Ultratrace voltammetric method for the detection of DNA sequence related to human immunodeficiency virus type 1

An ultra-trace voltammetric method was developed for the determination of single strand DNA (ss-DNA) related to the human immunodeficiency virus type 1 (HIV-1). It is based on the signal amplification of carbon nanotubes loaded with silver nanoparticles and placed on a gold microelectrode. The capture ss-DNA (a 21-mer) possessing a thiol group at the 3?? end was self-assembled onto the surface of the gold microelectrode. It was then hybridized with target HIV-1 ss-DNA (a 42-mer) and further hybridized with the electrochemical probe (a 18-mer ss-DNA) tagged with multiwall carbon nanotubes and loaded with silver nanoparticles. The resulting formation of a DNA sandwich conjugate led to a strong electrochemical oxidation signal that was linearly proportional to the concentration of HIV-1 ss-DNA in the range from 1.0 to 100?pM. The detection limit was 0.5?pM (at an S/N of 3). This was equivalent to 0.05?fmol of HIV-1 ss-DNA in a volume of 20???L. The relative standard deviation was 4.0% at 1.0?pM (n?=?11). Non-complementary ss-DNA of HIV-1 ss-DNA was effectively discriminated. This work demonstrates that the employment of the microelectrode and a sandwich hybridization model is promising in terms of sensitive and selective electrochemical detection of DNA.

Water‐soluble cationic polypyrrole based probe for fluorometric and voltammetric detection of base pair mismatched oligonucleotides

Water‐soluble cationic polypyrrole, poly(N‐(4‐butyl‐(1‐methylimidazole)) pyrrole bromide) (PNBMIP‐Br), was synthesized and applied for base pair mismatched oligonucleotides detection. Interactions between PNBMIP‐Br and a series of oligonucleotides, including ss‐DNA and base pair mismatched ds‐DNA were studied by fluorometric spectra, circular dichroism spectra and voltammetric detection. The results showed that the electrostatic attraction and fluorescence resonance energy transfer of PNBMIP‐Br/DNA complexes resulted in an amplification and effective recognition of the fluorescence signals. The results of cyclic voltammograms indicate that voltammetric detection is an effective method to distinguish ss‐DNA and ds‐DNA. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 1600–1605     

Ferrocenylnaphthalene diimide-based electrochemical detection of methylated gene

Ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay was applied to the detection of methylated cytosine of DNA using the products obtained after treatment with bisulfite followed by polymerase chain reaction (PCR), where unmethylated cytosine is converted to thymine and methylated one to cytosine. Twenty-meric DNA probes for the methylated (cytosine) and unmethylated (thymine) types of the part of the promoter region of cyclin D-dependent protein kinase inhibitor, p16, gene (p16^Ink4a) were used to be immobilized on the electrochemical array (ECA) chip. Using 1 μL of 10 ng/μL of methylated sample obtained from the methylation-specific PCR of methylated genome containing 10-times excess of unmethylated one, the methylated PCR sample could be detected by the identical electrochemical signals from the two DNA probes under the settled optimum hybridization conditions.     

Indigo Carmine as New Label in PNA Biosensor for Detection of Short Sequence of p53 Tumor Suppressor Gene

A new electrochemical PNA hybridization biosensor for detection of a 15‐mer sequence unique to p53 using indigo carmine (IC) as an electrochemical detector is described in this work. This genosensor is based on the hybridization of target oligonucleotide with its complementary probe immobilized on the gold electrode by self‐assembled monolayer formation. Because this label is electroactive in acidic medium, the interaction between IC and short sequence of p53 is studied by differential pulse voltammety (DPV) in 0.1 M H₂SO₄. The results of electrochemical impedance spectroscopy and cyclic voltammetry in the solution of [Fe(CN)₆]^3?/4? shows no breakage in PNA‐DNA duplex. A decrease in the voltammetric peak currents of IC is observed upon hybridization of the probe with the target DNA. The influence of probe concentration on effective discrimination against non‐complementary oligonucleotides is investigated and a concentration of 10^?7 M is selected. The diagnostic performance of the PNA sensor is described and the detection limit is found to be 4.31×10^?12 M.     

Voltammetric Characterization of Micro‐ and Submicrometer‐Electrode Arrays of Conical Shape for Electroanalytical Use

Densely packed micro‐ and submicrometer electrode arrays of platinum and gold (the nominal number, N, of electrodes in each array varies between 225 and 3600) are fabricated by photolithographic technique and vapor deposition processes of metal films. The electrodes are conical‐shaped and only their apexes are exposed to the electrolytic solution. The electrode arrays are characterized electrochemically in Ru(NH₃)₆Cl₃ aqueous solutions by using cyclic voltammetry at low scan rates, to establish the number of electrochemically active electrodes (N_ac) in each array; the geometric characterization is performed by scanning electron microscopy. All the investigated arrays provide steady‐state voltammograms, indicating diffusionally independent behavior of each microelectrode. The number of microelectrodes that are active in the fabricated arrays depends on microelectrode density. In particular, for the arrays with N=3600 and N=225, the fraction of active sites is about 45% and 90%, respectively. The analytical performance of some of the Pt version of the arrays is tested in hydrogen peroxide solutions, allowing verifying that linear calibration plots over the concentration range (0.1–20 mM) are obtained. This dynamic range is larger than that typically recorded at smooth polycrystalline platinum electrodes (0.5–5 mM), and the better performance is attributed to both the higher aspect ratio of the cone geometry and the higher mass transport associated to each microelectrode of the array. Reproducibility (within 3.5%, r.s.d.) and long‐term stability (within 5%, r.s.d., after 8 h continuous use) of the electrode systems are satisfactory. A low detection limit, based on the signal to noise ratio equal to 3, of 0.05 mM is found, which is adequate for a rapid monitoring of H₂O₂ in real samples and industrial processes.     

Development of a voltammetric electronic tongue for discrimination of edible oils

In this paper, we propose a novel strategy to perform cyclic voltammetric measurements with a platinum microelectrode directly in edible oil samples. The microelectrode was employed as an electronic tongue that, along with the application of chemometrics to the current–potential responses, proved useful for discriminating oils on the basis of their quality and geographical origin. The method proposed here is based on the use of suitable room temperature ionic liquids, added to oils as supporting electrolytes to provide conductivity to the low-polarity samples. The entire voltammograms, recorded directly on the oil/RTIL mixtures, were processed via principal component analysis and a classification technique (K nearest neighbors), to extract information on samples characteristics. Data processing showed that oils having different nature (i.e. maize and olive) or geographical origin (i.e. olive oils coming from different regions) can be distinguished.     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

A Subtractively Optimized DNA Microarray Using Non-sequenced Genomic Probes for the Detection of Food-Borne Pathogens

In this study, we present the successful detection of food-borne pathogens using randomly selected non-sequenced genomic DNA probes-based DNA microarray chips. Three food-borne pathogens, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), and Bacillus cereus, were subjected for the preparation of the DNA microarray probes. Initially, about 50 DNA probes selected randomly from non-sequenced genomic DNA of each pathogen were prepared by using a set of restriction enzyme pairs. The proto-type of DNA microarray chip for detecting three different pathogens simultaneously was fabricated by using those DNA probes prepared for each pathogen. This proto-type DNA microarray has been tested with three target pathogens and additional seven bacteria, and successfully verified with a few cross-hybridized probes. After this primary verification of the DNA microarray hybridization, this proto-type DNA microarray chip was redesigned and successfully optimized by eliminating a few cross-hybridized probes. The specificity of this redesigned DNA microarray chip to each pathogen was confirmed without any serious cross-hybridizations, and its multiplexing capability in its pathogen detection was found to be possible. This randomly selected non-sequenced genomic DNA probes-based DNA microarray was successfully proved to be the high-throughput simultaneous detection chip for the detection of food-borne pathogens, without knowing the exact sequence information of the target bacteria. This could be the first fabrication of DNA microarray chip for the simultaneous detection of different kinds of food-borne pathogens.     

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.     

Portable chemiluminescence multiplex biosensor for quantitative detection of three B19 DNA genotypes

A miniaturized multiplex biosensor exploiting a microfluidic oligonucleotide array and chemiluminescence (CL) lensless imaging detection has been developed for parvovirus B19 genotyping. The portable device consists of a reaction chip, comprising a glass slide arrayed with three B19 genotype-specific probes and coupled with a polydimethylsiloxane microfluidic layer, and a charge-coupled device camera modified for lensless CL imaging. Immobilized probes were used in DNA hybridization reactions with biotin-labeled targets, and then hybrids were measured by means of an avidin-horseradish peroxidase (HRP) conjugate and CL detection. All hybridization assay procedures have been optimized to be performed at room temperature through the microfluidic elements of the reaction chip, with sample and reagents delivery via capillary force exploiting adsorbent pads to drive fluids along the microchannels. The biosensor enabled multiplex detection of all B19 genotypes, with detectability down to 80 pmol?L^?1 for all B19 genotype oligonucleotides and 650 pmol?L^?1 for the amplified product of B19 genotype 1, which is comparable with that obtained in traditional PCR-ELISA formats and with notably shorter assay time (30 min vs. 2 h). The specificity of the assay has been evaluated by performing DNA–DNA hybridization reactions among sequences with different degrees of homology, and no cross hybridizations among B19 genotypes have been observed. The clinical applicability has been demonstrated by assaying amplified products obtained from B19 reference serum samples, with results completely consistent with the reference PCR-ELISA method. The next crucial step will be integration in the biosensor of a miniaturized PCR system for DNA amplification and for heat treatment of amplified products.

Prostate Cancer Biomarker Detection with Carbon Nanotubes Modified Screen Printed Electrodes

DNA hypermethylation is an epigenetic alteration and a promising biomarker for early prostate cancer detection. Simple, sensitive, easy to handle and rapid detection methodologies are imperative for point of care diagnostics especially for cancer. Herein, we describe for the first time a regenerable and compatible electrochemical biosensor for detection of Glutathione S‐Transferase P‐1 (GSTP‐1) gene hypermethylation related to prostate cancer via DNA hybridization onto the disposable Carbon and Multi Walled Carbon Nanotubes (MWCNT) Screen Printed Electrodes (SPEs). In the study, capture probes were adsorbed onto the SPEs by simple passive adsorption and then hybridization was achieved by sending the complementary target onto the probe‐modified electrodes. The selectivity of the biosensor was proved by control studies. Differential Pulse Voltammetry (DPV) technique was used to detect hybridization via guanine oxidation signals changes. The total time of the optimized method was nearly 1h, measurements took for less than 1 min, and the biosensor response was stable up to 40 days of storage period at 4 °C. The main advantages of the biosensor are very low detection limit (picomolar range) and capability of reusing the biosensor for at least 3 times after very simple regeneration process that is a unique property to reduce the cost of the assay. In addition, this is the first study that demonstrates the detection of GSTP‐1 hypermethylation electrochemically by using SPEs in order to create point of care diagnostics. The optimum parameters for the biosensor, as well as its future prospects to enhance the performance of DNA biosensors were also presented.     

Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> gene

A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ^INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ^INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.     

Evaluation of Poly(o‐phenylenediamine) Films for Application as Insulating Layers upon Carbon Substrates for Use within Sonochemically Fabricated Microelectrode Arrays

The formation of insulating layers of poly(o‐phenylenediamine) via electropolymerization was performed on single carbon screen‐printed electrodes. The effects of experimental parameters on the properties of the film were investigated, allowing for technique optimization. These conditions were then used to deposit films upon surfaces of 100 interconnected electrodes, with cyclic voltammetry used to study the electroactivity of the resulting electrodes. The insulating property of the film, for use in the formation of microelectrode array amperometric sensors, was evaluated. Finally, the insulated sensors were exposed to ultrasonic ablation to form microelectrode arrays, and these were subsequently assessed using optical and electrochemical techniques.     

Electrochemical Oxidation of Catechols in the Presence of Triethyl Phosphite

The mechanism of electrochemical oxidation of catechol and some of its derivatives have been studied in the presence of triethyl phosphite as a nucleophile in aqueous solution. Voltammetric studies indicate that the quinones derived from catechol, and its derivatives, participate in Michael addition reaction with triethyl phosphite. The reaction mechanism consists of electron transfer followed by a chemical reaction which is named as an EC mechanism. The homogeneous rate constants (k_obs) were estimated by comparing the experimental cyclic voltammograms with the digitally simulated voltammograms based on EC mechanism. Also the effects of nucleophile concentration and substituted group of catechols on voltammetric behavior and the rate constants of chemical reactions were examined.     

Rapid Electrochemical Assessment of Tumor Suppressor Gene Methylations in Raw Human Serum and Tumor Cells and Tissues Using Immunomagnetic Beads and Selective DNA Hybridization

We report a rapid and sensitive electrochemical strategy for the detection of gene‐specific 5‐methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5‐methylcytosines (5‐mC) are used for the capture of any 5‐mC methylated single‐stranded (ss)DNA sequence. A flanking region next to the 5‐mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen‐printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O‐6‐methylguanine‐DNA methyltransferase (MGMT) promoter region. The method is applied in the 45‐min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U‐87 glioblastoma cells and paraffin‐embedded brain tumor tissues without any amplification and pretreatment step.     

Comparison of Boron‐Doped Diamond and Glassy Carbon Electrodes for Determination of Procaine Hydrochloride

The electrochemical oxidation of procaine hydrochloride (PC?HCL, 2‐diethylaminoethyl 4‐aminobenzoate hydrochloride) was investigated at as‐deposited boron‐doped diamond (ad‐BDD) electrode, anodically oxidized BDD (ao‐BDD) electrode and glassy carbon (GC) electrode using cyclic voltammetry (CV). Well‐defined cyclic voltammograms were obtained for PC?HCL oxidation with high signal‐to‐background (S/B) ratio, low tendency for adsorption, good reproducibility and long‐term stability at ad‐BDD electrode, demonstrating its superior electrochemical behavior and significant advantages in contrast to ao‐BDD and GC electrode. At 100 μM PC?HCL, the voltammetric S/B ratio was nearly one order of magnitude higher at an ad‐BDD electrode than that at a GC electrode. In a separate set of experiments for oxidation of 100 μM PC?HCL, 96%, 92% and 84% of the initial oxidation peak current was retained at the ad‐BDD, ao‐BDD and GC electrode, respectively, by stirring the solution after the tenth cycle. The current response was linearly proportional to the square root of the scan rate within the range 10–1000 mV s^?1 in 10 μM PC?HCL solutions, indicating that the oxidation process was diffusion‐controlled with negligible adsorption at an ad‐BDD surface. The good linearity was observed for a concentration range from 5 to 200 μM with a linear equation of y=0.03517x+0.65346 (r=0.999), and the detection limit was 0.5 μM for oxidation of PC?HCL at the ad‐BDD electrode. The ad‐BDD electrode could maintain 100% of its original activity after intermittent use for 3 months.     

Voltammetric Label‐free Detection of DNA Hypermethylation Using Polypyrrole‐modified Microelectrode Array

An alternative strategy for the label‐free electrochemical detection of DNA hypermethylation using a microelectrode array as an oligodinucleotide (ODN) detector is presented. It relies on the oligonucleotide dependent electrostatic affinity interaction firstly with unmethylated ODN and then follow‐up with methylated DNA. The methylated cytosine status is quantified by monitoring the relative change in the exchange current at the ODN‐detector before and after the bisulfite treated DNA samples. This novel aproach displays unique advantages such as small working volumes of the analytes, low damage to DNA samples, easy integration of oligonucleides on the detector and signal evaluation.     

Electrochemical Detection of DNA Melting Curves by Means of Heated Biosensors

This article reports about the detection of DNA melting curves at heated electrochemical biosensors. Osmium tetroxide‐bipyridine‐labeled target oligonucleotides are hybridized with probe oligonucleotides immobilized on gold electrodes. Then, the gold electrode is successively heated in order to measure a complete melting curve consisting of alternating current voltammetric signals. Melting temperatures ?_m, determined at various ionic strengths and in dependence on different numbers of base pair mismatches, have been compared with those obtained by means of UV spectrophotometry. The proposed method holds great promise for the fast and easy parallel detection of nucleic acids sequences on selectively heated electrode arrays. A stringent hybridization temperature can be easily adjusted in order to discriminate base pair mismatches.     

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.     

Long voltammetric microelectrode for in vivo monitoring of acetaminophen in primate species

A long (45 cm) voltammetric microelectrode for in vivo monitoring of acetaminophen in primate species is described. Due to its length, intravascular insertion is feasible into regions of effective blood circulation. The working electrode is made of carbon paste, placed at the end of a Teflon tubing. Acetaminophen is detected in vivo, in rhesus monkey blood stream, following its intravenous (i.v.) administration. Differential pulse voltammetry with a relatively fast scan rate (2 V/min) is employed to allow rapid assessment of blood concentration of a drug (up to 90 measurements per hour). Well defined peak voltammograms are obtained. In the anodic potential range of whole blood voltammograms, no differential pulse peaks are observed. Despite the obvious problems associated with voltammetric measurements in a complex environment such as whole blood, the method appears to be suited for in vivo monitoring of oxidizable drugs.