Conformational switching immobilized hairpin DNA probes following subsequent expanding of gold nanoparticles enables visual detecting sequence-specific DNA

A simple, rapid, and sensitive method for visual detection of sequence-specific DNA was developed using hairpin DNA as the recognition element and hydroxylamine-enlarged gold nanoparticles (Au-NPs) as the signal producing component. In the assay, we employed a hairpin DNA probe dually labeled with amine and biotin at the 5'- and 3'-end, respectively. The probe was coupled with reactive N-oxysuccinnimide in a DNA-bind 96-well plate. Without the target DNA, the immobilized hairpin probe was in a "closed" state, which kept the streptavidin-gold off the biotin. The hybridization between the loop sequence and the target broke the short stem duplex upon approaching the target DNA. Consequently, biotin was forced away from the 96-well plate surface and available for conjugation with the streptavidin-gold. The hybridization could be detected visually after the HAuCl(4)-NH(2)OH redox reaction catalyzed by the Au-NPs. Under the optimized conditions, the visual DNA sensor could detect as low as 100 amol of DNA targets with excellent differentiation ability and even a single-base mismatch.     

Improved sensitivity for the electrochemical biosensor with an adjunct probe

Despite their promising applications in the biomedical research, the development of electrochemical biosensors with improved sensitivity and low detection limit has remained a great challenge. Here, we demonstrate a new approach to improve the sensitivity of the electrochemical biosensor by simply introducing an adjunct probe into its construction. This signal-on biosensor consists of a thiol-functionalized capture probe attached on the gold electrode surface, an electrochemical sign (methyl blue, MB)-modified reporter probe which is complementary to the capture probe, and an adjunct probe attached nearby the capture probe. The adjunct probe functions as a fixer to immobilize the element of reporter probe which is displaced by the target DNA and protein, increasing the chance of the dissociative reporter probe to collide with the electrode surface and facilitating the electron transfer. The biosensor with an adjunct probe exhibits improved sensitivity and a large dynamic range for DNA and the thrombin assay and can even distinguish 1-base mismatched target DNA. Importantly, the use of this biosensor is not limited to such and is viable for sensitive detection of numerous biomolecules, including RNA, proteins, and small molecules such as cocaine.     

DNA-wrapped carbon nanotubes as sensitive electrochemical labels in controlled-assembly-mediated signal transduction for the detection of sequence-specific DNA

A novel electrochemical strategy that uses DNA-wrapped carbon nanotubes (CNTs) as electrochemical labels is developed for sensitive and selective detection of sequence-specific DNA. The presence of target DNA mediates the formation of a sandwiched complex between the DNA-wrapped CNT and a hairpin DNA capture probe immobilized on magnetic beads. This allows target-selective collection of the CNT labels by magnetic separation and transfer on the electrode surface modified with an insulating self-assembled monolayer (SAM). After treatment with N,N-dimethylformamide, the collected sandwiched complex releases the bare CNTs and facilitates the removal of magnetic beads from the electrode surface. The bare CNTs can then assemble on the SAM-modified electrode surface and mediate efficient electron transfer between the electrode and the electroactive species in the solution with a strong current signal generated. The results indicate that the developed strategy shows a sensitive response to target DNA with a desirable signal gain and a low detection limit of 0.9 pM. This strategy is also demonstrated to provide excellent differentiation of single-base mismatch in target DNA. It is expected that this electrochemical strategy may hold great potential as a novel platform for clinical diagnostics and genetic analysis.     

Electrochemical biosensors employing an internal electrode attachment site and achieving reversible, high gain detection of specific nucleic acid sequences

Electrochemical DNA (E-DNA) sensors, which are rapid, reagentless, and readily integrated into microelectronics and microfluidics, appear to be a promising alternative to optical methods for the detection of specific nucleic acid sequences. Keeping with this, a large number of distinct E-DNA architectures have been reported to date. Most, however, suffer from one or more drawbacks, including low signal gain (the relative signal change in the presence of complementary target), signal-off behavior (target binding reduces the signaling current, leading to poor gain and raising the possibility that sensor fouling or degradation can lead to false positives), or instability (degradation of the sensor during regeneration or storage). To remedy these problems, we report here the development of a signal-on E-DNA architecture that achieves both high signal gain and good stability. This new sensor employs a commercially synthesized, asymmetric hairpin DNA as its recognition and signaling probe, the shorter arm of which is labeled with a redox reporting methylene blue at its free end. Unlike all prior E-DNA architectures, in which the recognition probe is attached via a terminal functional group to its underlying electrode, the probe employed here is affixed using a thiol group located internally, in the turn region of the hairpin. Hybridization of a target DNA to the longer arm of the hairpin displaces the shorter arm, allowing the reporter to approach the electrode surface and transfer electrons. The resulting device achieves signal increases of ～800% at saturating target, a detection limit of just 50 pM, and ready discrimination between perfectly matched sequences and those with single nucleotide polymorphisms. Moreover, because the hairpin probe is a single, fully covalent strand of DNA, it is robust to the high stringency washes necessary to remove the target, and thus, these devices are fully reusable.     

Electrochemical detection of DNA hybridization using biometallization

We demonstrate the amplified detection of a target DNA based on the enzymatic deposition of silver. In this method, the target DNA and a biotinylated detection DNA probe hybridize to a capture DNA probe tethered onto a gold electrode. Neutravidin-conjugated alkaline phosphatase binds to the biotin of the detection probe on the electrode surface and converts the nonelectroactive substrate of the enzyme, p-aminophenyl phosphate, into the reducing agent, p-aminophenol. The latter, in turn, reduces metal ions in solutions leading to deposition of the metal onto the electrode surface and DNA backbone. This process, which we term biometallization, leads to a great enhancement in signal due to the accumulation of metallic silver by a catalytically generated enzyme product and, thus, the electrochemical amplification of a biochemically amplified signal. The anodic stripping current of enzymatically deposited silver provides a measure of the extent of hybridization of the target oligomers. This biometallization process is highly sensitive, detecting as little as 100 aM (10 zmol) of DNA. We also successfully applied this method to the sequence-selective discrimination between perfectly matched and mismatched target oligonucleotides including a single-base mismatched target.     

Highly sensitive electrochemiluminescence detection of single-nucleotide polymorphisms based on isothermal cycle-assisted triple-stem probe with dual-nanoparticle label

We report here a new electrochemiluminescence (ECL) approach for detection of single nucleotide polymorphisms (SNPs) based on isothermal cycle-assisted triple-stem probe labeled with Au nanoparticles (NPs) and CdTe NPs. The system is composed of a CdS nanocrystals (NCs) film on glassy carbon electrode (GCE) as ECL emitter attached a double-stem DNA probe labeled with Au NPs. Then, the third stem labeled with CdTe NPs hybridizes with the double-stem DNA to form a triple-stem probe with the two labels near the CdS NCs film. A dual-quenched ECL of CdS NCs film is achieved due to energy transfer (ET) from CdS NCs to Au NPs and CdTe NPs, which makes the sensor exhibit relatively low background. Once the one base mutant DNA (mDNA) sequence as target of SNPs analysis displaces the third stem and hybridizes with the double-stem probe, forcing Au NPs away from the CdS NCs film, an ECL enhancement by the ECL-induced surface plasmon resonance of Au NPs is observed. Furthermore, after an isothermal cycle induced by primer, polymerase, and nicking endonuclease (NEase), a further enhancement of ECL is obtained. Taking advantages of the isothermal circular amplification system and the triple-stem probe architecture which enables turning its high selectivity toward specific target sequences, the reported biosensor shows excellent discrimination capabilities of SNPs with high selectivity and low detection limit (35 aM).     

Sensitive electrochemiluminescence detection of c-Myc mRNA in breast cancer cells on a wireless bipolar electrode

We report an ultrasensitive wireless electrochemiluminescence (ECL) protocol for the detection of a nucleic acid target in tumor cells on an indium tin oxide bipolar electrode (BPE) in a poly(dimethylsiloxane) microchannel. The approach is based on the modification of the anodic pole of the BPE with antisense DNA as the recognition element, Ru(bpy)(3)(2+)-conjugated silica nanoparticles (RuSi@Ru(bpy)(3)(2+)) as the signal amplification tag, and reporter DNA as a reference standard. It employs the hybridization-induced changes of RuSi@Ru(bpy)(3)(2+) ECL efficiency for the specific detection of reporter DNA released from tumor cells. Prior to ECL detection, tumor cells are transfected with CdSe@ZnS quantum dot (QD)-antisense DNA/reporter DNA conjugates. Upon the selective binding of antisense DNA probes to intracellular target mRNA, reporter DNA will be released from the QDs, which indicates the amount of the target mRNA. The proof of concept is demonstrated using a proto-oncogene c-Myc mRNA in MCF-7 cells (breast cancer cell line) as a model target. The wireless ECL biosensor exhibited excellent ECL signals which showed a good linear range over 2 × 10(-16) to 1 × 10(-11) M toward the reporter DNA detection and could accurately quantify c-Myc mRNA copy numbers in living cells. C-Myc mRNA in each MCF-7 cell and LO2 cell was estimated to be 2203 and 13 copies, respectively. This wireless ECL strategy provides great promise in a miniaturized device and may facilitate the achievement of point of care testing.     

Embedded DNA-polypyrrole biosensor for rapid detection of Escherichia Coli

The principal objective of this paper was to present the design and fabrication of a single-strand (ss) DNA biosensor for the detection of Escherichia coli (E. coli) DNA synthetic oligonucleotides as a model of rapid detection of bacterial select bioterrorism agents. Molecular biology and chemical electrodeposition techniques, such as cyclic voltammetry (CV), were combined to develop and test a model DNA-based biosensor on a platinum (Pt) electrode electropolimerized with polypyrrole (PPY). The hybridization on embedded DNA into PPY with complementary DNA samples was determined. The recognition element was a 25 base pair (bp) oligonucleotide specific for E. coli derived from the uidA gene that codes for the enzyme /spl beta/-D glucuronidase. CV scans between 0.0 and +0.70 V at a 50-mV/s scanning rate generated current versus potential graphs. A standard DNA concentration of 1 /spl mu/g//spl mu/L was used to determine the hybridization signal of the biosensor. The model biosensor generated distinctive CV signals between complementary and noncomplementary DNA oligonucleotides. The biosensor proved to be effective in the detection of complementary uidA 25-bp oligonucleotide for E. coli K-12.     

Electrochemiluminescent biosensor for hypoxanthine based on the electrically heated carbon paste electrode modified with xanthine oxidase

A new electrochemiluminescent (ECL) biosensor based on an electrically heated carbon paste electrode (HCPE) that was surface modified by xanthine oxidase (XOD) was designed and constructed in this work. It was found that the ECL intensity of luminol could be enhanced at the surface of XOD/HCPE by adding hypoxanthine (HX) to the solution, and there was a linear relationship between the ECL intensity and the concentration of HX. On the basis of this, an ECL enzyme biosensor can thus be developed to detect HX. However, because the activity of XOD is highly dependent on temperature, the biosensor is very sensitive to the temperature of the electrode. Also, because the temperature of the electrode may also affect the diffusion and convection of the luminescent compounds near the electrode surface, a suitable temperature for XOD/HCPE has to be controlled to achieve the best ECL signal. The key feature of the designed biosensor is that the temperature of the electrode is controllable so the most suitable temperature for the enzyme reaction can be obtained. The obtained results showed that the ECL enzyme biosensor exhibited the best sensitivity at an electrode temperature of 35 degrees C for the detection of HX. The detection limit was 30-fold lower than that at room temperature (25 degrees C).     

Electrochemical DNA biosensor based on conducting polyaniline nanotube array

Most of the recent developments in ultrasensitive detection of nucleic acid are based on the gold nanoparticles and carbon nanotubes as a medium of signal amplification. Here, we present an ultrasensitive electrochemical nucleic acid biosensor using the conducting polyaniline (PANI) nanotube array as the signal enhancement element. The PANI nanotube array of a highly organized structure was fabricated under a well-controlled nanoscale dimension on the graphite electrode using a thin nanoporous layer as a template, and 21-mer oligonucleotide probes were immobilized on these PANI nanotubes. In comparison with gold nanoparticle- or carbon nanotube-based DNA biosensors, our PANI nanotube array-based DNA biosensor could achieve similar sensitivity without catalytic enhancement, purification, or end-opening processing. The electrochemical results showed that the conducting PANI nanotube array had a signal enhancement capability, allowing the DNA biosensor to readily detect the target oligonucleotide at a concentration as low as 1.0 fM (approximately 300 zmol of target molecules). In addition, this biosensor demonstrated good capability of differentiating the perfect matched target oligonucleotide from one-nucleotide mismatched oligonucleotides even at a concentration of 37.59 fM. This detection specificity indicates that this biosensor could be applied to single-nucleotide polymorphism analysis and single-mutation detection.     

Determination of DNA methylation using electrochemiluminescence with surface accumulable coreactant

Cytosine methylation in DNA was determined by an enzyme linked immunosorbent assay (ELISA) with electrochemiluminescence (ECL) detection and employed for the DNA methylation assay of a long and real genomic sample for the first time. The developed method employed an antimethyl cytosine antibody labeled with acetylcholinesterase, which was added to recognize single methylated cytosine in a DNA oligomer. The acetylcholinesterase converted acetylthiocholine (substrate) to thiocholine (product), which was accumulated on a gold electrode surface via gold-thiol binding. This surface accumulated preconcentration made it possible to observe bright and distinctive ECL by applying a potential to the gold electrode in the presence of a tris(2,2-bipyridyl)ruthenium complex luminophore when the analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1-100 pmol range, which exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction (PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine, thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method using a real DNA bacteriophage sample (48,502 base pairs).     

Charge transfer through DNA: A selective electrochemical DNA biosensor

The charge-transfer properties of DNA duplexes are exploited to produce a fast, simple, sensitive, and selective DNA biosensor by exposing the DNA recognition interface to a sample containing target DNA and the redox-active intercalator, anthraquinonemonosulfonic acid (AQMS). Electrochemistry from electron transfer through the DNA to AQMS intercalated into DNA duplexes can be differentiated from electrochemistry due to direct access of the AQMS to the electrode surface due to the difference in the environment of the AQMS giving a shift in the potential at which the molecule is reduced. The ability to distinguish between the two electrochemical signals enables DNA hybridization to be monitored in real time. This in situ detection scheme has good selectivity, being able to differentiate between a complementary target DNA sequence and one containing either C-A or G-A single-base mismatches. The concentration detection limit of the biosensor is 0.5 nM (1 pmol) with an assay time of 1 h. The fact that the end user is only required to simultaneously add the sample containing the target DNA and AQMS gives a DNA biosensor that is highly compatible with PCR on chip technologies.     

Single-mismatch position-sensitive detection of DNA based on a bifunctional ruthenium complex

A ruthenium complex, pentaamine ruthenium [3-(2-phenanthren-9-yl-vinyl)-pyridine] (which we refer to as RuL in the text) generated in situ has been used as a sensitive and selective electrochemical indicator in DNA sensing. The complex incorporates dual functionalities with the Ru center providing a redox probe and the ligand (L) providing a fluorescent tag. The presence of the aromatic groups in the ligand endows the complex with an intercalative character and makes it capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Combining spectroscopic and electrochemical techniques, we have elucidated the nature of the interactions. From these data we conclude that the binding mode is fundamentally intercalative. The ligand-based fluorescence allows characterization of the complex formation as well as for melting experiments to be carried out. The metal-based redox center is employed as an electrochemical indicator to detect the hybridization event in a DNA biosensor. The biosensor has been developed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. With the use of this approach, complementary target sequences of Helicobacter can be quantified over the range of 106 to 708 pmol with a detection limit of 92+/-0.4 pmol and a linear correlation coefficient of 0.995. In addition, this approach allows the detection, without the need for a hybridization suppressor in solution, such as formamide, of not only a single mismatch but also its position in a specific sequence of H. pylori, due to the selective interaction of this bifunctional ruthenium complex with dsDNA.     

Bifunctional colorimetric oligonucleotide probe based on a G-quadruplex DNAzyme molecular beacon

A label-free bifunctional colorimetric oligonucleotide probe for DNA and protein detection has been developed on the basis of a novel catalytic molecular beacon consisting of two hairpin structures and a split G-quadruplex DNAzyme in the middle. The two loops of this molecular beacon consist of thrombin aptamer sequence and the complementary sequence of target DNA, which are utilized to sense single-stranded DNA and thrombin. The G-quadruplex DNAzyme can effectively catalyze the H(2)O(2)-mediated oxidation of 3,3',5,5'-tetramethylbenzidine sulfate to generate colorimetric signal. Upon addition of the target, the DNA or protein combines with one loop of the hairpin structures, and meanwhile drives the middle G-quadruplex DNAzyme to dissociate. This results in a decrease of catalytic activity, enabling the separate analysis of DNA and thrombin.     

Ultrasensitive flow injection chemiluminescence detection of DNA hybridization using signal DNA probe modified with Au and CuS nanoparticles

A novel and sensitive flow injection chemiluminescence assay for sequence-specific DNA detection based on signal amplification with nanoparticles (NPs) is reported in the present work. The "sandwich-type" DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe labeled with CuS NPs and Au NPs on the 3'- and 5'-terminus, respectively. The hybridization events were monitored by the CL intensity of luminol-H2O2-Cu(2+) after the cupric ions were dissolved from the hybrids. We demonstrated that the incorporation of Au NPs in this sensor design significantly enhanced the sensitivity and the selectivity because a single Au NP can be loaded with hundreds of signal DNA probe strands, which were modified with CuS NPs. The ratios of Au NPs, signal DNA probes, and CuS NPs modified on the gold electrode were approximately 1/101/103. A preconcentration process of cupric ions performed by anodic stripping voltammetry technology further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar target DNA and exhibited excellent selectivity against two-base mismatched DNA. Under the optimum conditions, the CL intensity was increased with the increase of the concentration of target DNA in the range of 2.0 x 10(-14)-2.0 x 10(-12) M. A detection limit of 4.8 x 10(-15) M target DNA was achieved.     

Characterization of Nanoporous Silicon-Based DNA Biosensor for the Detection of Salmonella Enteritidis

A biosensor for the detection of food-borne pathogens (Salmonella Enteritidis) was fabricated based on nanoporous silicon (NPS). P-type silicon wafers (100, 0.01 ) were anodized electrochemically in an electrochemical Teflon cell, containing ethanoic hydrofluoric acid solution to produce the porous layer on the silicon surface. The porous silicon surface was functionalized with DNA probes specific to the insertion element (Iel) gene of Salmonella Enteritidis. A biotin-streptavidin system was utilized to characterize the availability of the nanopores and the specificity of the DNA probe. Based on the electrical property of DNA, redox indicators and cyclic voltammetry were used for the characterization of the biosensor. Results showed that the DNA probe was specific to the target DNA, and the porous silicon-based biosensor had more active surface area and higher sensitivity (1 ng/mL) than the planar silicon-based biosensor. This simple, label-free porous silicon-based biosensor has potential applications in high-throughput detection of pathogens.     

Preparation and characterization of an electrogenerated chemiluminescence sensor by electrochemical grafting of 4-nitrophenyl diazonium salts onto a glass carbon electrode

An ECL sensor was fabricated by immobilization of a tris(2,2'-bipyridyl)ruthenium (II) complex (Ru(bpy)3(2+)) to an amine group-modified GC electrode (NH2-GC electrode). Here, the NH2-GC electrode was prepared by electrochemical reduction of a nitro group-modified GC electrode in 0.1 M KCl ethanol solution under H2 gas, which was followed by electrochemical grafting of 4-nitrophenyl diazonium salts in 0.1 M NBu4BF4 acetonitrile solution onto the GC electrode. The prepared ECL sensor was successfully confirmed via cyclic voltammetry, contact angle, scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and ECL spectrometry. The contact angle for the surface of the GC electrode, NO2-GC electrode, and NH2-GC electrod was 88.4 degrees, 67.4 degrees, and 52.4 degrees, respectively. The stability of the ECL sensor was investigated under continuous cyclic potential scanning for 55 cycles and the ECL intensity remained at 55%. The prepared ECL electrode can be expected to immobilize enzymes for preparation of the ECL biosensor to detect target molecules.     

Monitoring molecular beacon/DNA interactions using atomic force microscopy

The molecular beacon (MB) is a new fluorescence probe containing a single-stranded oligonucleotide with a probe sequence embedded in complementary sequences that form a hairpin stem. Due to the inherent fluorescent signal transduction mechanism, an MB functions as a sensitive probe with a high signal-to-background ratio for real-time monitoring and provides a variety of exciting opportunities in DNA, RNA, and protein studies. To better understand the properties of MBs, the specific interactions between MB and target DNA (complementary and one-base mismatch) have been directly investigated by atomic force microscopy. The interaction force between a linear DNA probe and the target DNA was also detected and compared to that between MB and target DNA. The results demonstrate the high specificity of the MB/target DNA compared to the linear DNA/target DNA interaction.     

Versatile electrochemiluminescence assays for cancer cells based on dendrimer/CdSe-ZnS-quantum dot nanoclusters

In this work, a novel dendrimer/CdSe-ZnS-quantum dot nanocluster (NC) was fabricated and used as an electrochemiluminescence (ECL) probe for versatile assays of cancer cells for the first time. A large number of CdSe-ZnS-quantum dots (QDs) were labeled on the NCs due to the many functional amine groups within the NCs, which could significantly amplify the QD's ECL signal. Capture DNA was specially designed as a high-affinity aptamer to the target cell; a novel ECL biosensor for cancer cells was directly accomplished by using the biobarcode technique to avoid cross-reaction. Moreover, magnetic beads (MBs) for aptamers immobilization were combined with the dendrimer/QD NCs probe for signal-on ECL assay of cancer cells, which greatly simplified the separation procedures and favored for the sensitivity improvement. In particular, a novel cycle-amplifying technique using a DNA device on MBs was further employed in the ECL assay of cancer cells, which greatly improved the sensitivity. To the best of our knowledge, this is the first study that the novel dendrimer/QD NCs probe combined with a DNA device cycle-amplifying technique was employed in the ECL assays of cells. Excellent discrimination against target and control cells is demonstrated, indicating that the ECL assays have great potential to provide a sensitive, selective, cost-effective, and convenient approach for early and accurate detection of cancer cells.     

Electrochemical biosensor for detection of BCR/ABL fusion gene using locked nucleic acids on 4-aminobenzenesulfonic acid-modified glassy carbon electrode

In this study, an electrochemical DNA biosensor was developed for detection of the breakpoint cluster region gene and the cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia by using 18-mer locked, nucleic acid-modified, single-stranded DNA as the capture probe. The capture probe was covalently attached on the sulfonic-terminated aminobenzenesulfonic acid monolayer-modified glassy carbon electrode through the free amines of DNA bases based on the acyl chloride cross-linking reaction. The covalently immobilized capture probe could selectively hybridize with its target DNA to form double-stranded DNA (dsDNA) on the LNA/4-ABSA/GCE surface. Differential pulse voltammetry was used to monitor the hybridization reaction on the capture probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated that, in pH 7.0 Tris-HCl buffer solution, the peak current was linear with the concentration of complementary strand in the range of 1.0 x 10 (-12)1.1 x 10 (-11) M with a detection limit of 9.4 x 10 (-13) M. This new method demonstrates its excellent specificity for single-base mismatch and complementary dsDNA after hybridization, and this probe has been used for assay of PCR real sample with satisfactory results.