Electrochemical strategy for ultrasensitive detection of microRNA based on MNAzyme-mediated rolling circle amplification on a gold electrode

Microchimica Acta - The authors describe an electrochemical strategy for ultrasensitive and specific detection of microRNA (miRNA). It is based on both multicomponent nucleic acid enzyme (MNAzyme)...     

A disposable amperometric enzyme immunosensor for rapid detection of Vibrio parahaemolyticus in food based on agarose/Nano-Au membrane and screen-printed electrode

Recent outbreaks of foodborne illnesses continue to support the need for rapid and sensitive methods for detection of foodborne pathogens. A disposable electrochemical immunosensor for detection of Vibrio parahaemolyticus (VP) based on the screen-printed electrode (SPE) coated with agarose/Nano-Au membrane and horseradish peroxidase (HRP) labeled VP antibody (HRP-anti-VP) has been developed in this paper. Then, the immunosensor was characterized by cyclic voltammetry (CV) and laser scanning confocal microscope (LSCM). The immunosensor was incubated with the one-step immunoassay format involving VP for 30 min at room temperature (25 ± 0.5 °C). The access of the active center of HRP catalyzing the oxidation reaction of thionine by H₂O₂ was partly inhibited by VP, which connected on the surface of the immunosensor by immunoreaction. VP could be quantificationally detected according to the shift of reduction current while CV was used as electrochemical means to detect the products of the enzymatic reaction. Under the optimum conditions of immunoreaction and electrochemical detection, VP was rapidly detectable by sigmoidal curve method and form a linear calibration between 10⁵ and 10⁹ cfu/ml with an associated detection limit of 7.374 × 10⁴ cfu/ml (S/N = 3). The immunosensor had acceptable specificity, reproducibility, stability and accuracy, indicating that the immunosensor could satisfy the need of practical sample detection.     

Label-free aptamer biosensor for selective detection of thrombin

We fabricated a novel fluorescence biosensor for the selective detection of thrombin by using bovine serum albumin-capped CdS quantum dots (BSA-CdS QDs). Two kinds of designed DNA (DNA1 and DNA2) could bind to CdS QDs through the electrostatic interaction between DNA and Cd²⁺ on the surface of CdS QDs. The obtained DNA/BSA-CdS QDs kept stable in the solution with the fluorescence intensity obviously enhanced. Hairpin structure of DNA1contained two domains, one is the aptamer sequence of thrombin and the other is the complementary sequence of DNA2. When thrombin was added, it would bind to DNA1 and induce the hairpin structure of DNA1 changed into G-quadplex structure. Meanwhile, DNA2 would transfer from the surface of CdS QDs to DNA1 via hybridization, which resulted in the removal of DNA1 and DNA2 from the surface of CdS QDs, and led to the fluorescence intensity of CdS QDs reduced. Thus, the determination of thrombin could be achieved by monitoring the change of the fluorescence intensity of CdS QDs. The present method is simple and fast, and exhibits good selectivity for thrombin over other proteins. We have successfully detected thrombin in human serum samples with satisfactory results.     

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.     

An ultrasensitive electrochemical biosensor for detection of DNA species related to oral cancer based on nuclease-assisted target recycling and amplification of DNAzyme

A simple, label-free, ultra-highly sensitive and selective electrochemical sensor based on nuclease-assisted target recycling and DNAzyme for the detection of DNA species related to oral cancer in saliva is developed.     

Conducting polymer-based impedimetric aptamer biosensor for in situ detection

We have successfully developed the first prototype of an electrochemical protein biosensor using polypyrrole as the substrate and doped aptamer as the probe. The sensitivity for a specific target is 10 ng/ml. Two targets, platelet-derived growth factor and immunoglobulin E, have been tested. In both cases the sensor exhibited high specificity and the signal was found to increase as the target concentration increased.     

A novel amperometric biosensor for the detection of nitrophenol

We report on a novel amperometric biosensor for detecting phenolic compounds based on the co-immobilization of horseradish-peroxidase (HRP) and methylene blue (MB) with chitosan on Au-modified TiO₂ nanotube arrays. The titania nanotube arrays were directly grown on a Ti substrate using anodic oxidation first; a gold thin film was then coated onto the TiO₂ nanotubes by an argon plasma technique. The morphology and composition of the fabricated Au-modified TiO₂ nanotube arrays were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). Cyclic voltammetry and amperometry were used to study the proposed electrochemical biosensor. The effect of pH, applied electrode potential and the concentration of H₂O₂ on the sensitivity of the biosensor have been systemically investigated. The performance of the proposed biosensor was tested using seven different phenolic compounds, showing very high sensitivity; in particular, the linearity of the biosensor for the detection of 3-nitrophenol was observed from 3 × 10⁻⁷ to 1.2 × 10⁻⁴ M with a detection limit of 9 × 10⁻⁸ M (based on the S/N = 3).     

One-step separation-free amperometric biosensor for the detection of protein

A novel enzyme biosensor for the detection of protein is presented. The biosensor was made from a screen-printed three-electrode configuration. Amino acid oxidase was immobilized with glutaraldehyde and polyethylenimine on a working electrode made of rodinised carbon. A protease was immobilized on an immunodyne membrane and was placed on the electrode. A protein sample was deposited on the membrane, and was subsequently hydrolyzed to amino acids in the presence of the protease. This in turn produced hydrogen peroxide by the immobilized amino acid oxidase. The oxidation of hydrogen peroxide was then detected at +400 mV vs. an Ag/AgCl reference electrode. The method was very effective at detecting a very low level of protein. The sensor does not require any washing step. The sensor works with only 40 μl of sample per detection, and may be used on-site as a disposable sensor using a hand-held meter. The electrodes are also stable for more than 6 weeks.     

A bienzymatic amperometric immunosensor exploiting supramolecular construction for ultrasensitive protein detection

Self-assembly of a ferrocene-appended polymer bearing an antigen fragment and lactate oxidase on a cyclodextrin-modified surface provides a highly sensitive, easy-to-operate and self-sufficient immunosensor.     

CuO/Cu2O nanowire array photoelectrochemical biosensor for ultrasensitive detection of tyrosinase

Photoelectrochemical(PEC) biosensors have shown great promise in bioanalysis and diagnostic applications in recent years. In this work, the CuO/Cu_2O nanowire array(CuO/Cu_2O Nanowire) supported on copper foam was prepared as a photocathode for detection of tyrosinase though quinone-chitosan conjugation chemistry method. The in-situ generated quinones that were the catalytic product of tyrosinase acted as electron acceptors, which were captured by the chitosan deposited on the surface of the electrode. Direct immobilization of electron acceptor on the electrode surface improved the photocurrent conversion efficiency and thus sensitivity. The as-prepared biosensor can realize a rapid response in a wide linear range of 0.05 U/mL to 10 U/mL with the detection limit as low as 0.016 U/mL of tyrosinase. The current work provides a new perspective to design and develop highly sensitive and selective PEC biosensor.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

An ultrasensitive fluorescent aptasensor based on truncated aptamer and AGET ATRP for the detection of bisphenol A

Given the gigantic harmfulness of bisphenol A (BPA), a novel and ultrasensitive aptasensor, which employs the truncated BPA aptamer, click chemistry, and activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP), was developed herein for the quantitative determination of BPA. Firstly, hairpin DNAs (hairpins) with a thiol at the 5′ end and an azide group at the 3′ end were conjugated with aminated magnetic beads (MBs) through heterobifunctional cross-linkers. BPA truncated aptamer (ssDNA-A) hybridizes with its complementary single-stranded DNA (ssDNA-B) to form double-stranded DNA. In the presence of BPA, ssDNA-A specifically captures BPA, and then ssDNA-B is released. Subsequently, the ssDNA-B hybridizes with hairpins to expose the azide group near the surface of the MBs. Then, propargyl-2-bromoisobutyrate (PBIB), the initiator of AGET ATRP containing alkynyl group, was conjugated with azide group of hairpins via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Consequently, a large number of fluorescein-o-acrylate (FA) were introduced to the MBs through AGET ATRP, resulting in that the fluorescence intensity was increased dramatically. Obviously, the fluorescence intensity was especially sensitive to the change of BPA concentration, and this method can be used in quantitative determination of BPA. Under optimal conditions, a broad liner range from 100 fM to 100 nM and a low limit of detection (LOD) of 6.6 fM were obtained. Moreover, the method exhibits not only excellent specificity for BPA detection over BPA analogues but high anti-interference ability in real water sample detection, indicating that it has huge application prospect in food safety and environment monitoring.

     

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.     

A label-free and high sensitive aptamer biosensor based on hyperbranched polyester microspheres for thrombin detection

In this paper, we have synthesized hyperbranched polyester microspheres with carboxylic acid functional groups (HBPE-CA) and developed a label-free electrochemical aptamer biosensor using thrombin-binding aptamer (TBA) as receptor for the measurement of thrombin in whole blood. The indium tin oxide (ITO) electrode surface modified with HBPE-CA microspheres was grafted with TBA, which has excellent binding affinity and selectivity for thrombin. Binding of the thrombin at the modified ITO electrode surface greatly restrained access of electrons for a redox probe of [Fe(CN)₆]^3−/4−. Moreover, the aptamer biosensor could be used for detection of thrombin in whole blood, a wide detection range (10 fM–100 nM) and a detection limit on the order of 0.90 fM were demonstrated. Control experiments were also carried out by using bull serum albumin (BSA) and lysozyme in the absence of thrombin. The good stability and repeatability of this aptamer biosensor were also proved. We expect that this demonstration will lead to the development of highly sensitive label-free sensors based on aptamer with lower cost than current technology. The integration of the technologies, which include anticoagulant, sensor and nanoscience, will bring significant input to high-performance biosensors relevant to diagnostics and therapy of interest for human health.     

Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate

A novel amperometric biosensor for the determination of lactate was constructed by first immobilizing lactate oxidase and an osmium redox polymer ([Os(bpy)(2)(PVP)(10)Cl]Cl; abbreviated Os-polymer) on the surface of a glassy carbon electrode, followed by coating with a sol-gel film derived from methyltriethoxysilane (MTEOS). The electrooxidation current of this electrode was found to be diffusion controlled. In the presence of lactate, a clear electrocatalytic oxidation wave was observed, and lactate could be determined amperometrically at 400 mV versus Ag AgCl . The concentration range of linear response, slope of linear response and detection limit were 0.1-9 mM, 1.02 microA mM(-1), and 0.05 mM, respectively. Although L-ascorbate was electrooxidized at this potential, uric acid, paracetamol and glucose were found not to interfere.     

Laccase-based amperometric biosensor enhanced by Ni/Au/PPy-COOH multisegmental nanowires for selective dopamine detection

One-dimensional Ni/Au/PPy-COOH nanowires with multiple segments were synthesized in this study. Smooth surfaces and magnetic properties of nanowires were investigated by scanning transmission electron microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), and Electron Spin Resonance (ESR) techniques. The nanowires were used to modify the screen-printed electrode surface and as a micro-environment for Trametes versicolor laccase. The ability of this enzyme biosensor to detect dopamine change in human biological samples was demonstrated by a wide linear range (0.01–50 μM) and a low LOD (2.265 nM). In addition, the biosensor exhibited excellent selectivity allowing the detection of dopamine in the presence of ascorbic acid, uric acid, L-Cys, serotonin, and glucose, with high sensitivity of reduction currents obtained at −0.2 V (vs. Ag/AgCl). The proposed biosensor allowed the detection of dopamine in commercial serum and artificial urine with recovery values close to 100 %. It also demonstrated reproducibility, reusability, and long-term storage stability. The sensitivity, K_m^app, and I_max values of the biosensor were determined as 2.05 μM and 1.03 μA, respectively. The LAC-Ni/Au/PPy-COOH/NAF/SPE biosensor is a reliable design for detecting dopamine with a wide linear range.     

Fabricating electrodes for amperometric detection in hybrid paper/polymer lab-on-a-chip devices

We present a novel, low-resource fabrication and assembly method for creating disposable amperometric detectors in hybrid paper-polymer devices. Currently, mere paper-based microfluidics is far from being able to achieve the same level of process control and integration as state-of-the-art microfluidic devices made of polymers. To overcome this limitation, in this work both substrate types are synergistically combined through a hybrid, multi-component/multi-material system assembly. Using established inkjet wax printing, we transform the paper into a profoundly hydrophobic substrate in order to create carbon electrodes which are simply patterned from carbon inks via custom made adhesive stencils. By virtue of the compressibility of the paper substrate, the resulting electrode-on-paper hybrids can be directly embedded in conventional, 3D polymeric devices by bonding through an adhesive layer. This manufacturing scheme can be easily recreated with readily available off-the-shelf equipment, and is extremely cost-efficient and rapid with turn-around times of only a few hours.