Highly sensitive electrochemical impedance spectroscopic detection of DNA hybridization based on Aunano-CNT/PANnano films

A polyaniline nanofibers (PAN_nano)/carbon paste electrode (CPE) was prepared via dopping PAN_nano in the carbon paste. The nanogold (Au_nano) and carbon nanotubes (CNT) composite nanoparticles were bound on the surface of the PAN_nano/CPE. The immobilization and hybridization of the DNA probe on the Au_nano-CNT/PAN_nano films were investigated with differential pulse voltammetry (DPV) and cyclic voltammetry (CV) using methylene blue (MB) as indicator, and electrochemical impedance spectroscopy (EIS) using [Fe(CN)₆]^3−/4− as redox probe. The voltammetric peak currents of MB increased dramatically owing to the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films, and then decreased obviously owing to the hybridization of the DNA probe with the complementary single-stranded DNA (cDNA). The electron transfer resistance (R_et) of the electrode surface increased after the immobilization of the probe DNA on the Au_nano-CNT/PAN_nano films and rose further after the hybridization of the probe DNA. The remarkable difference between the R_et value at the DNA-immobilized electrode and that at the hybridized electrode could be used for the label-free EIS detection of the target DNA. The loading of the DNA probe on Au_nano-CNT/PAN_nano films was greatly enhanced and the sensitivity for the target DNA detection was markedly improved. The sequence-specific DNA of phosphinothricin acetyltransferase (PAT) gene and the polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from transgenically modified beans were determined with this label-free EIS DNA detection method. The dynamic range for detecting the PAT gene sequence was from 1.0 × 10⁻¹² mol/L to 1.0 × 10⁻⁶ mol/L with a detection limit of 5.6 × 10⁻¹³ mol/L.     

Nucleic acid biosensor for detection of hepatitis B virus using 2,9-dimethyl-1,10-phenanthroline copper complex as electrochemical indicator

In this study, an electrochemical DNA biosensor was developed based on the recognition of target DNA by hybridization detection. The study was carried out using glassy carbon electrode (GCE) modified with lable-free 21-mer single-stranded oligonucleotides related to hepatitis B virus sequence via covalent immobilization and [Cu(dmp)(H₂O)Cl₂] (dmp = 2,9-dimethyl-1,10-phenanthroline) as an electrochemical indicator, whose sizes are comparable to those of the small groove of native double-duplex DNA. The method, which is simple and low cost, allows the accumulation of copper complex within the DNA layer. Electochemical detection was performed by cyclic voltammetry and differential pulse voltammetry over the potential range where the [Cu(dmp)(H₂O)Cl₂] was active. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed the assay time. With this approach, a sequence of the hepatitis B virus could be quantified over the ranges from 8.82 × 10⁻⁸ to 8.82 × 10⁻⁷ M with a linear correlation of r = 0.9937 and a detection limit of 7.0 × 10⁻⁸ M. The [Cu(dmp)(H₂O)Cl₂] signal observed from probe sequence before and after hybridization with four bases mismatch containing sequence is lower than that observed after hybridization with complementary sequence.     

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.     

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.     

A nano-porous CeO(2)/Chitosan composite film as the immobilization matrix for colorectal cancer DNA sequence-selective electrochemical biosensor

CeO₂/Chitosan (CHIT) composite matrix was firstly developed for the single-stranded DNA (ssDNA) probe immobilization and the fabrication of DNA biosensor related to the colorectal cancer gene. Such matrix combined the advantages of CeO₂ and chitosan, with good biocompatibility, nontoxicity and excellent electronic conductivity, showing the enhanced loading of ssDNA probe on the surface of electrode. The preparation method is quite simple and inexpensive. The hybridization detection was accomplished by using methylene blue (MB), an electroactive lable, as the indicator. The differential pulse voltammetry (DPV) was employed to record the signal response of MB and determine the amount of colorectal cancer target DNA sequence. The experimental conditions were optimized. The established biosensor has high detection sensitivity, a relatively wide linear range from 1.59 × 10⁻¹¹ to 1.16 × 10⁻⁷ mol L⁻¹ and the ability to discriminate completely complementary target sequence and four-base-mismatched sequence.     

Synergistic effects of nano-ZnO/multi-walled carbon nanotubes/chitosan nanocomposite membrane for the sensitive detection of sequence-specific of PAT gene and PCR amplification of NOS gene

The remarkable synergistic effects of the zinc oxide (ZnO) nanoparticles and multi-walled carbon nanotubes (MWNTs) were developed for the ssDNA probe immobilization and fabrication of the electrochemical DNA biosensor. The ZnO/MWNTs/chitosan nanocomposite membrane-modified glassy carbon electrode (ZnO/MWNTs/CHIT/GCE) was fabricated and the ssDNA probes were immobilized on the modified electrode surface. The preparation method is quite simple and inexpensive. The hybridization events were monitored by differential pulse voltammetry (DPV) using methylene blue (MB) as an indicator. As compared with previous MWNTs-based DNA biosensors, this composite matrix combined the attractive biocompatibility of ZnO nanoparticles with the excellent electron-transfer ability of MWNTs and fine membrane-forming ability of CHIT increased the DNA attachment quantity and complementary DNA detection sensitivity. The approach described here can effectively discriminate complementary DNA sequence, noncomplementary sequence, single-base mismatched sequence and double-base mismatched sequence related to phosphinothricin acetyltransferase (PAT) gene in transgenic corn. Under optimal conditions, the dynamic detection range of the sensor to PAT gene complementary target sequence was from 1.0 × 10⁻¹¹ to 1.0 × 10⁻⁶ mol/L with the detection limit of 2.8 × 10⁻¹² mol/L. The polymerase chain reaction (PCR) amplification of nopaline synthase (NOS) gene from the real sample of one kind of transgenic soybeans was also satisfactorily detected with this electrochemical DNA biosensor, suggesting that the ZnO/MWNTs/CHIT nanocomposite hold great promises for sensitive electrochemical biosensor applications.     

Electrochemical deoxyribonucleic acid biosensor based on carboxyl functionalized graphene oxide and poly-l-lysine modified electrode for the detection of tlh gene sequence related to vibrio parahaemolyticus

A carboxyl functionalized graphene oxide (GO-COOH) and electropolymerized ploy-l-lysine (PLLy) modified glassy carbon electrode (GCE) was fabricated and used for the construction of an electrochemical deoxyribonucleic acid (DNA) biosensor. The NH₂ modified probe ssDNA sequences were immobilized on the surface of GO-COOH/PLLy/GCE by covalent linking with the formation of amide bonds, which was stable and furthur hybridized with the target ssDNA sequence. Differential pulse voltammetry (DPV) was used to monitor the hybridization events with methylene blue as electrochemical indicator, which gave a sensitive reduction peak at −0.287 V (vs. SCE). Under the optimal conditions the reduction peak current was proportional to the concentration of tlh gene sequence in the range from 1.0 × 10⁻¹² to 1.0 × 10⁻⁶ mol L⁻¹ with a detection limit as 1.69 × 10⁻¹³ mol L⁻¹ (3σ). The polymerase chain reaction products of tlh gene from oyster samples were detected with satisfactory results, indicating the potential application of this electrochemical DNA sensor.     

Label-free and sequence-specific DNA detection down to a picomolar level with carbon nanotubes as support for probe DNA

This study describes a simple and label-free electrochemical impedance spectroscopic (EIS) method for sequence-specific detection of DNA by using single-walled carbon nanotubes (SWNTs) as the support for probe DNA. SWNTs are confined onto gold electrodes with mixed self-assembly monolayers of thioethanol and cysteamine. Single-stranded DNA (ssDNA) probe is anchored onto the SWNT support through covalent binding between carboxyl groups at the nanotubes and amino groups at 5′ ends of ssDNA. Hybridization of target DNA with the anchored probe DNA greatly increases the interfacial electron-transfer resistance (R_et) at the double-stranded DNA (dsDNA)-modified electrodes for the redox couple of Fe(CN)₆^3−/4−, which could be used for label-free and sequence-specific DNA detection. EIS results demonstrate that the utilization of SWNTs as the support for probe DNA substantially increases the surface loading of probe DNA onto electrode surface and thus remarkably lowers the detection limit for target DNA. Under the conditions employed here, R_et is linear with the concentration of target DNA within a concentration range from 1 to 10 pM with a detection limit down to 0.8 pM (S/N = 3). This study may offer a novel and label-free electrochemical approach to sensitive sequence-specific DNA detection.     

Electrochemiluminescence of CdTe quantum dots as labels at nanoporous gold leaf electrodes for ultrasensitive DNA analysis

A new electrochemiluminescence (ECL) DNA assay is developed using quantum dots (QDs) as DNA labels. When nanoporous gold leaf (NPGL) electrodes are used, sensitivity of the ECL assay is remarkably increased due to ultra-thin nanopores. In this assay, target DNA (t-DNA) is hybridized with capture DNA (c-DNA) bound on the NPGL electrode, which is fabricated by conjugating amino-modified c-DNA to thioglycolic acid (TGA) modified at the activated NPGL electrode. Following that, amino-modified probe DNA is hybridized with the t-DNA, yielding sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acid-capped CdTe QDs are labeled to the amino group end of the sandwich hybrids. Finally, in the presence of S₂O₈²⁻ as coreactant, ECL emission of the QD-labeled DNA hybrids on the NPGL electrode is measured by scanning the potential from 0 to −2 V to record the curve of ECL intensity versus potential. The maximum ECL intensity (I_m,ECL) on the curve is proportional to t-DNA concentration with a linear range of 5 × 10⁻¹⁵ to 1 × 10⁻¹¹ mol/L. The ECL DNA assay can be used to determine DNA corresponding to mRNA in cell extracts in this study.     

Electrochemical detection of DNA hybridization based on polypyrrole/ss-DNA/multi-wall carbon nanotubes paste electrode

A sensitive electrochemical detection of DNA hybridization using a paste electrode assembled by multi-wall carbon nanotubes (MWNT) and immobilizing DNA probe within electropolymerized polypyrrole (ppy) was developed. The detection approach relied on entrapping of DNA probe within electropolymerized ppy film on the MWNT paste electrode and monitoring the current change generated from an electroactive intercalator of ethidium bromide (EB) after DNA hybridization. As a consequence of DNA hybridization, significant changes in the current of EB intercalated with double-stranded DNA (ds-DNA) on the MWNT paste electrode were observed. Based on the response of EB, only the complementary DNA sequence gave an obvious current signal compared with the five-point mismatched and non-complementary sequences. The oxidation peak current was linearly related to the logarithm of the concentration of the complementary DNA sequence from 1.0 × 10⁻¹⁰ to 1.0 × 10⁻⁸ M with a detection limit of 8.5 × 10⁻¹¹ M. This work demonstrates that the incorporation of MWNT paste electrode with electropolymerization is a promising strategy of functional interfaces for the immobilization of biological recognition elements.     

The interface behavior of hemoglobin at carbon nanotube and the detection for H(2)O(2)

Direct electrochemistry of hemoglobin (Hb) is observed at carbon nanotube (CNT) interface. The adsorbing Hb can transfer electron directly at CNT interface compared with common carbon material. The heterogeneous electron transfer rate constant k of Hb can be calculated as 0.062 s⁻¹, the transfer coefficient α is 0.21 and the average surface coverage of Hb on CNT surface is 3.58 × 10⁻⁹ ± 2.7 × 10⁻¹⁰ mol/cm². It is found that the adsorbing Hb still keeps its catalytic activity to H₂O₂. This sensor was used to detect H₂O₂. The apparent Michaelis-Menten constant is calculated as 6.75 × 10⁻⁴ mol L⁻¹.     

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.     

Simple and label-free electrochemical assay for signal-on DNA hybridization directly at undecorated graphene oxide

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.     

DNA impedance biosensor for detection of cancer, TP53 gene mutation,based on gold nanoparticles/aligned carbon nanotubes modified electrode

For the first time, a new platform based on electrochemical growth of Au nanoparticles on aligned multi-walled carbon nanotubes (A-MWCNT) was developed for sensitive lable-free DNA detection of the TP53 gene mutation, one of the most popular genes in cancer research. Electrochemical impedance spectroscopy (EIS) was used to monitor the sequence-specific DNA hybridization events related to TP53 gene. Compared to the bare Ta or MWCNT/Ta electrodes, the synergistic interactions of vertically aligned MWCNT array and gold nanoparticles at modified electrode could improve the density of the probe DNA attachment and resulting the sensitivity of the DNA sensor greatly. Using EIS, over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship in respect to the logarithm of the complementary oligonucleotides sequence concentrations in the wide range of 1.0 × 10⁻¹⁵ − 1.0 × 10⁻⁷ M, with a detection limit of 1.0 × 10⁻¹⁷ M (S/N = 3). The prepared sensor also showed good stability (14 days), reproducibility (RSD = 2.1%) and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining gold nanoparticles with the on-site fabricated aligned MWCNT array represents a promising platform for achieving sensitive biosensor for fast mutation screening related to most human cancer types.     

Electrochemical behaviors of guanosine on carbon ionic liquid electrode and its determination

The electrochemical behaviors of guanosine on the ionic liquid of N-butylpyridinium hexafluorophosphate (BPPF₆) modified carbon paste electrode (CPE) was studied in this paper and further used for guanosine detection. Guanosine showed an adsorption irreversible oxidation process on the carbon ionic liquid electrode (CILE) with the oxidation peak potential located at 1.12 V (vs. SCE) in a pH 4.5 Britton-Robinson (B-R) buffer solution. Compared with that on the traditional carbon paste electrode, small shift of the oxidation peak potentials appeared but with a great increment of the oxidation peak current on the CILE, which was due to the presence of ionic liquid in the modified electrode adsorbed the guanosine on the surface and promoted the electrochemical response. The electrochemical parameters such as the electron transfer coefficient (α), the electron transfer number (n), and the electrode reaction standard rate constant (k_s) were calculated as 0.74, 1.9 and 1.26 × 10⁻⁴ s⁻¹, respectively. Under the optimal conditions the oxidation peak current showed a good linear relationship with the guanosine concentration in the range from 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol/L by cyclic voltammetry with the detection limit of 2.61 × 10⁻⁷ mol/L (3σ). The common coexisting substances showed no interferences to the guanosine oxidation. The CILE showed good ability to distinguish the electrochemical response of guanosine and guanine in the mixture solution. The urine samples were further detected by the proposed method with satisfactory results.     

Sensitive DNA biosensor improved by Luteolin copper(II) as indicator based on silver nanoparticles and carbon nanotubes modified electrode

A novel and sensitive electrochemical DNA biosensor has been developed for the detection of DNA hybridization. The biosensor was proposed by using copper(II) complex of Luteolin C₃₀H₁₈CuO₁₂ (CuL₂) as an electroactive indicator based on silver nanoparticles and multi-walled carbon nanotubes (Ag/MWCNTs) modified glassy carbon electrode (GCE). In this method, the 4-aminobenzoic acid (4-ABA) and Ag nanoparticles were covalently grafted on MWCNTs to form Ag/4-ABA/MWCNTs. The proposed method dramatically increased DNA attachment quantity and complementary ssDNA detection sensitivity for its large surface area and good charge-transport characteristics. DNA hybridization detection was performed using CuL₂ as an electroactive indicator. The CuL₂ was synthesized and characterized using elemental analysis (EA) and IR spectroscopy. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction between CuL₂ and ds-oligonucleotides (dsDNA). It was revealed that CuL₂ presented high electrochemical activity on GCE, and it could be intercalated into the double helices of dsDNA. The target ssDNA of the human hepatitis B virus (HBV) was quantified in a linear range from 3.23 × 10⁻¹² to 5.31 × 10⁻⁹ M (r = 0.9983). A detection limit of 6.46 × 10⁻¹³ M (3σ, n = 11) was achieved.     

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.     

Influence of hydrofluoric acid on extraction of thorium using a commercially available extraction chromatographic resin

The dependence of Th recovery on hydrofluoric acid (HF) concentration in nitric acid (HNO₃) solutions (1–5 mol/dm³) containing 1 × 10⁻⁶ mol/dm³ of Th and various concentrations of HF and the elution behavior were studied using a commercially available UTEVA (for uranium and tetravalent actinide) resin column. Thorium recovery decreased with an increase in HF concentration in the sample solutions. The concentration of HF at which Th recovery started to decrease was ∼1 × 10⁻⁴ mol/dm³ in 1 mol/dm³ HNO₃ solution, ∼1 × 10⁻³ mol/dm³ in 3 mol/dm³ HNO₃ solution, and ∼1 × 10⁻² mol/dm³ in 5 mol/dm³ HNO₃ solution. When Al(NO₃)₃ (0.2 mol/dm³) or Fe(NO₃)₃ (0.6 mol/dm³) was added as a masking agent for F⁻ to the Th solution containing 1 × 10⁻¹ mol/dm³ HF and 1 mol/dm³ HNO₃, Th recovery improved from 1.4 ± 0.3% to 95 ± 5% or 93 ± 3%. Effective extraction of Th using UTEVA resin was achieved by selecting the concentration of HNO₃ and/or adding masking agents such as Al(NO₃)₃ according to the concentration of HF in the sample solution.     

Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N-dG adducts by capillary electrophoresis immunoassay

The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5′-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K_b: 3.57 × 10⁸ M⁻¹) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(−)-anti-BPDE-dG displays lowest affinity (K_b: 1.14 ×10⁷ M⁻¹). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90mers (K_b: 6.36 ± 0.54 × 10⁸ M⁻¹) than trans-(−)-anti-BPDE-90mers (K_b: 4.52 ± 0.52 × 10⁸ M⁻¹). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K_b: 3.74 × 10⁸ M⁻¹), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA.     

Electrochemical DNA biosensor for the detection of short DNA species of Chronic Myelogenous Leukemia by using methylene blue

A novel assay for the voltammetric detection of 18-bases DNA sequences relating to Chronic Myelogenous Leukemia (CML, Type b3a2) using methylene blue (MB) as the hybridization indicator was reported. DNA was covalently attached onto a glassy carbon electrode (GCE) through amines of the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamion)propyl-N′-ethyl carbodiimidehydrochloride (EDC). The covalently immobilized single-stranded DNA (ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. A significant increase of the peak current for methylene blue upon the hybridization of immobilized ssDNA with cDNA in the solution was observed. This peak current change was used to monitor the recognition of CML DNA sequence. This electrochemical approach is sequence specific as indicated by the control experiments in which no peak current change was observed if a non-complementary DNA sequence was used. Factors, such as DNA target concentration and hybridization conditions determining the sensitivity of the electrochemical assay were investigated. Under optimal conditions, this sensor has a good calibration range between 1.25 × 10⁻⁷ and 6.75 × 10⁻⁷ M, with CML DNA sequence detection limit of 5.9 × 10⁻⁸ M.