Ultrasensitive electrogenerated chemiluminescence detection of DNA hybridization using carbon-nanotubes loaded with tris(2,2′-bipyridyl) ruthenium derivative tags

An ultrasensitive electrogenerated chemiluminescence (ECL) detection method of DNA hybridization based on single-walled carbon-nanotubes (SWNT) carrying a large number of ruthenium complex tags was developed. The probe single strand DNA (ss-DNA) and ruthenium complex were loaded at SWNT, which was taken as an ECL probe. When the capture ss-DNA with a thiol group was self-assembled onto the surface of gold electrode, and then hybridized with target ss-DNA and further hybridized with the ECL probe to form DNA sandwich conjugate, a strong ECL response was electrochemically generated. The ECL intensity was linearly related to the concentration of perfect-matched target ss-DNA in the range from 2.4 × 10⁻¹⁴ to 1.7 × 10⁻¹² M with a detection limit of 9.0 × l0⁻¹⁵ M. The ECL signal difference permitted to discriminate the perfect-matched target ss-DNA and two-base-mismatched ss-DNA. This work demonstrates that SWNT can provide an amplification platform for carrying a large number of ECL probe and thus resulting in an ultrasensitive ECL detection of DNA hybridization.     

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.     

Electrochemical Detection of DNA Hybridization Based on the Probe Labeled with Carbon‐Nanotubes Loaded with Silver Nanoparticles

A sensitive electrochemical method for the detection of DNA hybridization based on the probe labeled with multiwall carbon‐nanotubes (MWNTs) loaded with silver nanoparticles (Ag‐MWNTs) has been developed. MWNTs were electroless‐plated with a large number of silver nanoparticles to form Ag‐MWNTs. Probe single strand DNA (ss‐DNA) with a thiol group at the 3′‐terminal labeled with Ag‐MWNTs by self‐assembled monolayer (SAM) technique was employed as an electrochemical probe. Target ss‐DNA with a thiol group was immobilized on a gold electrode by SAM technique and then hybridized with the electrochemical probe. Binding events were monitored by differential pulse voltammetric (DPV) signal of silver nanoparticles. The signal difference permitted to distinguish the match of two perfectly complementary DNA strands from the near perfect match where just three base pairs were mismatched. There was a linear relation between the peak current at +120 mV (vs. SCE) and complementary target ss‐DNA concentration over the range from 3.1×10^?14 to 1.0×10^?11 mol/L with a detection limit of 10 fmol/L of complementary target ss‐DNA. The proposed method has been successfully applied to detection of the DNA sequence related to cystic fibrosis. This work demonstrated that the MWNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of DNA hybridization.     

Electrical detection of deoxyribonucleic acid hybridization based on carbon-nanotubes/nano zirconium dioxide/chitosan-modified electrodes

A novel and sensitive electrochemical DNA biosensor based on nanoparticles ZrO₂ and multi-walled carbon nanotubes (MWNTs) for DNA immobilization and enhanced hybridization detection is described. The MWNTs/nano ZrO₂/chitosan-modified glassy carbon electrode (GCE) was fabricated and oligonucleotides were immobilized to the GCE. The hybridization reaction on the electrode was monitored by differential pulse voltammetry (DPV) analysis using electroactive 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 increased DNA attachment quantity and complementary DNA detection sensitivity. The response signal increases linearly with the increase of the logarithm of the target DNA concentration in the range of 1.49 × 10⁻¹⁰ to 9.32 × 10⁻⁸ mol L⁻¹ with the detection limit of 7.5 × 10⁻¹¹ mol L⁻¹ (S/N = 3). The linear regression equation is I = 32.62 + 3.037 log C_DNA (mol L⁻¹) with a correlation coefficient value of 0.9842. This is the first application of carbon nanotubes combined with nano ZrO₂ to the fabrication of an electrochemical DNA biosensor with a favorable performance for the rapid detection of specific hybridization.     

Nanoparticle-based electrochemical DNA detection

Nanoscale architectures of DNA-linked particle networks are attractive for electrical detection of DNA hybridization. This article reviews a variety of new nanoparticle/polynucleotide assemblies for advanced electrical detection of DNA sequences. Recent activity has led to innovative and powerful nanoparticle-based electrochemical DNA hybridization assays based on a variety of detection schemes. Such protocols rely on the use of colloidal gold tags, semiconductor quantum dot tracers, polymeric carrier (amplification) beads, or magnetic (separation) beads. Particularly useful have been protocols based on capturing of metal nanoparticle tracers followed by dissolution and anodic-stripping voltammetric measurement of the metal tag. Remarkable sensitivity is achieved by coupling particle-based amplification units and various amplification processes. The use of nanoparticle tracers for designing multi-target electrochemical coding protocols will also be documented.     

Electrochemical DNA Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

Electrochemical stripping detection of DNA hybridization based on cadmium sulfide nanoparticle tags

We report on the detection of DNA hybridization in connection to cadmium sulfide nanoparticle tracers and electrochemical stripping measurements of the cadmium. A nanoparticle-promoted cadmium precipitation is used to enlarge the nanoparticle tag and amplify the stripping DNA hybridization signal. In addition to measurements of the dissolved cadmium ion we demonstrate solid-state measurements following a ‘magnetic’ collection of the magnetic-bead/DNA-hybrid/CdS-tracer assembly onto a thick-film electrode transducer. The new protocol combines the amplification features of nanoparticle/polynucleotides assemblies and highly sensitive stripping potentiometric detection of cadmium, with an effective magnetic isolation of the duplex. The low detection limit (100 fmol) is coupled to good reproducibility (RSD=6%). Prospects for using binary inorganic colloids for multi-target detection are discussed.     

Electrochemical DNA biosensors based on platinum nanoparticles combined carbon nanotubes

Platinum nanoparticles were used in combination with multi-walled carbon nanotubes (MWCNTs) for fabricating sensitivity-enhanced electrochemical DNA biosensor. Multi-walled carbon nanotubes and platinum nanoparticles were dispersed in Nafion, which were used to fabricate the modification of the glassy carbon electrode (GCE) surface. Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated daunomycin. Due to the ability of carbon nanotubes to promote electron-transfer reactions, the high catalytic activities of platinum nanoparticles for chemical reactions, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.0 × 10⁻¹¹ mol l⁻¹.     

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)₂₅ (DBT) with covalently bound (dT)₂₅ probe are used for the hybridization with target DNA containing adenine stretches. Target DNA is modified with osmium tetroxide,2,2′-bipyridine (Os,bipy) and the immunogenic DNA-Os,bipy adduct is determined by the enzyme-linked immunoassay with electrochemical detection. Electroinactive 1-naphthyl phosphate is used as a substrate and the electroactive product (1-naphthol) is measured on the carbon electrodes. Alternatively Os,bipy-modified target DNA can be determined directly by measuring the osmium signal on the pyrolytic graphite electrode (PGE). A comparison between determinations of the 67-mer oligodeoxynucleotide on carbon electrodes using (a) the guanine oxidation signal, (b) direct determination of the DNA-Os,bipy adduct and (c) its electrochemical immunoassay showed immunoassay to be the most sensitive method. In combination with DBT, the DNA hybridization of long target deoxyoligonucleotides (such as 67- and 97-mers) and a DNA PCR product (226-base pairs) have been detected by immunoassay at high sensitivity and specificity.     

Synthesis of functionalized CdTe/CdS QDs for spectrofluorimetric detection of BSA

Luminescent quantum dots (QDs)-semiconductor nanocrystals are a promising alternative to organic dyes for fluorescence-based applications. We have developed procedures to use CdS to encapsulate CdTe and synthesize a new kind of functionalized CdTe/CdS QDs for the quantitative and selective determination of bovine serum albumin (BSA). Maximum fluorescence intensity was produced at pH 6.83, with excitation and emission wavelengths at 336 and 524 nm, respectively. Under optimal conditions, the straight line equation: DeltaF=6.84+62.29C (10(-6) mol dm(-3)) was found between the relative fluorescence intensity and the concentration of BSA in the range of 0-1.2 x 10(-6) mol dm(-3), and the limit of detection was 5.4 x 10(-8) mol dm(-3). Based on this approach, a novel quantitative method for the determination of BSA is presented in this paper.     

Quantum dot-enhanced detection of dual short RNA sequences via one-step template-dependent surface hybridization

A novel multiplexed method for short RNA detection is reported that employs a design strategy in which capture and reporter probes anneal to each other in the presence of a short RNA target via the formation of a stable three-component complex. Quantum dots (QDs) functionalized with reporter DNA are thus specifically bound onto a capture probe-modified 96-well plate by one-step hybridization for simple RNA detection. In comparison with conventional organic dye-modified reporter probes, the use of reporter DNA-modified QD conjugates increase the melting temperature and lead to the detection of short RNA without the need for a ligation reaction. Moreover, QD properties allow multiple short RNA sequences to be simultaneously determined via rapid and simple one-step hybridization, as exemplified herein. The present results clearly demonstrate that this new strategy can be used to detect dual-short RNA sequence at concentrations of 10 pM in 100 μL.     

Ratiometric fluorescence detection of mercuric ion based on the nanohybrid of fluorescence carbon dots and quantum dots

A novel nanohybrid ratiometric fluorescence probe comprised of carbon dots (C-dots) and hydrophilic CdSe@ZnS quantum dots (QDs) has been developed by simply mixing the blue-emission C-dots with red-emission carboxylmethyldithiocarbamate modified CdSe@ZnS QDs (GDTC-QDs). The nanohybrid ratiometric fluorescence probe exhibits dual emissions at 436 nm and 629 nm under a single excitation wavelength. Due to the strong chelating ability of GDTC on the surface of QDs to mercuric ion (Hg²⁺), the fluorescence of the GDTC-QDs in the nanohybrid system could be selectively quenched in the presence of Hg²⁺ while the fluorescence of the C-dots remained constant, resulting in an obviously distinguishable fluorescence color evolution (from red to blue) of the nanohybrid system. The detection limit of this method was found to be as low as 0.1 μM. Furthermore, the recovery result for Hg²⁺ in real samples including tap water and lake water by this method was satisfying, suggesting its potential application for Hg²⁺ sensing.     

Simultaneous electrochemical DNA hybridization assay for PAT and FMV 35S gene sequence using quantum dots as labels

An electrochemical method for the simultaneous detection of two different DNA sequences from PAT and FMV 35S gene sequence using CdS and PbS quantum dots （QDs） as labels was described. The QDs were readily functionalized with oligonucleotides as electrochemical DNA probes and selectively hybridized to the complementary sequences immobilized on the microplate. The QDs anchored on the hybrids were dissolved in the solution by the oxidation of HNO3 and further detected by a sensitive differential pulse anodic stripping voltammetric method （DPASV）. The DPASV signals of the oxidation of Cd^2＋ and Pb^2＋ ions present in the solution were different and reflected the identity of corresponding ssDNA targets sequences.     

DNA detection based on Mn-doped ZnS quantum dots/methylene blue nanohybrids

Nanohybrids were formed from 3-mercaptopropionic acid(MPA)-coated Mn-doped ZnS quantum dots(QDs) and methylene blue(MB) via electrostatic interaction, and then used in the detection of trace DNA.The principle of detection is as follows: MB binds with Mn-doped ZnS QDs via electrostatic interaction,and then quenches the room temperature phosphorescence(RTP) of the QDs through photoinduced electron-transfer(PIET). After the addition of DNA, MB binds with DNA through intercalation and electrostatic interaction, and desorbs from the surfaces of Mn-doped ZnS QDs, which recovers the RTP of the QDs. On this basis, a DNA detection method based on the properties of RTP was set up. This method shows a detection range of 0.2–20 mg/L, and a detection limit of 0.113 mg/L. Since this method is based on the RTP of QDs, it is not interfered by the background fluorescence or scattering light in vivo, and thus,avoids complex sample pretreatment. Thus, this method is very feasible for detection of trace DNA in biofluids.     

Electrochemical biosensing of DNA hybridization by ferrocenyl groups functionalized polypyrrole

We have developed a new material based on polypyrrole functionalized with both ferrocenyl group and DNA probe. We have developed a precursor polymer based on a pyrrole 3-acetic acid and a pyrrole bearing ferrocenyl groups substituted by easy leaving group in which amino-labeled oligonocleotides probe were directly linked. The electrochemical response of the modified electrode was examined both in aqueous and organic media and shows high electroactivity in both media. A large modification of redox activity of ferrocenyl groups was obtained upon addition of ODN target to the electrolytic media, which is determined by amperometric methods. The detection limit of this electrochemical biosensor is about 10⁻¹⁴ mol without any signal processing.     

Applications of Carbon Nanotubes in Electrochemical DNA Biosensors

The discovery of carbon nanotubes (CNTs) about a decade ago has brought fascinating evolutions in electronics, material industry, as well as bio-techniques for DNA analysis, gene therapy, drug delivery etc. It has also dramatically promoted the development of DNA biosensing techniques, especially electrochemical DNA biosensor. The application of CNTs in electrochemical DNA biosensors includes two main aspects: on one hand, using CNTs as a novel substrate not only enables immobilization of DNA molecules but also serves as a powerful amplifier to amplify signal transduction event of DNA hybridization. On the other hand, CNTs can also be employed as a powerful carrier to pre-concentrate enzymes or electroactive molecules for electrochemical sensing of DNA hybridization as a novel indicator. In this review, we place emphasis on recent studies of CNTs-based electrochemical DNA biosensors based on these two aspects, with advantages and disadvantages of each aspect introduced herein.     

Interactions between CdTe quantum dots and DNA revealed by capillary electrophoresis with laser‐induced fluorescence detection

Quantum dots (QDs) are one of the most promising nanomaterials, due to their size‐dependent characteristics as well as easily controllable size during the synthesis process. They are promising label material and their interaction with biomolecules is of great interest for science. In this study, CdTe QDs were synthesized under optimal conditions for 2 nm size. Characterization and verification of QDs synthesis procedure were done by fluorimetric method and with CE. Afterwards, QDs interaction with chicken genomic DNA and 500 bpDNA fragment was observed employing CE‐LIF and gel electrophoresis. Performed interaction relies on possible matching between size of QDs and major groove of the DNA, which is approximately 2.1 nm.     

Rapid DNA electrochemical biosensing platform for label-free potentiometric detection of DNA hybridization

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-G_NP) layer-by-layer films. A precursor film of 3-mercaptopropionic acid (MPA) was firstly self-assembled on the Au electrode surface. CA-G_NP was covalently deposited on the Au/MPA electrode to obtain a stable substrate. SPAN nanofibre and CA-G_NP 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-G_NP/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-G_NP) 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-G_NP/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.     

Optimized DNA hybridization detection on nanocolloidal particles by dielectrophoresis

Oligonucleotides of varying surface coverage are functionalized onto the surface of 100 nm silica particles and the corresponding hybridization reaction with target ssDNA is studied using dielectrophoresis (DEP). The measured DEP cross‐over frequency (cof) is found to be sensitive to the oligonucleotide surface conformation. Zeta potential and particle size measurements suggest that at low oligo surface concentrations, non‐specific binding of oligo to the particle surface prevents efficient hybridization. At high surface coverage, steric hindrance due to the fully stretched, tightly packed oligo conformation prevents diffusion of DNA molecules to the particle surface. The optimum surface coverage exists at intermediate coverage where the particle is found to be the least electrically conductive, and hence exhibits the lowest measured cof. A simple DEP cof measurement hence allows one to determine the optimal oligo surface coverage for increased hybridization efficiency and detection sensitivity.