Label-free Electrochemical Detection Research Articles

In situ DNA immobilization on stainless steel microelectrodes in a microchannel

DNA immobilization at specific locations on the inner wall of microchannels could lead to many interesting applications. In this paper, three procedures (electrostatic adsorption, thiol—Au covalent bonding, and electrochemical entrapment) were carried out for the immobilization of DNA probes onto stainless steel microdot electrodes inside a polydimethylsiloxane (PDMS) microchannel. The microdot electrodes were fabricated via crossed microwire moulding, a modified soft lithographic technique, to be naked on the inner wall of the PDMS microchannel. The immobilization of DNA probes onto the surface of the microdot electrodes was performed by microfluidics. It was found that, for DNA electrostatic adsorption, different electrode behaviours were exhibited inside and outside the microchannel; in the microchannel, the negatively charged DNA probes would undergo abnormal electrostatic adsorption onto the cathode. Furthermore, DNA immobilization through thiol—Au covalent bonding and DNA entrapment in enhanced polypyrrole (PPy)-modified dots inside microchannels was demonstrated. Further work is needed, but present results are promising and suggest a route by which microfluidics may be married with label-free electrochemical detection and with very low-cost DNA diagnostics, which could prove invaluable.

E-DNA sensors for convenient, label-free electrochemical detection of hybridization

We review the development of reagentless, electrochemical sensors for the sequence-specific detection of nucleic acids that are based on the target-induced folding or unfolding of electrode-bound oligonucleotides. These devices, which are sometimes termed E-DNA sensors, are comprised of an oligonucleotide probe modified on one terminus with a redox reporter and attached to an electrode at the other. Hybridization of this probe DNA to a target oligonucleotide influences the rate at which the redox reporter collides with the electrode, leading to a detectable change in redox current. Because all sensing elements of this method are strongly linked to the interrogating electrode, E-DNA sensors are label-free, operationally convenient and readily reusable. As E-DNA signaling is predicated on a binding-specific change in the dynamics of the probe DNA (rather than simply monitoring the adsorption of a target to the sensor surface) and because electroactive contaminants (interferents) are relatively rare, this class of sensors is notably resistant to false positives arising from the non-specific adsorption of interferents, and performs well even when challenged directly with blood serum, soil and other complex sample matrices. We review the history of and recent advances in this promising DNA and RNA detection approach.

Label-free electrochemical detection of nanomolar adenosine based on target-induced aptamer displacement

In this work, a label-free electrochemical sensor based on target-induced displacement is reported with adenosine as the model analyte. The sensing substrate is prepared using a gold electrode modified with a self-assembled monolayer of 1,6-hexanedithiol that mediates the assembly of a gold nanoparticle film, which can increase the surface loading of capture probe and enhance the signal. An aptamer for adenosine is applied to hybridizing with the capture probe, yielding a double-stranded complex of the aptamer and the capture probe on the surface. The interaction of adenosine with the aptamer displaces the aptamer sequence and causes it to dissociate from the interface. This results in a decrease in the amount of aptamer/capture probe duplex form, and, accordingly, the desorption of methylene blue, an electroactive indicator bound to the duplex, from the electrode. Then, the redox current of the indicator can reflect the concentration of the analyte. The fabricated sensor is shown to exhibit high sensitivity, desirable selectivity and a three-decade wide linear range.

An Overview of Label-free Electrochemical Protein Sensors.

Electrochemical-based protein sensors offer sensitivity, selectivity and reliabilityat a low cost, making them very attractive tools for protein detection. Although the sensorsuse a broad range of different chemistries, they all depend on the solid electrode surface,interactions with the target protein and the molecular recognition layer. Traditionally, redoxenzymes have provided the molecular recognition elements from which target proteins haveinteracted with. This necessitates that the redox-active enzymes couple with electrodesurfaces and usually requires the participation of added diffusional components, or assemblyof the enzymes in functional chemical matrices. These complications, among many others,have seen a trend towards non-enzymatic-based electrochemical protein sensors. Severalelectrochemical detection approaches have been exploited. Basically, these have fallen intotwo categories: labeled and label-free detection systems. The former rely on a redox-activesignal from a reporter molecule or a label, which changes upon the interaction of the targetprotein. In this review, we discuss the label-free electrochemical detection of proteins,paying particular emphasis to those that exploit intrinsic redox-active amino acids.

Label-Free DNA Detection of Hepatitis C Virus Based on Modified Conducting Polypyrrole Films at Microelectrodes and Atomic Force Microscopy Tip-Integrated Electrodes

We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82x10(-21) mol L(-1). Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.

Label-free electrochemical detection of DNA using ferrocene-containing cationic polythiophene and PNA probes on nanogold modified electrodes

A label-free electrochemical method for the detection of DNA–PNA hybridization using a water-soluble, ferrocene-functionalized polythiophene transducer and single-stranded PNA probes on the nanogold modified electrode is investigated. Nanogold modified electrodes can largely increase the immobilization amount of ss-PNA capture probe and lead to an increase of the electrical signal. The ferrocene-containing cationic polythiophene do not interact electrostatically with the PNA probes due to the absence of the anionic phosphate groups on the PNA probes. But after DNA–PNA hybridization, cationic polythiophene is adsorbed on the DNA backbone, giving a clear hybridization detection signal in differential pulse voltammetry (DPV). Very good discrimination against non-complementary DNA and four-base mismatch DNA is observed. These studies show that the proposed method can provide an alternative for expanding the range of detection methods available for DNA hybridization.

Label-Free Electrochemical Detection of DNA in Blood Serum via Target-Induced Resolution of an Electrode-Bound DNA Pseudoknot

We have demonstrated a fully covalent, signal-on E-DNA architecture based on the target-induced resolution of a DNA pseudokont. In the absence of target, the electrode-bound DNA probe adopts a pseudoknot conformation that segregates an attached methylene blue (MB) from the electrode. Upon target binding, the pseudoknot is resolved, leading to the formation of a single-stranded DNA element that supports electron transfer from the methylene blue to the electrode.

Electrochemical probe DNA design in PCR amplicon sequence for the optimum detection of microbiological diseases

Direct electrochemical genosensor was developed for the detection of a probe sequence relative position in a PCR amplicon for the optimum detection of bacterial and microbiological diseases, in this study. The genosensor relies on a label-free electrochemical detection. The amino-linked inosine modified (guanine-free) coequal capture probes which were chosen from different parts of a PCR amplicon, immobilized on to disposable pencil graphite electrodes (PGE) by electrostatically and covalently. As a model case Hepatitis B virus (HBV) genome amplicon was used for the detection and specification. Hybridization was occurred after surface coverage with denatured amplicons. After hybridization, optimum probe sequence position was identified by using the differences between the responses of guanine oxidation signals. The results of this study might have a great convenience for the microbiological diseases detection applications such as DNA micro arrays.

Label-free electrochemical detection of the phosphorylated and non-phosphorylated forms of peptides based on tyrosine oxidation

Identification of protein phosphorylation is an important goal in proteomics, because of the central role played by phosphorylation in the regulation of cellular activities. An exciting consequence of tyrosine (Tyr) oxidation is that it allows a clear distinction between the phosphorylated and non-phosphorylated forms of peptides using electrochemical analysis. In this report, we monitored the effect of phosphorylation on the electro-oxidation of Tyr in connection with differential pulse voltammetry (DPV) using a screen-printed carbon electrode (SPCE). First, we monitored the electrochemical current responses of Tyr and o-phospho- l-Tyrosine ( l-3-(4-hydroxyphenyl)alanine 4′-phosphate, Tyr-P). The detection limit for Tyr was determined as 10 nM on the SPCE surface ( S/ N = 3). We observed that the phosphorylation caused a significant suppression on the electro-oxidation of Tyr. We also monitored the electrochemical responses of Src peptide 521–533 (H–Thr-Ser-Thr-Glu-Pro-Gln-Tyr-Gln-Pro-Gly-Glu-Asn-Leu–OH), both in the non-phosphorylated and phosphorylated forms. The detection limit for Src peptide was determined as 100 nM ( S/ N = 3). By monitoring the current signals obtained from the Tyr kinase substrate peptides, we suggest that label-free electrochemical in vitro detection of Tyr phosphorylation can be performed in a rapid and cost-effective format.

Label-free bioelectronic immunoassay for the detection of human telomerase reverse transcriptase in urine

The aim of our study was to prospectively evaluate the potential diagnostic value and clinical applicability of label-free electrochemical detection of human telomerase reverse transcriptase (hTERT). The expression of human telomerase reverse transcriptase (hTERT) was analyzed by adsorptive transfer differential pulse stripping voltammetry (DPSV) from the cell lysates prepared from the urine samples of consenting adults. The principle of the electrochemical determination relies on the intrinsic electro-activity of the proteins. Tyrosine (Tyr), Tryptophan (Trp) and Cysteine (Cys) residues in proteins are oxidizable at carbon electrodes. We employ the direct oxidation of these electro-active amino acids in a label-free voltammetric immunoassay. Monitoring the changes in the electrochemical signals of antibodies (Abs), before and after the binding of the antigens (Ags), provides the basis for our label-free immunoassay. We have also performed reverse-transcription-polymerase chain reaction (RT-PCR) as the conventional method for the confirmation of the electrochemical data. Positive control tests were performed with DPSV and RT-PCR using the exfoliated T24 cell lines. The electrochemical oxidation peak current of the hTERT-mAb was detected at ∼0.60 V (vs. Ag/AgCl). The binding of hTERT expressed by T24 cells caused a decrease in the peak signal of hTERT-mAb. There was a linear relationship between the number of cells in the lysates, and the drop in the current peak. The label-free electrochemical method offers a rapid, simple and reproducible non-invasive assay for hTERT expression in the cell lysates. This assay has a promising potential to provide a useful and new tool for future investigations, facilitating the analysis of hTERT expression in the cell lysates of urine samples for cancer diagnosis and follow-up.

Label-Free Electrochemical Immunoassay for the Detection of Human Chorionic Gonadotropin Hormone

Here we report on a new and rapid immunoassay for the label-free voltammetric detection of human chorionic gonadotropin hormone (hCG) in urine. Monitoring the changes in the current signals of antibodies (Abs) before and after the binding of the antigen (Ag) provides the basis for an immunoassay that is simple, rapid, and cost-effective. Since hCG is found at highly elevated levels in pregnant female urine with the range of 30,000-200,000 mIU/mL (approximately 30-200 nM) by 8-10 weeks into pregnancy, its label-free electrochemical detection was achieved by using our method. The coverage of the electrode surface with the Ab and the incubation time with the target Ag were optimized for the detection of hCG. The limit of detection of our method was calculated to be 15 pM (n = 3, approximately 15 mIU/mL) in synthetic hCG samples and 20 pM (n = 3, approximately 20 mIU/mL) in human urine. The electrochemical results for the detection of hCG in the urine samples were in agreement with the results obtained using a reference system, enzyme-linked immunosorbent assay. Further research about the intrinsic electroactivity of Abs and their target molecules would surely provide new and sensitive screening assays, as well as extensive data regarding their interaction mechanisms.

Label-Free Electrochemical Detection of Protein Based on a Ferrocene-Bearing Cationic Polythiophene and Aptamer

Two label-free electrochemical methods for the detection of human alpha-thrombin using a water-soluble, ferrocene-functionalized polythiophene transducer and a single-stranded oligonucleotide aptamer probe are described. The first approach is a direct method in which the recorded current decreases upon addition of the targeted protein. The second one requires more steps and the additional utilization of PNA probes and nuclease enzyme. This indirect method leads to an increase of the electrical signal as a function of the concentration of human alpha-thrombin with a detection limit of 75 fmol.

Metallic oxide CdIn2O4 films for the label free electrochemical detection of DNA hybridization

DNA functionalised semiconductor metallic oxide electrodes have been developed for the direct electrochemical detection of DNA hybridization, without labelling or the introduction of a redox couple. Conductive CdIn(2)O(4) thin films with controlled properties were deposited on glass substrates using an aerosol pyrolysis technique. The films exhibit a polycrystalline microstructure with a surface roughness of 1.5 nm (r.m.s.) and an electrical resistivity ranging between 1 and 3 x 10(-3) Omega cm. These electrodes were functionalised using hydroxylation and silanisation steps, to allow the binding of DNA probe sequences (20 bases). The electrical detection of DNA hybridization with complementary sequences has been performed using electrochemical impedance spectrometry (EIS) measuring the variation of impedance before and after hybridization. Two set-ups were used, a standard set-up including three electrodes and a set-up including two symmetrical electrodes. In both configurations, a significant increase of the impedance modulus, more particularly of the real part of the impedance (160-225% according to the electrochemical cell used) has been obtained over a frequency range of 10-10(5)Hz. DNA hybridization has also been systematically confirmed using the fluorescence spectrometry. This study emphasizes the high sensitivity of the CdIn(2)O(4) as a working electrode for the detection of biological events occurring at the electrode surface.

Label-Free DNA Detection Based on Modified Conducting Polypyrrole Films at Microelectrodes

A label-free electrochemical detection method for DNA hybridization based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes is reported. Synthetic single-stranded 27-mer oligonucleotides (probe) have been immobilized at 2,5-bis(2-thienyl)-N-(3-phosphorylpropyl)pyrrole film formed by electropolymerization on the previously formed polypyrrole layer. The 27- or 18-mer target oligonucleotides were monitored via the electrochemically driven anion exchange of the inner polypyrrole film. The performance of the miniaturized DNA biosensor system was studied in respect to selectivity, sensitivity, reproducibility, and regeneration of the sensor. Control experiments were performed with a noncomplementary target of 27-mer DNA and 12 base-pair mismatched 18-mer sequences, respectively, and did not show any unspecific binding. Under optimized experimental conditions, the label-free electrochemical biosensor enabled the detection limits of 0.16 and 3.5 fmol for the 18- and 27-mer DNA strand, respectively. Furthermore, we demonstrate reusability of the electrochemical DNA biosensor after successful recovery of up to 100% of the original signal by regenerating the DNA "label-free" electrode with 50 mM HCl at room temperature.

Poly(hydroquinone)-coated electrode for immobilizing of 5′-amine functioned capture probe DNA and electrochemical response to DNA hybridization

Enzymatically polymerized hydroquinone, PHQ, was applied for polymer-coated electrode whose surface was further modified with 5′-amine dodecamer DNAs (capture probe DNA, CP-DNA) by taking advantage of Michael reaction. The film-forming property of PHQ on graphite substrate surfaces was confirmed to be satisfactory by AFM imaging. Cyclic voltammetric (CV) measurements showed that the PHQ-modified graphite electrode gave well-defined redox waves showing characteristics for surface process. The pH dependence of the formal potential, E1/2, suggested that the electrode reaction occurred by two-proton and two-electron mechanism (- 59 mV per a pH decade). CVs also gave the specific amount of PHQ adsorption of 0.52 or 0.83 nmol cm−2 (monomer unit) for the different electrode preparations. This was indicative of two- or three-monolayer adsorption of PHQ. For applications of gene detection, the immobilization reaction including the CP–DNA hybridization was studied by microgravimetric analysis using a quartz-crystal microbalance (QCM). Summaries for the two different runs were 1.01 and 0.69 nmol cm−2 (monomer unit) for PHQ adsorption on gold surfaces, 0.19 and 0.15 nmol cm−2 for CP-DNA attachment on the PHQ/Au, and 0.14 and 0.11 nmol cm−2 for hybridization with the complementary DNA on the CP-DNA/PHQ/Au, respectively. Importantly, monitoring of the series of the experiments were possible by measuring the voltammetric properties of the electrode; the distinct redox waves due to PHQ-electrode reaction are suppressed upon immobilizing of the CP-DNA, and its hybridization further suppressed the redox activity. Neither treating of the PHQ-electrode with native DNAs nor treating of CP-DNA/PHQ-electrode with non-target DNA gave no noticeable responses. A possible mechanism for the electrode response was discussed briefly based on electrochemical QCM measurements. We think these observations are important as the basis of DNA hybridization sensor that enables totally, label-free electrochemical detection of the target DNA.

Electrical recognition of label-free oligonucleotides upon streptavidin-modified electrode surfaces

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in Fe(CN)6 3−/Fe(CN)6 4− solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.

Genomagnetic assay based on label-free electrochemical detection using magneto-composite electrodes

A very sensitive genomagnetic assay based on a label-free electrochemical detection of the DNA target by using graphite-epoxy composite (GEC) and magneto-GEC electrodes as electrochemical transducers has been developed. The assay is based on the capture of DNA target on streptavidin magnetic beads by its hybridization with biotinylated inosine-substituted probe and its electrochemical detection achieved by the measurement of the signal coming from the guanine oxidation of the DNA target. The genomagnetic assay involves the following steps: the immobilization of biotinylated inosine-substituted capture probe onto streptavidin coated magnetic beads following by hybridization with its target DNA or in the presence of non-complementary (NC) DNA. Two different genomagnetic strategies have been developed for the selective electrochemical detection of DNA target. Firstly, an alkaline treatment was performed in order to dissociate DNA hybrids from the magnetic beads, followed by the voltammetric measurement of guanine oxidation signal using GEC electrode as an electrochemical transducer. In order to simplify the genomagnetic procedure, in the second strategy the magnetic separation was directly achieved by using a novel magneto-electrochemical transducer based on GEC electrode containing a small magnet (m-GEC). The genomagnetic assay based on label-free electrochemical detection was developed for the specific detection of a sequence related with Salmonella spp. with a target concentration of 20 μg/mL in 20 min of hybridization time. The main features related with these genomagnetic assays, such as detection limit and the reproducibility, are discussed and compared with other genomagnetic assays.

Ferrocene‐Functionalized Cationic Polythiophene for the Label‐Free Electrochemical Detection of DNA

Ferrocene‐Functionalized Cationic Polythiophene for the Label‐Free Electrochemical Detection of DNA

DNA‐Directed Attachment of Carbon Nanotubes for Enhanced Label‐Free Electrochemical Detection of DNA Hybridization

AbstractMulti‐walled carbon nanotubes (MWNTs) were used as nanowires, which combined DNA molecules to a carbon paste electrode (CPE). The attachment of MWNT on the electrode surface was controlled by a hybridization assay between adenine and thymine containing oligonucleotides. The appearance of guanine oxidation signal after hybridization with target DNA greatly simplified the specific sequence DNA detection mechanism. Combination of sidewall‐ and end‐functionalization of MWNT provided a significant enhancement in the voltammetric signal of guanine oxidation in comparison with the signals obtained from only end‐oxidized MWNT modified CPE and a bare CPE. A control experiment involving adenine containing polynucleotide (poly(A)) instead of adenine probe modified MWNT was performed. The effect of target and noncomplementary DNA concentration on the guanine signal was also monitored. Discrimination against single‐base mismatch and noncomplementary DNA was achieved by surfactant containing washing solution. The promising conductivity of carbon nanotubes, and the creation of a larger surface area for DNA immobilization by sidewall‐ and end‐oxidation of MWNT provided a detection limit down to 10 pg/mL, which is compatible with the demand of the genetic tests.

Label-free electrochemical recognition of DNA hybridization by means of modulation of the feedback current in SECM.

Stranded on the surface: The modulation of the amperometric feedback current in scanning electrochemical microscopy (SECM) through coulomb interactions between the phosphate groups of immobilized oligonucleotides and a freely diffusing, negatively charged redox mediator is used for imaging DNA spots (see picture) and establishing a simple and efficient label-free electrochemical detection of complementary hybridization on DNA microarrays.