Signal Amplification Platform Research Articles

MicroRNA-triggered, cascaded and catalytic self-assembly of functional "DNAzyme ferris wheel" nanostructures for highly sensitive colorimetric detection of cancer cells.

The construction of DNA nanostructures with various sizes and shapes has significantly advanced during the past three decades, yet the application of these DNA nanostructures for solving real problems is still in the early stage. On the basis of microRNA-triggered, catalytic self-assembly formation of the functional "DNAzyme ferris wheel" nanostructures, we show here a new signal amplification platform for highly sensitive, label-free and non-enzyme colorimetric detection of a small number of human prostate cancer cells. The microRNA (miR-141), which is catalytically recycled and reused, triggers isothermal self-assembly of a pre-designed, G-quadruplex sequence containing hairpin DNAs into "DNAzyme ferris wheel"-like nanostructures (in association with hemin) with horseradish peroxidase mimicking activity. These DNAzyme nanostructures catalyze an intensified color transition of the probe solution for highly sensitive detection of miR-141 down to 0.5 pM with the naked eye, and the monitoring of as low as 283 human prostate cancer cells can also, theoretically, be achieved in a colorimetric approach. The work demonstrated here thus offers new opportunities for the construction of functional DNA nanostructures and for the application of these DNA nanostructures as an effective signal amplification means in the sensitive detection of nucleic acid biomarkers.

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 0.005–10μM with the detection limit of 2nM. 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.

A graphene oxide-based enzyme-free signal amplification platform for homogeneous DNA detection.

A graphene oxide (GO) based enzyme-free signal amplification platform for homogeneous DNA sensing is developed with simplicity and high sensitivity. In the absence of the target DNA, labeled hairpin probe 1 (H1) and probe 2 (H2) were adsorbed on the surface of GO, resulting in the fluorescence quenching of the dyes and minimizing the background fluorescence. The addition of the target DNA facilitated the formation of double-stranded DNA (dsDNA) between H1 and H2, causing the probes to separate from GO and release the target DNA through a strand displacement reaction. Meanwhile, the whole reaction started anew. This is an excellent isothermal signal amplification technique without the involvement of enzymes. By monitoring the change of the fluorescence intensity, the target DNA not only can be determined in buffer solution, but also can be detected in 1% serum solution spiked with a series of concentrations of the target DNA. In addition, the consumption amount of the probes in this method is lower than that in traditional molecular beacon methods.

Facile fabrication of an ultrasensitive sandwich-type electrochemical immunosensor for the quantitative detection of alpha fetoprotein using multifunctional mesoporous silica as platform and label for signal amplification

A novel and ultrasensitive sandwich-type electrochemical immunosensor was designed for the quantitative detection of alpha fetoprotein (AFP) using multifunctional mesoporous silica (MCM-41) as platform and label for signal amplification. MCM-41 has high specific surface area, high pore volume, large density of surface silanol groups (SiOH) and good biocompatibility. MCM-41 functionalized with 3-aminopropyltriethoxysilane (APTES), gold nanoparticles (Au NPs) and toluidine blue (TB) could enhance electrochemical signals. Moreover, primary antibodies (Ab1) and secondary antibodies (Ab2) could be effectively immobilized onto the multifunctional MCM-41 by the interaction between Au NPs and amino groups (–NH2) on antibodies. Using multifunctional MCM-41 as a platform and label could greatly simplify the fabrication process and result in a high sensitivity of the designed immunosensor. Under optimal conditions, the designed immunosensor exhibited a wide liner range from 10−4ng/mL to 103ng/mL with a low detection limit of 0.05pg/mL for AFP. The designed immunosensor showed acceptable selectivity, reproducibility and stability, which could provide potential applications in clinical monitoring of AFP.

Amplified impedimetric aptasensor based on gold nanoparticles covalently bound graphene sheet for the picomolar detection of ochratoxin A

An amplified electrochemical impedimetric aptasensor for ochratoxin A (OTA) was developed with picomolar sensitivity. A facile route to fabricate gold nanoparticles covalently bound reduced graphene oxide (AuNPs–rGO) resulted in a large number of well-dispersed AuNPs on graphene sheets with tremendous binding sites for DNA, since the single rGO sheet and each AuNP can be loaded with hundreds of DNA strands. An aptasensor with sandwich model was fabricated which involved thiolated capture DNA immobilized on a gold electrode to capture the aptamer, then the sensing interface was incubated with OTA at a desired concentration, followed by AuNPs–rGO functionalized reporter DNA hybridized with the residual aptamers. By exploiting the AuNPs–rGO as an excellent signal amplified platform, a single hybridization event between aptamer and reporter DNA was translated into more than 107 redox events, leading to a substantial increase in charge-transfer resistance (Rct) by 7∼ orders of magnitude compared with that of the free aptamer modified electrode. Such designed aptasensor showed a decreased response of Rct to the increase of OTA concentrations over a wide range of 1pgmL−1–50ngmL−1 and could detect extremely low OTA concentration, namely, 0.3pgmL−1 or 0.74pM, which was much lower than that of most other existed impedimetric aptasensors. The signal amplification platform presented here would provide a promising model for the aptamer-based detection with a direct impedimetric method.

(4‐Ferrocenylethyne) phenylamine on Graphene as the Signal Amplificator to Determinate Dopamine and Acetaminophen Simultaneously

AbstractA novel (4‐ferrocenylethyne) phenylamine functionalized graphene sheets (FEPA‐GR), coupling with chitosan (CS) were used as a signal amplification platform for simultaneous and sensitive determination of dopamine (DA) and acetaminophen (AC). In this work, FEPA used as electron transfer mediator can be immobilized on GR surface via strong π‐π stacking interaction between the conjugate chain of FEPA and GR, which effectively prevents FEPA electron mediator leaking from the electrode surface and amplified the signal. Transmission electron microscopy, FT‐IR spectroscopy, UV‐vis spectroscopy and electrochemical experiments results are all demonstrated the strong π‐π stacking interaction between FEPA and GR. The resulted biosensor exhibited a fast response, remarkable electrocatalytic activity, perfect anti‐interference ability and good stability for simultaneous detection of DA and AC. Under the optimum conditions, the oxidation peak currents of DA and AC were linearly correlated to their concentrations in the range of 2.0–135.0 µmol·L−1 and 0.3–80.0 µmol·L−1, respectively. The lower detection limits for DA and AC were 0.30 and 0.05 µmol·L−1, respectively. The feasibility of the proposed method was validated by successfully applied to the simultaneous determination of DA and AC in serum samples with the standard addition method.

A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid, dopamine, uric acid and acetaminophen based on a nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet

A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid (AA), dopamine (DA), uric acid (UA) and acetaminophen (AC) was fabricated by a nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet. The platform was constructed by coating a newly synthesized phenylethynyl ferrocene thiolate (Fc-SAc) modified Fe3O4@Au NPs coupling with graphene sheet/chitosan (GS-chitosan) on a glassy carbon electrode (GCE) surface. The Fe3O4@Au–S–Fc/GS-chitosan modified GCE exhibits a synergistic catalytic and amplification effect toward AA, DA, UA and AC oxidation. The oxidation peak currents of the four compounds on the electrode were linearly dependent on AA, DA, UA and AC concentrations in the ranges of 4–400μM, 0.5–50μM, 1–300μM and 0.3–250μM in the individual detection of each component, respectively. By simultaneously changing the concentrations of AA, DA, UA and AC, their electrochemical oxidation peaks appeared at −0.03, 0.15, 0.24 and 0.35V, and good linear current responses were obtained in the concentration ranges of 6–350, 0.5–50, 1–90 and 0.4–32μM with the detection limits of 1, 0.1, 0.2 and 0.05μM (S/N=3), respectively.

A universal mismatch-directed signal amplification platform for ultra-selective and sensitive DNA detection under mild isothermal conditions

We describe a novel mismatch-directed signal amplification method which can be performed easily under mild isothermal conditions for the ultra-selective and sensitive detection of any DNA sequence of interest. By taking advantage of the unique discrimination properties of an endonuclease IV and lambda exonuclease coupling system in cleavage reactions of DNA duplexes containing apurinic/apyrimidinic sites with different adjacent mismatched bases, the established assay enables sensitive detection of 1.0 fmol of target strand and selective differentiation of 0.5% target strand from single-base different sequences at 37 °C without the need for temperature adjustment.

An enzyme-responsive nanocontainer as an intelligent signal-amplification platform for a multiple proteases assay

Based on selective pore-opening in the presence of protease, we have developed a novel signal amplification assay for multiple proteases detection and their inhibition using protein-capped mesoporous scaffolding as the substrate.

Simple detection of nucleic acids with a single-walled carbon-nanotube-based electrochemical biosensor

We report for the first time a simple approach to fabricate an electrochemical DNA (E-DNA) biosensor by introducing the single-walled carbon nanotubes (SWNTs). The SWNTs combine with the electrochemical label (methyl blue, MB)-modified single-stranded DNA (ssDNA) probes to generate a nanomaterial–biomolecule composite, which functions as a signal amplification platform to facilitate the electron-transfer between the electrochemical label and the electrode. This SWNT-based E-DNA biosensor produces a high square wave voltammetry (SWV) signal in the absence of target DNA. In the presence of target DNA, the MB-labeled ssDNA probes are removed from the SWNT-modified electrode due to the formation of a double-stranded DNA (dsDNA), generating a relatively low SWV signal. This signal-off SWNT-based E-DNA biosensor exhibits improved sensitivity and large linear dynamic range with low detection limit; it can even distinguish 1-base mismatched target DNA. Further experiments demonstrate that the SWNT-based E-DNA biosensor is superior to the multi-walled carbon nanotube (MWNT)-based one for DNA detection. Moreover, the introduction of aptamer into the SWNT-based biosensor might be further extended to detect small biomolecules such as adenosine.

Signal amplification of target protein on heparin glycan microarray

A heparin glycan chip (HepGlyChip) with a 4800-fold enhanced signal-to-noise ratio as compared with the control without heparin was developed for high-throughput analysis of heparin–protein interactions for new drug development and for screening biological samples in diagnostic applications. As a proof of concept, a heparin glycan microarray was prepared on a poly(styrene- co-maleic anhydride) (PS–MA)-coated glass slide. Heparin was covalently immobilized on poly-l-lysine (PLL) layer with multiple binding sites by sulfo-ethylene glycol bis(succinimidylsuccinate) (sulfo-EGS), increasing the signal-to-noise ratio, minimizing nonspecific binding of target proteins, and resulting in a three-dimensional (3D) structure on the HepGlyChip. This on-chip signal amplification platform was successfully demonstrated by probing the heparin microarray with the highly specific heparin-binding protein antithrombin III (AT III).

SERUM ENZYME STUDIES IN MUSCLE DISEASE. II. SERUM CREATINE KINASE ACTIVITY IN MUSCULAR DYSTROPHY AND IN OTHER MYOPATHIC AND NEUROPATHIC DISORDERS.

<h3>SUMMARY</h3> Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling, however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here we show that the NF-ĸB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-ĸB to the nucleus. TNF induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-ĸB is locally activated and transported to the nucleus by mitochondria, resulting in an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN.