Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

Ionic Liquid Modified Single‐use Electrode Developed for Voltammetric Detection of miRNA‐34a and its Application to Real Samples

The ionic liquid (IL) modified chemically activated (CA) pencil graphite electrodes (PGEs) were developed for label‐free voltammetric detection of miRNA‐34a, and implemented to the real samples. Firstly, the electrochemical characterization of unmodified PGE, CA‐PGE, IL‐PGE and IL‐CA‐PGE was performed by cyclic voltammetry (CV) as well as their DNA binding capacity was studied by electrochemical impedance spectroscopy (EIS) technique. The microscopic characterization of the surface of each electrodes was investigated by scanning electron microscopy (SEM). Differential pulse voltammetry (DPV) technique was used for measuring the oxidation signal of guanine in order to perform a label‐free voltammetric monitoring of a full‐match hybridization specific to miRNA‐34a. The selectivity of biosensor was tested against to miRNA‐155, miRNA‐660 as well as to the mismatch sequence of miRNA‐34a. The further selectivity of this proposed biosensor was studied in the mixture of samples containing miRNA‐34a with other miRNAs (1 : 1). The voltammetric detection of miRNA‐34a was also explored in the artificial serum medium as fetal bovine serum (FBS) and also in total RNA samples isolated from HUH‐7 human hepatocellular carcinoma cell line.     

Hydroxyapatite Nanoparticles Modified Graphite Electrodes for Electrochemical DNA Detection

Hydroxyapatite nanoparticles (HaNP) modified pencil graphite electrodes (PGEs) were developed for the first time in the literature, and accordingly they were applied for electrochemical monitoring of sequence‐selective DNA hybridization. The experimental conditions for HaNP modification of PGE, and DNA hybridization related to Hepatitis B Virus (HBV) DNA sequence were optimized. The microscopic and electrochemical characterization of HaNP‐PGE in contrast to the unmodified one was utilized. Under optimized experimental conditions, the selectivity of HBV DNA probe immobilized biosensor was tested against to non‐complementary (NC), mismatch (MM) sequences and the mixture of target:NC (1 : 1) or target: MM (1 : 1).     

Differential Pulse Voltammetric Detection of Hepatitis C Virus 1a Oligonucleotide Chain by a Label‐Free Electrochemical DNA Hybridization Biosensor Using Consensus Sequence of Hepatitis C Virus 1a Probe on the Pencil Graphite Electrode

The short sequence related to hepatitis C virus (HCV1) is detected by a label‐free DNA hybridization biosensor. The sensor relies on the immobilization of a 20‐mer oligonucleotide containing 2 guanine and 11 cytosine bases denoted PHCV1 as probe on the pencil graphite electrode (PGE). The hybridization event was monitored by differential pulse voltammetry (DPV) using the guanine signal. The selectivity of the biosensor was studied using some noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit was found to be 6.5 nM.     

An Electrochemical DNA Biosensor for the Detection of the Apa I Polymorphism in the Vitamin D Receptor Gene Using Meldola's Blue as a Hybridization Indicator

Electrochemical detection of nucleic acid base mismatches related to Apa I single nucleotide polymorphism (SNP) in the vitamin D receptor gene was performed successfully using 7‐dimethyl‐amino‐1,2‐benzophenoxazinium salt (Meldola's blue, MDB) with 10.9 pmol/100 μL of detection limit. MDB reduction signals obtained from probe, mismatch(probe‐SNP containing target) and hybrid(probe‐target) modified pencil graphite electrode(PGE) increased respectively. The sensor was able to clearly distinguish perfect match from mismatch DNA in a 30 min. detection time. Several factors affecting on the hybridization and indicator response are studied to maximize sensitivity and selectivity. The advantages of the biosensor are discussed in comparison with previous electrochemical assays for DNA hybridization.     

Chitosan/Nitrogen Doped Reduced Graphene Oxide Modified Biosensor for Impedimetric Detection of microRNA

The development of a low‐cost and disposable biosensor platform for the sensitive and rapid detection of microRNAs (miRNAs) is of great interest for healthcare, pharmaceuticals, and medical science. We designed an impedimetric biosensing platform using Chitosan (CHIT)/nitrogen doped reduced graphene oxide (NRGO) conductive composite to modify the surface of pencil graphite electrodes (PGE) for the sensitive detection of miRNAs. An initial optimisation protocol involved investigation of the effect of NRGO concentration and miR 660 DNA probe concentration on the response of the modified electrode. After the optimization protocol, the sequence‐selective hybridization between miR 660 DNA probe and its RNA target was evaluated by measuring changes on charge transfer resistance, R_ct values. Moreover, the selectivity of impedimetric biosensor was tested in the presence of non‐complementary miRNA (NC) sequences, such as miR 34a and miR 16. The hybridization process was examined both in phosphate buffer (PBS) and in PBS diluted fetal bovine serum (FBS:PBS) solutions. The biosensor demonstrated a detection limit of 1.72 μg/mL in PBS and 1.65 μg/mL in FBS:PBS diluted solution. Given the easy, quick and disposable attributes, the proposed conductive nanocomposite biosensor platform shows great promise as a low‐cost sensor kit for healthcare monitoring, clinical diagnostics, and biomedical devices.     

Quantitative Biodetection of Anticancer Drug Rituxan with DNA Biosensor Modified PAMAM Dendrimer/Reduced Graphene Oxide Nanocomposite

PAMAM dendrimer/reduced graphene oxide nanocomposite modified pencil graphite electrode (PAMAM/RGO/PGE) was used to fabricate an electrochemical DNA biosensor for determination of Rituxan (RTX) at low concentrations, for the first time. The fabricated biosensor was characterized with FE‐SEM, EIS, and CV techniques. The ds‐DNA/PAMAM/RGO/PGE was used as a working electrode to study the interaction between the RTX and salmon sperm ds‐DNA by DPV technique. Because of the interaction between the drug and DNA leads to a decrease in the guanine oxidation peak current, it was used as an indicator for the determination of the RTX. Under the optimized experimental conditions, a wide linear relationship between RTX concentration and guanine signal was obtained within the range of 7.0 to 60.0 μmol L⁻¹ and 60.0 to 300.0 μmol L⁻¹ with a low detection limit (0.56 μmol L⁻¹). To clarify the interaction mechanism between the RTX and the ds‐DNA, DPV and UV‐Vis measurements were used. The reproducibility, stability, and performance of the constructed biosensor was examined by quantitative measuring RTX in pharmaceutical and human serum samples with good precision (RSD; 2.0–6.0 %) and acceptable recoveries (100.04–101.95 %).     

A Novel Electrochemical DNA Biosensor Fabricated with Layer‐by‐Layer Covalent Attachment of Multiwalled Carbon Nanotubes and Gold Nanoparticles

A novel DNA biosensor has been fabricated for the detection of DNA hybridization based on layer‐by‐layer (LBL) covalent assembly of gold nanoparticles (GNPs) and multiwalled carbon nanotubes (MWCNTs). The stepwise LBL assembly process was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization events were monitored by differential pulse voltammetry (DPV) measurement of the intercalated doxorubicin, and the factors influencing the performance of the DNA hybridization was investigated in detail. The signal was linearly changed with target DNA concentration increased from 0.5 to 0.01 nM, and had a detection limit of 7.5 pM (signal/noise ratio of 3). In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label

Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its complementary sequence (chIL-2) as the target was studied by square wave voltammetry (SWV) of MB accumulated on the PGE. In this approach the extent of hybridization is evaluated on the basis of the difference between SWV signals of MB accumulated on the probe-PGE and MB accumulated on the probe-target-PGE. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. Some experimental variables affecting the performance of the biosensor including: polishing of PGE, its electrochemical activation conditions (i.e., activation potential and activation time) and probe immobilization conditions on the electrodes (i.e., immobilization potential and time) were investigated and the optimum values of 1.80 V and 300 s for PGE activation, and −0.5 V and 400 s for the probe immobilization on the electrode were suggested.     

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.     

An Electropolymerized Membrane Biosensor for Speciffic DNA Recognition

A sensitive electrochemical biosensor for detecting the sequence of short DNA oligomers is represented. The biosensor is based on a platinum electrode covered a polymerized membrane of conductive monomer N‐[6‐(thien‐3‐yl)acetoxy]‐pyrrolidine‐2, 5‐dione (TAPD). The membrane of TAPD immobilizes a probe DNA on the electrode. The hybridization of the probe with a sequence‐specific DNA in sample solutions is monitored by a self‐synthesized electroactive indicator, which specifically intercalates in the hybrids on the electrode surface. The current signal of the biosensor is proportional to the concentration of the target DNA in samples, and a very low detection limit of 5 × 10^?10 mol/L is found. The biosensor has been used to detect the short oligomers containing of HTV‐1 and mycobacterrium nucleotide sequences.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

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

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

Impedance DNA Biosensor Using Electropolymerized Polypyrrole/Multiwalled Carbon Nanotubes Modified Electrode

In this paper, we present an electrochemical impedance‐based DNA biosensor by using a composite material of polypyrrole (PPy) and multiwalled carbon nanotubes (MWNTs) to modify glassy carbon electrode (GCE). The polymer film was electropolymerized onto GCE by cyclic voltammetry (CV) in the presence of carboxylic groups ended MWNTs (MWNTs‐COOH). Such electrode modification method is new for DNA hybridization sensor. Amino group ended single‐stranded DNA (NH₂‐ssDNA) probe was linked onto the PPy/MWNTs‐COOH/GCE by using EDAC, a widely used water‐soluble carbodiimide for crosslinking amine and carboxylic acid group. The hybridization reaction of this ssDNA/PPy/MWNTs‐COOH/GCE resulted in a decreased impedance, which was attributed to the lower electronic transfer resistance of double‐stranded DNA than single‐stranded DNA. As the result of the PPy/MWNTs modification, the electrode obtained a good electronic transfer property and a large specific surface area. Consequently, the sensitivity and selectivity of this sensor for biosensing DNA hybridization were improved. Complementary DNA sequence as low as 5.0×10^?12 mol L^?1 can be detected without using hybridization marker or intercalator. Additionally, it was found that the electropolymerization scan rate was an important factor for DNA biosensor fabrication. It has been optimized at 20 mV s^?1.     

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

Graphene Oxide Modified Chemically Activated Graphite Electrodes for Detection of microRNA

In our study, graphene oxide (GO) modified graphite electrodes were used for sensitive and selective impedimetric detection of miRNA. After chemical activation of pencil graphite electrode (PGE) surface using covalent agents (CA), GO modification was performed at the surface of chemically activated PGE. Then, CA‐GO‐PGEs were applied for impedimetric miRNA detection. The microscopic and electrochemical characterization of CA‐GO‐PGEs was performed by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The optimization of experimental conditions; such as GO concentration, DNA probe concentration and miRNA target concentration was performed by using EIS technique. After the hybridization occurred between miRNA‐34a RNA target and its complementary DNA probe, the hybrid was immobilized onto the surface of CA‐GO‐PGEs. Then, the impedimetric detection of miRNA‐DNA hybridization was performed by EIS. The selectivity of our assay was also tested under the optimum experimental conditions.     

Direct DNA Hybridization on the Single‐Walled Carbon Nanotubes Modified Sensors Detected by Voltammetry and Electrochemical Impedance Spectroscopy

Carboxylic acid functionalized single‐walled carbon nanotubes modified graphite sensors (SWCNT‐PGEs) were developed for electrochemical monitoring of direct DNA hybridization related to specific sequence of Hepatitis B virus, which substantially enhance the electrochemical transduction resulting from guanine oxidation signal comparison to bare PGEs. The performance characteristics of DNA hybridization on disposable CNT‐PGE were explored measuring the guanine signal in terms of optimum analytical conditions; probe and target concentration, hybridization time, and selectivity. The voltammetric results were also complemented with electrochemical impedance spectroscopy (EIS), that was used to characterize the successful construction of carbon nanotubes modification onto the surface of PGEs.     

A Peptide Nucleic Acid (PNA)‐DNA Ferrocenyl Intercalator for Electrochemical Sensing

A ferrocenyl intercalator was investigated to develop an electrochemical DNA biosensor employing a peptide nucleic acid (PNA) sequence as capture probe. After hybridization with single strand DNA sequence, a naphthalene diimide intercalator bearing ferrocene moieties (FND) was introduced to bind with the PNA‐DNA duplex and the electrochemical signal of the ferrocene molecules was used to monitor the DNA recognition. Electrochemical impedance spectroscopy was used to characterize the different modification steps. Differential pulse voltammetry was employed to evaluate the electrochemical signal of the FND intercalator related to its interaction with the complementary PNA‐DNA hybrid. The ferrocene oxidation peaks were utilised for the target DNA quantification. The developed biosensor demonstrated a good linear dependence of FND oxidation peak on DNA concentration in the range 1 fM to 100 nM of target DNA, with a low detection limit of 11.68 fM. Selectivity tests were also investigated with a non‐complementary DNA sequence, indicating that the FND intercalator exhibits a selective response to the target PNA‐DNA duplex.     

Label‐Free Bioelectronic Detection of Point Mutation by Using Peptide Nucleic Acid Probes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes with a label‐free protocol is described. The detection of PNA‐DNA and DNA‐DNA hybridizations were accomplished based on the oxidation signal of guanine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE). It was observed that the oxidation signals of guanine obtained from the PNA and DNA probe modified CPEs were higher than those obtained from the PNA‐DNA and DNA‐DNA hybrid modified CPEs due to the accessible unbound guanine bases. The detection of hybridization between PNA probe and point mutation containing DNA target sequences was clearly observed due to the difference of the oxidation signals of guanine bases, because the point mutation was guanine nearly at the middle of the sequence. The effect of the DNA target concentration on the hybridization signal was also observed. The PNA probe was also challenged with excessive and equal amount of noncomplementary DNA and also mixtures of point mutation and target DNA.     

An Electrochemical DNA Sensor Based on Electrodepositing Aluminum Ion Films on Stearic Acid‐Modified Carbon Paste Electrode and Its Application for the Detection of Specific Sequences Related to Bar Gene and CP4 Epsps Gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid‐modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well‐defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single‐stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the specific sequence related to the target bar gene with the dynamic range comprised between 1.0×10^?7 mol/L to 1.0×10^?4 mol/L. A detection limit of 2.25×10^?8 mol/L of oligonucleotides can be estimated.