A highly sensitive, multiplex immunoassay using gold nanoparticle-enhanced signal amplification

We describe a highly sensitive, multiplex immunoassay utilizing gold nanoparticles for conjugation of detection antibodies with signal generators. The method has been compared with conventional methods and evaluated for simultaneous detection of two different biomarkers in human serum.     

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.

     

Ultrasensitive electrochemical biosensor for uranium using deoxyribozymes with amplification by gold nanoparticles

A novel highly sensitive and specific electrochemical biosensor for detecting uranium based on specific Deoxyribozymes and gold nanoparticles (AuNPs) is reported. In this work, AuNPs provide excellent electrochemical signal transduction and a large surface area for immobilising numerous Deoxyribozymes, so a low detection limit of 3.24 ng L^?1 uranium and a good linear relationship over the range 5.94–35.1 ng L^?1 (r = 0.994) were obtained. The proposed biosensor presents high specificity and selectivity for uranium and is not affected by other metal ions. Thus, the biosensor protocol offers good selectivity, rapid speed and operational convenience for detection uranium in liquid waste.     

Microgravimetric lectin biosensor based on signal amplification using carbohydrate-stabilized gold nanoparticles

A highly sensitive microgravimetric lectin biosensor has been developed using carbohydrate-stabilized Au nanoparticles as a signal amplifier; mannose-stabilized Au nanoparticles formed a sandwich-type complex with the target Con A specifically bound to a mannose-modified Au QCM electrode to give an amplified frequency response.     

Incubation-free electrochemical immunoassay for diethylstilbestrol in milk using gold nanoparticle-antibody conjugates for signal amplification

We report on a novel enzyme-enhanced label for the electrochemical determination of diethylstilbestrol (DES). The label was obtained by orientation-controlled immobilization of a multiplex horseradisch peroxidase (HRP) conjugated polymer on gold nanoparticles (AuNPs) using the Envision reagent (EV) which is an enzyme-polymer complex that contains HRP and anti-IgG antibody in a polydextrin amine skeleton. The AuNPs were modified with Concanavalin A (Con A) and served as a carrier for immobilization of the EV?DES antibody composite. This resulted in a bioconjugate of the type AuNP?Con A?EV?DES Ab which was employed as the label. On exposure to samples containing DES, a sandwich immunocomplex is formed between antibody against DES (which was immobilized on a glassy carbon electrode and is acting as a capture probe), DES (the analyte), and the above label as the signal tracer. Hemin was used as an electronic mediator in the reaction of HRP. The HRP on the label catalyzes the oxidative formation of hydrogen peroxide at pH 7.0, and this induces an increased reductive current in the presence of hemin as an electron mediator. Under optimal conditions, the current increases linearly with increasing concentrations of DES in the range from 5 to 500 pg?·?mL^?1, with a detection limit as low as 2 pg?·?mL^?1 (at an S/N of 3). The method exhibits high selectivity and good stability. It works without incubation so that the time for an assay is shortened to 5 min. The assays was successfully applied to the determination of DES in milk samples.

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.

Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification

MicroRNAs (miRNAs) play an important regulatory role in cells and dysregulation of miRNA has been associated with a variety of diseases, making them a promising biomarker. In this work, a novel biosensing strategy has been developed for label-free detection of miRNA using surface plasmon resonance (SPR) coupled with DNA super-sandwich assemblies and biotin–strepavidin based amplification. The target miRNA is selectively captured by surface-bound DNA probes. After hybridization, streptavidin is employed for signal amplification via binding with biotin on the long DNA super-sandwich assemblies, resulting in a large increase of the SPR signal. The method shows very high sensitivity, capable of detecting miRNA at the concentration down to 9 pM with a wide dynamic range of 6 orders of magnitude (from 1 × 10⁻¹¹ M to 1 × 10⁻⁶ M) in 30 min, and excellent specificity with discriminating a single base mismatched miRNA sequence. This biosensor exhibits good reproducibility and precision, and has been successfully applied to the detection of miRNA in total RNA samples extracted from human breast adenocarcinoma MCF-7 cells. It, therefore, offers a highly effective alternative approach for miRNA detection in biomedical research and clinical diagnosis.     

A sensitive electrochemical biosensor for detection of DNA methyltransferase activity by combining DNA methylation-sensitive cleavage and terminal transferase-mediated extension

A novel electrochemical biosensor was developed for activity assay of DNA methyltransferase and its inhibitor based on methylation-sensitive cleavage, which activated a primer for terminal transferase-mediated extension of biotinylated dUTP followed by sensitive detection via enzymatic amplification.     

Molecular beacon based biosensor for the sequence-specific detection of DNA using DNA-capped gold nanoparticles-streptavidin conjugates for signal amplification

We describe a highly sensitive and selective molecular beacon-based electrochemical impedance biosensor for the sequence-specific detection of DNA. DNA-capped conjugates between gold nanoparticles (Au-NPs) and streptavidin are used for signal amplification. The molecular beacon was labeled with a thiol at its 5′ end and with biotin at its 3′ end, and then immobilized on the surface of a bare gold electrode through the formation of Au-S bonds. Initially, the molecular beacon is present in the “closed” state, and this shields the biotin from being approached by streptavidin due to steric hindrance. In the presence of the target DNA, the target DNA molecules hybridize with the loop and cause a conformational change that moves the biotin away from the surface of the electrode. The biotin thereby becomes accessible for the reporter (the DNA-streptavidin capped Au-NPs), and this results in a distinct increase in electron transfer resistance. Under optimal conditions, the increase in resistance is linearly related to the logarithm of the concentration of complementary target DNA in the range from 1.0 fM to 0.1 μM, with a detection limit of 0.35 fM (at an S/N of 3). This biosensor exhibits good selectivity, and acceptable stability and reproducibility.

Current signal amplification strategies in aptamer-based electrochemical biosensor:A review

Due to their high specificity and affinity towards various targets,along with other unique advantages such as stability and low cost,aptamers are widely applied in analytical techniques.A typical aptamerbased electrochemical biosensor is composed of a aptamer as the biological recognition element and transducer conve rting the biologic interaction into electrical signals for the quantitative measure ment of targets.Improvement of the sensitivity of a biosensor is significantly important in order to achieve the detection of biomolecules with low abundance,and different amplification strategies have been explored.The strategies either employ nanomaterials such as gold nanoparticles to construct electrodes which can trans fer the biological reactions more efficiently,or attempt to obtain enha nced signal through multi-labeled carriers or utilize enzyme mimics to catalyze redox cycling.This review discusses recent advances in signal amplification methods and their applications.Critical assessment of each method is also considered.     

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.     

Salmonella typhi determination using voltammetric amplification of nanoparticles: A highly sensitive strategy for metalloimmunoassay based on a copper-enhanced gold label

A highly sensitive electrochemical amplification immunoassay for Salmonella typhi (S. typhi) determination has been developed for the first time by using a copper-enhanced gold nanoparticle label coupled with anodic stripping voltammetry. Monoclonal antibodies for S. typhi were first immobilized on polystyrene microwells and then captured by S. typhi bacteria. After an immunoreaction occurred, a polyclonal, antibody-colloidal gold conjugate was added to bind to the S. typhi bacteria. Next, a copper-enhancer solution containing ascorbic acid and copper (II) sulfate was added into the polystyrene microwells. The ascorbic acid was employed to reduce the copper (II) ions to copper (0), which was subsequently deposited onto the gold nanoparticle tags. After the copper was dissolved in nitric acid, the released copper ions were detected by anodic stripping voltammetry. The amount of deposited copper was related to the amount of gold nanoparticle tag present, which was controlled by the amount S. typhi attached to the polyclonal antibody-colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration over concentration range of 1.30 × 10² cfu/mL to 2.6 × 10³ cfu/mL in a logarithmic plot, with a detection limit as low as 98.9 cfu/mL. The influences of the relevant experimental variables, such as the concentration of copper and the reaction time of S. typhi with antibody, were investigated. We also successfully applied this method to determine the presence of S. typhi in human serum. Our results are a step towards developing more sensitive and reliable nanoparticle immunoassays.     

Electrochemical aptasensor for highly sensitive determination of cocaine using a supramolecular aptamer and rolling circle amplification

Microchimica Acta - We report on a novel strategy for the electrochemical detection of cocaine. It is based on the use of a supramolecular aptamer, rolling circle amplification (RCA), and multiplex...     

Graphene oxide-based biosensor for sensitive fluorescence detection of DNA based on exonuclease III-aided signal amplification

Based on the super fluorescence quenching efficiency of graphene oxide and exonuclease III aided signal amplification, we develop a facile, sensitive, rapid and cost-effective method for DNA detection. In the presence of target DNA, the target-probe hybridization forms a double-stranded structure and exonuclease III catalyzes the stepwise removal of mononucleotides from the blunt 3′ termini of probe, resulting in the recycling of the target DNA and signal amplification. Therefore, our proposed sensor exhibits a high sensitivity towards target DNA with a detection limit of 20 pM, which was even lower than previously reported GO-based DNA sensors without enzymatic amplification, and provides a universal sensing platform for sensitive detection of DNA.     

Aptamer-based highly sensitive electrochemical detection of thrombin via the amplification of graphene

Herein, we successfully fabricated a highly sensitive label-free electrochemical aptasensor for thrombin based on the amplification of graphene (Gra). The excellent electrochemical probe of nickel hexacyanoferrate nanoparticles (NiHCFNPs) was introduced to form Nafion-Graphene-NiHCFNPs (Nf-Gra-NiHCFNPs) nanocomposites membrane on the gold electrode. The employment of graphene not only enhanced the surface area of the electrode with increased NiHCFNPs immobilization, but also improved the conductivity of the electrode, which further effectively improved the sensitivity of this proposed aptasensor. Subsequently, AuNPs layer was formed to immobilize the thrombin aptamer (TBA) and enhance the stability of the composite monolayer mentioned above. Then, thiol-modified TBA was assembled onto the AuNPs layer. Thereafter, hexanethiol (HT) was employed to block the possible remaining active sites. With the dual amplification of Gra and AuNPs, the resulting aptasensor exhibited good current response to target thrombin with a wide linear range extended from 1 pM to 80 nM (the detection limit was 0.3 pM). Additionally, the morphologies of bare Au substrate, nickel hexacyanoferrate nanoparticles (NiHCFNPs) and nanocomposites were successfully characterized by atomic force microscopy (AFM).