Highly sensitive electrochemical sensor for estradiol based on the signal amplification strategy of Cu-BDC frameworks

Using 1,4-Benzenedicarboxylic acid (H₂BDC) as the ligand, a kind of copper-based metal-organic frameworks (MOFs) were prepared and characterized using transmission electron microscopy, scanning electron microscopy, infrared spectroscopy, and X-ray diffraction. After that, the prepared Cu-BDC frameworks were used to modify the carbon paste electrode, constructing a novel electrochemical sensor for estradiol (E2). The prepared Cu-BDC frameworks are much more active for the oxidation of E2, and greatly increase the oxidation signals of E2. The results from chronocoulometry indicate that the Cu-BDC frameworks modified electrode exhibit much higher accumulation efficiency toward E2. Based on the signal amplification strategy of Cu-BDC frameworks, a sensitive and rapid electrochemical method was developed for the determination of E2. The linear range was from 5.00 to 650.0 nM, and the detection limit was as low as 3.80 nM. It was used in different water samples, and the values of recovery were over the range from 96.5 to 101%. The practical applications reveal that this new sensing system is accurate and convenient, and has great potential applications in the environmental monitoring.     

一次性信号放大电化学阻抗RNA传感器研究

以自制的丝网印刷碳电极(SPCE)为基体电极,利用DNA四面体纳米探针和酶催化信号放大构建了一个一次性电化学阻抗型RNA传感器.固定在AuNPs修饰的SPCE表面的DNA四面体结构能确保DNA探针具有可控的密度和方向,结合辣根过氧化物酶(HRP)催化H_2O_2氧化4-氯-1-萘酚(CN)的反应,生成不溶物沉积在电极表面,有效地放大电化学阻抗信号,实现了miRNA的高灵敏阻抗测定.检测限可以低至1.0 pmol/L,阻抗值和miRNA-141浓度的对数在3.0～1 000 pmol/L之间具有良好的定量关系.     

Cascaded DNA circuits-programmed self-assembly of spherical nucleic acids for high signal amplification

Signal amplification is an important issue in DNA nanotechnology and molecular diagnostics. In this work, we report a strategy for the catalytic self-assembly of spherical nucleic acids(SNAs) programmed by two-layer cascaded DNA circuits through integrating an entropy-driven catalytic network, a catalytic hairpin assembly circuit, and a facile SNA assembly-based reporter system. This integrated system could implement ～100,000-fold signal amplification in the presence of 1 p M of input target.Possessing powerful amplification ability of nucleic acid signal, our strategy should be of great potential in fabricating more robust dynamic networks to be applied for signal transduction, DNA computing, and nucleic acid-based diagnostics.     

Enzyme-catalyzed signal amplification for electrochemical DNA detection with a PNA-modified electrode

The signal amplification technique of peptide nucleic acid (PNA)-based electrochemical DNA sensor was developed in a label-free and one-step method utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. The negatively charged mediator, ferrocenedicarboxylic acid, cannot diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of the breast cancer susceptibility gene BRCA1 at a sample concentration lower than 10(-9) M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.     

An electrochemical microRNA biosensor based on protein p19 combining an acridone derivate as indicator and DNA concatamers for signal amplification

The new acridone derivative 5, 7-dinitro-2-sulfo-acridone (DSA) with excellent electrochemical activity was synthesized and reported for the first time in this paper. Then an electrochemical biosensor was fabricated for the signal amplified detection of microRNA (miRNA) via applying home-made DSA as signal unit. The p19 protein-functionalized magnetic beads (PFMBs) for specific recognition and enrichment of miRNA. Then DSA is combined with the long DNA concatamers, which functions as a signal enhancement platform to facilitate the high selectivity and sensitivity determination of miRNA. The usage of this novel electrochemical activity made a contribution to the performance of the approach, such as achieving a detection limit of 6 aM. To the best of our knowledge, this is the first attempt to apply DSA, PFMBs and long DNA concatamers for the fabrication of the electrochemical biosensors, which may represent a promising path toward early diagnosis of cancer at the point of care.     

Ultrasensitive electrochemical immunosensor for zeranol detection based on signal amplification strategy of nanoporous gold films and nano-montmorillonite as labels

Nano-montmorillonites belong to aluminosilicate clay minerals with innocuity, high specific surface area, ion exchange, and favorable adsorption property. Due to the excellent properties, montmorillonites can be used as labels for the electrochemical immunosensors. In this study, nano-montmorillonites were converted to sodium montmorillonites (Na-Mont) and further utilized for the immobilization of thionine (TH), horseradish peroxidase (HRP) and the secondary anti-zeranol antibody (Ab₂). The modified particles, Na-Mont-TH-HRP-Ab₂ were used as labels for immunosensors to detect zeranol. This protocol was used to prepare the immunosensor with the primary antibody (Ab₁) immobilized onto the nanoporous gold films (NPG) modified glassy carbon electrode (GCE) surface. Within zeranol concentration range (0.01–12 ng mL⁻¹), a linear calibration plot (Y = 0.4326 + 8.713 X, r = 0.9996) was obtained with a detection limit of 3 pg mL⁻¹ under optimal conditions. The proposed immunosensor showed good reproducibility, selectivity, and stability. This new type of immunosensors with montmorillonites and NPG as labels may provide potential applications for the detection of zeranol.     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

DNA detection and signal amplification via an engineered allosteric enzyme

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 fmol DNA in less than 3 min under physiological conditions.     

Polymerization-assisted signal amplification for electrochemical detection of biomarkers

We present a novel immunosensor by using polymerization-assisted signal amplification strategy coupled with electrochemical detection. A sandwich immunoassay process was used to immobilize a polymerization reaction center, the initiator-conjugated polyclonal prostate specific antigen (PSA) or polyclonal carcinoembryonic antigen (CEA) antibodies on the surface of the electrode. Activator generated electron transfer for atom transfer radical polymerization (AGET ATRP) subsequently triggered the local accumulation of glycidyl methacrylate (GMA) monomers. Growth of long chain polymers provided excess epoxy groups for electrochemical tags aminoferrocene (FcNH(2)) coupling, which in turn significantly increased the loading of the signal molecules and enhanced the electrochemical readouts. The detection limit was ～0.14 pg mL(-1) for PSA and ～0.10 pg mL(-1) for CEA in PBS buffers. The proposed immunosensor was highly sensitive, selective and has a good match to the clinical electrochemiluminescent method. This suggested that the polymerization-assisted immunosensing strategy could be used as an effective method to significantly enhance signal output of the sandwich immunoassays and acted as a promising platform for the clinical screening of cancer biomarkers.     

An exonuclease-assisted amplification electrochemical aptasensor of thrombin coupling “signal on/off” strategy

In this work, a dual-signaling electrochemical aptasensor based on exonuclease-catalyzed target recycling was developed for thrombin detection. The proposed aptasensor coupled “signal-on” and “signal-off” strategies. As to the construction of the aptasensor, ferrocene (Fc) labeled thrombin binding aptamer (TBA) could perfectly hybridize with the methylene blue (MB) modified thiolated capture DNA to form double-stranded structure, hence emerged two different electrochemical signals. In the presence of thrombin, TBA could form a G-quadruplex structure with thrombin, leading to the dissociation of TBA from the duplex DNA and capture DNA formed hairpin structure. Exonuclease could selectively digest single-stranded TBA in G-quadruplex structure and released thrombin to realize target recycling. As a consequence, the electrochemical signal of MB enhanced significantly, which realized “signal on” strategy, meanwhile, the deoxidization peak current of Fc decreased distinctly, which realized “signal off” strategy. The employment of exonuclease and superposition of two signals significantly improved the sensitivity of the aptasensor. In this way, an aptasensor with high sensitivity, good stability and selectivity for quantitative detection of thrombin was constructed, which exhibited a good linear range from 5 pM to 50 nM with a detection limit of 0.9 pM (defined as S/N = 3). In addition, this design strategy could be applied to the detection of other proteins and small molecules.     

Graphitized carbon black in polyethylene as an electrochemical sensor

The electrochemical behaviour of a material obtained by moulding graphitized carbon black and polyethylene at 100–150°C is described. The material can be used as a pellet electrode in voltammetric procedures. As a tubular anode held in a teflon body, the material is valuable as a sensor for high-performance liquid chromatography. Its properties are comparable with those of glassy carbon with better signal-to-noise ratios. It is applied for the determination of several phenols, chlorophenols and hydroquinone in the low mg l^?1 range or less.     

Surface chemistry effects on the performance of an electrochemical DNA sensor

E-DNA sensors are a reagentless, electrochemical oligonucleotide sensing platform based on a redox-tag modified, electrode-bound probe DNA. Because E-DNA signaling is linked to hybridization-linked changes in the dynamics of this probe, sensor performance is likely dependent on the nature of the self-assembled monolayer coating the electrode. We have investigated this question by characterizing the gain, specificity, response time and shelf-life of E-DNA sensors fabricated using a range of co-adsorbates, including both charged and neutral alkane thiols. We find that, among the thiols tested, the positively charged cysteamine gives rise to the largest and most rapid response to target and leads to significantly improved storage stability. The best mismatch specificity, however, is achieved with mercaptoethanesulfonic and mercaptoundecanol, presumably due to the destabilizing effects of, respectively, the negative charge and steric bulk of these co-adsorbates. These results demonstrate that a careful choice of co-adsorbate chemistry can lead to significant improvements in the performance of this broad class of electrochemical DNA sensors.     

Effect of molecular crowding on the response of an electrochemical DNA sensor

E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stem-loop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.     

Synergetic signal amplification based on electrochemical reduced graphene oxide-ferrocene derivative hybrid and gold nanoparticles as an ultra-sensitive detection platform for bisphenol A

In this paper, a novel electro-active graphene oxide (GO) nanocomposite was firstly prepared by covalently grafted (4-ferrocenylethyne) phenylamine (Fc-NH₂) onto the surface of GO. The synthesized hybridized nanocomposite of GO-Fc-NH₂ coupled with HAuCl₄ simultaneously electrodeposited on the glassy carbon electrodes (GCE) to obtain rGO-Fc-NH₂/AuNPs/GCE. The covalently grafted material of the rGO-Fc-NH₂/AuNPs film can effectively prevent the electron mediator leaking from the electrode surface, which can hold the advantage of both the nanomaterials and electron mediator. By employing the catalysis effect of the nanomaterial and electron mediator coupling with large active surface area and high accumulation capacity of rGO-Fc-NH₂/AuNPs, a synergetic signal amplification platform for ultra-sensitive detection of bisphenol A (BPA) was successfully established. With this novel sensor, the oxidation peak currents of BPA were linearly dependent on the BPA concentrations in the range of 0.005–10 μM with the detection limit of 2 nM. Modification of electron mediators on nanomaterials can greatly enhance the electrochemical performance of the sensors and will provide a new concept for fabricating newly electro-active nanomaterials-based electrochemical biosensors.     

Understanding signal amplification strategies of nanostructured electrochemical sensors for environmental pollutants

Nanomaterials used in electrochemical sensors can significantly improve the analytical performance to environmental pollutants. This review mainly discusses the strategies for signal amplification by the rational design of nanoelectrode materials from the perspective of mass and electron transfer processes of electrode/solution interface. First, the advantages and features of nanostructured electrochemical sensors for environmental pollutants are summarized. Then, the detailed discussions are focused on the signal amplification strategies by regulating dimensionality, atomic arrangement, and composition of electrode materials. This review gives a unique insight about the influences of electrode material design on mass and electron transfer processes of electrochemical sensors. Finally, on the basis of the current achievements in the field of nanomaterials, the perspectives on the challenges and opportunities for the exploration of nanostructured electrochemical sensors are put forward.     

A small molecule sensor for fluoride based on an autoinductive, colorimetric signal amplification reaction

This article describes a small molecule reagent that is capable of detecting fluoride down to 0.12 mM (2.3 ppm) in water. The reagent reveals this level of fluoride through a novel autoinductive signal amplification reaction that produces an unambiguous colorimetric readout.     

Disposable electrochemical immunosensor by using carbon sphere/gold nanoparticle composites as labels for signal amplification

This work designed a simple, sensitive, and low-cost immunosensor for the detection of protein marker by using a carbon sphere/gold nanoparticle (CNS/AuNP) composite as an electrochemical label. The nanoscale carbon spheres, prepared with a hydrothermal method by using glucose as raw material, were used to load AuNPs for labeling antibody by electrostatic interaction, which provided a feasible pathway for electron transfer due to the remarkable conductivity. The disposable immunosensor was constructed by coating a polyethylene glycol (PEG) film on a screen-printed carbon-working electrode and then immobilizing capture antibody on the film. With a sandwich-type immunoassay format, the analyte and then the CNS/AuNP-labeled antibody were successively bound to the immunosensor. The bound AuNPs were finally electro-oxidized in 0.1 M HCl to produce AuCl(4)(-) for differential pulse voltammetric (DPV) detection. The high-loading capability of AuNPs on CNS for the sandwich-type immunorecognition led to obvious signal amplification. By using human immunoglobulin?G (IgG) as model target, the DPV signal of AuNPs after electro-oxidized at optimal potential of +1.40?V for 40?s showed a wide linear dependence on the logarithm of target concentration ranging from 10?pg mL(-1) to 10?ng mL(-1). The detection limit was around 9?pg mL(-1). The immunosensor showed excellent analytical performance with cost effectivity, good fabrication reproducibility, and acceptable precision and accuracy, providing significant potential application in clinical analysis.     

A glucose dehydrogenase biosensor as an additional signal amplification step in an enzyme-flow immunoassay

Both the antibody affinity and the detectability of the label are essential in deciding the final characteristics of a heterogeneous immunoassay. This paper describes an approach to obtain a supplementary enhancement of the signal generated by using an enzyme label, e.g., by including the product of the enzymatic reaction in an additional amplification cycle during the detection step performed with an amperometric biosensor based on glucose dehydrogenase (GDH). An immunoassay format with a labelled analyte derivative that competes with the analyte present in the sample for a limited amount of antibody binding sites was employed. The beta-galactosidase label hydrolyses the substrate aminophenyl-beta-galactopyranoside, and the generated aminophenol enters then into a bioelectrocatalytic amplification cycle at the GDH biosensor. The principle was applied for determination of 4-nitrophenol, with the best minimal concentration of 1.5 microM and a midpoint of the calibration of 24 microM. The potentials and limitations of such a system are discussed.