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.     

Label-Free Detection of DNA Hybridization Based on MnO2 Nanoparticles

Abstract

Nano-MnO₂/chitosan composite film modified glassy carbon electrode (MnO₂/CHIT/GCE) was fabricated and a DNA probe was immobilized on the electrode surface. The immobilization and hybridization events of DNA were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The EIS was applied to the label-free detection of the target DNA. The human immunodeficiency virus (HIV) gene fragment was successfully detected by this DNA electrochemical sensor. The dynamic detection range was from 2.0 × 10^?11 to 2.0 × 10^?6 mol/L, with a detection limit of 1.0 × 10^?12 mol/L.     

Voltammetric Separation and Determination of Glutathione and L‐tyrosine with Chlorogenic Acid as an Electrocatalytic Mediator

A novel and sensitive voltammetric method was proposed for separation and determination of glutathione (GSH) and L‐tyrosine (Tyr) at acetylene black and chitosan modified glassy carbon electrode (AB‐CS/GCE). By introducing chlorogenic acid (CGA) as a new electrocatalytic mediator, GSH could be detected at much lower potential with symmetric peak shape. Acetylene black and chitosan composite served as current signal amplifier for sensitive detection. The electrochemical behavior of GSH and Tyr in the presence of CGA was studied at AB‐CS/GCE and complete separation of anodic peaks was achieved. Under the optimum conditions, the electrocatalytic oxidation peak current of GSH showed a linear dependence on its concentration in the ranges of 2.0×10⁻⁷‐4.0×10⁻⁵ M with the detection limit of 5.8×10⁻⁸ M (S/N=3), while the oxidation peak current of Tyr was linear to its concentration from 2.5×10⁻⁶ to 4.3×10⁻⁴ M with the detection limit of 9.2×10⁻⁷ M (S/N=3) by differential pulse voltammetry (DPV). The established method has been applied to the simultaneous determination of GSH and Tyr in human urine with satisfactory results.     

A Biosensor for Genetic Modified Soybean DNA Determination via Adsorption of Anthraquinone‐2‐sulphonic Acid in Reduced Graphene Oxide

An electrochemical DNA biosensor for DNA determination of genetically modified (GM) soybean (CaMV 35S target genes) was developed utilizing a new detection concept based on the adsoption of anthraquinone‐2‐sulphonic acid (AQMS) on the reduced graphene oxide nano‐particles (rGO) during DNA hybridization events. The aminated DNA probe for CaMV 35S was immobilized onto poly(n‐butyl acrylate) film modified with succinimide functional groups [poly(nBA‐NAS)] via peptide covalent bond. Nanosheets of rGO were entrapped in the poly(nBA‐NAS) film to form a conducting [poly(nBA‐NAS)‐rGO] film of the DNA biosensor. Besides facilitating the electron transfer reactions, the rGO also functioned as an adsorbent for AQMS. The sensing mechanism of the proposed DNA biosensor involved measuring the oxidation current of the AQMS adsorbed on the electrode surface at −0.50 V using differential pulse voltammetry (DPV) before and after a DNA hybridization event. Under optimum conditions, the DNA biosensor demonstrated a linear proportionality between AQMS oxidation signal and logarithm cDNA concentration from 1.0×10⁻¹⁵ M to 1.0×10⁻⁸ M target DNA with a detection limit of 6.3×10⁻¹⁶ M. The electrochemical DNA biosensor possessed good selectivity and a shelf life of about 40 days with relative standard deviation of reproducibility obtained in the range of 3.7–4.6% (n=5). Evaluation of the DNA biosensor using GM soybean DNA extracts showed excellent recovery percentages of 97.2–104.0.     

Development of electrochemical DNA biosensor for Trichoderma harzianum based on ionic liquid/ZnO nanoparticles/chitosan/gold electrode

Electrochemical DNA biosensor was successfully developed by depositing the ionic liquid (e.g., 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM][Otf])), ZnO nanoparticles, and chitosan (CHIT) nanocomposite membrane on a modified gold electrode (AuE). The electrochemical properties of the [EMIM][Otf]/ZnO/CHIT/AuE for detection of DNA hybridization were studied. Under optimal conditions using cyclic voltammetry, the target DNA sequences could be detected in the concentration range of 1.0 × 10⁻¹⁸ to 1.82 × 10⁻⁴ mol L⁻¹, and with the detection limit of 1.0 × 10⁻¹⁹ mol L⁻¹. This DNA biosensor detection approaches provide a quick, sensitive, and convenient method to be used in the identification of Trichoderma harzianum.     

Highly Sensitive in vitro Biosensor for Enterotoxigenic Escherichia coli Detection Based on ssDNA Anchored on PtNPs‐Chitosan Nanocomposite

Detection of Enterotoxigenic Escherichia coli in various biological samples has tremendous importance in human health. In this direction, we have designed a label free electrochemical biosensor for highly selective detection of Escherichia coli through detecting ST gene. The ability of sensor probe to detect ST_G was confirmed using polymerase chain reaction. The biosensor was fabricated based on ST_G specific probes immobilized on platinum nanoparticles chitosan nanocomposite on screen printed carbon electrode, which was characterized by cyclic voltammetry, transmission electron microscopy, and fourier transform infrared spectroscopy. A highly sensitive label free sensing was achieved by analyzing ST_G hybridization using electrochemical impedance spectroscopy (EIS) technique. The EIS analysis showed a significant increase in charge transfer resistance after ST_G interaction with the highly selective ssDNA probe immobilized on the nanocomposite film. The increase in charge transfer resistance was evaluated for varying concentrations of ST_G, which shows a dynamic range between 1.0×10⁻¹² and 1.0×10⁻⁴ with the detection limit of 3.6×10⁻¹⁴ M (RSD<4.5 %). The regeneration of sensor probe was also studied and interference due to non‐target sequences was evaluated to ensure the selectivity of the designed sensor. The practical applicability of sensor probe was also analyzed by detecting the ST_G from the bacteria present in surface water.     

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.     

Electro‐oxidized Monolayer CVD Graphene Film Transducer for Ultrasensitive Impedimetric DNA Biosensor

We report the effect of electrochemical anodization on the properties of monolayer graphene as the main aim of this research and consequently using the resulting label‐free impedimetric biosensor for DNA sequences detection. Monolayer graphene was grown by chemical vapor deposition (CVD) with methane as precursor on copper foil, transferred onto a glassy carbon electrode and electrochemically anodized. Raman spectroscopy and X‐Ray photo electron spectroscopy revealed enhancement of defect density, roughness and formation of C−O−C, C−O−H and C=O functional groups after anodization. Amine‐terminated poly T probe was linked covalently to the carboxylic groups of anodized graphene by the zero‐length linker to fabricate the impedance‐based DNA biosensor. The anodized graphene electrode demonstrated a superior performance for electrochemical impedance detection of DNA. The DNA biosensor showed a large linear dynamic range from 2.0×10⁻¹⁸ to 1.0×10⁻¹² M with a limit of detection of 1.0×10⁻¹⁸ M using electrochemical impedance spectroscopy (EIS) method. Equivalent circuit modeling shows that DNA hybridization is detected through a change in charge transfer resistance.     

Disposable Amperometric Biosensor Based on Poly‐L‐lysine and Fe3O4 NPs‐chitosan Composite for the Detection of Tyramine in Cheese

An amperometric tyramine biosensor based on poly‐L‐lysine (PLL) and Fe₃O₄ nanoparticles (Fe₃O₄NP) modified screen printed carbon electrode (SPCE) was developed. PLL was formed on the SPCE by the electropolymerization of L‐lysine. Subsequently, Fe₃O₄NP suspension prepared in chitosan (CH) solution was casted onto the PLL/SPCE. Tyrosinase (Ty) enzyme was immobilized onto the modified Fe₃O₄?CH/PLL/SPCE and the electrode was coated with Nafion to fabricate the Ty/Fe₃O₄?CH/PLL/SPCE. Different techniques including scanning electron microscopy, chronoamperometry (i–t curve), cyclic voltammetry and electrochemical impedance spectroscopy were utilized to study the fabrication processes, electrochemical characteristics and performance parameters of the biosensor. The analytical performance of the tyramine biosensor was evaluated with respect to linear range, sensitivity, limit of detection, repeatability and reproducibility. The response of the biosensor to tyramine was linear between 4.9×10^?7–6.3×10^?5 M with a detection limit of 7.5×10^?8 M and sensitivity of 71.36 μA mM^?1 (595 μA mM^?1 cm^?2). The application of the developed biosensor for the determination of tyramine was successfully tested in cheese sample and mean analytical recovery of added tyramine in cheese extract was calculated as 101.2±2.1 %. The presented tyramine biosensor is a promising approach for tyramine analysis in real samples due to its high sensitivity, rapid response and easy fabrication.     

Multilayer Assembly of Hemoglobin and Colloidal Gold Nanoparticles on Multiwall Carbon Nanotubes/Chitosan Composite for Detecting Hydrogen Peroxide

Chitosan (CS) was chosen for dispersing multi‐wall carbon nanotubes (MWNTs) to form a stable CS‐MWNTs composite, which was first coated on the surface of a glassy carbon electrode to provide a containing amino groups interface for assembling colloidal gold nanoparticles (GNPs), followed by the adsorption of hemoglobin (Hb). Repeating the assembly step of GNPs and Hb resulted in {Hb/GNPs}_n multilayers. The assembly of GNPs onto CS‐MWNTs composites was confirmed by transmission electron microscopy. The consecutive growth of {Hb/GNPs}_n multilayers was confirmed by cyclic voltammetry and UV‐vis absorption spectroscopy. The resulting system brings a new platform for electrochemical devices by using the synergistic action of the electrocatalytic activity of GNPs and MWNTs. The resulting biosensor displays an excellent electrocatalytic activity and rapid response for hydrogen peroxide. The linear range for the determination of H₂O₂ was from 5.0×10^?7 to 2.0×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelis–Menten constant K_M^app value of 0.19 mM.     

A sensitive and selective sensor‐coated molecularly imprinted sol–gel film incorporating β‐cyclodextrin‐multi‐walled carbon nanotubes and cobalt nanoparticles‐chitosan for oxacillin determination

A sensitive and selective imprinted electrochemical sensor for the determination of oxacillin was developed based on indium tin oxide electrode. The proposed sensor was decorated with imprinted sol–gel film and cobalt nanoparticles‐chitosan/β‐cyclodextrin‐multiwalled carbon nanotubes nanocomposites. The surface morphologies of the modified electrodes were characterized by scanning electron microscopy and transmission electron microscope. The stepwise assembly process and electrochemical behavior of the novel sensor were characterized by differential pulse voltammetry, cyclic voltammetry and Amperometric i‐t response. The imprinted sensor displayed excellent selectivity toward oxacillin. Meanwhile, the introduced cobalt nanoparticles‐chitosan and β‐cyclodextrin‐multi‐walled carbon nanotubes exhibited noticeable amplified electrochemical response signal. The differential voltammetric anodic peak current was linear to oxacillin concentration in the range from 2.0 × 10^?7 to 1.0 × 10^?4 mol·l^?1, and the detection limit was 6.9 × 10^?9 mol·l^?1. The proposed imprinted sensor was applied to the determination of oxacillin in human blood serum samples successfully. Copyright © 2011 John Wiley & Sons, Ltd.     

Synergistic Effect of MWNTs/CeO2/CHIT Film for Detection of CdSe Nanoparticle Labeled Sequence‐Specific of 35S Promoter of Cauliflower Mosaic Virus Gene

A porous composite film was fabricated combining the advantages of multiwalled carbon nanotubes, CeO₂ and chitosan. The synergistic effect of the film improved the immobilization of probe ssDNA. The loaded probe ssDNA was used for detection of CdSe quantum dots labeled target DNA. The DNA hybridization reaction was detected by differential pulse anodic stripping voltammetry of Cd²⁺ after the oxidative release of labeled CdSe quantum dots. The established DNA biosensor can discriminate different target sequences associated with 35S promoter of cauliflower mosaic virus gene with relatively wide linear range and low detection limit (2.4×10^?13 mol/L).     

A Selective Voltammetric Method for Uric Acid Detection at a Glassy Carbon Electrode Modified with Electrodeposited Film Containing DNA and Pt‐Fe(III) Nanocomposites

A novel biosensor by electrochemical codeposited Pt‐Fe(III) nanocomposites and DNA film was constructed and applied to the detection of uric acid (UA) in the presence of high concentration of ascorbic acid (AA). Based on its strong catalytic activity toward the oxidation of UA and AA, the modified electrode resolved the overlapping voltammetric response of UA and AA into two well‐defined peaks with a large anodic peak difference (ΔE_pa) of about 380mV. The catalytic peak current obtained from differential pulse voltammetry (DPV) was linearly dependent on the UA concentration from 3.8×10^?6 to 1.6×10^?4 M (r=0.9967) with coexistence of 5.0×10^?4 M AA. The detection limit was 1.8×10^?6 M (S/N=3) and the presence of 20 times higher concentration of AA did not interfere with the determination. The modified electrode shows good sensitivity, selectivity and stability.     

Electrochemical DNA Biosensor Based on Graphene and TiO2 Nanorods Composite Film for the Detection of Transgenic Soybean Gene Sequence of MON89788

Based on graphene (GR), TiO₂ nanorods, and chitosan (CTS) nanocomposite modified carbon ionic liquid electrode (CILE) as substrate electrode, a new electrochemical DNA biosensor was effectively fabricated for the detection of the transgenic soybean sequence of MON89788. By using methylene blue (MB) as hybridization indicator for monitoring the hybridization with different ssDNA sequences, the differential pulse voltammetric response of MB on DNA modified electrodes were recorded and compared. Due to the synergistic effects of TiO₂ nanorods and GR on the electrode surface, the electrochemical responses of MB were greatly increased. Under optimal conditions the differential pulse voltammetric response of the target ssDNA sequence could be detected in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 7.21×10^?13 mol/L (3σ). This electrochemical DNA biosensor was further applied to the polymerase chain reaction (PCR) product of transgenic soybeans with satisfactory results.     

Selective Electroanalysis of Ascorbic Acid Using a Nickel Hexacyanoferrate and Poly(3,4‐ethylenedioxythiophene) Hybrid Film Modified Electrode

The mixed‐valent nickel hexacyanoferrate (NiHCF) and poly(3,4‐ethylenedioxythiophene) (PEDOT) hybrid film (NiHCF‐PEDOT) was prepared on a glassy carbon electrode (GCE) by multiple scan cyclic voltammetry. The films were characterized using atomic force microscopy, field emission scanning electron microscopy, energy dispersive spectroscopy, X‐ray diffraction, and electrochemical impedance spectroscopy (AC impedance). The advantages of these films were demonstrated for the detection of ascorbic acid (AA) using cyclic voltammetry and amperometric techniques. The electrocatalytic oxidation of AA at different electrode surfaces, such as the bare GCE, the NiHCF/GCE, and the NiHCF‐PEDOT/GCE modified electrodes, was determined in phosphate buffer solution (pH 7). The AA electrochemical sensor exhibited a linear response from 5×10⁻⁶ to 1.5×10⁻⁴ M (R²=0.9973) and from 1.55×10⁻⁴ to 3×10⁻⁴ M (R²=0.9983), detection limit=1×10⁻⁶ M, with a fast response time (3 s) for AA determination. In addition, the NiHCF‐PEDOT/GCE was advantageous in terms of its simple preparation, specificity, stability and reproducibility.     

Amperometric Biosensor for Detection of Phenolic Compounds Based on Tyrosinase,N‐Acetyl‐L‐cysteine‐capped Gold Nanoparticles and Chitosan Nanocomposite

A novel biosensor was fabricated based on the immobilization of tyrosinase and N ‐acetyl‐L ‐cysteine‐capped gold nanoparticles onto the surface of the glassy carbon electrode via the film forming by chitosan. The NAC‐AuNPs (N ‐acetyl‐L ‐cysteine‐capped gold nanoparticles) with the average size of 3.4 nm had much higher specific surface area and good biocompatibility, which were favorable for increasing the immobilization amount of enzyme, retaining the catalytic activity of enzyme and facilitating the fast electron transfer. The prepared biosensor exhibited suitable amperometric responses at −0.2 V for phenolic compounds vs. saturated calomel electrode. The parameters of influencing on the working electrode such as pH , temperature, working potential were investigated. Under optimum conditions, the biosensor was applied to detect catechol with a linear range of 1.0 × 10⁻⁷ to 6.0 × 10⁻⁵ mol•L⁻¹ , and the detection limit of 5.0 × 10⁻⁸ mol•L⁻¹ (S /N =3). The stability and selectivity of the proposed biosensor were also evaluated.     

Graphene/Poly(vinylferrocene) Composite Based Amperometric Biosensor for L‐lysine Determination

A novel and sensitive amperometric biosensor for L‐lysine determination based on a glassy carbon electrode (GCE) modified with graphene (GR) and redox polymer poly(vinylferrocene) (PVF) was constructed. L‐lysine‐α‐oxidase was immobilized onto the modified GCE by a glutaraldehyde/bovine serum albumin cross‐linking procedure. SEM, CV and EIS were used for the characterization of the surface morphology and stepwise fabrication processes of PVF/GR composite. Optimal composition of the biosensor and experimental conditions that affect the performance of the biosensor are discussed. The effect of buffer pH on biosensor response was studied in detail over a wide pH range. L‐lysine biosensor displayed a linear range of 9.9×10⁻⁷ ‐ 3.1×10⁻⁴ M with a low detection limit of 2.3×10⁻⁷ M and K_M ^app value of 0.4 mM. The L‐lysine biosensor was tested using pharmaceutical sample and cheese with satisfactory results.     

A Novel Hydrogen Peroxide Biosensor Based on the PDDA‐GNPs/MWNTs Nanocomposite Matrix

A novel nanocomposite designed by the assembly of the positively charged poly(diallyldimethylammonium chloride) protected gold nanoparticles (PDDA‐GNPs), and the negatively charged multi‐walled carbon nanotubes (MWCNTs) on ITO electrode via electrostatic interaction, was used as a supporting matrix for immobilizing hemoglobin (Hb) to develop a high‐performance hydrogen peroxide (H₂O₂) biosensor. The cyclic voltammetrys of immobilized Hb showed a pair of well‐defined and quasi‐reversible redox peaks with the formal potential of ‐0.205V (vs. SCE) and the peak‐to‐peak potential separation of 44 mV at a scan rate of 100 mV×s^?1 in 0.1 mol×L^?1 pH 7.0 PBS. Under the optimized experimental conditions, a linearity range for determination of H₂O₂ was from 2.0 × 10^?6 to 5.2 × 10^?4 mol×L^?1 with a correlation coefficient of 0.9994 (n = 37) and a detection limit of 8.4 × 10^?7 mol×L^?1. The biosensor displayed excellent electrochemical and electrocatalytic response to the reduction of H₂O₂, high sensitivity, long‐term stability, good bioactivity and selectivity.     

A Newly Competitive Electrochemical Sensor for Sensitive Determination of Chrysin Based on Electrochemically Activated Ta2O5 Particles Modified Carbon Paste Electrode

A novel electrochemically activated doped Ta₂O₅ particles modified carbon paste electrode (EA‐Ta₂O₅‐CPE) was prepared and applied for selective and sensitive determination of chrysin. X‐ray diffraction (XRD), scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) techniques and cyclic voltammetry (CV) were used to characterize the Ta₂O₅ particles and investigate the electrochemical response of the sensor. Compared with bare CPE, the doped Ta₂O₅ modified electrode got much more porous by electrochemical treatment and exhibited larger effective surface area, more reactive site and excellent electrochemical catalytic activity toward the oxidation of chrysin. Under optimum conditions by LSV, the oxidation peak currents responded to chrysin linearly over a concentration range from 5.0×10⁻⁸ to 7.0×10⁻⁶ mol L⁻¹ with a detection limit of 2.0×10⁻⁸ mol L⁻¹ (5.08 ng mL⁻¹). The fabricated sensor showed anti‐interference ability against the biological common interferents (i.e. baicalein, baicalin) and provided to be reliable for the determination of chrysin in Chinese medicinal herb Oroxylum indicum and chrysin capsules samples with satisfactory results.     

Fabrication of CeO2 Nanoparticle Modified Glassy Carbon Electrode for Ultrasensitive Determination of Trace Amounts of Uric Acid in Urine

The preparation of a glassy carbon electrode modified by CeO2 nanoparticles was described, which was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. In pH 6.0 buffer, the CeO2 nanoparticle modified electrode （CeO2 NP/GC） gave an excellent electrocatalytic activity for the oxidation of uric acid （UA）. The catalytic current of UA versus its concentration had a good linear relation in the range of 2.0 × 10^-7-5.0 × 10^- 4 mol/L, with the correlation coefficient of 0.9986 and detection limit of 1.0 ×10^-7 mol/L. The modified electrode can be used for the determination of UA in urine, which can tolerate the interference of ascorbic acid up to 1000-fold. The method was simple, quick and sensitive.