基于硫化镉纳米团簇标记DNA电化学传感的研究

合成了表面具有自由羧基的硫化镉纳米团簇，以乙基-（3-二甲基丙基）碳二 亚胺盐酸盐为偶联活化剂，将其标记于人工合成的5'端氨基修饰的寡聚核苷酸片段 上，制备成CdS纳米团簇标记DNA探针，该寡聚核苷酸片段与大肠杆菌肠毒素基因相 关。在一定的条件下，使基与固定晨玻碳电极表面的待测DNA序列进行杂交反应， 利用阳极溶出示差脉冲伏安法（ASDPV）间接测定Cd的量，实现对互补、非互补 DNA片段的识别和电化学检测，从而对大肠杆菌肠毒素基因片段识别和检测。     

An electrochemical DNA hybridization detection assay based on a silver nanoparticle label

A novel, sensitive electrochemical DNA hybridization detection assay, using silver nanoparticles as the oligonucleotide labeling tag, is described. The assay relies on the hybridization of the target DNA with the silver nanoparticle-oligonucleotide DNA probe, followed by the release of the silver metal atoms anchored on the hybrids by oxidative metal dissolution and the indirect determination of the solubilized Ag(I) ions by anodic stripping voltammetry (ASV) at a carbon fiber ultramicroelectrode. The influence of the relevant experimental variables, including the surface coverage of the target oligonucleotide, the duration of the silver dissolution steps and the parameters of the electrochemical stripping measurement of the silver(I) ions, is examined and optimized. The combination of the remarkable sensitivity of the stripping metal analysis at the microelectrode with the large number of silver(I) ions released from each DNA hybrid allows detection at levels as low as 0.5 pmol L(-1) of the target oligonucleotides.     

Lead Sulfide Nanoparticle as Oligonucleotides Labels for Electrochemical Stripping Detection of DNA Hybridization

We report a method for the detection of DNA hybridization in connection to lead sulfide (PbS) nanoparticle tags and electrochemical stripping measurement of the lead. A kind of lead sulfide nanoparticle with free carboxyl groups on its surface was synthesized in aqueous solution. The nanoparticle was used as a marker to label a sequence‐known oligonucleotide, which was then employed as a DNA probe for identifying a target ssDNA immobilized on a PPy modified electrode based on a specific hybridization reaction. The hybridization events were monitored by the oxidation dissolution of the lead sulfide anchored on the hybrids and the indirect determination of the lead ions by anodic stripping voltammetry (ASV). The detection limit is 0.3 pmol L^?1 of target oligonucleotides. The PbS nanoparticle combining its easy conjugation to the DNA molecule with the highly sensitive stripping voltammetry detection of lead shows its promising application in the electrochemical DNA hybridization analysis assay.     

Sensitive DNA-hybridization biosensors based on gold nanoparticles for testing DNA damage by Cd(II) ions

A DNA biosensor was constructed by immobilizing a 20-mer oligonucleotide probe and hybridizing it with its complementary oligomer on the surface of a glassy carbon electrode modified with gold nanoparticles. The properties of the biosensor and its capability of recognizing its complementary sequence were studied by electrochemical impedance spectroscopy. The oxidative stress caused by cadmium ions can be monitored by differential pulse voltammetry using the cobalt(III)tris(1,10-phenanthroline) complex and methylene blue as electrochemical indicators. The biosensor is capable of indicating damage caused by Cd(II) ions in pH 6.0 solution. The results showed that the biosensor can be used for rapid screening for DNA damage.

Application of cadmium sulfide nanoparticles as oligonucleotide labels for the electrochemical detection of NOS terminator gene sequences

A mercaptoacetic acid (MAA)-modified cadmium sulfide (CdS) nanoparticle was synthesized in aqueous solution and used as an oligonucleotide label for the electrochemical detection of nopaline synthase (NOS) terminator gene sequence. The carboxyl groups on the surface of the CdS nanoparticle can be easily covalently linked with NH₂-modified NOS oligonucleotide probe sequences. The target ssDNA sequence was fixed onto the electrode surface by covalently linking to a mercaptoethanol self-assembled gold electrode, and the DNA hybridization of target ssDNA with probe ssDNA was accomplished on the electrode surface. The CdS nanoparticles anchored on the hybrids were dissolved in the solution by the oxidation with HNO₃ and further detected by a sensitive differential pulse anodic stripping voltammetric method. The detection results can be used for monitoring the hybridization, and the NOS target sequence was satisfactorily detected in the approximate range from 8.0 × 10⁻¹² to 4.0 × 10⁻⁹ mol L⁻¹ with a detection limit of 2.75 × 10⁻¹² mol L⁻¹ (3σ). The established method extended the nanoparticle-labeled electrochemical DNA analysis to genetically modified organisms (GMOs) specific sequence samples with higher sensitivity and selectivity.     

Label-free oligonucleotide detection method based on a new L-cysteine-dihydroartemisinin complex electroactive indicator

A novel base-mismatched oligonucleotide assay method based on label-free electrochemical biosensor was developed, in which the L-cysteine (Cys)-dihydroartemisinin (DHA) complex was used as a new electroactive indicator. In DNA sensor, Cys-DHA complex was initially formed on electrode surface by cathodic scanning, and target oligonucleotide was conjugated with Cys-terminated DHA indicator through electrostatic interaction under optimal pH. The subsequent sequence assay was responsive to hybridization recognition, which target oligonucleotide was captured by the surface-anchored DNA/Cys-DHA probe. The electrochemical signals of biosensor before and after hybridization were compared basing the measurements of semi-derivative linear scan voltammetry (SDLSV) and electrochemical impedance spectroscopy (EIS). On the basis of signal amplification of electroactive indicator and specific recognition of DNA probe, five target oligonucleotides with different mismatched bases were assayed, and a detection limit reached 0.3 nM. Furthermore, atomic force microscopy (AFM) was used to visually characterize specific recognition spots of biosensor at nanoscale. This study demonstrated a new electroactive molecule-based, biomolecule-involved electroactive indicator and its application in recognition and detection of complementary and base-mismatched oligonucleotide.     

Electrochemical and Electrochemiluminescence Determination of Cancer Cells Based on Aptamers and Magnetic Beads

The electrochemical and electrochemiluminescence (ECL) detection of cell lines of Burkitt’s lymphoma (Ramos) by using magnetic beads as the separation tool and high‐affinity DNA aptamers for signal recognition is reported. Au nanoparticles (NPs) bifunctionalized with aptamers and CdS NPs were used for electrochemical signal amplification. The anodic stripping voltammetry technology employed for the analysis of cadmium ions dissolved from CdS NPs on the aggregates provided a means to quantify the amount of the target cells. This electrochemical method could respond down to 67 cancer cells per mL with a linear calibration range from 1.0×10² to 1.0×10⁵ cells mL^?1, which shows very high sensitivity. In addition, the assay was able to differentiate between target and control cells based on the aptamer used in the assay, indicating the wide applicability of the assay for diseased cell detection. ECL detection was also performed by functionalizing the signal DNA, which was complementary to the aptamer of the Ramos cells, with tris(2,2‐bipyridyl) ruthenium. The ECL intensity of the signal DNA, replaced by the target cells from the ECL probes, directly reflected the quantity of the amount of the cells. With the use of the developed ECL probe, a limit of detection as low as 89 Ramos cells per mL could be achieved. The proposed methods based on electrochemical and ECL should have wide applications in the diagnosis of cancers due to their high sensitivity, simplicity, and low cost.     

Tris(2,2′-bipyridyl)cobalt(III)-doped silica nanoparticle DNA probe for the electrochemical detection of DNA hybridization

A new and sensitive electrochemical DNA hybridization detection assay, using tris(2,2′-bipyridyl)cobalt(III) [Co(bpy)₃³⁺]-doped silica nanoparticles as the oligonucleotide (ODN) labeling tag, and based on voltammetric detection of Co(bpy)₃³⁺ inside silica nanoparticles, is described. Electro-active Co(bpy)₃³⁺ is not possible for directly linking with DNA, it is doped into the silica nanoparticles in the process of nanoparticles synthesis for DNA labeling with trimethoxysilylpropydiethylenetriamine (DETA) and glutaraldehyde as linking agents. The Co(bpy)₃³⁺ labeled DNA probe is used to hybridize with target DNA immobilized on the surface of glassy carbon electrode. Only the complementary sequence DNA (cDNA) could form a double-stranded DNA (dsDNA) with the DNA probe labeled with Co(bpy)₃³⁺ and give an obvious electrochemical response. A three-base mismatch sequence and non-complementary sequence had negligible response. Due to the large number of Co(bpy)₃³⁺ molecules inside silica nanoparticles linked to oligonucleotide DNA probe, the assay showed a high sensitivity. It allows the detection at levels as low as 2.0×10⁻¹⁰ mol l⁻¹ of the target oligonucleotides.     

Electrochemical biosensor based on nanogold-modified poly-eriochrome black T film for BCR/ABL fusion gene assay by using hairpin LNA probe

A novel electrochemical biosensor is described for detection of breakpoint cluster region gene and a cellular abl (BCR/ABL) fusion gene in chronic myelogenous leukemia (CML) by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe. The hairpin LNA probe was immobilized on the nanogold (NG)/poly-eriochrome black T (EBT) film-modified glassy carbon electrode (GCE). The immobilized LNA probe could selectively hybridize with its target DNA on LNA/NG/EBT/GCE surface. The immobilization and hybridization of the LNA probe were characterized with cyclic voltammetry and electrochemical impedance spectroscopy. The hybridization of the immobilized LNA probe with the target DNA was detected by differential pulse voltammetry with the electroactive methylene blue as an indicator. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This novel electrochemical biosensor has been used for assay of PCR real sample with satisfactory result.     

Electrochemical quantification of single-nucleotide polymorphisms using nanoparticle probes

We report a new approach for electrochemical quantification of single-nucleotide polymorphisms (SNPs) using nanoparticle probes. The principle is based on DNA polymerase I (Klenow fragment)-induced coupling of the nucleotide-modified nanoparticle probe to the mutant sites of duplex DNA under the Watson-Crick base pairing rule. After liquid hybridization events occurred among biotinylated DNA probes, mutant DNA, and complementary DNA, the resulting duplex DNA helixes were captured to the surface of magnetic beads through a biotin-avidin affinity reaction and magnetic separation. A cadmium phosphate-loaded apoferritin nanoparticle probe, which is modified with nucleotides and is complementary to the mutant site, is coupled to the mutant sites of the formed duplex DNA in the presence of DNA polymerase. Subsequent electrochemical stripping analysis of the cadmium component of coupled nanoparticle probes provides a means to quantify the concentration of mutant DNA. The method is sensitive enough to detect 21.5 attomol of mutant DNA, which will enable the quantitative analysis of nucleic acid without polymerase chain reaction preamplification. The approach was challenged with constructed samples containing mutant and complementary DNA. The results indicated that it was possible to accurately determine SNPs with frequencies as low 0.01. The proposed approach has a great potential for realizing an accurate, sensitive, rapid, and low-cost method of SNP detection.     

Reaction of Cd(II)-Morin with dsDNA for biosensing of ssDNA oligomers with complementary, GCE-immobilized ssDNA

In this work, the complex cadmium(II)-morin was synthesized and its interaction with double-stranded salmon sperm DNA was studied by electrochemical methods on glassy carbon electrode (GCE). It was shown that Cd(II)-Morin with high electrochemical activity can intercalate into the double-helix DNA, and the binding stoichiometry and equilibrium dissociation constant according to the Hill model for cooperative binding were calculated to be 1.761 and 2.5 x 10(-5) M, respectively. Using Cd(II)-Morin as a novel hybridization indicator, the hybridization between the probe and its complementary and mismatched sequence was investigated by differential pulse voltammetry (DPV), which was to access the selectivity of the developed electrochemical DNA biosensor. The complementary target ssDNA could be quantified over the range from 2.69 x 10(-8) M to 9.16 x 10(-7) M with a linear correlation of 0.9971 and a detection limit of 9.30 x 10(-9) M. These results demonstrated that the Cd(II)-Morin indicator provides great promise for the rapid and selective measurement of the target DNA.     

DNA Hybridization at Magnetic Nanoparticles with Electrochemical Stripping Detection

A simple and practical method for electrochemical DNA hybridization assay has been developed to take advantage of magnetic nanoparticles for ssDNA immobilization and zinc sulfide nanoparticle as oligonucleotide label. Magnetic nanoparticles were prepared by coprecipitation of Fe²⁺ and Fe³⁺ with NH₄OH, and then amino silane was coated onto the surface of magnetite nanoparticles. The magnetic nanoparticles have the advantages of easy preparation, easy surface modification and low cost. The target ssDNA with the phosphate group at the 5′ end was then covalently immobilized to the amino group of magnetite nanoparticles by forming a phosphoramidate bond in the presence of 1‐ethyl‐3‐(3‐dimeth‐ylaminopropyl)carbodiimide (EDAC). The zinc sulfide (ZnS) nanoparticle‐labeled oligonucleotides probe was used to identify the target ssDNA immobilized on the magnetic nanoparticles based on a specific hybridization reaction. The hybridization events were assessed by the dissolution of the zinc sulfide nanoparticles anchored on the hybrids and the indirect determination of the dissolved zinc ions by anodic stripping voltammetry (ASV) at a mercury film glassy carbon electrode (GCE). The proposed method couples the high sensitivity of anodic stripping analysis for zinc ions with effective magnetic separation for eliminating nonspecific adsorption effects and offers great promise for DNA hybridization analysis.     

Microplate electrochemical DNA detection for phosphinothricin acetyltransferase gene sequence with cadmium sulfide nanoparticles

An electrochemical DNA detection method for the phosphinothricin acetyltransferase (PAT) gene sequence from the transgenetic plants was established by using a microplate hybridization assay with cadmium sulfide (CdS) nanoparticles as oligonucleotides label. The experiment included the following procedures. Firstly target PAT ssDNA sequences were immobilized on the polystyrene microplate by physical adsorption. Then CdS nanoparticle labeled oligonucleotide probes were added into the microplate and the hybridization reaction with target ssDNA sequences took place in the microplate. After washing the microplate for three times, certain amounts of HNO₃ were added into the microplate to dissolve the CdS nanoparticles anchored on the hybrids and a solution containing Cd²⁺ ion was obtained. At last differential pulse anodic stripping voltammetry (DPASV) was used for the sensitive detection of released Cd²⁺ ion. Based on this principle a sensitive electrochemical method for the PAT gene sequences detection was established. The voltammetric currents of Cd²⁺ were in linear range with the target ssDNA concentration from 5.0 × 10^− 13 to 1.0 × 10^− 10 mol/L and the detection limit was estimated to be 8.9 × 10^− 14 mol/L (3σ). The proposed method showed a good promise for the sensitive detection of specific gene sequences with good selectivity for the discrimination of the mismatched sequences.     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

The application of β-cyclodextrin derivative functionalized aligned carbon nanotubes for electrochemically DNA sensing via host-guest recognition

We functionalized aligned carbon nanotubes (ACNTs) electrode with a new kind of β-cyclodextrin (β-CD) derivative through diazotization reaction. The resulting β-CD/ACNTs electrode was used to detect DNA hybridization in homogeneous solution based on host–guest recognition technology. In the sensing protocol, one special DNA probe was designed with a stem-loop structure and both ends modified, which we called dually labeled DNA probe (DLP). One end of the DLP was labeled with dabcyl as guest molecule for β-CD/ACNTs electrode capture, and the other end was labeled with a CdS nanoparticle as an electrochemical tag to indicate the occurrence of DNA hybridization. In the absence of the target DNA sequence, the DLP maintains its hairpin structure in solution phase and would not be captured and detected by the β-CD/ACNTs electrode. In the presence of the complementary target sequence, the conformational structure of the DLP was altered and a double-stranded DNA (dsDNA) molecule was formed by the hybridization of DLP and complementary DNA sequence. Consequently, the dsDNA was captured by the β-CD/ACNTs electrode owing to guest–host recognition between β-CD and dabcyl. The electrochemical signal from the CdS nanoparticle–dsDNA/β-CD/ACNTs was then measured. Under optimized detection conditions, the proposed method showed high sensitivity and selectivity with a detection limit of 5.0 × 10⁻¹³ M for complementary DNA sequence.     

Nano-ZnO/Chitosan Composite Film Modified Electrode for Voltammetric Detection of DNA Hybridization

Abstract

A sensitive electrochemical DNA biosensor based on nano-ZnO/chitosan composite matrix for DNA hybridization detection was developed. The Nano-ZnO was synthesized by the hydrothermal method and dispersed in chitosan, which was used to fabricate the modification of the glassy carbon electrode (GCE) surface. The ZnO/chitosan-modified electrode exhibited good biocompatibility and excellent electrochemical conductivity. The hybridization detection was monitored with differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. The established biosensor can effectively discriminate complementary target sequence and two-base-mismatched sequence, with a detection limit of 1.09 × 10^?11 mol L^?1 of complementary target.     

Carbon nanotube-enhanced electrochemical DNA biosensor for DNA hybridization detection

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.     

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 detection of DNA hybridization based on carbon-nanotubes loaded with CdS tags

An effective method for amplifying electrical detection of DNA hybridization based on carbon-nanotubes (CNT) carrying a large number of CdS particle tracers is described. Such use of CNT amplification platforms is combined with an ultrasensitive stripping-voltammetric detection of the dissolved CdS tags following dual hybridization events of a sandwich assay on a streptavidin modified 96-well microplate. Anchoring of the monolayer-protected quantum dots to the acetone-activated CNT was accomplished via hydrophobic interactions. SEM images indicate that the nanocrystals are attached along the CNT sidewall, with a loading of around 500 particles per CNT. A substantial (500-fold) lowering of the detection limit is obtained compared to conventional single-particle stripping hybridization assays, reflecting the CdS loading on the CNT carrier. A large excess (250-fold) of non-complimentary oligonucleotides have minimal effect on the response. Such use of CNT as carriers for multiple electrochemical tags offers great promise for ultrasensitive detection of other biorecognition events.     

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.