Electroactive Label Research Articles

Ultrasensitive electrochemical biosensor for detection of microRNA-155 as a breast cancer risk factor

Due to the stability of microRNAs (miRNAs) in serum and other body fluids, they are known as promising cancer biomarkers. Recent studies have indicated higher expression of miRNA-155 (miR-155) in patients with breast cancer compared to healthy people. In the present report, a rapid and sensitive electrochemical biosensor has been developed for detection of miR-155 as a breast cancer risk factor. At first, a thiolated probe was immobilized on the gold electrode surface. Then, the target (miR-155) was exposed to the probe. In the next step, the positively charged polyethyleneimine-silver nanoparticles as electroactive labels were absorbed onto the negatively charged probe-target hybrid. In the third step, the anodic peak current which was produced due to the oxidation of silver nanoparticles was recorded as the electrochemical signal. The designed biosensor provided an ultrasensitive method for the detection of miR-155 with the detection limit of 20 zmol and a wide linear range from 2 × 10−20 to 2 × 10−12 mol. Moreover, the biosensor was able to detect miR-155 in real serum samples with satisfactory results.

A novel G-quadruplex DNA-based biosensor for sensitive electrochemical determination of thallium(I) ions

We used electroactive label crystal violet (CVi) as a G-quadruplex (G4) DNA intercalator for sensitive electrochemical sensing of Tl+. Differential pulse voltammetry (DPV) and cyclic voltammetry (CV) techniques were applied for electrochemical determination of Tl+ at the surface of carbon paste electrode as working electrode. Under optimized conditions, the difference in the DPV reduction peak currents of CVi before and after its intercalation into the formed G4 (ΔI) used an analytical signal for determination of Tl+ was linearly related to the concentration of Tl+ over the ranges of 0.4–50.0 nM and 0.1–10 μM. Limit of detection was calculated 0.341 nM (S/N = 3). The biosensor showed good selectivity and satisfactory results in real sample analysis of Tl+.

Immunomagnetic bead-based bioassay for the voltammetric analysis of the breast cancer biomarker HER2-ECD and tumour cells using quantum dots as detection labels.

An electrochemical magnetic immunosensing strategy was developed for the determination of HER2-ECD, a breast cancer biomarker, and breast cancer cells in human serum. A sandwich assay was performed on carboxylic acid-functionalized magnetic beads (MBs) using a screen-printed carbon electrode (SPCE) as transducer surface. The affinity process was detected using electroactive labels; core/shell streptavidin-modified CdSe@ZnS Quantum Dots (QDs). Cd2+ ions, released from the QDs, were determined by differential pulse anodic stripping voltammetry (DPASV). An assay time of 90min, with an actual hands-on time of about 20min, a linear range between 0.50-50ng·mL-1 of HER2-ECD and a limit of detection of 0.29ng·mL-1 were achieved. Analysis of live breast cancer cells was also performed using the optimized assay. Breast cancer cell lines SK-BR-3 (a HER2-positive cell line), MDA-MB-231 (a HER2-negative cell line) and MCF-7 (a cell line with low HER2 expression) were tested. The selectivity of the assay towards SK-BR-3 cells was confirmed. A concentration-dependent signal that was 12.5× higher than the signal obtained for the HER2-negative cells (MDA-MB-231) and a limit of detection of 2 cells·mL-1 was obtained. Graphical abstractSchematic representation of the electrochemical immunomagnetic assay for the determination of the breast cancer biomarker HER2-ECD and cancer cells using magnetic beads (MBs), a screen-printed carbon electrode (SPCE) as transducer surface and quantum dots (QD) as electroactive labels.

AuNPs/methylene blue dual-signal nanoimmunoconjugates and electrode activation for electrochemical biosensors

The use of nanoimmunoconjugates displaying multiple electroactive labels for enhanced analyte quantification is scarce in the literature. In this work, new nanoimmunoconjugates, based on gold nanoparticles (AuNPs) and either methylene blue (MB) or an NHS ester MB derivative (MBNHS), have been developed by two chemical paths. They have then been used as dual-signal nanolabels in electrochemical biosensors by means of the MB oxidation and AuNPs reduction peaks. The optimization on bare screen-printed carbon electrodes (SPCE) revealed that the use of Fe(CN)63−/4- in the measurement solution enhanced the currents displayed by the MB tag. Additionally, electrochemical SPCE activation in 70% ethanol improved significantly the electrode electrochemical properties compared with the untreated ones. Using 1 mM Fe(CN)63−/4-, we found that this activation increased 6–10 times the peaks registered by CV and the electrode active area, and reduced significantly the charge-transfer resistance (Rct) obtained from the impedance curve (from 11.4 to 0.3 kΩ). This SPCE activation process allowed detecting the nanoimmunoconjugate in a model immunosensor using the MB tags nearly as sensitively as when measuring the AuNPs counterpart tags (limits of detection of 0.77 and 0.55 nM, respectively). However, while MB detection was faster and simpler, Au detection was less prone to sample matrix interference. This provided sensor efficient performance in a wider range of experimental conditions than when measuring each label independently.

Electroactive NPs and D-amino acids oxidase engineered electrochemical chiral sensor for D-alanine detection

A simple and accurate magnetic electrochemical sensor is developed for the ultrasensitive chiral recognition of D-amino acids (D-AAs). Fe3O4@Au@Ag@CuxO nanoparticles (NPs) (x = 1, 2) are controllable prepared and used as the electrochemical beacons. The controllable integration of plasmonic metal NPs, semiconductors and magnetic NPs enables multilayered Fe3O4@Au@Ag@CuxO NPs to display a sharp copper stripping peak at -0.16 V, owning to the electron transfer from Cu+ to Cu2+ and the synergistic electron transfer of Au and Ag double layers. D-AAs can be catalyzed by D-amino acid oxidase (DAAO) to produce H2O2. The autocatalytic oxidation reaction of CuxO shell with the existence of H2O2 induces the decrease of electrochemical signals of Fe3O4@Au@Ag@CuxO NPs. Fe3O4@Au@Ag@CuxO NPs served as electrochemical beacons achieve the sensitive and accurate enantioselective recognition of D-alanine (D-Ala) in the range from 100 pM to 10 μM. The limit of detection (LOD) is as low as 52 pM. This developed protocol can be extended to the fabrication of a large set of electroactive labels by the introduction of plasmonic metal NPs with high electron transfer efficiency, for the reliable enantioselective recognition with high sensitivity.

Quantum dots as nanolabels for breast cancer biomarker HER2-ECD analysis in human serum

Early detection of cancer increases the possibility for an adequate and successful treatment of the disease. Therefore, in this work, a disposable electrochemical immunosensor for the front-line detection of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2-ECD), a breast cancer biomarker, in a simple and efficient manner is presented. Bare screen-printed carbon electrodes were selected as the transducer onto which a sandwich immunoassay was developed. The affinity process was detected through the use of an electroactive label, core/shell CdSe@ZnS Quantum Dots, by differential pulse anodic stripping voltammetry in a total time assay of 2 h, with an actual hands-on time of less than 30 min. The proposed immunosensor responded linearly to HER2-ECD concentration within a wide range (10–150 ng/mL), showing acceptable precision and a limit of detection (2.1 ng/mL, corresponding to a detected amount (sample volume = 40 μL) of 1.18 fmol) which is about 7 times lower than the established cut-off value (15 ng/mL). The usefulness of the developed methodology was tested through the analysis of spiked human serum samples. The reliability of the presented biosensor for the selective screening of HER2-ECD was confirmed by analysing another breast cancer biomarker (CA15-3) and several human serum proteins.

Ultrasensitive Electrochemical Immunoassay Based on Cargo Release from Nanosized PbS Colloidosomes.

Colloidosome is a novel nanostructure composed of millions of colloid particles. In this work, nanosized PbS colloidosomes were initially prepared and applied as nanoprobes for an ultrasensitive immunoassay. The colloidosomes were simply prepared in mild conditions by assembling the elementary approximately 8 nm PbS nanoparticles at the water-in-oil interface of emulsion droplets. To enhance the rigidity and biocompatibility of the colloidosomes, interfacial polymer was introduced by utilizing self-polymerization of dopamine. By treating with dilute nitric acid, a bursting release of lead ions from the colloidosomes occurred and the lead ions can be detected easily by anodic stripping voltammetry. In this way, a colloidosome-based electrochemical immunoassay was developed by using the nanosized PbS colloidosomes as electroactive labels. The proposed method featured a linear calibration range from 10 fg·mL-1 to 100 ng·mL-1 with a low detection limit of 3.4 fg·mL-1 for the detection of human epididymis protein 4. This work introduced a new member for the family of colloidosomes and offered a novel perspective for the rational implementation of various colloidosomes for novel low-abundance cancer biomarkers analysis.

Ag/CdO NP-Engineered Magnetic Electrochemical Aptasensor for Prostatic Specific Antigen Detection.

A simple magnetic electrochemical aptasensor was established for the detection of prostatic specific antigen (PSA). Ag/CdO nanoparticles (NPs) were fabricated and exhibited strong electroreduction peaks at -1.07 V, attributing to the electron transfer from Cd2+ to Cd0 and the superior electron transportation of Ag. Aptamer-modified Ag/CdO NPs were assembled on the surface of superparamagnetic Fe3O4/graphene oxide nanosheets (GO/Fe3O4 NSs) through the hydrophobic and π-π stacking interaction of aptamers and GO NSs. These assemblies possessed superior electroactive properties, efficient electron transfer, and superparamagnetic response and could serve as sensing units for PSA detection with the aid of a magnetic electrode. With increasing concentrations of PSA, the high affinity of aptamers to PSA enabled the dissociation of Ag/CdO NPs from GO/Fe3O4 NSs, decreasing the intensity of electroreduction peaks. The Ag/CdO NP-engineered magnetic electrochemical aptasensor achieved sensitive and accurate detection of PSA in the range of 50 pg/mL to 50 ng/mL. The limit of detection (LOD) was as low as 28 pg/mL. This developed protocol can be extended to a large set of strong electroactive labels for reliable tumor biomarker detection with high sensitivity and specificity.

A voltammetric assay for microRNA-25 based on the use of amino-functionalized graphene quantum dots and ss- and ds-DNAs as gene probes

The authors describe a DNA based voltammetric assay for the cancer biomarker microRNA-25. A glassy carbon electrode (GCE) was modified with amino-functionalized graphene quantum dots and used as an amplifier of electrochemical signals. p-Biphenol is introduced as a new electroactive probe with a fairly low working potential of 0.3V (vs. Ag/AgCl). The stages of fabricating the electrode were characterized by cyclic voltammetry and electrochemical impedance spectroscopy. ss-Probe DNA was immobilized on the modified GCE and then exposed to a sample containing microRNA-25. The results indicated that the electrode can distinguish complementary microRNA-25 from a single-base mismatch. The increase in the electrochemical response of PBP and the positive shift in the potential peak indicate that PBP is intercalated between two strands. Under optimized experimental conditions, the current of the electrode increases linearly with the logarithm of the microRNA-25 concentration in the range from 0.3nM to 1.0μM, and the detection limit is 95.0 pM. The assay was successfully employed to the determination of microRNA-25 in spiked human plasma. Graphical abstract A novel electrochemical nanogenosensor is introduced for simple and sensitive determination of microRNA-25, as a biomarker, based on amino-functionalized graphene quantum dots (as a surface modifier) and p-biphenol (as an electroactive label).

Tunable electrochemistry of gold-silver alloy nanoshells

The widespread and increasing interest in enhancing biosensing technologies by increasing their sensitivities and lowering their costs has led to the exploration and application of complex nanomaterials as signal transducers and enhancers. In this work, the electrochemical properties of monodispersed AuAg alloy nanoshells (NSs) with finely tunable morphology, composition, and size are studied to assess their potential as electroactive labels. The controlled corrosion of their silver content, caused by the oxidizing character of dissolved oxygen and chlorides of the electrolyte, allows the generation of a reproducible electrochemical signal that is easily measurable through voltammetric techniques. Remarkably, the underpotential deposition of dissolved Ag+ catalyzed on AuAg NS surfaces is observed and its dependence on the nanoparticle morphology, size, and elemental composition is studied, revealing a strong correlation between the relative amounts of the two metals. The highest catalytic activity is found at Au/Ag ratios higher than ≈ 10, showing how the synergy between both metals is necessary to trigger the enhancement of Ag+ reduction. The ability of AuAg NSs to generate an electrocatalytic current without the need for any strong acid makes them an extremely promising material for biosensing applications.

DNA-fueled target recycling-induced two-leg DNA walker for amplified electrochemical detection of nucleic acid

Taking advantage of the homogeneous and heterogeneous electrochemical biosensors, a simple, sensitive, and selective electrochemical biosensor is constructed by combining entropy-driven amplification (EDA) with DNA walker. This electrochemical biosensor realizes the biorecognition and EDA operation in homogeneous solution, which is beneficial to improve the recognition and amplification efficiency. A two-leg DNA walker generated by EDA can walk on the surface of gold electrode for cleaving the immobilized substrate DNA and releasing the electroactive labels, giving rise to a significant decrease of the electrochemical signal. The immobilization of the electroactive labels ensures the reproducibility and reliability of the biosensor. The present cascade amplification assay can be applied to detect target DNA with a detection limit of 0.29 fM, and base mutations can be easily distinguished. Moreover, the proposed electrochemical biosensor shows a satisfactory performance for the detection of target DNA in human serum. Thus, the novel electrochemical biosensor holds promising potential for a future application in disease diagnosis.

Electrochemical primer extension based on polyoxometalate electroactive labels for multiplexed detection of single nucleotide polymorphisms

Polyoxymetalates (POMs) ([SiW11O39{Sn(CH2)2CO)}]4- and [P2W17O61{Sn(CH2)2CO)}]6-) were used to modify dideoxynucleotides (ddNTPs) through amide bond formation, and applied to the multiplexed detection of single nucleotide polymorphisms (SNPs) in an electrochemical primer extension reaction. Each gold electrode of an array was functionalised with a short single stranded thiolated DNA probe, specifically designed to extend with the POM-ddNTP at the SNP site to be interrogated. The system was applied to the simultaneous detection of 4 SNPs within a single stranded 103-mer model target generated using asymmetric PCR, highlighting the potential of POM-ddNTPs for targeted, multiplexed SNP detection. The four DNA bases were successfully labelled with both ([SiW11O39{Sn(CH2)2CO)}]4- and [P2W17O61{Sn(CH2)2CO)}]6-), and [SiW11O39{Sn(CH2)2CO)}]4- demonstrated to be the more suitable due to its single oxidation peak, which provides an unequivocal signal. The POM-ddNTP enzymatically incorporated to the DNA anchored to the surface was visualised by AFM using gold coated mica. The developed assay has been demonstrated to be highly reproducible, simple to carry out and with very low non-specific background signals. Future work will focus on applying the developed platform to the detection of SNPs associated with rifampicin resistance in real samples from patients suffering from tuberculosis.

Sensitive electrochemical DNA-based biosensors for the determination of Ag+ and Hg2+ ions and their application in analysis of amalgam filling

In the present paper, we used single-stranded poly-T (100% thymine bases) and poly-C (100% cytosine bases) nucleic acids as DNA probes for selective and sensitive individual electrochemical determination of Hg2+ and Ag+, respectively, on the multi-walled carbon nanotube paste electrodes (MWCNTPEs) using [Fe(CN)6]3−/4− as electroactive labels. In the presence of Hg2+ and Ag+, the probe–Hg2+/Ag+ interactions through T–Hg2+–T and C–Ag+–C complexes formation could cause the formation of a unimolecular hybridized probe. This structure of probe led to its partial depletion from electrode surface and facilitation of electron transfer between [Fe(CN)6]3−/4− redox couple and electrode surface, resulting in the enhanced differential pulse voltammetry (DPV) oxidation current of [Fe(CN)6]3−/4− at the probe-modified electrode surface. We applied the difference in the oxidation peak currents of [Fe(CN)6]3−/4− before and after Hg2+/Ag+–DNA probe bonding (∆I) for electrochemical determination of these heavy metal ions. Detection limits were 8.0 × 10−12 M and 1.0 × 10−11 M for Hg2+ and Ag+ ions determination, respectively. The biosensors were utilized to determine the weight percent of toxic metals, i.e., silver and mercury in dental amalgam filling composition. The results of their practical applicability in analysis of the amalgam sample were satisfactory.

Preparation and application of AgNPs/SWCNTs nanohybrid as an electroactive label for sensitive detection of miRNA related to lung cancer

An electrochemical nanogenosensor based on silver nanoparticles/single-walled carbon nanotubes nanohybrid (AgNPs/SWCNTs nanohybrid), as a label, was developed for the analysis of miRNA (miR-25) related to lung cancer. The preparation steps of the proposed nanogenosensor were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The two components of the AgNPs/SWCNTs nanohybrid have different roles in the nanogenosensor performance. SWCNTs are responsible for interaction with the probe, while AgNPs produce electroanalytical signals. It was shown that the π-π interaction between the AgNPs/SWCNTs nanohybrid and the nitrogenous bases related to double-stranded nucleic acid is weaker than that for single-stranded nucleic acid. The different abilities of AgNPs/SWCNTs nanohybrid to bind with the probe hybridized with complementary (miRNA related to lung cancer), non-complementary and one-base mismatch targets led to identification of the nature and the concentration of miRNA through a simple and fast approach. The developed nanogenosensor was used to determine miR-25 in two concentration ranges of 1.0 × 10−12 to 1.0 × 10−10 M and 1.0 × 10−10 to 1.0 × 10−8 M with a limit of detection (LOD) of 3.13 × 10−13 M without PCR amplification. Furthermore, the nanogenosensor was employed to detect miR-25 in human plasma. This electrochemical procedure can be valuable in genetic analysis and early clinical diagnosis.

Ultrasensitive DNAzyme based amperometric determination of uranyl ion using mesoporous silica nanoparticles loaded with Methylene Blue

The authors describe an ultrasensitive amperometric enzymatic assay for uranyl ion. It is based on the use of mesoporous silica nanoparticles (mesoSiNPs) loaded with Methylene Blue (MB) and functionalized with an UO2(II)-dependent DNAzyme. The electroactive label MB was sealed in the inner pores of the mesoSiNPs along with double stranded DNA (containing the DNAzyme and the substrate strand). In the presence of UO2(II), the DNAzyme is actived to cleave the substrate strands. This leads to the cleavage of the caps and the release of MB from the mesoSiNPs. The amount of released MB depends on the concentration of UO2(II) and can be determined amperometrically, best at a working voltage of −0.25 V (vs SCE), by using a chitosan coated carbon paste electrode. Response is linear in the 20 pM to 0.1 nM UO2(II) concentration range, and the detection limit is as low as 0.15 pM. Recoveries from spiked samples varied from 91.3 to 99.4%. The assay is highly specific, selective, and not interfered by other metal ions.

Amplified detection of hepatitis B virus using an electrochemical DNA biosensor on a nanoporous gold platform

In the present study, a nanoporous gold platform was applied for the amplified detection of Hepatitis B virus (HBV) by an electrochemical DNA biosensor. Ferrocene as a redox reporter was covalently attached to the DNA probe and its electrochemical signal was recorded as the biosensor response. For real samples, DNA was firstly extracted from blood of patients and then amplified by polymerase chain reaction (PCR) for 5cycles. Sensitivity of this biosensor was enhanced by using nanoporous gold electrode, therefore this sensor can discriminate the genome of HBV in real sample with low PCR cycles. By this strategy and signal amplification using nanoporous platform and covalently attached electroactive label, the biosensor can distinguish between healthy and HBV patients with limited PCR cycles. Moreover, the errors of PCR with large cycles can be disregarded. A linear dynamic range of 0.4 to 10nmol of mutant DNA was achieved, with reliable reproducibility (RSD) 8.9%.

Novel Approaches in DNA Methylation Studies – MS-HRM Analysis and Electrochemistry

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Highly Selective and Sensitive Electrochemical Immunoassay of Cry1C Using Nanobody and π-π Stacked Graphene Oxide/Thionine Assembly.

Cry1C is one of the emerging toxin proteins produced by the Bacillus thuringiensis in the genetically modified crops for pest control in agriculture; thus, it is vital to measure the Cry1C level in crops for the healthy and environmental concerns. Current detections of Cry1C mainly rely on instrumental analysis such as high-performance liquid chromatography, which are time-consuming and are generally cost-prohibitive. Herein, a simple nanobodies (Nbs)-based electrochemical immunosensor has been first proposed for highly selective and sensitive detection of Cry1C. The Nbs pair, i.e., Nb51 and Nb54, which bind to different epitopes on Cry1C, was screened out from an immunized Bactrian camel, with an extra benefit of higher stability compared with conventional antibodies. Further, by using a π-π stacked graphene oxide/thionine assembly that had fast electron transfer kinetics as an electroactive label, the immunoreaction that occurred between the two Nbs and Cry1C can be highly sensitively quantified by square wave voltammetry. The linear detection range was from 0.01 to 100 ng·mL-1, and the low detection limit was 3.2 pg·mL-1. This method was further successfully applied for sensing Cry 1C in spiked samples with recoveries ranging from 100.17% to 106.69% and relative standard deviation less than 4.62%. This proposed assay would provide a simple highly sensitive and selective approach for the Cry1C toxin detection and be applicable to be extended to other toxin proteins sensing in foods.

Protein p53 Binding to Cisplatin‐modified DNA Targets Evaluated by Modification‐specific Electrochemical Immunoprecipitation Assay

AbstractStudies of protein interactions with chemically modified nucleic acids are of importance in various areas of biomolecular and biomedical research, including investigations of the binding of proteins important in medicine with DNA modified with drugs and diagnostic applications of modified DNAs in biosensing and bioanalysis. Chemical modification of DNA substrates with various species inside or outside specific protein binding sites can affect the protein‐DNA recognition. In this paper we present a simple electrochemical immunoprecipitation technique designed for evaluation of the effects of antitumor drug cisplatin on the p53‐DNA binding. The cisplatin‐DNA adducts are utilized as electroactive labels allowing a facile determination of the p53‐bound modified DNA. Effects observed using this technique accord with results of previous biochemical assays. This approach is potentially applicable in studies that deal with the influence of any electroactive DNA modifications on the protein‐DNA binding.

Development of electrochemical genosensor for MYCN oncogene detection using rhodamine B as electroactive label

A novel electrochemical genosensor based on a graphite electrode modified with poly(4-aminophenol) has been constructed for prognostic of neuroblastoma, a malignant tumor originating from embryonic precursor cells of the sympathetic nervous system and associated with the amplification of the MYCN oncogene. The genosensor exhibited distinct electrical and morphological properties using rhodamine B as indicator of DNA hybridization. The detection limit was evaluated to be 0.47 μmol L−1 (n = 3), and the electrochemical genosensor was selective for the complementary DNA, using serum sample. This DNA sensing platform was successfully applied to detect MYCN, an important biomarker for neuroblastoma.