Preparation of SiO2/(PMMA/Fe3O4) nanoparticles using linolenic acid as crosslink agent for nucleic acid detection using chemiluminescent method

Usually, magnetic nanoparticles (MNPs) are prepared based on the famous St?ber process in which divinylbenzene (DVA) is often used as a crosslink agent to synthesize SiO2/(PMMA/Fe3O4) nanoparticles. Compared with DVA, linolenic acid (LNA) is innoxious and can polymerize more easily for it has three unsaturated double bonds. In this paper, LNA was used as a new crosslink agent instead of DVA to synthesize the SiO2/(PMMA/Fe3O4) nanoparticles. The results showed that the core-shell structure could be observed obviously. The sizes of nanoparticles with core-shell structure range from 200 to 500 nm. The DNA probes which was immobilized on the surface of MNPs were used to capture the biotin modified complementary sequence of the probe, and the formed complexes were bonded with streptavidin-modified alkaline phosphatase (SA-AP). Finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane (AMPPD) which was the substrate reagent of AP. The specificity and sensitivity of this approach were investigated in this paper.     

Electrochemical genosensor based on colloidal gold nanoparticles for the detection of Factor V Leiden mutation using disposable pencil graphite electrodes

Electrochemical genosensors for the detection of the Factor V Leiden mutation from polymerase chain reaction (PCR) amplicons using the oxidation signal of colloidal gold (Au) is described. A pencil graphite electrode (PGE) modified with target DNA, when hybridized with complementary probes conjugated to Au nanoparticles, responded with the appearance of a Au oxide wave at approximately +1.20 V. Specific probes were immobilized onto the Au nanoparticles in two different modes: (a) Inosine-substituted probes were covalently attached from their amino groups at the 5' end using N-(3-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) as a coupling agent onto a carboxylate-terminated l-cysteine self-assembled monolayer (SAM) preformed on the Au nanoparticles, and (b) probes with a hexanethiol group at their 5' phosphate end formed a SAM on Au nanoparticles. The genosensor relies on the hybridization of the probes with their complementary targets, which are covalently immobilized at the PGE surface. Au-tagged 23-mer capture probes were challenged with the synthetic 23-mer target, 131-base single-stranded DNA or denatured 256-base polymerase chain reaction (PCR) amplicon. The appearance of the Au oxidation signal shortened the assay time and simplified the detection of the Factor V Leiden mutation from PCR amplified real samples. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the Au signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized. The detection limit for the PCR amplicons was found to be as low as 0.78 fmol; thus, it is suitable for point-of-care applications.     

Quadruple-analyte chemiluminometric hybridization assay. Application to double quantitative competitive polymerase chain reaction

We developed a highly sensitive quadruple-analyte chemiluminometric hybridization assay for simultaneous quantification of four nucleic acid sequences. The targets are amplified by the polymerase chain reaction (PCR) and captured to microtiter wells coated with streptavidin. The immobilized fragments are hybridized with specific probes containing a sequence complementary to the target and a sequence or a hapten that allows linkage with a chemiluminescent reporter. We prepared a mixture of four reporters conjugated to complementary oligonucleotides or antihapten antibodies. The reporters were aequorin-(dT)(30), galactosidase-oligonucleotide, horseradish peroxidase-antifluorescein, and alkaline phosphatase-antidigoxigenin conjugates. The four chemiluminescent reactions were triggered sequentially. The signals were linearly related to the concentration of target sequences. The entire quadruple-analyte bioluminometric hybridization assay is complete in 75 min. We have demonstrated the applicability of the proposed assay to high-throughput quantitative competitive PCR of two target sequences in the presence of the corresponding competitors. The assay is universal since the same reporter conjugates can be used for multianalyte quantification of any sequences with properly designed probes.     

Fabrication and application of single nucleotide polymorphisms library on magnetic nanoparticles using adaptor PCR

We have developed a novel approach to fabricate single nucleotide polymorphisms (SNPs) library on magnetic nanoparticles (MNPs) based on adaptor PCR. Each SNP locus in the library was interrogated by hybridization with a pair of allele specific dual-color fluorescence (Cy3, Cy5) probes to determine SNP. Two SNPs loci (M235T and A-6G) associated with essential hypertension in the angiotensinogen (AGT) gene were detected by this method and their fluorescent signals were quantified. The fluorescent ratios (match probe: mismatch probe signal) of homozygous genotypes were over 3.0, whereas heterozygous genotypes had ratios near to 1.0. Without any complex multiplex PCR procedure, it is a simple, efficient and reliable method for the multiplex SNPs detection using limited amount of DNA samples from individuals.     

Immobilization of aromatic aldehyde molecules on indium tin oxide surface using acetalization reaction

We demonstrated an acetalization reaction as a versatile method to immobilize aromatic aldehyde molecules on surfaces of metal oxides, silicon dioxide, and indium tin oxide. First, a trimethylsily (TMS) terminated surface was formed using a silylation reaction between a chloride group of trimethylsilychloride and a hydroxyl group of the substrate surfaces. Second, terephthalaldehyde (TPA) was immobilized on the surfaces using an acetalization reaction between the TMS-terminated surface and an aldehyde group of TPA. Results of contact angle, X-ray photoelectron, and ultraviolet absorption spectra revealed that the TPA molecules on the surfaces were well-packed with a high surface density.     

Surface modification of gamma-Fe2O3@SiO2 magnetic nanoparticles for the controlled interaction with biomolecules

Modifying the surface of magnetic nanoparticles (MNPs) to allow for controlled interaction with biomolecules enables their implementation in biomedical applications such as contrast agents for magnetic resonance imaging, labels in magnetic biosensing or media for magnetically assisted bioseparation. In this paper, self-assembly of trialkoxysilanes is used to chemically functionalize the surface of gamma-Fe2O3@SiO2 core-shell particles. First, the silane deposition procedure was optimized using infrared analysis in order to obtain maximum packing density of the silanes on the particles. The surface coverage was determined to be approximately 8 x 10(14) molecules/cm2. It was shown that the magnetic, crystalline, and morphological properties of the MNPs were not altered by deposition of a thin silane coating. The optimized procedure was transferred for the deposition of aldehyde and poly(ethylene glycol) (PEG) presenting silanes. The presence of both silanes on the particle surface was confirmed using XPS and FTIR. The interaction of proteins with silane-modified MNPs was monitored using a Bradford protein assay. Our results demonstrate that, by introducing aldehyde functions, the MNPs are capable of covalently binding human IgG while retaining their specific binding capacity. Maximum surface coverage occurs at 46 microg antibodies per mg particle, which corresponds to 35 antibodies bound to an average sized MNP (54 nm in diameter). The human IgG functionalized MNPs exhibit a high degree of specificity (approximately 90%) and retained a binding capacity of 32%. Using the same approach, streptavidin was coupled onto the MNPs and the biotin binding capacity was determined using biotinylated fluorescein. At maximum surface coverage, a biotin binding capacity of 1500 pmol/mg was obtained, corresponding to a streptavidin activity of 76%. On the other hand, by introducing PEG functions the non-specific adsorption of serum proteins could be significantly suppressed down to approximately 3 microg/mg. We conclude that self-assembly of silane films creates a generic platform for the controlled interactions of MNPs with biomolecules.     

Silver nanoparticle-based ultrasensitive chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms

A new nanoparticle-based chemiluminescent (CL) method has been developed for the ultrasensitive detection of DNA hybridization. The assay relies on a sandwich-type DNA hybridization in which the DNA targets are first hybridized to the captured oligonucleotide probes immobilized on polystyrene microwells and then the silver nanoparticles modified with alkylthiol-capped oligonucleotides are used as probes to monitor the presence of the specific target DNA. After being anchored on the hybrids, silver nanoparticles are dissolved to Ag+ in HNO3 solution and sensitively determined by a coupling CL reaction system (Ag+-Mn2+-K2S2O8-H3PO4-luminol). The combination of the remarkable sensitivity of the CL method with the large number of Ag+ released from each hybrid allows the detection of specific sequence DNA targets at levels as low as 5 fM. The sensitivity increases 6 orders of magnitude greater than that of the gold nanoparticle-based colorimetric method and is comparable to that of surface-enhanced Raman spectroscopy, which is one of the most sensitive detection approaches available to the nanoparticle-based detection for DNA hybridization. Moreover, the perfectly complementary DNA targets and the single-base mismatched DNA strands can be evidently differentiated through controlling the temperature, which indicates that the proposed CL assay offers great promise for single-nucleotide polymorphism analysis.     

DNA covalent immobilization onto screen-printed electrode networks for direct label-free hybridization detection of p53 sequences

A new electrochemical biochip for the detection of DNA sequences was developed. The entire biochip-i.e., working, reference, and counter electrodes-was constructed based on the screen-printing technique and exhibits eight working electrodes that could be individually addressed and grafted through a simple electrochemical procedure. Screen-printed electrode networks were functionalized electrochemically with 1-ethyl-3-(3dimethylaminopropyl)carbodidiimide according to a simple procedure. Single-stranded DNA with a C6-NH(2) linker at the 5'-end was then covalently bound to the surface to act as probe for the direct, nonlabeled, detection of complementary strands in a conductive liquid medium. In the present system, the study was focused on a particular codon (273) localized in the exon 8 of the p53 gene (20 mer, TTGAGGTGCATGTTTGTGCC). The integrity of the immobilized probes and its ability to capture target sequences was monitored through chemiluminescent detection following the hybridization of a peroxidase-labeled target. The grafting of the probe at the electrode surface was shown to generate significant shifts of the Nyquist curves measured in the 10-kHz to 80-Hz range. These variations of the faradaic impedance were found to be related to changes of the double layer capacitance of the electrochemical system's equivalent circuit. Similarly, hybridization of complementary strands was monitored through the measurements of these shifts, which enabled the detection of target sequences from 1 to 200 nM. Discrimination between complementary, noncomplementary, and single-nucleotide mismatch targets was easily accomplished.     

Detection of attomole quantities [correction of quantitites] of DNA targets on gold microelectrodes by electrocatalytic nucleobase oxidation

The electrochemical detection of nucleic acid targets at low concentrations has a number of applications in diagnostics and pharmaceutical research. Self-assembled monolayers of alkanethiol-derivatized oligonucleotides on gold electrodes provide a useful platform for such detectors, and the electrocatalytic oxidation of nucleobases included in the DNA targets is a particularly sensitive method of electrochemical detection. A strategy has been developed for combining these two aspects by substituting either 7,8-dihydro-8-oxoguanine (8G) or 5-aminouridine (5U) into DNA targets. Upon hybridization of targets containing these modified nucleobases, electrocatalytic signals at probe-modified gold electrodes are observed in the presence of Os(bpy)(3)(2+), which oxidizes both 8G and 5U upon oxidation to the Os(III) state. Self-assembled monolayers were prepared on both macro (1.6 mm) and micro (25 microm) gold electrodes using published procedures involving C6-terminated alkanethiol oligonucleotides and mercaptohexanol as the diluent. The extent of electrode modification by the modified probe was assessed using radiolabeling and a standard chronocoulometry method; both approaches gave loading levels within expected ranges ((1-6) x 10(12) molecules/cm(2)). Hybridization of the modified targets where the non-native nucleobase was incorporated by solid-phase synthesis produced electrocatalytic signals from strands that were independently detected using radiolabeling and chronocoulometry. This result was used as a basis to develop an on-electrode amplification scheme where Taq polymerase was used to extend the immobilized DNA probes from solution-phase polymeric templates using modified nucleotriphosphates. This reaction produced an electrode that was modified with extended DNA containing the appropriate modified nucleotide. Radiolabeled nucleotide triphosphates were used to confirm the desired on-electrode DNA synthesis. When these electrodes were cycled in the presence of Os(bpy)(3)(2+), electrocatalytic signals were observed when as little as 40 amol (400 fM) of the desired target was present in the hybridization solution.     

Magnetic nanoparticle-enhanced biosensor based on grating-coupled surface plasmon resonance

A highly sensitive surface plasmon resonance (SPR) biosensor employing magnetic nanoparticle (MNP) assays is presented. In the reported approach, MNPs simultaneously served as "vehicles" for rapid delivery of target analyte from a sample to the sensor surface and as labels increasing the measured refractive index changes that are associated with the binding of target analyte. An optical setup based on grating-coupled surface plasmon resonance (GC-SPR) was used with a magnetic field gradient applied through the sensor chip for manipulating with MNPs on its surface. Iron oxide MNPs and a sensor surface with metallic diffraction grating were modified with antibodies that specifically recognize different epitopes of the analyte of interest. The sensitivity of the biosensor was investigated as a function of mass transport of the analyte to the sensor surface driven by diffusion (free analyte) or by the magnetic field gradient (analyte bound to MNPs). Immunoassay-based detection of β human chorionic gonadotropin (βhCG) was implemented to evaluate the sensitivity of the MNP-enhanced GC-SPR biosensor scheme. The results reveal that the sensitivity of βhCG detection was improved by 4 orders of magnitude compared with the regular SPR sensor with direct detection format, and a limit of detection below pM was achieved.     

微流控芯片表面修饰及在蛋白质富集中的应用

应用分子自组装技术，在SiO2表面衍生活泼醛基，在SiO2表面有125nm的衍生物，衍生的醛基和氨基发生共价反应而将入免疫球蛋白G固定在二氧化硅表面，抗原抗体反应显示固定的抗体有活性。应用微加工技术加工含交叉排列的椭圆形微柱阵列的微流控芯片，有效增加内表面积和流体接触机会，用同样修饰方法修饰微流控芯片内表面并固定人免疫球蛋白G，流经管道的相应抗抗体和其发生反应而被吸附在管道表面，实现对该抗抗体的亲和富集，富集后荧光密度增加15倍。表面修饰技术能实现蛋白质在二氧化硅表面的固定并保持其生物活性，结合微流控芯片能实现对相应蛋白质的微量富集。     

Functionalized surfaces for optical biosensors: Applications to in vitro pesticide residual analysis

Functionalized biosensing surfaces were developed for chemiluminescent immunoassay of pesticides. Two approaches to construct functionalized surfaces were tested: (i) pesticide is immobilized to the surface and interacts with a labeled antibody; (ii) antibody is immobilized and interacts with a labeled pesticide. As labels alkaline phosphatase and peroxidase were used with their corresponding substrates CSPD and luminol, respectively. Light produced by chemiluminescent substrate was detected by a thermoelectrically cooled CCD camera or a photomultiplier. The best detection limit 0.00001 ng/ml was obtained using antibodies immobilized to dextran-enhanced surface. Completely renewable surface was obtained using reversible lectin-monosaccharide interaction, one surface was used for 200 analyses without any loss of binding capacity. Most favorable stability and cost per analysis was achieved with molecularly imprinted polymer (MIP) instead of antibody. The functionalized biosensing surfaces were prepared to detect 2,4-dichlorophenoxyacetic (2,4-D) acid as a model pesticide. The developed concepts are, however, generally applicable to other pesticides and to other optical formats, e.g. optical fiber.     

Preparation and characterization of iminodiacetic acid-functionalized magnetic nanoparticles and its selective removal of bovine hemoglobin

In this study, a novel route for the preparation of magnetite (Fe(3)O(4)) nanoparticles (NPs) with immobilized metal affinity ligand iminodiacetic acid (IDA) charged with Cu(2+) was developed. First, magnetite nanoparticles were synthesized by a hydrothermal method. Charged with Cu(2+), the magnetic nanoparticles (MNPs) were applied to separate a model protein mixture of bovine hemoglobin (BHb) and bovine serum albumin (BSA). They could be separated completely and showed low non-specific adsorption. The morphology, structure and composition of the magnetite MNPs were characterized by transmission electron microscopy, power x-ray diffraction, x-ray photoelectron spectrometry and Fourier transform infrared spectroscopy. The resulting magnetite MNPs charged with Cu(2+) show not only a strong magnetic response to externally applied magnetic field, but are also highly specific to protein BHb. It is interesting that MNPs modified with metal ligands showed a property of magnetic colloid photonic crystals. Furthermore, they could efficiently remove the abundant protein bovine hemoglobin from bovine blood. They have potential application in removing abundant protein in proteomic analysis.     

Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule

We describe herein a detection and quantification system for on-chip phosphorylation of peptides by surface plasmon resonance (SPR) imaging techniques using a newly synthesized phosphate capture molecule (i.e., biotinylated zinc(II) complex). The biotinylated compound is a dinuclear zinc(II) complex that is suitable for accessing phosphate anions as a bridging ligand on the two zinc(II) ions. The compound was exposed on the peptide array and detected with streptavidin (SA) via a biotin-SA interaction by SPR imaging. In the conventional method using antibody, both anti-phosphoserine and anti-phosphotyrosine antibodies were required for phosphoserine and phosphotyrosine detection, respectively. Detection of the phosphate group by the zinc(II) complex, however, was independent of the phosphorylated amino acid residues. The calibration curve for the phosphorylation ratios was established with a calibration chip, on which phosphoserine-containing peptide probes were immobilized. The peptide probes, which were phosphorylated on the surface by protein kinase A, were detected and quantified by SPR imaging using the zinc(II) complex, SA, and anti-SA antibody. The reaction rate and the kinetics of on-chip phosphorylation were also evaluated with the peptide array. The phosphorylation ratio was saturated at approximately 20% in 2 h in this study.     

Design of electrochemical biosensor systems for the detection of specific DNA sequences in PCR-amplified nucleic acids related to the catechol-O-methyltransferase Val108/158Met polymorphism based on intrinsic guanine signal

Psychiatric disorders are common and complex diseases that show polygenic and multifactorial heredity. A single nucleotide polymorphism (Val108/158Met) in the catechol-O-methyl transferase (COMT) gene is related to many psychiatric disorders such as schizophrenia, alcoholism, bipolar disorder, and obsessive-compulsive disorder. Schizophrenia is a complex disorder and a single nucleotide polymorphism (Val108/158Met) at the COMT gene is related to schizophrenia susceptibility. A novel hybridization-based disposable electrochemical DNA biosensor for the detection of a common functional polymorphism in the COMT gene from polymerase chain reaction (PCR) amplicons has been described without using an external label. This developed technology combined with a disposable carbon graphite electrode and differential pulse voltammetry was performed by using short synthetic oligonucleotides and PCR amplicons in length 203 bp to measure the change of guanine oxidation signal obtained at approximately +1.0 V after DNA hybridization between probe and target (synthetic target or denatured PCR samples). COMT-specific oligonucleotides were immobilized onto the carbon surface with a simple adsorption method in two different modes: (a) Guanine-containing targets were attached or (b) inosine-substituted probes were attached onto an electrode. By controlling the surface coverage of the target DNA, the hybridization event between the probes and their synthetic targets or specific PCR products was optimized. The wild-type or polymorphic allele-specific probes/targets were also interacted with an equal amount of noncomplementary and one-base mismatch-containing DNAs in order to measure the sensor selectivity. The decrease or appearance in the intrinsic guanine signal simplified the detection procedure and shortened the assay time because protocol eliminates the label-binding step. The nonspecific binding effects were minimized by using sodium dodecyl sulfate with different washing methods. The Val108/158Met COMT genotype detection were performed with real samples containing wild-type (healthy controls), polymorphic (mutant type), and heterozygous PCR products. The detection limit (S/N = 3) of the biosensor was 2.44 pmol of target sequence in the 30-muL samples. Analytical performance of the sensor is described, along with future prospects.     

Multifunctional label-free electrochemical biosensor based on an integrated aptamer

Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A Au electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA). The unimolecular MBA contained small-molecule ATP binding aptamer (ABA) and also protein alpha-thrombin binding aptamer (TBA). Thus, the aptasensor could be used for detection of ATP and alpha-thrombin both. The detection limit of ATP was 1 x 10(-8) M, and its detection range could extend up to 10(-4) M, whereas the detection limit of alpha-thrombin was 1 x 10(-11) M, and its detection range was from 1 x 10(-11) to 1 x 10(-7) M. Meanwhile, after detecting alpha-thrombin, the sensing interface could be used for ATP recognition as well. The aptasensor regeneration could be realized by rehybridizing of the MBA strand with the partly complementary strand immobilized on the Au surface after ATP detection or by treating with a large amount of ATP and then rehybridizing the MBA strand with the partly complementary strand immobilized on the Au surface after alpha-thrombin detection. The aptasensor fabricated exhibited several advantages such as label-free detection, high sensitivity, regeneration, and multifunctional recognition. It also showed the detectability in biological fluid. Therein it held promising potential for integration of the sensing ability such as the simultaneous detection for multianalysis in the future.     

Thiol-yne reaction on boron-doped diamond electrodes: application for the electrochemical detection of DNA-DNA hybridization events

Boron-doped diamond (BDD) interfaces were chemically functionalized through the catalyst free thiol-yne reaction. Different thiolated precursors (e.g., perfluorodecanethiol, 6-(ferrocenyl)-hexanethiol, DNA) were successfully "clicked" to alkynyl-terminated BDD by irradiating the interface at 365 nm for 30 min. Thiolated oligonucleotide strands were immobilized using the optimized reaction conditions, and the surface concentration was tuned to obtain a surface coverage of 3.1 × 10(12) molecules cm(-2). Electrochemical impedance spectroscopy (EIS) was employed to follow the kinetics of hybridization and dehybridization events. The sensitivity of the oligonucleotide modified BDD interface was assayed, and a detection limit of 1 nM was obtained.     

Sensitive quantification of Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni by combining stopped polymerase chain reaction with chemiluminescence flow-through DNA microarray analysis

Rapid analysis of pathogenic bacteria is essential for food and water control to preserve the public health. Therefore, we report on a chemiluminescence (CL) flow-through DNA microarray assay for the rapid and sensitive quantification of the pathogenic bacteria Escherichia coli O157:H7, Salmonella enterica , and Campylobacter jejuni in water. Using the stopped polymerase chain reaction (PCR) strategy, the amount of amplified target DNA was strongly dependent on the applied cell concentration. The amplification was stopped at the logarithmic phase of the PCR to quantify the DNA products on the DNA microarray chip. The generation of single-stranded DNA sequences is essential for DNA hybridization assays on microarrays. Therefore, the DNA strands of the PCR products were separated by streptavidin-conjugated magnetic nanoparticles. This was achieved by introducing a reverse primer labeled with biotin together with a digoxigenin labeled forward primer for CL microarray imaging. A conjugate of an antidigoxigenin antibody and horseradish peroxidase recognized the digoxigenin-labeled antistrands bound to the probes on the microarray surface and catalyzed the reaction of luminol and hydrogen peroxide. The generated light emission was recorded by a sensitive charge-coupled device (CCD) camera. The quantification was conducted by a flow-through CL microarray readout system. The DNA microarrays were based on an NHS-activated poly(ethylene glycol)-modified glass substrate. The DNA probes which have the same DNA sequence as the reverse primer were immobilized on this surface. The full assay was characterized by spiking experiments with heat-inactivated bacteria in water. The total assay time was 3.5 h, and the detection limits determined on CL microarrays were for E. coli O157:H7, S. enterica , and C. jejuni 136, 500, and 1 cell/mL, respectively. The results of the DNA microarray assay were comparable to the SYBR green-based assays analyzed with a real-time PCR device. The advantage of the new microarray analysis method is seen in the ability of a high multiplex degree on DNA microarrays, the high specificity of DNA hybridization on DNA microarrays, and the possibility to get quantitative results on an automated CL flow-through microarray analysis system.     

Allele-specific genotype detection of factor V Leiden mutation from polymerase chain reaction amplicons based on label-free electrochemical genosensor

An electrochemical genosensor for the genotype detection of allele-specific factor V Leiden mutation from PCR amplicons using the intrinsic guanine signal is described. The biosensor relies on the immobilization of the 21-mer inosine-substituted oligonucleotide capture probes related to the wild-type or mutant-type amplicons, and these probes are hybridized with their complementary DNA sequences at a carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The guanine signal was monitored as a result of the specific hybridization between the probe and amplicon at the CPE surface. No label-binding step was necessary, and the appearance of the guanine signal shortened the assay time and simplified the detection of the factor V Leiden mutation from polymerase chain reaction (PCR)-amplified amplicons. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the guanine signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 51.14 fmol/mL target DNA. With the help of the appearance of the guanine signal, the yes/no system is established for the electrochemical detection of allele-specific mutation on factor V for the first time. Features of this protocol are discussed and optimized.     

Nanofabrication of bio-self assembled monolayer and its electrochemical property for toxicant detection

Cyclic voltammetry (CV) has been used to investigate the electrochemical behavior of a glutathione (GSH) self assembled monolayer on modified gold electrodes (Bio-SAM). The GSH monolayer exhibits an influence on electrode surface activity. Electrochemically immobilized dsDNA onto a Cyt c/GSH-SAM/Au electrode, which is useful for the fabrication of a nanobiosensing device. The immobilized Cyt c followed by dsDNA immobilized films maintained its surface activity and finally dsDNA/Cyt c/GSH-SAM/Au electrode, targeted for the detection of toxicants. The films were characterized by CV, DPV, and AFM. The differential pulse voltammetry (DPV) technique was applied to detect three kinds of common toxins, 2-aminoanthracene (2-AA), 3-bromobenzanthrone (3-BBA) and bisphenol A (BPhA). The electrochemical signals showed good inverse relationship with the increase of concentrations of toxicants. Our proposed system based on electrochemical method with nanoscale film technology can be applied at highly sensitive biosensor for detecting various toxic chemicals.