Electrochemical detection of 17beta-estradiol using DNA aptamer immobilized gold electrode chip

An electrochemical detection method for chemical sensing has been developed using a DNA aptamer immobilized gold electrode chip. DNA aptamers specifically binding to 17beta-estradiol were selected by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random ssDNA library, composed of approximately 7.2 x 10(14) DNA molecules. Gold electrode chips were employed to evaluate the electrochemical signals generated from interactions between the aptamers and the target molecules. The DNA aptamer immobilization on the gold electrode was based on the avidin-biotin interaction. The cyclic voltametry (CV) and square wave voltametry (SWV) values were measured to evaluate the chemical binding to aptamer. When 17beta-estradiol interacted with the DNA aptamer, the current decreased due to the interference of bound 17beta-estradiol with the electron flow produced by a redox reaction between ferrocyanide and ferricyanide. In the negative control experiments, the current decreased only mildly due to the presence of other chemicals.     

QCM-based DNA biosensor for detection of genetically modified organisms (GMOs)

Development of a mass sensitive quartz crystal microbalance (QCM)-based DNA biosensor for the detection of the hybridization of CaMV 35S promoter sequence (P35S) was investigated for the screening of genetically modified organisms (GMOs). Attention was focused on the choice of the coating chemistry that could be used for the immobilization of probe sequences on the gold surface of the quartz crystal. Two immobilization procedures were tested and compared considering the amount of the immobilized P35S probe and the extent of the hybridization reaction with the target oligonucleotide. In wet chemistry procedure, the interaction between the thiol and gold for the immobilization of a thiolated probe was employed. Direct surface functionalization of piezoelectric quartz crystals were achieved in 13.56 MHz plasma polymerization reactor utilising ethylenediamine (EDA) precursors for the immobilization of amined probes. Results indicated that immobilization of a thiolated probe provides better immobilization characteristics and higher sensitivity for the detection of the hybridization reaction. The thiolated probe was used for the detection of P35S sequence in PCR-amplified DNAs and in real samples of pflp (ferrodoxin like protein)-gene inserted tobacco plants. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and ultrasonication. The results obtained from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM-based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label-free, direct detection of DNA samples for the screening of GMOs.     

New immobilization method for immunoaffinity biosensors by using thiolated proteins

A new immobilization method for immunoaffinity (IA) biosensors that ensures the high surface density and the stability of the IA layer was developed. For the immobilization of biomolecules, the molecular recognition protein was first thiolated by covalent conjugation of mercaptopropionic acid, and then the thiolated protein was attached on the gold surface of the transducer. In this work, horseradish peroxidase (HRP) and its antibody were used as a model antigen-antibody, and the following properties of the IA layer prepared by thiolated protein were estimated: (i) biological integrity of HRP after the immobilization process by using activity assay, (ii) charge transfer resistance by immobilization, (iii) mass loading by the surface plasmon resonance (SPR) biosensor, (iv) number of binding sites, and (v) feasibility test for the measurement of capacitive change by the antigen-antibody interaction. Based on these parameters, the immobilization method by using thiolated protein was determined to be feasible for application to IA biosensors.     

Integrated capillary fluorescence DNA biosensor

Covalent attachment of dsDNA molecules inside a glass capillary without the need for hybridization is described. It is shown that the glass capillary has a surface density of 2.5 x 10(13) molecules/cm(2) with specific binding capacity of 62.5%. The resulting substrate was used to develop a biosensor for determining fluorescent organic analytes and metal binding with DNA. The biosensor combines highly specific immobilization chemistry with a capillary-geometry flow cell arrangement. The results show that fluorescent dyes are retained in the dsDNA-modified surface and that exposure to concentrations of nickel and lead ions resulted in a recoverable, highly reproducible diminishment of the fluorescence intensity.     

Development of DNA electrochemical biosensor based on covalent immobilization of probe DNA by direct coupling of sol-gel and self-assembly technologies

A new procedure for fabricating deoxyribonucleic acid (DNA) electrochemical biosensor was developed based on covalent immobilization of target single-stranded DNA (ssDNA) on Au electrode that had been functionalized by direct coupling of sol-gel and self-assembled technologies. Two siloxanes, 3-mercaptopropyltrimethoxysiloxane (MPTMS) and 3-glycidoxypropyltrimethoxysiloxane (GPTMS) were used as precursors to prepare functionally self-assembly sol-gel film on Au electrode. The thiol group of MPTMS allowed assembly of MPTMS sol-gel on gold electrode surface. Through co-condensation between silanols, GPTMS sol-gel with epoxide groups interconnected into MPTMS sol-gel and enabled covalent immobilization of target NH(2)-ssDNA through epoxide/amine coupling reaction. The concentration of MPTMS and GPTMS influenced the performance of the resulting biosensor due to competitive sol-gel process. The linear range of the developed biosensor for determination of complementary ssDNA was from 2.51 x 10(-9) to 5.02 x 10(-7)M with a detection limit of 8.57 x 10(-10)M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of target ssDNA on self-assembled sol-gel matrix could serve as a versatile platform for DNA immobilization and fabrication of biosensors.     

Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces

The immobilization of short ss-DNA (18- and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (approximately 12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (x 10(11)) globules/cm2 for the 18- and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (x10(11)) average pore density corresponding to the 18- and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.     

Label-free genosensor based on immobilized DNA hairpins on gold surface

In this report, we demonstrate a label-free genosensor based on DNA hairpins coupled to gold coated sensor surfaces. The hairpin probes were labeled with a thiolated moiety for immobilization at the 5' end and with a fluorophore for signal transduction at the 3' end. In the absence of the complement, the fluorophore is quenched by energy transfer to the gold surface. Addition of the target sequence leads to the hairpin unfolding, and releases the fluorescent signal. This built-in property, using a gold film as both the immobilizing substrate and quenching agent, has the advantage of simplicity in design and ease of further integration. Our results showed that lengths of both the stem and the loop structures have significant effects on the sensor performance. Hybridization kinetics was investigated for various probe/target lengths and concentrations. An optimized hairpin probe gave a fluorescent signal increase of 39 folds after hybridization, which is much higher than the earlier reported results. A limit of detection (LOD) down to 0.3 nM for the complementary target DNA detection has been achieved. The developed sensor was further successfully applied for the detection of single-base mismatch targets, as well as for the direct detection of PCR products.     

Electric potential control of DNA immobilization on gold electrode

The assembly of synthetic, controllable molecules is one of the goals in nanotechnology. The primary objective of this contribution is to selectively immobilize DNA on gold via electric potential control. The self-assembly monolayer (SAM) was prepared with 2-aminoethanethiol (AET) on the gold electrode. A new approach based on electric potential was firstly used to control DNA immobilization covalently onto the SAM with the activation of 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) in low ionic strength solution. The influence of electric potential on DNA immobilization was investigated by means of cyclic voltammogram, A.C. impedance, auger electron spectrometer as well as atomic force microscope (AFM) on template-stripped gold surface. The result proves that controlled potential can affect the course of DNA immobilization. More negative potential can restrain the DNA immobilization, while the more positive potential can accelerate the DNA immobilization. It is of great significance for the control of DNA self-assembly and will find wide application in the fields of DNA-based devices.     

Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti-Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature-time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4+/-3.8% on the gold surface which then allowed an immobilization of 42.1+/-2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6+/-0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to microg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only approximately 8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S. typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI-GA immobilization. The frequency decreases with S. typhimurium concentration of approximately 1.5 x 10(9) CFU/ml were 50+/-2 Hz and 44+/-3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.     

Effect of surface coverage of gold(111) electrode with cysteine on the chiral discrimination of DOPA

The enantioselectivity imparted to a gold electrode by modifying its surface with a self-assembled monolayer (SAM) of cysteine (Cys) was investigated for the electrochemical redox reaction of 3,4-dihydroxyphenylalanine (DOPA). A cyclic voltammetric study of the redox reaction revealed that the enantioselectivity was determined by the surface coverage of the gold electrode with Cys molecules. The electrode modified with approximately 1.8 x 10(14) Cys molecules cm(-2) exhibited enantioselectivity in the voltammogram for the oxidation and reduction of DOPA, while the voltammograms obtained by the electrodes with either more or less surface coverages did not exhibit significant enantioselectivity. It is suggested that the accessibility of DOPA to that area of the gold surface which is not blocked by Cys molecules at an optimum surface coverage, is required for the enantioselective redox reaction of DOPA to proceed.     

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.     

Attachment of gold nanoparticles to glassy carbon electrode and its application for the direct electrochemistry and electrocatalytic behavior of hemoglobin

Gold nanoparticles have been attached onto glassy carbon electrode surface through sulfhydryl-terminated monolayer and characterized by X-ray photoelectron spectroscopy, atomic force microscopy, electrochemical impedance spectroscopy and cyclic voltammetry. The gold nanoparticles-attached glassy carbon electrodes have been applied to the immobilization/adsorption of hemoglobin, with a monolayer surface coverage of about 2.1 x 10(-10) mol cm(-2), and consequently obtained the direct electrochemistry of hemoglobin. Gold nanoparticles, acting as a bridge of electron transfer, can greatly promote the direct electron transfer between hemoglobin and the modified glassy carbon electrode without the aid of any electron mediator. In phosphate buffer solution with pH 6.8, hemoglobin shows a pair of well-defined redox waves with formal potential (E0') of about -0.085 V (versus Ag/AgCl/saturated KCl). The immobilized hemoglobin maintained its biological activity, showing a surface controlled electrode process with the apparent heterogeneous electron transfer rate constant (ks) of 1.05 s(-1) and charge-transfer coefficient (a) of 0.46, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide. A potential application of the hemoglobin-immobilized gold nanoparticles modified glassy carbon electrode as a biosensor to monitor hydrogen peroxide has been investigated. The steady-state current response increases linearly with hydrogen peroxide concentration from 2.0 x 10(-6) to 2.4 x 10(-4) M. The detection limit (3sigma) for hydrogen peroxide is 9.1 x 10(-7) M.     

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.     

Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.

The movements of E-cadherin, epidermal growth factor receptor, and transferrin receptor in the plasma membrane of a cultured mouse keratinocyte cell line were studied using both single particle tracking (SPT; nanovid microscopy) and fluorescence photobleaching recovery (FPR). In the SPT technique, the receptor molecules are labeled with 40 nm-phi colloidal gold particles, and their movements are followed by video-enhanced differential interference contrast microscopy at a temporal resolution of 33 ms and at a nanometer-level spatial precision. The trajectories of the receptor molecules obtained by SPT were analyzed by developing a method that is based on the plot of the mean-square displacement against time. Four characteristic types of motion were observed: (a) stationary mode, in which the microscopic diffusion coefficient is less than 4.6 x 10(-12) cm2/s; (b) simple Brownian diffusion mode; (c) directed diffusion mode, in which unidirectional movements are superimposed on random motion; and (d) confined diffusion mode, in which particles undergoing Brownian diffusion (microscopic diffusion coefficient between 4.6 x 10(-12) and 1 x 10(-9) cm2/s) are confined within a limited area, probably by the membrane-associated cytoskeleton network. Comparison of these data obtained by SPT with those obtained by FPR suggests that the plasma membrane is compartmentalized into many small domains 300-600 nm in diameter (0.04-0.24 microns2 in area), in which receptor molecules are confined in the time scale of 3-30 s, and that the long-range diffusion observed by FPR can occur by successive movements of the receptors to adjacent compartments. Calcium-induced differentiation decreases the sum of the percentages of molecules in the directed diffusion and the stationary modes outside of the cell-cell contact regions on the cell surface (which is proposed to be the percentage of E-cadherin bound to the cytoskeleton/membrane-skeleton), from approximately 60% to 8% (low- and high-calcium mediums, respectively).     

Comprehensive study of interactions between DNA and new electroactive Schiff base ligands. Application to the detection of singly mismatched Helicobacter pylori sequences

N,N'-Bis(3,4-dihydroxybenzylidene)-1,2-diaminobenzene (3,4-DHS) and N,N'-bis(2,5-dihydroxybenzylidene)-1,2-diaminobenzene (2,5-DHS) have been used as electrochemical probes in DNA sensing. These ligands, containing ortho and para quinone functional groups, respectively, as well as planar aromatic domains, are capable of binding to double stranded DNA (ds-DNA) more efficiently than to single stranded DNA (ss-DNA). Emphasis has been placed on the elucidation of the nature of the interaction by combining spectroscopic and electrochemical techniques. From spectrophotometric titration experiments, the binding constants of 3,4-DHS and 2,5-DHS with ds-DNA were found to be (9.0+/-0.3) x 10(3) and (3.3+/-0.2) x 10(3)M(-1), respectively. These values are consistent with a binding mode dominated by interactions with the minor groove of ds-DNA. The electroactivity of the quinone moiety in 3,4-DHS bound to DNA could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. After hybridization with the complementary target sequence, 3,4-DHS was accumulated within the double stranded DNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range where the quinone moiety is redox active. Using this approach, complementary target sequences of H. pylori can be quantified over the range of 8.9-22.2 microM with a detection limit of 8.3+/-0.4 microM and a linear correlation coefficient of 0.989. In addition this approach is capable of detecting hybridization of complementary sequences containing a single mismatch.     

A new scheme of hybridization based on the Au(nano)-DNA modified glassy carbon electrode

DNA hybridization on the Au(nano)-DNA modified glassy carbon electrode (GCE) was investigated. The thiol modified probe oligonucleotides (SH-ssDNA) at the 5' phosphate end were assembled on the Au(nano)-DNA modified GCE surface. The electrochemical response of the probe immobilization and hybridization with target DNA was measured by differential pulse voltammetry (DPV) using methylene blue (MB) as the electroactive indicator. Gold nanoparticles can be dispersed effectively on the GCE surface in the presence of calf thymus DNA. Au(nano)-DNA modified GCE could greatly increase the active sites and enhance the response signal during immobilization and hybridization. The hybridization amount of target DNA could be greatly increased. The linear detection range of Au(nano)-DNA electrode for the complementary 21-mer oligonucleotide (cDNA) was achieved from 1.52 x 10(-10) to 4.05 x 10(-8) mol L(-1). The detection limit could reach the concentration of 10(-10) mol/L.     

High-sensitivity detection for model organophosphorus and carbamate pesticide with quartz crystal microbalance-precipitation sensor

The effect of acetylcholinesterase (AChE) immobilization over the surface of a quartz crystal microbalance (QCM), by chemisorption of the AChE thiolated with a heterobifunctional cross-linker, sulfo-succinimidyl-6-[3-(2-pyridyldithio)propionamido]hexanoate, and carboxyl-amine coupling of AChE to 3-mercaptopropionic acid self-assembled monolayer, on the responses of a batch-type QCM-precipitation sensor was compared, resulting in a better sensitivity and binding efficiency in the former method. When an inhibition study with the developed sensor was undertaken at the optimized AChE immobilization with varying concentrations of a model organophosphorus pesticide EPN and carbamate one carbofuran, a sensitive detection for them was possible with the limit of detection corresponding to 1.55 x 10(-8) and 1.30 x 10(-9)M, respectively.     

Piezoelectric biosensors: strategies for coupling nucleic acids to piezoelectric devices

The development of a piezoelectric biosensor based on nucleic acids interaction is presented focusing on the methodology for probe immobilization. This is a key step in any DNA biosensor development. Often, the detection limits and, in general, the analytical performances of the biosensor can be improved by optimizing the immobilization of the receptor on the transducer surface. DNA must be attached to the solid support, retaining native conformation, and binding activity. This attachment must be stable over the course of a binding assay and, in addition, sufficient binding sites must be presented to the solution phase to interact with the analyte. In this paper, the optimization of the coating of the gold quartz crystal surface, to immobilize an oligonucleotide probe, is reported. Two immobilization procedures are illustrated in details with a comparison regarding the immobilization of the probe, the detection of the hybridization reaction, and the possibility of regeneration. The two procedures are based on the use of biotinylated or thiolated DNA probes. Specific applications will be also presented.     

Detection of DNA hybridization and extension reactions by an extended-gate field-effect transistor: characterizations of immobilized DNA-probes and role of applying a superimposed high-frequency voltage onto a reference electrode

As we have already shown in a previous publication [Kamahori, M., Ihige, Y., Shimoda, M., 2007. Anal. Sci. 23, 75-79], an extended-gate field-effect transistor (FET) sensor with a gold electrode, on which both DNA probes and 6-hydroxyl-1-hexanethiol (6-HHT) molecules are immobilized, can detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to a reference electrode. However, kinetic parameters such as the dissociation constant (K(d)(s)) and the apparent DNA-probe concentration (C(probe)(s)) on a surface were not clarified. In addition, the role of applying the superimposed high-frequency voltage was not considered in detail. In this study, the values of K(d)(s) and C(probe)(s) were estimated using a method involving single-base extension reaction combined with bioluminescence detection. The value of K(d)(s) on the surface was 0.38 microM, which was about six times that in a liquid phase. The value of C(probe)(s), which expressed the upper detection limit for the solid phase reaction, was 0.079 microM at a DNA-probe density of 2.6 x 10(12)molecules/cm(2). We found that applying the superimposed high-frequency voltage accelerated the DNA molecules to reach the gold surface. Also, the distance between the DNA-probes immobilized on the gold surface was controlled to be over 6 nm by applying a method of competitive reaction with DNA probes and 6-HHT molecules. This space was sufficient to enable the immobilized DNA-probes to lie down on the 6-HHT monolayer in the space between them. Thus, the FET sensor could detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to the DNA-probes density-controlling gold surface.