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

Microchimica Acta - The authors describe an ultrasensitive amperometric enzymatic assay for uranyl ion. It is based on the use of mesoporous silica nanoparticles (mesoSiNPs) loaded with...     

Surface-initiated DNA self-assembly as an enzyme-free and nanoparticle-free strategy towards signal amplification of an electrochemical DNA sensor

Surface-initiated DNA polymerization has been employed in this work as an appealing signal amplification strategy for electrochemical DNA sensors. This strategy is especially superior in that enzymes, colloidal particles and other bulky structures are not involved in order to achieve amplified signals, and thus is highly promising in circumventing problems due to uncontrolled nucleation, adsorption, aggregation or disassembly of nanoparticles, liposomes and proteins, as well as enzyme deactivations. Our preliminary results have shown that a decrease (as compared to an amplification-free system) in detection limit by a factor greater than 300 can be easily achieved by cyclic voltammetry under still not optimized conditions, with an ability of differentiating a single base mutation.     

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.     

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide,graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0?×?10^?14 and 5.0?×?10^?9 M concentration range, and the limit of detection is 2.2?×?10^?15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Ultrasensitive electrochemiluminescent immunosensor based on dual signal amplification strategy of gold nanoparticles-dotted graphene composites and CdTe quantum dots coated silica nanoparticles

A facile and ultrasensitive electrochemiluminescent (ECL) immunosensor for detection of prostate-specific antigen (PSA) was designed by using CdTe quantum dots coated silica nanoparticles (SiO₂@QDs) as bionanolabels. To construct such an electrochemiluminescence immunosensor, gold nanoparticles-dotted graphene composites were immobilized on the working electrode, which can increase the surface area to capture a large amount of primary antibodies as well as improve the electronic transmission rate. The as-prepared SiO₂@QDs used as bionanolabels, showed good ECL performance and good ability of immobilization for secondary antibodies. The approach provided a good linear response ranging from 0.005 to 10 ng?mL^?1 with a low detection limit of 0.0032 ng?mL^?1. Such immunosensor showed good precision, acceptable stability, and reproducibility. Satisfactory results were obtained for determination of PSA in human serum samples. Therefore, the proposed method provides a new promising platform of clinical immunoassay for other biomolecules.     

DNA nanosensors based on the use of single gold nanowire electrodes and Methylene Blue as an intercalator

The authors describe an electrochemical DNA nanosensor based on the use of single gold nanowire electrodes (AuNWEs). The probe DNA is immobilized on the AuNWE via Au-S bonds that are formed between thiol-terminated DNA and the gold surface. Single AuNWEs were prepared by an improved laser-assisted pulling method and hydrofluoric acid etching. The nanoelectrodes were characterized by cyclic voltammetry and COMSOL simulation. Square wave voltammetry was used to monitor the DNA hybridization event between probe DNA and target DNA by using Methylene Blue (MB) as an intercalator of dsDNA. Under optimal conditions, the peak current for MB (best measured at a potential of ?0.2 V vs. Ag/AgCl) increases linearly with the logarithm of the analyte concentration in the 1.0 f. to 10 nM range, with a 0.48 fmM detection limit at an S/N ratio of 3. The assay is highly selective, reproducible and stable. Considering the small overall dimensions and high sensitivity, this nanoelectrode potentially can be applied to in-vivo sensing of DNA inside living cells

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Graphical abstract Schematic presentation of an electrochemical DNA nanosensor using single gold nanowire electrodes and based on the interaction of thiol-terminated DNA and gold surface. It was used to detect complementary DNA with high selectivity and sensitivity.

     

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo⁻), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.     

Ultrasensitive electrochemiluminescent detection of pentachlorophenol using a multiple amplification strategy based on a hybrid material made from quantum dots,graphene, and carbon nanotubes

We report on a highly sensitive and selective electrochemiluminescence (ECL) based method for the determination of pentachlorophenol (PCP). It is based on a new hybrid material composed of CdS quantum dots (QDs), graphene, and carbon nanotubes (CNTs), and uses peroxodisulfate as the coreactant. The use of this system results in a nearly 18-fold increase in ECL intensity. On interaction between PCP and the QDs, a decrease in ECL intensity is observed at PCP in a concentration as low as 1.0 pM and over a wide linear range (from 1.0 pM to 1.0 nM). The method is hardly affected by other chlorophenols and nitrophenols, and the electrode can be recycled.

Sensitive determination of sialic acid expression on living cells by using an ITO electrode modified with graphene,gold nanoparticles and thionine for triple signal amplification

Microchimica Acta - The authors demonstrate a multivalent recognition and triple signal amplification strategy for amperometric cytosensing and dynamic evaluation of cell surface sialic acid (SA)....     

Non-invasive determination of glucose directly in raw fruits using a continuous flow system based on microdialysis sampling and amperometric detection at an integrated enzymatic biosensor

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h⁻¹) and ease of automation.     

Quadruple signal amplification strategy based on hybridization chain reaction and an immunoelectrode modified with graphene sheets,a hemin/G-quadruplex DNAzyme concatamer,and alcohol dehydrogenase: ultrasensitive determination of influenza virus subtype H7N9

We report on a new amplification strategy for use in an immunoassay for influenza virus subtype H7N9. Graphene sheets were first placed on a glassy carbon electrode (GCE), and gold nanoparticles were then electrodeposited as a support for a layer of alcohol dehydrogenase (ADH) in a sol–gel containing thiol groups. Protein A was used to properly orientate immobilized antibody against H7N9 on the sol–gel, and this is shown to result in strongly improved specificity of the antigen-antibody binding. Thus, a sensitive and specific immunosensor was obtained in which a quadruple signal amplification strategy is employed, viz. (a) via the use of graphene sheets, (b) via a hybridization chain reaction, (c) the use of hemin/G-quadruplex DNAzyme concatamers, and (d) the use of ADH. The hemin/G-quadruplex is a typical DNAzyme, which simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. The hybridization chain reaction-based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system. Sandwich immunoreactions occurred between the capture antibody on the electrode and the secondary antibody labeled with MWCNTs. Positively charged Methylene Blue (MB) was then used as an intercalator to detect the DNAzyme concatamer formed. The differential pulse voltammetric signals for MB are related to the concentration of H7N9 in the range from 8 to 60 pg · mL⁻¹, and the detection limit is 0.81 pg · mL⁻¹ (at an S/N ratio of 3). This immunoassay is very sensitive, specific and robust.

An electrochemical sandwich immunosensor has been developed for sensitive and specific detection of influenza virus subtype H7N9. Protein A was used to properly orientate antibody. The hybridization chain reaction based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system.

     

Colorimetric determination of the microcystin leucine-arginine based on the use of a hairpin aptamer,graphene oxide,and Methylene Blue acting as an optical probe

Microchimica Acta - The authors describe an aptamer-based colorimetric assay for the specific detection of the toxic microcystin leucine-arginine (MC-LR). The method is based on the use of graphene...     

Fluorometric aptamer based determination of adenosine triphosphate based on deoxyribonuclease I-aided target recycling and signal amplification using graphene oxide as a quencher

Microchimica Acta - The authors describe an aptamer based fluorometric assay for the determination of ATP. It is based on deoxyribonuclease I-aided target recycling and signal amplification. The...     

Sensitive and selective DNA detection based on the combination of hairpin-type probe with endonuclease/GNP signal amplification using quartz-crystal-microbalance transduction

In order to develop a highly sensitive and selective piezoelectric transducer for the detection of DNA, the bio-recognizing probe is for the first time designed by introducing a hairpin structure and a recognition site for EcoRI into an oligonucleotide sequence and signal amplifiers are prepared by modifying gold nanoparticles (GNPs) with biomolecules, deepening the application and understanding of biomaterials. The piezoelectric transducer is prepared by immobilizing designed hairpin recognition probe onto the quartz-crystal-microbalance (QCM). In the absence of target DNA, the hairpin probe is removed from the QCM surface after exposure to endonuclease, inhibiting the subsequent signaling reaction. In contrast, introduction of target DNA can open the hairpin probe due to the probe/target hybridization, dissociating the cleavable double-stranded portion. In this case, even if being treated with endonuclease, the integrated hairpin probe is maintained. Subsequent introduction of GNPs modified with detection probes that can hybridize to the terminal sequence of hairpin probe results in a many-folds increase of the frequency response. Utilizing the proposed transduction scheme, the reliable target DNA detection can be accomplished. The detection limit of 2 pM and dynamic response range for target DNA from 2 to 300 pM are obtained. Furthermore, single-base mismatched DNAs can be easily identified. The developed proof-of-principle of a novel piezoelectric transduction scheme is expected to establish a potential platform for the disease-associated mutation analysis and DNA hybridization detection in biotechnology and medical diagnostics.     

A universal electrochemical biosensor for the highly sensitive determination of microRNAs based on isothermal target recycling amplification and a DNA signal transducer triggered reaction

MicroRNAs (miRNAs) play a considerable role in cancer occurrence and development, and have been identified as promising noninvasive biomarkers. The authors describe a voltammetric method for the determination of the cancer biomarker microRNA-21 (miRNA). It is based on a combination of a universal DNA signal transducer and isothermal target recycling amplification. A hairpin capture probe is bound to the target miRNA to form a duplex structure and to create a toehold in the transducer for initiating the target recycling amplification reaction. In contrast to traditional capture probes, a mismatched site is introduced to improve its ability to capture the target. In order to reduce the complex design procedures of the sequence and widen the applicability of this method, a signal transducer is introduced. Under optimal conditions, response to target miRNA is linear in the 0.5 to 2000 pM concentration range, with a 56 fM. detection limit (at an S/N ratio of 3). In order to characterize the process of target recycling and the stepwise modification of the electrode, real-time fluorescence, agarose gel electrophoresis, cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry were used. The results indicate that this isothermal target recycling amplification results in an electrochemical biosensing scheme with wide potential for sensing other bioanalytes.

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Graphical abstract Schematic illustration of the electrochemical biosensing platform for miRNA-21 detection based on isothermal target recycling amplification and DNA signal transducer triggered strategy.

     

Electrochemiluminescence based determination of micro-RNA using target-guided assembly of gold nanoparticles on an electrode modified with Nafion,carbon nanotubes and polyvinylpyrrolidone

Microchimica Acta - The authors describe a sensing interface that is capable of selectively adsorbing gold nanopartices (AuNPs). It was applied to electrochemiluminescent (ECL) detection of...     

Aptamer based strategy for cytosensing and evaluation of HER-3 on the surface of MCF-7 cells by using the signal amplification of nucleic acid-functionalized nanocrystals

The electrochemical detection of cell lines of MCF-7 (human breast cancer) has been reported, using magnetic beads for the separation tool and high-affinity DNA aptamers for signal recognition. The high specificity was obtained by using the magnetic beads and aptamers, and the good sensitivity was realized with the signal amplification of DNA capped CdS or PbS nanocrystals. The ASV (anodic stripping voltammetry) technology was employed for the detection of cadmic cation and lead ions, for electrochemical assay of the amount of the target cells and biomarkers on the membrane of target cells, respectively. This electrochemical method could respond to as low as 100 cells mL⁻¹ of cancer cells with a linear calibration range from 1.0 × 10² to 1.0 × 10⁶ cells mL⁻¹, showing very high sensitivity. Moreover, the amounts of HER-3 which were overexpressed on MCF-7 cells were calculated correspond to be 3.56 × 10⁴ anti-HER-3 antibody molecules. In addition, the assay was able to differentiate between different types of target and control cells based on the aptamers and magnetic beads used in the assay, indicating the wide applicability of the assay for early and accurate diagnose of cancers.