A novel and simple biomolecules immobilization method: electro-deposition ZrO2 doped with HRP for fabrication of hydrogen peroxide biosensor

For the first time, a very novel and simple immobilization method for fabrication of hydrogen peroxide biosensor was reported in this paper. The biocompatible composite HRP-ZrO(2) thin films were synthesized on gold electrode surface based on electro-deposition zirconia doped with horseradish peroxidase (HRP) by cyclic voltammetry scanning in KCl solution containing ZrO(2) and HRP. The fabricated process of biosensor was characterized by electrochemical impedance spectroscopy (EIS) and the surface topography of the prepared films was imaged by atomic force microscope (AFM). The HRP in HRP-ZrO(2) thin films kept its bioactivity and exhibited excellent electrocatalytical response to the reduction of H(2)O(2). Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP-ZrO(2)/Au electrode) showed a linear response to H(2)O(2) over a concentration range from 0.02 to 9.45mM with a detection limit of 2muM based on a signal-to-noise ratio of 3 under optimized conditions. The apparent Michaelis-Menten constant (K(M)(app)) was evaluated to be 8.01mM, which indicated the HRP in HRP-ZrO(2) thin films kept its native bioactivity and had high affinity for H(2)O(2). Moreover, the proposed biosensor showed high sensitivity, good reproducibility and long-term stability. What is more, this immobilization methodology widened biosensor application in biomolecules immobilization and could further develop for other protein and biomolecules immobilization.     

Ultrasensitive DNA sensor based on gold nanoparticles/reduced graphene oxide/glassy carbon electrode

We have designed a simple and novel electrochemical biosensor based on glassy carbon electrode (GCE) for DNA detection. GCE was modified with reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) by the electrochemical method, which is helpful for immobilization of thiolated bioreceptors. The electrode modification processes were characterized by scanning electron microscopy (SEM) and electrochemical methods. Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time. The experimental conditions, such as probe immobilization time and target DNA (complementary DNA) hybridization time and temperature with probe DNA, were optimized using electrochemical methods. The electrochemical response for DNA hybridization and synthesis was measured using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) methods. The calibration graph contains two linear ranges; the first part is in the range of 3.0 × 10⁻²⁰ to 1.0 × 10⁻¹² M, and the second segment part is in the range of 1.0 × 10⁻¹² to 1.0 × 10⁻⁷ M. The biosensor showed excellent selectivity for the detection of the complementary sequences from noncomplementary sequences, so it can be used for detection of breast cancer.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

A sputtered thin film of nanostructured Ni/Pt/Ti on Al2O3 substrate for ethanol sensing

A novel thin film ethanol sensor using sputtered Ni/Pt/Ti on an Al2O3 substrate as the working electrode in an alkaline solution was developed. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to characterize the nanostructure of nickel films. Sputtering deposition conditions for maximum catalytic efficiency, electrode selectivity, and reproducibility were discussed. The results showed that ethanol oxidation was more efficient on the sputtered Ni/Pt/Ti on an Al2O3 substrate electrode than that on the conventional nickel electrode. The optimal operating conditions to generate the sputtered Ni/Pt/Ti on the Al2O3 substrate electrode were: 45 min of Ni sputtering deposition time, and 50 W of Ni sputtering power. The results also indicated that the response time of the prepared ethanol sensor is 27 s and the best sensitivity is 3.08 microA microM(-1) cm(-2).     

Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode

The preparations and performances of the novel amperometric biosensors for glucose based on immobilized glucose oxidase (GOD) on modified Pt electrodes are described. Two types of modified electrodes for the enzyme immobilization were used in this study, polyvinylferrocene (PVF) coated Pt electrode and gold deposited PVF coated Pt electrode. A simple method for the immobilization of GOD enzyme on the modified electrodes was described. The enzyme electrodes developed in this study were called as PVF-GOD enzyme electrode and PVF-Au-GOD enzyme electrode, respectively. The amperometric responses of the enzyme electrodes were measured at constant potential, which was due to the electrooxidation of enzymatically produced H2O2. The electrocatalytic effects of the polymer, PVF, and the gold particles towards the electrooxidation of the enzymatically generated H2O2 offers sensitive and selective monitoring of glucose. The biosensor based on PVF-Au-GOD electrode has 6.6 times larger maximum current, 3.8 times higher sensitivity and 1.6 times larger linear working portion than those of the biosensor based on PVF-GOD electrode. The effects of the applied potential, the thickness of the polymeric film, the amount of the immobilized enzyme, pH, the amount of the deposited Au, temperature and substrate concentration on the responses of the biosensors were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. Finally the effects of interferents, stability of the biosensors and applicability to serum analysis of the biosensor were also investigated.     

Disposable biosensor based on enzyme immobilized on Au-chitosan-modified indium tin oxide electrode with flow injection amperometric analysis

Indium tin oxide (ITO) electrode is used to fabricate a novel disposable biosensor combined with flow injection analysis for the rapid determination of H2O2. The biosensor is prepared by entrapping horseradish peroxidase (HRP) enzyme in colloidal gold nanoparticle-modified chitosan membrane (Au-chitosan) to modify the ITO electrode. The biosensor is characterized by scanning electron microscope, atomic force microscope, and electrochemical methods. Parameters affecting the performance of the biosensor, including concentrations of o-phenylenediamine (OPD) and pH of substrate solution, were optimized. Under the optimal experimental conditions, H2O2 could be determined in the linear calibration range from 0.01 to 0.5 mM with a correlation coefficient of 0.997 (n=8). The amperometric response of the biosensor did not show an obvious decrease after the substrates were injected continuously 34 times into the flow cell. The prepared biosensor not only is economic and disposable, due to the low-cost ITO film electrode obtained from industrial mass production, but also is capable with good detection precision, acceptable accuracy, and storage stability for the fabrication in batch.     

Third-generation superoxide anion sensor based on superoxide dismutase directly immobilized by sol-gel thin film on gold electrode

A third-generation biosensor for superoxide anion (O(2)*-) was developed based on superoxide dismutase (SOD) immobilized by thin silica-PVA sol-gel film on gold electrode surface. A rapid and direct electron transfer of SOD in the thin sol-gel film at the gold electrode was realized without any mediators or promoters. The characterization of the SOD electrodes showed a quasi-reversible electrochemical redox behavior with a formal potential of 80 + 5 mV (versus SCE) in 50 mmol l(-1) phosphate buffer solution (PBS), pH 7.0. The heterogeneous electron transfer rate constant was evaluated to be about 2.1s(-1). The anodic and cathodic transfer coefficients are 0.6 and 0.4, respectively. Based on biomolecular recognition for specific reactivity of SOD toward O(2)*- the SOD electrode was applied to a sensitive and selective measurement of O(2)*- with the low operation potential (-0.15 V versus SCE) in phosphate buffer solution, pH 7.0. The amperometric response was proportional to O(2)*- concentration in the range of 0.2-1.6 micromol l(-1) and the detection limit was 0.1 micromol l(-1) at a signal-to-noise ration of 3. The preparation of SOD electrode is easy and simple. The uniform porous structure of the silica-PVA sol-gel matrix results in a fast response rate of immobilized SOD and is very efficient for stabilizing the enzyme activity.     

Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor

A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 μm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12) M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.     

Influence of immobilization strategies on biosensing response characteristics: A comparative study

The immobilization technique plays an important role in fabrication of a biosensor. NiO based cholesterol biosensor has been used to study the effect of various immobilization techniques on the biosensing response characteristics. The biosensors were fabricated by immobilizing cholesterol oxidase on NiO thin films by three different immobilization techniques viz. physisorption, cross-linking and covalent binding. The study reveals a strong dependence of biosensing response on corresponding immobilization technique. The biosensor based on immobilization by covalent bonding shows superior response characteristics as compared to others owing to its zero length. The results highlight the significance of immobilization technique for biosensor fabrication.     

A novel hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase onto Au-modified titanium dioxide nanotube arrays

In this study, we report on a promising H(2)O(2) biosensor based on the co-immobilization of horseradish peroxidase (HRP) and chitosan onto Au-modified TiO(2) nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then uniformly coated onto the TiO(2) nanotube arrays by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO(2) nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and chronoamperometry were used to study and to optimize the performance of the resulting electrochemical biosensor. The effect of pH, applied electrode potential, the presence of the electron-mediator methylene blue, and the anodic oxidation time of the Ti substrate on the electrochemical biosensor has been systemically studied. Our electrochemical measurements show that the Au-modified TiO(2) nanotube arrays provide excellent matrices for the immobilization of HRP and that the optimized electrochemical biosensor exhibits long linearity, a low detection limit, high stability and very good reproducibility for the detection of H(2)O(2). Under the optimized conditions the linearity of the developed biosensor for the detection of H(2)O(2) is observed from 5 x 10(-6) to 4 x 10(-4) moll(-1) with a detection limit of 2 x 10(-6) moll(-1) (based on the S/N=3).     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

QCM detection of DNA targets with single-base mutation based on DNA ligase reaction and biocatalyzed deposition amplification

A novel biosensing technique for highly specific identification of gene with single-base mutation is proposed based on the implementation of the DNA ligase reaction and the biocatalyzed deposition of an insoluble product. The target gene mediated deposition of an insoluble precipitate is then transduced by quartz crystal microbalance (QCM) measurements. In this method, the DNA target hybridizes with a capture DNA probe tethered onto the gold electrode and then with a biotinylated allele-specific detection DNA. A ligase reaction is performed to generate the ligation between the capture and the detection probes, provided there is perfect match between the DNA target and the detection probe. Otherwise even when there is an allele mismatch between them, no ligation would take place. After thermal treatment at an elevated temperature, the formed duplex melts apart that merely allows the detection probe perfectly matched with the target to remain on the electrode surface. The presence of the biotinylated allele-matched probe is then detected by the QCM via the binding to streptavidin-peroxide horseradish (SA-HRP), which catalyzes the oxidative precipitation of 3,3-diaminobenzidine (DAB) by H2O2 on the electrode and provides an amplified frequency response. The proposed approach has been successfully implemented for the identification of single-base mutation in -28 site of the beta-thalassemia gene with a detection limit of 0.1 nM, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.     

Triplex signal amplification for electrochemical DNA biosensing by coupling probe-gold nanoparticles-graphene modified electrode with enzyme functionalized carbon sphere as tracer

An ultrasensitive electrochemical DNA biosensor was constructed by assembling probe labeled gold nanoparticles (ssDNA-AuNP) on electrochemically reduced graphene oxide (ERGO) modified electrode with thiol group tagged (GT) DNA strand (d(GT)(29)SH) and coupling with horseradish peroxidase (HRP) functionalized carbon sphere (CNS) as tracer. The heteronanostructure formed on the biosensor surface appeared relatively good conductor for accelerating the electron transfer, while the HRP tagged CNS provided dual signal amplification for electrochemical biosensing. The triplex signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to attomolar level (5 aM) with a linear range of 5 orders of magnitude (from 1 × 10(-17)M to 1 × 10(-13)M), and appeared high selectivity to differentiate single-base mismatched and three-base mismatched sequences of DNA. The proposed approach provided a simple and reliable method for DNA detection with high sensitivity and specificity, indicating promising application in bioanalysis and biomedicine.     

Fluorescein-labeled “arch-like” DNA probes for electrochemical detection of DNA on gold nanoparticle-modified gold electrodes

In the present study, a gold nanoparticle-modified gold electrode (nanogold electrode) was used to develop a novel fluorescein electrochemical DNA biosensor based on a target-induced conformational change. The nanogold electrode was obtained by electrodepositing gold nanoparticles onto a bare gold electrode. This modification not only immobilized probe oligonucleotides, but also adsorbed fluorescein onto the surface of the gold nanoparticles to form an “arch-like” structure. This article compares the electrochemical signal changes caused by the hybridization of “arch-like” DNA on nanogold electrode and linear DNA on bare gold electrode. The results showed that the adsorption effect of nanogold can enhance the sensitivity of the sensor. The linear range of target ssDNA is from 2.0 × 10⁻⁹ M to 2.0 × 10⁻⁸ M with a correlation coefficient of 0.9956 and detection limit (3σ) of 7.10 × 10⁻¹⁰ M. Additionally, the specificity and hybridization response of this simple sensor were investigated.     

Amperometric third-generation hydrogen peroxide biosensor based on the immobilization of hemoglobin on multiwall carbon nanotubes and gold colloidal nanoparticles

A convenient and effective strategy for preparation nanohybrid film of multi-wall carbon nanotubes (MWNT) and gold colloidal nanoparticles (GNPs) by using proteins as linker is proposed. In such a strategy, hemoglobin (Hb) was selected as model protein to fabricate third-generation H2O2 biosensor based on MWNT and GNPs. Acid-pretreated, negatively charged MWNT was first modified on the surface of glassy carbon (GC) electrode, then, positively charged Hb was adsorbed onto MWNT films by electrostatic interaction. The {Hb/GNPs}n multilayer films were finally assembled onto Hb/MWNT film through layer-by-layer assembly technique. The assembly of Hb and GNPs was characterized with cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and transmission electron microscopy (TEM). The direct electron transfer of Hb is observed on Hb/GNPs/Hb/MWNT/GC electrode, which exhibits excellent electrocatalytic activity for the reduction of H2O2 to construct a third-generation mediator-free H2O2 biosensor. As compared to those H2O2 biosensors only based on carbon nanotubes, the proposed biosensor modified with MWNT and GNPs displays a broader linear range and a lower detection limit for H2O2 determination. The linear range is from 2.1x10(-7) to 3.0x10(-3) M with a detection limit of 8.0x10(-8) M at 3sigma. The Michaelies-Menten constant KMapp value is estimated to be 0.26 mM. Moreover, this biosensor displays rapid response to H2O2 and possesses good stability and reproducibility.     

Electrochemical characterization and application of azurin-modified gold electrodes for detection of superoxide

A novel biosensor for superoxide radical (O(2)(*-)) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O(2)(*-) was found to be 10(5)M(-1)s(-1) in solution and five times lower, i.e., 0.2 x 10(5)M(-1)s(-1), for azurin coupled to gold by 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6s(-1). The sensitivity of this biosensor to O(2)(*-) was 6.8 x 10(2)Am(-2)M(-1). The response to the interference substances, such as uric acid, H(2)O(2), and dimethylsulfoxide was negligible below 10 microM. The electrode was applied in three O(2)(*-) generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO(2)), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O(2)(*-) in all three environments, and the signals were antagonized by superoxide dismutase.     

DNA microdevice for electrochemical detection of Escherichia coli 0157:H7 molecular markers

An electrochemical DNA sensor based on the hybridization recognition of a single-stranded DNA (ssDNA) probe immobilized onto a gold electrode to its complementary ssDNA is presented. The DNA probe is bound on gold surface electrode by using self-assembled monolayer (SAM) technology. An optimized mixed SAM with a blocking molecule preventing the nonspecific adsorption on the electrode surface has been prepared. In this paper, a DNA biosensor is designed by means of the immobilization of a single stranded DNA probe on an electrochemical transducer surface to recognize specifically Escherichia coli (E. coli) 0157:H7 complementary target DNA sequence via cyclic voltammetry experiments. The 21 mer DNA probe including a C6 alkanethiol group at the 5' phosphate end has been synthesized to form the SAM onto the gold surface through the gold sulfur bond. The goal of this paper has been to design, characterise and optimise an electrochemical DNA sensor. In order to investigate the oligonucleotide probe immobilization and the hybridization detection, experiments with different concentration of DNA and mismatch sequences have been performed. This microdevice has demonstrated the suitability of oligonucleotide Self-assembled monolayers (SAMs) on gold as immobilization method. The DNA probes deposited on gold surface have been functional and able to detect changes in bases sequence in a 21-mer oligonucleotide.     

Ultrasensitive electrochemical detection of Bacillus thuringiensis transgenic sequence based on in situ Ag nanoparticles aggregates induced by biotin-streptavidin system

A novel electrochemical biosensor was developed for detecting short DNA oligonucleotide of Bacillus thuringiensis (Bt) transgenic sequence based on Ag nanoparticle aggregates. To fabricate this DNA biosensor, the thiol-modified capture DNA (cDNA) was first anchored on gold (Au) electrode, and then the target DNA (tDNA) was hybridized with the immobilized cDNA. Subsequently, the probe DNA (pDNA) functionalized by biotinylated Ag nanoparticle was associated with the fixed tDNA, and the single Ag nanoparticle label was obtained (cited as SAg label). Finally, dissociative biotinylated Ag nanoparticle was bound to the resultant biotinylated SAg label assembled on Au electrode by virtue of bridge molecule streptavidin (SA) through biotin-SA specific interaction, which could lead to in situ aggregate of Ag nanoparticles on Au electrode and induce a novel tag including multiple Ag nanoparticles (cited as MAg tag). The novel tag exhibited excellent electroactive property in the solid-state Ag/AgCl process and was successfully applied to Bt transgenic sequence assay. A detection limit of 10 fM was achieved, which was improved by three orders of magnitude as compared to the SAg label. Furthermore, this novel DNA biosensor demonstrated a good selectivity towards tDNA.     

Single-base mismatch detection based on charge transduction through DNA

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based upon charge transport through DNA films. Double-helical DNA films on gold surfaces have been prepared and used to detect DNA mismatches electrochemically. The signals obtained from redox-active intercalators bound to DNA-modified gold surfaces display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Differential mismatch detection was accomplished irrespective of DNA sequence composition and mismatch identity. Single-base changes in sequences hybridized at the electrode surface are also detected accurately. Coupling the redox reactions of intercalated species to electrocatalytic processes in solution considerably increases the sensitivity of this assay. Reporting on the electronic structure of DNA, as opposed to the hybridization energetics of single-stranded oligonucleotides, electrochemical sensors based on charge transport may offer fundamental advantages in both scope and sensitivity.