Preparation of an electrochemical PNA biosensor for detection of target DNA sequence and single nucleotide mutation on p53 tumor suppressor gene corresponding oligonucleotide

Development of an electrochemical biosensor based on peptide nucleic acid (PNA) probe for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene corresponding oligonucleotide using methylene blue (MB) as an electrochemical indicator is described. The interaction between MB and short sequence of p53, one of the most important tumor suppressor genes due to its dysfunction in the majority of human cancers, was studied by differential pulse voltammety (DPV). Probe modified electrode was prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of gold electrode (GE). The hybridization of PNA probe with target DNA was performed in solution to form PNA-DNA hybrid on the surface of the GE. A significant increase in the reduction signal of MB was observed upon hybridization of the probe with the complementary DNA. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Furthermore, our results confirmed the ability of the sensor to detect single base mismatch in the sample oligonucleotide. The influence of probe concentration on the effective discrimination against noncomplementary sequence and point mutation was also investigated. Diagnostic performance of the biosensor is described and the detection limit is found 6.82 × 10⁻¹⁰ M. The electrochemical impedance spectroscopy was also employed to further investigate the sensor function.     

Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator

Benzoate binuclear copper (II) complex, [Cu₂(C₇H₅O₂)₄(C₂H₆O)₂] (abbreviated as CuR₂) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 7.4 phosphate buffer solution was studied by electrochemical experiments at the Au electrode (AuE). It was revealed that CuR₂ presented an excellent electrochemical activity on AuE and could bind with dsDNA by intercalation mode. The CuR₂ was further utilized as a new indicator in the fabrication of an electrochemical DNA biosensor for detection of BCR/ABL fusion gene. The biosensor based on nanogold (NG) modified AuE was developed by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe for hybridization with BCR/ABL fusion gene. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization. The constructed electrochemical DNA biosensor achieved a detection limit of 1.0 × 10⁻¹⁰ M for complementary target DNA with a good stability.     

Rapid parallelized and quantitative analysis of five pathogenic bacteria by ITS hybridization using QCM biosensor

We developed a 2 × 5 model quartz crystal microbalance (QCM) DNA biosensor array for detection of five bacteria, which based on hybridization analysis of bacterial 16S-23S rDNA internal transcribed spacer (ITS) region. A pair of universal primers was designed for PCR amplification of the ITSs. The PCR products were analyzed by the biosensor. We used gold nanoparticles to amplify the frequency shift signals. Fifty clinical samples were detected by both the biosensor and conventional bacteria culture method. We found a linear quantitative relationship between frequency shift and logarithmic concentration of synthesized oligonucleotides or bacteria cells. The measurable concentration ranged from 10⁻¹² to 10⁻⁸ M for synthesized oligonucleotides and 1.5 × 10² to 1.5 × 10⁸ CFU/mL for bacteria. The 10⁻¹² M of synthesized oligonucleotides or 1.5 × 10² CFU/mL of Pseudomonas aeruginosa could be detected by the biosensor system. The detection could be completed within 5 h including the PCR amplification procedure. Compared with bacteria culture method, the detection sensitivity and specificity of the biosensor system were 94.12% and 90.91%, respectively. There was no significant difference between these two methods (P = 0.625 > 0.05). The biosensor system provides a rapid and sensitive method for parallelized and quantitative analysis of multiple pathogenic bacteria in clinical diagnosis.     

Electrochemical behaviors and determination of isoniazid at ordered mesoporous carbon modified electrode

In this work, an electrochemical sensor based on ordered mesoporous carbon (OMC) for the amperometric detection of isoniazid was developed. OMC was dispersed in a solution of Nafion, and the suspension was modified onto the surface of glassy carbon (GC) electrode. Cyclic voltammetry and amperometry were used to investigate the electrochemical behaviors of isoniazid on Nafion-OMC modified electrode (Nafion-OMC/GC). The results indicate that OMC can facilitate the electrochemical oxidation of isoniazid with a great decrease of overpotential in pH 7.0 phosphate buffer solution. The proposed biosensor provides excellent performance towards the determination of isoniazid with a high sensitivity of 0.031 μA/μM, a low detection limit of 8.4 × 10⁻⁸ M and wide linear range from 1.0 × 10⁻⁷ M to 3.7 × 10⁻⁴ M at +0.20 V vs. Ag/AgCl. The method was successfully applied to the determination of isoniazid tablets with satisfying results. All the results suggest that Nafion-OMC/GC electrode is a potential candidate for a stable and efficient electrochemical sensor to detect isoniazid.     

An amperometric biosensor based on lactate oxidase immobilized in laponite-chitosan hydrogel on a glassy carbon electrode. Application to the analysis of l-lactate in food samples

A biosensor based on the immobilization of lactate oxidase (LOx) on a glassy carbon electrode modified with laponite/chitosan hydrogels for the quantification of l-lactate in alcoholic beverages and dairy products is presented. Ferrocene-methanol (FcMe) is used as artificial mediator. The purpose of this work is to determine the best hydrogel composition from the analytical point of view. The characterization of the hydrogels was carried out by CV, amperometry and EIS. According to permeabilities and charge transfer resistances for ferrocyanide (used as molecular probe) as well as the enzymatic behavior of the enzyme for l-lactate, the best laponite/chitosan mass ratio found was 25/50. The distinct features of the bioelectrode are its long stability, its ability to reject or minimize most interferents including ascorbic acid, and its excellent analytical response, which allowed the reduction of the enzyme content below 0.5 U, for a sensitivity of (0.326 ± 0.003) A cm⁻² M⁻¹, with a time response lower than 5 s and a detection limit of (3.8 ± 0.2) × 10⁻⁶ M. Our l-lactate biosensor was validated by comparison with a standard spectroscopic method.     

A stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene)-single walled carbon nanotubes/ascorbate oxidase/nafion films for detection of L-ascorbic acid

In this paper, a stable sandwich-type amperometric biosensor based on poly(3,4-ethylenedioxythiophene) (PEDOT)-single walled carbon nanotubes (SWCNT)/ascorbate oxidase (AO)/Nafion films for detection of l-ascorbic acid (AA) was successfully developed. PEDOT-SWCNT nanocomposite and Nafion films were used as inner and outer films, respectively. AO was immobilized between these two films. The PEDOT-SWCNT nanocomposite films were characterized by electrochemical impedance spectroscopy and scanning electron microscopy. The influence of detection potential and temperature on the biosensor performance was examined in detail. Despite the multilayer configuration, the biosensor exhibited a relatively fast response (less than 10 s) and a linear range from 1 μM to 18 mM (a correlation coefficient of 0.9974). The sensitivity of the biosensor was found to be 28.5 mA M⁻¹ cm⁻². Its experimental detection limit was 0.7 μM (S/N = 3) and the apparent Michaelis-Menten constant (K_m) was calculated to be 18.35 mM. Moreover, the biosensor exhibited good anti-interferent ability and excellent long-term stability. All the results showed that such sandwich-type PEDOT-SWCNT/AO/Nafion films could provide a promising platform for the biosensor designs for AA detection.     

Screen-printed carbon electrode for choline based on MnO2 nanoparticles and choline oxidase/polyelectrolyte layers

This paper presents the amperometric biosensor that determines choline and cholinesterase activity using a screen printed graphite electrode. In order to detect H₂O₂ we have blanket modified the electrode material with manganese dioxide nanoparticles layer. Using layer-by-layer technique on the developed hydrogen peroxide sensitive electrode surface choline oxidase was incorporated into the interpolyelectrolyte nanofilm. Its ability to serve as a detector of choline in bulk analysis and cholinesterase assay was investigated. We examined the interferences from red-ox species and heavy metals in the blood and in the environmental sample matrixes. The sensor exhibited a linear increase of the amperometric signal at the concentration of choline ranging from 1.3 × 10⁻⁷ to 1.0 × 10⁻⁴ M, with a detection limit (evaluated as 3σ) of 130 nM and a sensitivity of 103 mA M⁻¹ cm⁻² under optimized potential applied (480 mV vs. Ag/AgCl). The biosensor retained its activity for more than 10 consecutive measurements and kept 75% of initial activity for three weeks of storage at 4 °C. The R.S.D. was determined as 1.9% for a choline concentration of 10⁻⁴ M (n = 10) with a typical response time of about 10 s. The developed choline biosensor was applied for butyrylcholinesterase assay showing a detection limit of 5 pM (3σ). We used the biosensor to develop the cholinesterase inhibitor assay. Detection limit for chlorpyrifos was estimated as 50 pM.     

Aptamer-based colorimetric biosensing of dopamine using unmodified gold nanoparticles

A simple, sensitive and selective colorimetric biosensor for the detection of dopamine (DA) was demonstrated with a 58-mer dopamine-binding aptamer (DBA) as recognition element and unmodified gold nanoparticles (AuNPs) as probes. Upon the addition of DA, the conformation of DBA would change from a random coil structure to a rigid tertiary structure like a pocket and this change has been demonstrated by circular dichroism spectroscopic experiments. Besides, the conformational change of DBA could facilitate salt-induced AuNP aggregation and lead to the color change of AuNPs from red to blue. The calibration modeling showed that the analytical linear range covered from 5.4 × 10⁻⁷ M to 5.4 × 10⁻⁶ M and the corresponding limit of detection (LOD) was 3.6 × 10⁻⁷ M. Some common interferents such as 3,4-dihydroxyphenylalanine (DOPA), catechol, epinephrine (EP), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and ascorbic acid (AA) showed no or just a little interference in the determination of DA.     

Electrochemical studies of bovine serum albumin immobilization onto the poly-o-phenylenediamine and carbon-coated nickel composite film and its interaction with papaverine

A biosensor based on bovine serum albumin (BSA) and poly-o-phenylenediamine (PoPD)/carbon-coated nickel (C-Ni) nanobiocomposite film modified electrode has been developed to study the interaction of BSA with papaverine (PAP). The well-dispersed C-Ni nanoparticles were dripped onto the glassy carbon electrode (GCE) surface firstly, and PoPD films were subsequently electropolymerized by cyclic voltammetry (CV) to prepare PoPD/C-Ni/GCE. Finally, the BSA was easily immobilized on the PoPD films via electrostatic adsorption. The morphology and the electrochemical properties of the fabricated composite electrodes were examined by scanning electron microscope (SEM) and electrochemical impedance spectroscopy (EIS), respectively. The interaction of PAP with BSA was monitored by differential pulse voltammetry (DPV), using PoPD as the electrochemical indicator. The binding constant (K), obtained by DPV, was 1.7 × 10⁴ L/mol, which was consistent with the fluorescence analysis. This constructed biosensor also exhibited a fine linear correlation with PAP concentration range of 2.5 × 10⁻⁹-4.5 × 10⁻⁵ mol/L and a detection limit of 8.3 × 10⁻¹⁰ mol/L was achieved by DPV.     

A sensitive hydrazine electrochemical sensor based on electrodeposition of gold nanoparticles on choline film modified glassy carbon electrode

A highly sensitive hydrazine sensor was developed based on the electrodeposition of gold nanoparticles onto the choline film modified glassy carbon electrode (GNPs/Ch/GCE). The electrochemical experiments showed that the GNPs/Ch film exhibited a distinctly higher activity for the electro-oxidation of hydrazine than GNPs with 3.4-fold enhancement of peak current. The kinetic parameters such as the electron transfer coefficient (α) and the rate of electron exchange (k) for the oxidation of hydrazine were determined. The diffusion coefficient (D) of hydrazine in solution was also calculated by chronoamperometry. The sensor exhibited two wide linear ranges of 5.0 × 10⁻⁷-5.0 × 10⁻⁴ and 5.0 × 10⁻⁴-9.3 × 10⁻³ M with the detection limit of 1.0 × 10⁻⁷ M (s/n = 3). The proposed electrode presented excellent operational and storage stability for the determination of hydrazine. Moreover, the sensor showed outstanding sensitivity, selectivity and reproducibility properties. All the results indicated a good potential application of this sensor in the detection of hydrazine.     

High sensitive simultaneous determination of hydroquinone and catechol based on graphene/BMIMPF6 nanocomposite modified electrode

A novel nanocomposite, comprising of graphene sheet (GS) and ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆), was developed on the glassy carbon electrode (GCE) for the simultaneous determination of hydroquinone and catechol in 0.10 M acetate buffer solution (pH 5.0). At the GS/BMIMPF₆/GCE, both hydroquinone and catechol can cause a pair of quasi-reversible and well-defined redox peaks. In comparison with bare GCE and GS modified electrode, GS/BMIMPF₆/GCE showed larger peak currents, which was related to the higher specific surface area of graphene and high ionic conductivity of BMIMPF₆. Under the optimized condition, the cathodic peak current were linear over ranges from 5.0 × 10⁻⁷ M to 5.0 × 10⁻⁵ M for hydroquinone and from 5.0 × 10⁻⁷ M to 5.0 × 10⁻⁵ M for catechol, with the detection limits of 1.0 × 10⁻⁸ M and 2.0 × 10⁻⁸ M, respectively. The proposed method was successfully applied to the simultaneous determination of hydroquinone and catechol in artificial sample, and the results are satisfactory.     

Lead (II) carbon paste electrode based on derivatized multi-walled carbon nanotubes: Application to lead content determination in environmental samples

For the first time a novel derivatized multi-walled carbon nanotubes-based Pb²⁺ carbon paste electrode is reported. The electrode with optimum composition, exhibits an excellent Nernstian response to Pb²⁺ ion ranging from 5.9 × 10⁻¹⁰ to 1.0 × 10⁻² M with a detection limit of 3.2 × 10⁻¹⁰ M and a slope of 29.5 ± 0.3 mV dec⁻¹ over a wide pH range (2.5-6.5) with a fast response time (25 s) at 25 °C. Moreover, it also shows a high selectivity and a long life time (more than 3 months). Importantly, the response mechanism of the proposed electrode was investigated using AC impedance technique. Finally, the electrode was successfully applied for the determination of Pb²⁺ ion concentration in environmental samples, e.g. soils, waste waters, lead accumulator waste and black tea, and for potentiometric titration of sulfate anion.     

Electrochemical behavior of graphene doped carbon paste electrode and its application for sensitive determination of ascorbic acid

In present paper, the graphene doped carbon paste electrode (CPE) was firstly prepared with the addition of graphene into the carbon paste mixture. Compared with conventional CPE, an improved electrochemical response of graphene doped CPE toward the redox couple of Fe(CN)₆^3−/4− was demonstrated owing to the excellent electrical conductivity of graphene. The graphene doped CPE was further used for the successful determination of ascorbic acid (AA), and it showed an excellent electrocatalytic oxidation activity toward AA with a lower overvoltage, pronounced current response, and good sensitivity. Under the optimized experimental conditions, the proposed electrochemical AA sensor exhibited a rapid response to AA within 5 s and a linear calibration plot ranged from 1.0 × 10⁻⁷ to 1.06 × 10⁻⁴ M was obtained with a detection limit of 7.0 × 10⁻⁸ M.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized into poly (lactic-co-glycolic acid)/room temperature ionic liquid composite film

A promising material of poly(lactic-co-glycolic acid) (PLGA) and, room temperature ionic liquid (ILs) (1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF₄) was firstly used as an immobilization matrix to entrap proteins and its bioelectrochemical properties were studied. Direct electrochemistry and electrocatalytic behaviors of hemoglobin (Hb) entrapped in the PLGA/ILs composite film on the surface of glass carbon electrode were investigated. UV-vis spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the composite film. The obtained results demonstrated that the Hb molecule in the film kept its native structure and showed its good electrochemical behavior. A pair of well-defined redox peaks of Hb was obtained at the Hb/PLGA/ILs composite film-modified GC electrode through direct electron transfer between the protein and the underlying electrode. The proposed biosensor showed good reproducibility and high sensitivity to H₂O₂ with the detection limit of 2.37 × 10⁻⁷ M (S/N = 3). In the range of 5.0 × 10⁻⁶ to 8.05 × 10⁻³ M, the catalytic reduction current of H₂O₂ was proportional to its concentration. The apparent Michaelis-Menten constant of Hb in the PLGA/ILs composite film was estimated to be 0.069 mM, showing its high affinity.     

Voltammetric studies of the interaction of rutin with DNA and its analytical applications on the MWNTs-COOH/Fe3O4 modified electrode

Multi-walled carbon nanotubes functionalized with a carboxylic acid group (MWNTs-COOH)/iron oxide (Fe₃O₄) modified glassy carbon electrode (MWNTs-COOH/Fe₃O₄/GCE) and DNA/MWNTs-COOH/Fe₃O₄/GCE were prepared. The electrochemical behaviors of rutin (RU) were investigated on MWNTs-COOH/Fe₃O₄/GCE by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in britton-robinson buffer solution (B-R). The interaction of RU with DNA was also explored. Dramatic decrease of peak current without obvious peak potential shift were observed in both cases of DNA in the solution and immobilized on the electrode surface. In addition, the electron transfer coefficient (α) and the rate constant (k_s) kept unchanged in the absence and presence of DNA. So interaction of DNA with RU formed a non-electroactive complex. The binding constant and binding ratio was obtained in the process. The interaction was also confirmed by UV-visible spectroscopy. The reduction peak current was linear with the concentration of RU in the range of 2.50 × 10⁻⁸ to 1.37 × 10⁻⁶ M, with a detection limit of 7.5 nM. The MWNTs-COOH/Fe₃O₄/GCE showed comparatively low detection limit, rapid response, simplicity for the determination of RU.     

Electrocatalytic determination of hydrazine by a glassy carbon electrode modified with PEDOP/MWCNTs-Pd nanoparticles

The electrocatalysis of hydrazine oxidation by poly-ethylenedioxy pyrrole (PEDOP)-coated MWCNTs-palladium nanoparticles [PEDOP/MWCNTs-Pd] was investigated as an electrochemical sensor on the surface of glassy carbon electrode (GCE) in aqueous medium. Electrochemical oxidation of hydrazine in phosphate buffer (pH 7.4) was performed using cyclic voltammetry (CV) and chronoamperometry (CA) methods. Using the proposed electrode, the catalytic oxidation peak current of hydrazine was high and the overpotential of its oxidation decreased. Based on the obtained results, a mechanism for electrooxidation of hydrazine at [PEDOP/MWCNTs-Pd/GCE] demonstrated an irreversible diffusion-controlled electrode process and a four-electron transfer involved in the overall reaction. The experimental results showed that the mediated oxidation peak currents of the hydrazine were linearly dependent on the concentration of hydrazine in the range of 1.0 × 10⁻⁷ to 5.0 × 10⁻³ M. The detection limit (S/N = 3) was found to be 4 × 10⁻⁸ M with a fast response time of 10 s.     

Two-dimensional molecular imprinting approach for the electrochemical detection of trinitrotoluene

In this work, we demonstrated a sensitive and selective electrochemical sensing protocol for the detection of TNT prepared from alkanethiols self-assembled on AuNPs modified glassy carbon (GC) electrode with preadsorbed templates of TNT. It demonstrated that the 2D molecular imprinting monolayers (MIMs) can provide a better site accessibility and lower mass-transfer resistance, while the AuNPs can enhance electrode conductivity, facilitate the electron transfer and increase the amount of TNT-imprinted sites. The prepared sensor showed not only high selectivity toward TNT in comparison to other similar nitroaromatic compounds (NACs), but also a wide linear range over TNT concentration from 4.0 × 10⁻⁸ to 3.2 × 10⁻⁶ M with a detection limit of 1.3 × 10⁻⁸ M (S/N = 3). Moreover, the imprinted sensor has been applied to the determination of TNT in spiked environmental water samples and shows promise for fast and sensitive measurement of trace levels of TNT in real samples.     

Graphene-polyaniline composite film modified electrode for voltammetric determination of 4-aminophenol

An electrochemical sensor based on graphene-polyaniline (GR-PANI) nanocomposite for voltammetric determination of 4-aminophenol (4-AP) is presented. The electrochemical behavior of 4-AP at the GR-PANI composite film modified glassy carbon electrode (GCE) was investigated by cyclic voltammetry. 4-AP exhibits enhanced voltammetric response at GR-PANI modified GCE. This electrochemical sensor shows a favorable analytical performance for 4-AP detection with a detection limit of 6.5 × 10⁻⁸ M and high sensitivity of 604.2 μA mM⁻¹. Moreover, 4-AP and paracetamol can be detected simultaneously without interference of each other in a large dynamic range.     

Electrochemical genosensor based on modified octadecanethiol self-assembled monolayer for Escherichia coli detection

An electrochemical genosensor based on 1-fluoro-2-nitro-4-azidobenzene (FNAB) modified octadecanethiol (ODT) self-assembled monolayer (SAM) has been fabricated for Escherichia coli detection. The results of electrochemical response measurements investigated using methylene blue (MB) as a redox indicator reveal that this nucleic acid sensor has 60 s of response time, high sensitivity (0.5 × 10⁻¹⁸ M) and linearity as 0.5 × 10⁻¹⁸-1 × 10⁻⁶ M. The sensor has been found to be stable for about four months and can be used about ten times. It is shown that water borne pathogens like Klebsiella pneumonia, Salmonella typhimurium and other gram-negative bacterial samples has no significant effects in the response of this sensor.