Coupling of a Reagentless Electrochemical DNA Biosensor with Conducting Polymer Film and Nanocomposite as Matrices for the Detection of the HIV DNA Sequences

Abstract

This paper describes a reagentless electrochemical DNA biosensor applied to the detection of human immunodeficiency virus (HIV) sequences based on electrochemical impedance spectroscopy (EIS). The novel DNA biosensor has been elaborated by means of an opposite‐charged adsorption Au‐Ag nanocomposite to a conductive polymer polypyrrole (PPy) modified platinum electrode (Pt) and self‐assembly the mercapto oligonucleotide probes onto the surface of modified electrode via the nanocomposite. The duplex formation was detected by measuring the electrochemical impedance signal of nucleic acids in phosphate buffer solution (PBS). Such response is based on the concomitant conductivity changes of the PPy film and nanocomposite. The reagentless scheme has been characterised using 21‐mer synthetic oligonucleotides as models: parameters affecting the hybridization assay were explored and optimized. The detection limit is 5.0×10^?10 M of target oligonucleotides at 3σ. The potential for development of reagentless DNA hybridization analysis in the clinical diagnosis is being pursued.     

A Novel Bioelectrochemical Sensor Based on Immobilized Urease on the Surface of Nickel Oxide Nanoparticle and Polypyrrole Composite Modified Pt Electrode

Nickel oxide nanoparticle (NiO?NP) and polypyrrole (PPy) composite were deposited on a Pt electrode for fabrication of a urea biosensor. To develop the sensor, a thin film of PPy?NiO composite was deposited on a Pt substrate that serves as a matrix for the immobilization of enzyme. Urease was immobilized on the surface of Pt/PPy?NiO by a physical adsorption. The response of the fabricated electrode (Pt/PPy?NiO/Urs) towards urea was analyzed by chronoamperometry and cyclic voltammetry (CV) techniques. Electrochemical response of the bio‐electrode was significantly enhanced. This is due to electron transfer between Ni²⁺ and Ni³⁺ as the electro‐catalytic group and the reaction between polypyrrole and the urease‐liberated ammonium. The fabricated electrode showed reliable and demonstrated perfectly linear response (0.7–26.7 mM of urea concentration, R²= 0.993), with high sensitivity (0.153 mA mM^?1 cm^?2), low detection of limit (1.6 μM), long stability (10 weeks), and low response time (～5 s). The developed biosensor was highly selective and obtained data were repeatable and reproduced using PPy‐NiO composite loaded with immobilized urease as urea biosensors.     

Carbon Nanotubes‐Ionic Liquid and Chloropromazine Modified Electrode for Determination of NADH and Fabrication of Ethanol Biosensor

Potential cycling was used for oxidation of chloropromazine and producing an electroactive redox couples which strongly adsorbed on the electrode surface modified with carbon nanotubes and ionic liquid nanocomposite. The modified electrode shows excellent electrocatalytic activity toward NADH oxidation. The differential pulse voltammetry detection provided high sensitivity, 0.5835 A M^?1, low detection limit, 80 nM at concentration range up to 20 μM. An ethanol biosensor was also developed by immobilizing alcohol dehydrogenase enzyme onto nanocomposite. Differential pulse voltammetric detection of ethanol gives linear responses over the concentration range 40 μM–1.5 mM with detection limit 5 μM and sensitivity 1.97 μA mM^?1.     

A non‐covalent functionalization of copper tetraphenylporphyrin/chemically reduced graphene oxide nanocomposite for the selective determination of dopamine

A non‐covalent functionalization based on a copper tetraphenylporphyrin/chemically reduced graphene oxide (Cu‐TPP/CRGO) nanocomposite is demonstrated for selective determination of dopamine (DA) in pharmaceutical and biological samples. A homogeneous electron‐rich environment can be created on the graphene surface by Cu‐TPP due to the π–π non‐covalent stacking interaction. The synthesized Cu‐TPP/CRGO nanocomposite was characterized using scanning electron microscopy NMR, ultraviolet–visible and electrochemical impedance spectroscopies. The electrocatalytic activity of DA was evaluated using cyclic voltammetry and differential pulse voltammetry. The oxidation peak current (I_pa) of DA increased linearly with increasing concentration of DA in the range 2–200 μM. The detection limit was calculated as 0.76 μM with a high sensitivity of 2.46 μA μM^?1 cm ^{? 2}. The practicality of the proposed DA sensor was evaluated in DA hydrochloride injection, human urine and saliva, and showed satisfactory recovery results for the detection of DA. In addition, the Cu‐TPP/CRGO nanocomposite‐modified electrode showed excellent stability, repeatability and reproducibility towards the detection of DA. Copyright © 2015 John Wiley & Sons, Ltd.     

A Selective Cholesterol Biosensor Based on Composite Film Modified Electrode for Amperometric Detection

An amperometric cholesterol biosensor based on immobilization of cholesterol oxidase in a Prussian blue (PB)/polypyrrole (PPy) composite film on the surface of a glassy carbon electrode was fabricated. Hydrogen peroxide produced by the enzymatic reaction was catalytically reduced on the PB film electrode at 0 V with a sensitivity of 39 μA (mol/L)^?1. Cholesterol in the concentration range of 10^?5 ? 10^?4 mol/L was determined with a detection limit of 6 × 10^?7 mol/L by amperometric method. Normal coexisting compounds in the bio‐samples such as ascorbic acid and uric acid do not interfere with the determination. The excellent properties of the sensor in sensitivity and selectivity are attributed to the PB/PPy layer modified on the sensor.     

Controlled Electrodeposition of Au‐Copper Oxide Nanocomposite on a Renewable Carbon Ceramic Electrode for Sensitive Determination of NADH in Serum Samples

In this study, a sensitive nicotinamide adenine dinucleotide (NADH) biosensor based on Au‐Copper oxide nanocomposite modified carbon ceramic electrode (Au?CuO/CCE) was introduced. The developed NADH biosensor was prepared by controlled electrodeposition of copper and Au nanoparticles on the surface of a renewable CCE and was turned to Au?CuO/CCE by cycling the potential in alkaline media. The prepared electrode was carefully characterized with scanning electron microscopy, X‐ray diffraction, atomic force microscopy and cyclic voltammetry techniques. According to scan rate study, surface coverage (Γ) of the fabricated Au?CuO/CCE was calculated to be 1.54×10^?8 mol cm^?2 which was 3 time more than CuO/CCE. The fabricated electrode is well stable which could be reliably utilized for the determination of NADH with amperometry technique over the concentration range of 1–29 μM with sensitivity and detection limit (S/N=3) of 0.1025 μA μM^?1 and 0.09 μM respectively. The prepared biosensor was used for NADH determination in serum samples with fast response time and satisfactory analytical results.     

Fabrication of Glucose Biosensor Based on Encapsulation of Glucose‐Oxidase on Sol‐Gel Composite at the Surface of Glassy Carbon Electrode Modified with Carbon Nanotubes and Celestine Blue

A simple procedure was developed to prepare a glassy carbon electrode modified with multi walled carbon nanotubes (MWCNTs) and Celestin blue. Cyclic voltammograms of the modified electrode show stable and a well defined redox couple with surface confined characteristic at wide pH range (2–12). The formal potential of redox couple (E′) shifts linearly toward the negative direction with increasing solution pH. The surface coverage of Celestine blue immobilized on CNTs glassy carbon electrode was approximately 1.95×10^?10 mol cm^?2. The charge transfer coefficient (α) and heterogeneous electron transfer rate constants (k_s) for GC/MWCNTs/Celestine blue were 0.43 and 1.26 s^?1, respectively. The modified electrode show strong catalytic effect for reduction of hydrogen peroxide and oxygen at reduced overpotential. The glucose biosensor was fabricated by covering a thin film of sol‐gel composite containing glucose oxides (GOx) on the surface of Celestine blue /MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 0.3 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. The accuracy of the biosensor for glucose detection was evaluated by detection of glucose in a serum sample, using standard addition protocol. In addition biosensor can reach 90% of steady currents in about 3.0 sec and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) was eliminated. Furthermore, the apparent Michaelis–Menten constant 2.4 mM, of GOx on the nano composite exhibits excellent bioelectrocatalytic activity of immobilized enzyme toward glucose oxidation. Excellent electrochemical reversibility of redox couple, high stability, technically simple and possibility of preparation at short period of time are of great advantages of this procedure for modification of glucose biosensor.     

One‐pot Electro‐polymerized SDPAS/PPy/CNTs Modified Electrode for Selective Detection of Dopamine

To improve the performance of dopamine (DA) detection in the presence of ascorbic acid (AA) and uric acid (UA), sodium diphenylamine sulfonate/polypyrrole/multi‐walled carbon nanotubes (SDPAS/PPy/CNTs) film was fabricated on the surface of gold electrode through one‐pot polymerization initiated by electrochemical oxidation. SDPAS were covalently embedded into the backbone of PPy to endow the resultant film with numerous negative‐charged terminals, resulting in selective pre‐adsorption of protonated DA⁺ on the electrode and switching the following anodic reaction to be an adsorption‐controlled process. The detection of DA in the presence of AA and UA by square wave voltammetry method showed an outstanding repeatability with the relative standard deviation of 0.45 %. A good linear relationship was observed between the oxidative peak current and the concentration of DA in the range of 0.827–104 μM (R²=0.993), and the limit of detection (LOD) was calculated to be 0.105 μM (S/N=3).     

Development of glucose amperometric biosensor based on a novel attractive enzyme immobilization matrix: amino derivative of thiacalix[4]arene

Calixarenes and their derivatives may be a promising material for enzyme immobilization owing to their particular configuration, unique molecule recognition function and aggregation properties. In this paper, p-tert-butylthiacalix[4]arene tetra-amine (TC4TA) was first used as enzyme immobilization material. This attractive material was exploited for the mild immobilization of glucose oxidase (GOD) to develop glucose amperometric biosensor. GOD was strongly adsorbed on the TC4TA modified electrode to form TC4TA/GOD composite membrane. The adsorption mechanism was driven from the covalent bond between amino-group of TC4TA and carboxyl group of GOD and molecule recognition function of TC4TA. Amperometric detection of glucose was evaluated by holding the modified electrode at 0.60 V (versus SCE) to oxidize the hydrogen peroxide generated by the enzymatic reaction. The sensor (TC4TA/GOD) showed a relative fast response (response time was about 5 s), low detection limit (20 μM, S/N = 3), and high sensitivity (ca. 10.2 mA M⁻¹ cm⁻²) with a linear range of 0.08–10 mM of glucose, as well as a good operational and storage stability. In addition, optimization of the biosensor construction, the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.     

Chitosan Incorporating Cetyltrimethylammonium Bromide Modified Glassy Carbon Electrode for Simultaneous Determination of Ascorbic Acid and Dopamine

Simultaneous determination of a neurotransmitter, dopamine (DA), and ascorbic acid (AA) is achieved at neutral pH on a chitosan incorporating cetyltrimethylammonium bromide (CTAB) modified glassy carbon (GC) electrode. Differential pulse voltammetry (DPV) technique was used to investigate the electrochemical response of DA and AA at a glassy carbon electrode modified with chitosan incorporating CTAB. An optimum 6.0 mmol L^?1 of CTAB together with 0.5 wt% of chitosan was used to improve the resolution and the determination sensitivity. In 0.1 mol L^?1 aqueous phosphate buffer solution of pH 6.8, the chitosan‐CTAB modified electrode showed a good electrocatalytic response towards DA and AA. The anodic peak potential of DA shifted positively, while that of AA shifted negatively. Thus, the difference of the anodic peaks of DA and AA reached 0.23 V, which was enough to separate the two anodic peaks very well. The presented method herein could be applied to the direct simultaneous determination of DA and AA without prior treatment. The anodic peak currents (I_pa) of DPV are proportional to DA in the concentration range of 8 μM to 1000 μM, to that of AA 10 μM to 2000 μM, with correlation coefficients of 0.9930 and 0.9945, respectively. The linear range is much wider than previously reported.     

Electrochemically Prepared Three‐dimensional Porous Nitrogen‐doped Graphene Modified Electrode for Non‐enzymatic Detection of Hydrogen Peroxide

A facile and green electrochemical method for the fabrication of three‐dimensional porous nitrogen‐doped graphene (3DNG) modified electrode was reported. This method embraces two consecutive steps: First, 3D graphene/polypyrrole (ERGO/PPy) composite was prepared by electrochemical co‐deposition of graphene and polypyrrole on a gold foil. Subsequently, the ERGO/PPy composite modified gold electrode was annealed at high temperature. Thus 3DNG modified electrode was obtained. Scanning electron microscopy (SEM), X‐ray photoelectron spectroscopy (XPS) and Raman spectroscopy were used to characterize the structure and morphology of the electrode. The electrode exhibits excellent electroanalytical performance for the reduction of hydrogen peroxide (H₂O₂). By linear sweep voltammetric measurement, the cathodic peak current was linearly proportional to H₂O₂ concentration in the range from 0.6 μM to 2.1 mM with a sensitivity of 1.0 μA μM⁻¹ cm⁻². The detection limit was ascertained to be 0.3 μM. The anti‐interference ability, reproducibility and stability of the electrode were carried out and the electrode was applied to the detection of H₂O₂ in serum sample with recoveries from 98.4 % to 103.2 %.     

Electrochemically Deposited Porous Graphene−Polypyrrole−Polyphenol Oxidase for Dopamine Biosensor

We report a method for the fabrication of glassy carbon electrode modified porous graphene-polypyrrole-polyphenol oxidase (GCE−PG−PPy−PPO) modified electrode for the determination dopamine. The optimization of pH, concentration and detection limit of dopamine was employed by amperomatric technique. The detection limit of dopamine was found to be in a linear range of 2×10⁻⁸ to 4.6×10⁻⁵ M and lower limit detection is 4×10⁻⁹ M. Michealis – Menten constant (Km) and the activation energy were calculated as 31.32 μM and 37.4−Kj mol⁻¹, respectively. The developed biosensor was used to quantify the dopamine in human urine sample.     

Selective and sensitive determination of dopamine by composites of polypyrrole and graphene modified electrodes

A novel method is developed to fabricate the polypyrrole (PPy) and graphene thin films on electrodes by electrochemical polymerization of pyrrole with graphene oxide (GO) as a dopant, followed by electrochemical reduction of GO in the composite film. The composite of PPy and electrochemically reduced graphene oxide (eRGO)-modified electrode is highly sensitive and selective toward the detection of dopamine (DA) in the presence of high concentrations of ascorbic acid (AA) and uric acid (UA). The sensing performance of the PPy/eRGO-modified electrode is investigated by differential pulse voltammetry (DPV), revealing a linear range of 0.1-150 μM with a detection limit of 23 nM (S/N = 3). The practical application of the PPy/eRGO-modified electrode is successfully demonstrated for DA determination in human blood serum.     

Novel Designing of Chemically Modified Electrode (CME) of the Bio-MOF-1 for the Detection of Dopamine Based on Inhibition of [Ru(bpy)3]2+/DBAE System

Metal organic frameworks (MOFs) have attracted extensive attention in electrochemical research fields due to their high surface area and controlled porosity. Current study is design to investigate the ECL performance of the chemically modified electrode (CME) based on the bio-MOF-1, a porous zinc-adenine framework, which loaded ruthenium complex and employed for the detection of dopamine (DA). The composite material [Ru(bpy)₃]²⁺@bio-MOF-1 (Ru-bMOF) modified carbon glassy electrode (Ru-bMOF/GCE) exhibited an excellent ECL performance having a linear co-efficient response (R²=0.9968) for 2-(dibutyl amino) ethanol (DBAE), a classical ECL co-reactant was obtained over a concentration range of 1.0×10⁻⁹ M to 1.0×10⁻⁴ M in 0.10 M pH=6.0 phosphate buffer solution (PBS). Furthermore, DA was detected based on its inhibition effect on [Ru(bpy)₃]²⁺/DBAE system. Compared to traditional analytical methods, this method has various advantages such as simple electrode preparation, quick response, high reproducibility (RSD<2.0 %), low limit of detection (LOD=1.0×10⁻¹⁰ mol/L). This chemical investigated modified electrode had exploited potential for detection of DA.     

Electrocatalytic Reduction of H2O2 and Oxygen on the Surface of Thionin Incorporated onto MWCNTs Modified Glassy Carbon Electrode: Application to Glucose Detection

A new H₂O₂ enzymeless sensor has been fabricated by incorporation of thionin onto multiwall carbon nanotubes (MWCNTs) modified glassy carbon electrode. First 50 μL of acetone solution containing dispersed MWCNTs was pipetted onto the surface of GC electrode, then, after solvent evaporations, the MWCNTs modified GC electrode was immersed into an aqueous solution of thionin (electroless deposition) for a short period of time <5–50 s. The adsorbed thin film of thionin was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase enzyme. Also the modified electrode shows excellent catalytic activity for oxygen reduction at reduced overpotential. The rotating modified electrode shows excellent analytical performance for amperometric determination of hydrogen peroxide, at reduced overpotentials. Typical calibration at ?0.3 V vs. reference electrode, Ag/AgCl/3 M KCl, shows a detection limit of 0.38 μM, a sensitivity of 11.5 nA/μM and a liner range from 20 μM to 3.0 mM of hydrogen peroxide. The glucose biosensor was fabricated by covering a thin film of sol–gel composite containing glucose oxides on the surface of thionin/MWCNTs modified GC electrode. The biosensor can be used successfully for selective detection of glucose based on the decreasing of cathodic peak current of oxygen. The detection limit, sensitivity and liner calibration rang were 1 μM, 18.3 μA/mM and 10 μM–6.0 mM, respectively. In addition biosensor can reach 90% of steady currents in about 3.0 s and interference effect of the electroactive existing species (ascorbic acid–uric acid and acetaminophen) is eliminated. The usefulness of biosensor for direct glucose quantification in human blood serum matrix is also discussed. This sensor can be used as an amperometric detector for monitoring oxidase based biosensors.     

A Simple Layer‐by‐Layer Assembly Strategy for a Reagentless Biosensor Based on a Nanocomposite of Methylene Blue‐Multiwalled Carbon Nanotubes

A simple layer‐by‐layer (LBL) assembly strategy was established for constructing a novel reagentless biosensor based on a nanocomposite of methylene blue multiwalled carbon nanotubes (MB‐MWNTs). A nanocomposite of MB‐MWNTs was obtained by direct premixing and possessed good dispersion in barbital‐HCl buffer. Through electrostatic interactions, the nanocomposite of MB‐MWNTs could alternately be assembled with horseradish peroxidase (HRP) on the Au electrode modified with precursor films. UV/Vis spectra and scanning electron microscopy (SEM) were applied to reveal the formation of the nanocomposite of MB‐MWNTs. The LBL assembly process was also verified by electrochemical impedance spectroscopy (EIS). The MB is a well‐established mediator and efficiently facilitated the electron shuttle between the HRP and the electrode, as demonstrated by the cyclic voltammetry (CV) measurements. The as‐prepared reagentless biosensor exhibited a fast response for the determination of hydrogen peroxide (H₂O₂) and reached 95% of the steady‐state current within 3 s. It was found that the linear response range of the reagentless biosensor for H₂O₂ was from 4.0 μM to 3.78 mM with a detection limit of 1.0 μM and a sensitivity of 22.5 μA mM⁻¹. The biosensor exhibited a high reproducibility and stability.     

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Electrochemical Fabrication of Prussian Blue Nanocube‐decorated Electroreduced Graphene Oxide for Amperometric Sensing of NADH

In this study, Prussian blue (PB) film on the electroreduced graphene oxide (ERGO)‐modified Au electrode surface (ERGO/PB) is easily prepared by means of cyclic voltammetric technique in the mixture of K₃Fe(CN)₆ and FeCl₃. Its electrochemical behaviors for NADH biosensor are studied. The structural and morphological characters of modified electrode material are analyzed with using of XPS, XRD, Raman, EDS, and SEM techniques. ERGO/PB hybrid nanocomposite for NADH biosensor is exhibited to the higher catalytic effect (linear range from 1.0 to 100 μM, detection limit of 0.23 μM at S/N=3) compared to naked Au, ERGO‐modified Au, and PB‐modified Au electrodes. In addition to, ERGO/PB electrode was used to voltammetric and amperometric detection of H₂O₂. ERGO/PB electrodes also showed the same behavior as the NADH sensor. This ERGO/PB‐modified electrode supplied a simple, new, and low‐cost route for amperometric sensing of both NADH and H₂O₂.     

Reduced Graphene Oxide Non‐covalent Functionalized with Zinc Tetra Phenyl Porphyrin Nanocomposite for Electrochemical Detection of Dopamine in Human Serum and Rat Brain Samples

We described the use of a nanocomposite consisting of reduced graphene oxide and zinc tetraphenylporphyrin (RGO/Zn‐TPP) for electrochemical sensing of dopamine (DA). The surface of RGO was homogeneously functionalized with Zn‐TPP via non‐covalent π‐π interaction. The nanocomposite was characterized by scanning electron microscopy, UV‐Vis spectrometry, nuclear magnetic resonance spectroscopy and electrochemical impedance spectroscopy. The electroanalysis behavior of the nanocomposite was studied by cyclic voltammetry and amperometry. The excellent electrocatalytic activity is found for oxidation of DA, best at working voltage of 0.214 V (vs. Ag/AgCl) and linear response range of 0.04–238.8 μM. The sensitivity and detection limit were of 0.665 μA µM^?1 cm^?2 and 3 nM, respectrively. The electrode is well reproducible, stable, and represents a viable platform for the analysis of DA in DA injection, human serum and rat brain sample.     

Hemoglobin‐Titanate Composite Based Biosensor for the Amperometric Determination of Hydrogen Peroxide in Acidic Medium

A hemoglobin‐titanate composite based biosensor was chosen for determination of H₂O₂ in an acidic medium. CV results of the Hb‐titanate modified pyrolytic graphite electrode showed a pair of well‐defined, quasi‐reversible redox peaks centered at ?246 mV (vs. Ag/AgCl) in a pH 5.0 HAc‐NaAc buffer solution. The modified electrode exhibited good electrocatalytic response for monitoring H₂O₂ and had a large linear detection range from 20 μM to 3.2 mM with a detection limit of 8 μM (S/N=3) and a sensitivity of 29.7 mA M^?1 cm^?2 in the pH 5.0 solution. The biosensor also possessed good long term storage stability.