Electroactive Label Research Articles

An electrochemical DNA biosensor based on Oracet Blue as a label for detection of Helicobacter pylori

An innovative method of a DNA electrochemical biosensor based on Oracet Blue (OB) as an electroactive label and gold electrode (AuE) for detection of Helicobacter pylori, was offered. A single–stranded DNA probe with a thiol modification was covalently immobilized on the surface of the AuE by forming an Au–S bond. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of reduction of the OB binding to double–stranded DNA (ds–DNA). Our results showed that OB–based DNA biosensor has a decent potential for detection of single–base mismatch in target DNA. Selectivity of the proposed DNA biosensor was further confirmed in the presence of non–complementary and complementary DNA strands. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 0.3nmolL−1 to 240.0nmolL−1, and the detection limit was 0.17nmolL−1, whit a promising reproducibility and repeatability.

Refining the Structure of Graphite Oxide through Selective Labeling of Functional Groups

The various inherent functional groups within graphite oxide (GO) are of significant interest for numerous research applications exploiting their reactivity. While there has been much advance in the research of graphenes and GO over the years, our current understanding of GO structure is primarily derived using spectroscopic techniques such as XPS, NMR, FTIR and Raman spectroscopies. In contrast, we report an alternative electrochemical approach to investigate the composition of GO, previously developed to study surface functionalities within other carbon-based materials. In separate reactions, four electroactive labels were first employed to selectively label the various functional groups in GO, followed by their subsequent quantification using cyclic voltammetry. Distinct functional group compositions were consequently found depending on the choice of GO oxidation method, either via chlorate or permanganate oxidations. An additional advantage of the method is the ability to distinguishing the types of carbonyl groups such as quinonyl species over isolated ketones. Thus, we noted that permanganate oxidation produced GO with its carbonyls mainly existing as 1,2-quinones, while chlorate oxidation resulted primarily in aliphatic ketones. Furthermore, carboxylic groups that were erstwhile thought to be responsible for the acidic nature of GO were observed to be low in permanganate-GO and non-existent in chlorate-GO. We also deduced that epoxides likely exist as the main functionality within permanganate-GO, and discuss that the relatively low labelled-hydroxyl content may be due to previously proposed disproportionation reactions which result in formation of other groups. Overall, this selective labelling and electrochemical detection method has provided an improved and refined understanding of GO structures.

A novel electrochemical biosensor for selective determination of mercury ions based on DNA hybridization

An electrochemical biosensor was developed for Hg2+ determination based on DNA hybridization. In the presence of Hg2+, the target and probe DNAs with thymine–thymine (T–T) mismatches could hybridize by forming T–Hg2+–T complex. This induced DNA hybridization led to the decrease in reduction peak currents of ethyl green (EG) as electroactive label, which could be used for determination of Hg2+. The difference in the value of the peak currents of EG before and after DNA hybridization (ΔI) was linear with the concentration of Hg2+ in the range of 9.0 × 10−11–1.0 × 10−9 M. The detection limit was 3.08 × 10−11 M.

Design of an electroactive peptide probe for sensing of a protein

We designed a new electroactive peptide probe that has a molecular recognition function for the sensing of a protein. Ovalbumin (OVA) was the model protein, and when RNRCKGTDVQAW interacted with OVA, it conjugated with a tyrosine-rich peptide (Y4C). This peptide is electroactive, has a high degree of biocompatibility, and offers the possibility of gene expression. To measure the effect of a number of the tyrosine residues, voltammetric measurements were conducted using a series of tyrosine-rich peptides (YnC, n = 3–7) with sensitivities that ranged from 10−9 to 10−8 M. The electrode response of Y5C was the maximum value in the series. However, the peak current did not increase when the number of tyrosine residues was increased in a linear fashion. This may have been due to the micelles that are formed by a tyrosine-rich surfactant peptide. Thus, Y4C was suitable as an electroactive label for the construction of the peptide probe. The electrode response of Y4CRNRCKGTDVQAW obtained by a glassy carbon electrode was 100-fold that of tyrosine alone. The measurement of OVA via the peptide probe resulted in a detection on the order of 10−12 M. In contrast, the sensitivity of OVA using RCKGTDVQAWY4C probe was at the 10−11 M level, because the hydrophobic moiety gave it a molecular recognition function. The recoveries of the OVA using Y4CRNRCKGTDVQAW in a solution containing fetal bovine serum ranged between 98 and 101%. Consequently, the combination of a specific peptide and an electroactive element could be a powerful probe for the sensing of proteins.

Non-enzymatic electrochemical immunoassay using noble metal nanoparticles: a review

Electrochemical immunodetection has attracted considerable attention due to its high sensitivity, low cost and simplicity. Large efforts have recently made in order to design ultrasensitive assays. Noble metal nanoparticles (NM-NPs) offer advantages such as high conductivity and large surface-to-volume ratio. NM-NPs therefore are excellent candidates for developing electrochemical platforms for immunodetection and as signal tags. The use of biofunctionalized NM-NPs often results in amplified recognition via stronger loading of signal tags, and also in enhanced signal. This review (with 87 references) gives an overview on the current state in the use of NM-NPs in Non-enzymatic electrochemical immunosensing. We discuss the application of NM-NPs as electrode matrices and as electroactive labels (either as a carrier or as electrocatalytic labels), and compare the materials (mainly nanoparticles of gold, platinum, or of bimetallic materials) in terms of performance (for example by increasing sensitivity via label amplification or via high densities of capture molecules). A conclusion covers current challenges and gives an outlook. Rather than being exhaustive, the review focuses on representative examples that illustrate novel concepts and promising applications. NM-NPs based immunosensing opens a series of concepts for basic research and offers new tools for determination of trace amounts of protein-related analytes in environment and clinical applications.

Novel electrochemical DNA hybridization biosensors for selective determination of silver ions

In this work, novel electrochemical biosensors for Ag+ determination based on Ag+-induced DNA hybridization, using Ethyl green (EG) as an electroactive label on the surface of bare carbon paste electrode (CPE) and gold nanoparticles-modified carbon paste electrode (GN-CPE) are reported. Two single-strand poly-C (100% cytosine bases) DNAs are used as oligonucleotide probe and target. In the presence of Ag+, the target DNA with full cytosine–cytosine (C–C) mismatches could hybridize with the probe DNA by forming C–Ag+–C complex. The induced hybridization of the two oligonucleotides leads to the decrease in the reduction peak currents of EG, which could be used for electrochemical determination of Ag+. This difference in the values of the reduction peak current of EG before and after DNA hybridization (∆I) was linear with the concentration of Ag+ in the ranges from 3.0×10−10 to 1.0×10−9molL−1 and 9.0×10−11 to 1.0×10−9molL−1, for the biosensor and nanoparticles modified-biosensor, respectively. Calculated detection limits were 1.04×10−10 and 2.64×10−11molL−1 for biosensor and nanoparticles modified-biosensor, respectively. The biosensors demonstrated good selectivity towards Ag+ ions in the presence of some metal ions such as Pb2+, Cu2+, Ca2+, Zn2+, Fe2+ and Hg2+. The proposed biosensors were applied successfully to the voltammetric determination of Ag+ in real samples.

Mycotoxin Aptasensing Amplification by using Inherently Electroactive Graphene‐Oxide Nanoplatelet Labels

AbstractWe develop an electrochemical aptasensor for ochratoxin A (OTA) by utilizing graphene‐oxide nanoplatelets (GONPs) as electroactive labels. OTA aptamer (OTA‐apt), which serves as the recognition element, was first immobilized onto the electrode surface before the modified electrode underwent exposure to OTA. Subsequently, incubation with GONPs was performed and the nanoplatelets conjugated to the immobilized OTA‐apt through π–π interactions. The principle of detection lies in the ability of these nanoplatelets to be electrochemically reduced, thereby producing a well‐defined reduction peak that was then employed as the analytical signal. The fabricated aptasensor is capable of detecting OTA in the concentration range of 310 fM to 310 pM, with good selectivity against common interferences. The obtained results from this study have the potential to contribute towards the aim of developing a biosensor for OTA, with high selectivity and sensitivity.

Target-induced reconfiguration of DNA probes for recycling amplification and signal-on electrochemical detection of hereditary tyrosinemia type I gene.

By coupling target DNA-induced reconfiguration of the dsDNA probes with enzyme-assisted target recycling amplification, we describe the development of a signal-on electrochemical sensing approach for sensitive detection of hereditary tyrosinemia type I gene. The dsDNA probes are self-assembled on the sensing electrode, and the addition of the target DNA reconfigures and switches the dsDNA probes into active substrates for exonuclease III, which catalytically digests the probes and leads to cyclic reuse of the target DNA. The target DNA recycling and the removal of one of the ssDNA from the dsDNA probes by exonuclease III result in the formation of many hairpin structures on the sensor surface, which brings the electroactive methylene blue labels into proximity with the electrode and produces a significantly amplified current response for sensitive detection of the target gene down to 0.24 pM. This method is also selective to discriminate single-base mismatch and can be employed to detect the target gene in human serum samples. With the demonstration for the detection of the target gene, we expect the developed method to be a universal sensitive sensing platform for the detection of different nucleic acid sequences.

Application of Oracet Blue in a novel and sensitive electrochemical biosensor for the detection of microRNA

A simple, novel and sensitive electrochemical biosensor was developed based on thiolated capture probe on gold electrode and electroactive label Oracet Blue, for microRNA detection.

An ultrasensitive electrochemical immunosensor for the detection of human immunoglobulin G based on Ag@BSA microspheres

Ag@BSA microspheres were applied as electroactive labels for signal amplification and the ultimate immunosensor exhibited an ultralow detection limit.

A Label-Free Electrochemical Aptasensor for the Analysis of The Potassium Ion

Herein, a simple and novel electrochemical method for the detection of potassium ions (K+) was developed. In the presence of potassium ions, the potassium ions aptamer will form a G-quadruplex complex. Thus, further addition of hemin in the presence of potassium ions will lead to the formation of a recombined G-quadruplex. Then the electroactive label, hemin, will give an electrochemical response. The linear range of the method covered a large variation of K+ concentration from 0.1 nM to 0.1 μM and the detection limit of 0.1 nM was obtained. Moreover, this assay was able to detect K+ with high selectivity and had great potential applications.

Electrochemical analysis of nucleic acids, proteins and polysaccharides in biomedicine

Electrochemical analysis of nucleic acids, proteins and polysaccharides represents an interesting, although not widely spread alternative to current methods based predominantly on optical detection because it offers a relatively inexpensive, fast and instrumentally simple detection of parallel samples on miniaturized chips, ideal for personalized medicine of the 21st century. Nucleic acid electrochemistry enables, for example, detection of specific DNA sequences (for determination of genes or presence of bacteria and viruses, etc.), DNA damage analysis and interaction with other molecules, DNA methylation or detection of microRNAs as potential cancer bio-markers. In the electrochemistry of proteins, great emphasis is put on construction of immunosensors for capturing specific proteins (antigens) using antibodies, suitable for diagnostics. From a bio-physical point of view, intrinsic electrocatalytic signal of proteins sensitive to conformational changes could be useful in discrimination of mutant proteins (e. g. p53), native and aggregated forms (α-synuclein in Parkinsons disease) or for studies of protein interactions with low molecular weight ligands and DNA. Due to an increased interest of scientists in glycoproteins, new electrochemical papers emerged aiming at detection of oligosaccharides and polysaccharides (i.e. glycans, when part of the protein). These assays employ for instance electroactive labels specific for saccharides or lectin bio-sensors using lectins which strongly bind glycans. Electrochemical analysis thus appears as an interesting tool in current genomics, proteomics and glycomics, as well as for cancer diagnostics.

Nanomaterial-based biosensors for food toxin detection.

There is an increased interest toward the development of bioelectronic devices for food toxin (mycotoxins) detection. Mycotoxins are highly toxic secondary metabolites produced by fungi like Fusarium, Aspergillus, and Penicillium that are frequently found in crops or during storage of food including cereals, nuts, fruits, etc. The contamination of food by mycotoxins has become a matter of increasing concern. High levels of mycotoxins in the diet can cause adverse, acute, and chronic effects on human health and a variety of animal species. Side effects may particularly affect the liver, kidney, nervous system, endocrine system, and immune system. Among 300 mycotoxins known till date, there are a few that are considered to play an important part in food safety, and for these, a range of analytical methods have been developed. Some of the important mycotoxins include aflatoxins, ochratoxins, fumonisins, citreoviridin, patulin, citrinin, and zearalenon. The conventional methods of analysis of mycotoxins normally require sophisticated instrumentation, e.g., liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. Hence, new analysis tools are necessary to attain more sensitive, specific, rapid, and reliable information about the desired toxin. For the last about two decades, the research and development of simpler and faster analytical procedures based on affinity biosensors has aroused much interest due to their simplicity and sensitivity. The nanomaterials have recently had a great impact on the development of biosensors. The functionalized nanomaterials are used as catalytic tools, immobilization platforms, or as optical or electroactive labels to improve the biosensing performance to obtain higher sensitivity, stability, and selectivity. Nanomaterials, such as carbon nanomaterials (carbon nanotubes and graphene), metal nanoparticles, nanowires, nanocomposites, and nanostructured metal oxide nanoparticles are playing an increasing role in the design of sensing and biosensing systems for mycotoxin determination. Furthermore, these nanobiosystems are also bringing advantages in terms of the design of novel food toxin detection strategies. We will focus on some of the recent results related to fabrication of nanomaterial-based biosensors for food toxin detection obtained in our laboratories.

Enhanced detection of the potential electroactive label methylene blue by electrode nanostructuration with carbon nanotubes

Electrochemical sensors are developing very fast in the last decades due to the possibilities of generating miniaturized and simplified low-cost analytical devices. However, the search for highly detectable electroactive labels and sensitive detection strategies is still required. In this work, the electrochemical signal of a very promising molecule that can be employed as biolabel, methylene blue, is enhanced by recording it on nanostructured (MWCNTs-NH2) gold screen-printed electrodes. A dispersion of amino-functionalised CNTs in Nafion®/ethanol:water (1:1) is employed for modification by depositing a drop on the surface of a screen-printed gold working electrode. This produces a change in the electrochemical behavior of MB, with increase of both, reversibility and peak current. On the other hand, unlike what happens on bare electrodes where MB presents a diffusion controlled process, adsorption occurs on modified electrodes. This allows higher enhancement of the signal by introducing an accumulation time. In this way, and once parameters that affect the modification are optimized, improvements up to fifteen times are obtained. This is the first time such enhancement is produced for this molecule based on its adsorption on nanostructured electrodes. In this way, a detection limit of 100nM for differential pulse voltammetry or square wave voltammetry is obtained after 120s of accumulation time. The sensitivity increases almost by seven times (from 0.711 to 5.029μAμM−1) when calibration curves obtained from SW voltammograms recorded on bare and modified electrodes are compared. Modification is stable for at least 10 days.

Copper-doped titanium dioxide nanoparticles as dual-functional labels for fabrication of electrochemical immunosensors

Constructions of versatile electroactive labels are key issues in the development of electrochemical immunosensors. In this study, copper-doped titanium dioxide nanoparticle (Cu@TiO2) was synthesized and used as labels for fabrication of sandwich-type electrochemical immunosensors on glassy carbon electrode (GCE). Due to the presence of copper ions, Cu@TiO2 shows a strong response current when coupled to an electrode. The prepared nanocomposite also shows high electrocatalytic activity towards reduction of hydrogen peroxide (H2O2). The dual functionality of Cu@TiO2 enables the fabrication of immunosensor using different detection modes, that is, square wave voltammetry (SWV) or chronoamperometry (CA). While Cu@TiO2 was used as labels of secondary antibodies (Ab2), carboxyl functionalized graphene oxide (CFGO) was used as electrode materials to immobilize primary antibodies (Ab1). Using human immunoglobulin G (IgG) as a model analyte, the immunosensor shows high sensitivity, acceptable stability and good reproducibility for both detection modes. Under optimal conditions, a linear range from 0.1pg/mL to 100ng/mL with a detection limit of 0.052pg/mL was obtained for SWV analysis. For CA analysis, a wider linear range from 0.01pg/mL to 100ng/mL and a lower detection limit of 0.0043pg/mL were obtained. The proposed metal ion-based enzyme-free and noble metal-free immunosensor may have promising applications in clinical diagnoses and many other fields.

On the Use of Metal Purine Derivatives (M=Ir, Rh) for the Selective Labeling of Nucleosides and Nucleotides

The reactions of neutral or cationic IrIII and RhIII derivatives of phenyl purine nucleobases with unsymmetrical alkynes produce new metallacycles in a predictable manner, which allows for the incorporation of either photoactive (anthracene or pyrene) or electroactive (ferrocene) labels in the nucleotide or nucleoside moiety. The reported methodology (metalation of the purine derivative and subsequent marker insertion) could be used for the postfunctionalization and unambiguous labeling of oligonucleotides.

Ultrasensitive Telomerase Activity Detection in Circulating Tumor Cells Based on DNA Metallization and Sharp Solid‐State Electrochemical Techniques

Being considered a “liquid biopsy”, circulating tumor cell (CTC) quantification is of great interest for evaluating cancer dissemination, predicting patient prognosis, and also for the evaluation of therapeutic treatments, representing a reliable potential alternative to invasive biopsies and subsequent proteomic and functional genetic analysis. Compared to a biopsy, the gold standard of current cancer diagnosis, an important characteristic of a blood test is that it is safe and can be performed at many points during the disease, allowing the development of appropriate therapy modifications and potentially improving patient's quality of life. In this work, an ultrasensitive electrochemical telomerase activity‐sensing strategy is presented that utilizes DNA‐templated deposition of silver nanoparticles as electroactive labels through a highly sharp solid‐state Ag/AgCl reaction with DNA exonuclease III‐assisted background current suppression. This nanoparticle‐mediated signal amplification resulted in significantly decreased detection limit, which is better than the vast majority of reported methods and achieves a sensitivity comparable to the conventional telomeric repeat amplification protocol (TRAP). This work may pave a new PCR‐free way for the detection of telomerase activity in CTCs via a noninvasive routine blood test for point‐of‐care diagnosis and individualized treatment of cancer.

Molybdenum disulfide (MoS2) nanoflakes as inherently electroactive labels for DNA hybridization detection.

The detection of specific DNA sequences plays a critical role in the areas of medical diagnostics, environmental monitoring, drug discovery and food safety. This has therefore become a strong driving force behind the ever-increasing demand for simple, cost-effective, highly sensitive and selective DNA biosensors. In this study, we report for the first time, a novel approach for the utilization of molybdenum disulfide nanoflakes, a member of the transition metal dichalcogenides family, in the detection of DNA hybridization. Herein, molybdenum disulfide nanoflakes serve as inherently electroactive labels, with the inherent oxidation peak exploited as the analytical signal. The principle of detection is based on the differential affinity of molybdenum disulfide nanoflakes towards single-stranded DNA and double-stranded DNA. The employment of transition metal dichalcogenide nanomaterials for sensing and biosensing purposes represents an upcoming research area which holds great promise. Hence, our findings are anticipated to have significant contributions towards the fabrication of future DNA biosensors.

Methylene blue covalently attached to single stranded DNA as electroactive label for potential bioassays

Methylene blue is an electroactive molecule that has been employed for the detection of the DNA hybridization event in electrochemical sensors. However, its use as a covalent label is very scarce and in most of the cases, non-covalent interactions (hydrophobic, electrostatic) are employed. Although it has advantages as simplicity and fewer number of procedure steps, the covalent attachment is less exploited in the development of these sensors. In this article, the electrochemical behavior of methylene blue attached to different DNA-strands is studied. Several lengths (15- and 30-mer) and different degree of DNA modification (MB-DNA, MB-DNA-MB and MB-DNA-SH) have been studied. The highest signals were obtained for longer strands with two MB molecules. In all the cases the signal is enhanced by CNT-nanostructuration of the electrode. Adsorption on these modified screen-printed electrodes allowed the amplification by employing an accumulation time. In this way, a sensitivity of −0.2864μAμM−1 and a limit of detection of 800nM for a 120s accumulation time were obtained.

Preparation of Ag/NaA zeolite modified carbon paste electrode as a DNA biosensor

In this research, we reported a method for synthesis of NaA zeolite. NaA zeolite has been successfully prepared within 30min by microwave heating without using any conventional hydrothermal treatment and due to the exchange process the silver-loaded NaA microsized zeolites was produced. The synthesized zeolite was characterized using X-ray diffraction, scanning electronic microscopy and FT-IR techniques. The modified carbon paste electrode was prepared by incorporation of Ag(I) zeolite in the carbon paste matrix. Differential pulse voltammetry (DPV) was employed for development of electrochemical DNA hybridization biosensors based on Ag/NaA zeolite modified carbon paste electrode (Ag/ZMCPE) and methylene blue (MB) as electroactive label was used. The sensors rely on immobilization of a 15-mer single stranded oligonucleotide probe for detection of target DNA, as a model. The hybridization event was evaluated by DPV. Moreover, Ag/ZMCPE showed better advantages than NaA zeolite modified carbon paste electrode (ZMCPE). Under optimized experimental conditions, limit of detection was calculated 4.00×10−12M.