sequence-specific detection of nucleic acids that are based on the target-induced folding or unfolding of electrode-bound oligonucleotides. These devices, which are sometimes termed E-DNA sensors, are comprised of an oligonucleotide probe modified on one terminus with a redox reporter and attached to an electrode at the other. Hybridization of this probe DNA to a target oligonucleotide influences the rate at which the redox reporter collides with the electrode, leading to a detectable change in redox current. Because all sensing elements of this method are strongly linked to the interrogating electrode, E-DNA sensors are label-free, operationally convenient and readily reusable. As E-DNA signaling is predicated on a binding-specific change in the dynamics of the probe DNA (rather than simply monitoring the adsorption of a target to the sensor surface) and because electroactive contaminants (interferents) are relatively rare, this class of sensors is notably resistant to false positives arising from the non-specific adsorption of interferents, and performs well even when challenged directly with blood serum, soil and other complex sample matrices. We review the history of and recent advances in this promising DNA and RNA detection approach. 相似文献