Shu Jun Zhen

Carbon Nanotubes as a Low Background Signal Platform for a Molecular Aptamer Beacon on the Basis of Long-Range Resonance Energy Transfer

Although holding the advantages of both an aptamer and a molecular beacon (MB), a molecular aptamer beacon (MAB) needs complicated and expensive modifications at both of its ends and usually has a high background signal because of the low energy transfer efficiency between the donor and the acceptor. To overcome these shortcomings, in this study, we develop a long-range resonance energy transfer (LrRET) system by separating the donor from the acceptor, wherein only one end of the MAB is fluorescently labeled and acts as the energy donor and multiwalled carbon nanotubes (MWCNTs) are introduced as the energy acceptor. To test the feasibility of the newly designed MAB system, adenosine triphosphate (ATP) has been employed as a proof-of-concept target. It is found that the fluorescence of the designed MAB is completely quenched by MWCNTs, supplying a very low background signal. Then the quenched fluorescence is recovered significantly with the addition of ATP, so that ATP can be detected in the range of 0.8-80 μM with a limit of detection of 0.5 μM (3σ). Compared with the conventional fluorescence resonance energy transfer, the efficiency of LrRET between the dye and MWCNTs is much higher. Since only one end of the MAB needs the modification, the present strategy is simple and cost-effective. Furthermore, the use of MWCNTs can greatly reduce the fluorescence background of the MAB and supply a high sensitivity, showing its generality for detection of a variety of targets.

A gold nanoparticles-based colorimetric assay for alkaline phosphatase detection with tunable dynamic range

In this report, a simple and label-free colorimetric assay was developed for detecting alkaline phosphatase (ALP). Based on the conjugated gold nanoparticle/adenosine triphosphate (AuNP/ATP) sensing system, this assay is highly sensitive and selective. In this system, ATP induces the aggregation of cetyltrimethylammonium bromide (CTAB)-capped AuNPs and ALP stimulates the disaggregation of AuNPs by converting ATP into adenosine through an enzymatic dephosphorylation reaction. Hence, the presence of ALP can be visually observed (gray-to-red color change) and monitored by the shift of the surface plasmon resonance (SPR) absorption band of AuNPs. Furthermore, the dynamic range of the method can be varied by addition of different metal ions (e.g. 100-600unit/L to 5.0-100unit/L and 0.2-20unit/L in the presence of Ca(2+) and Pb(2+), respectively). The feasibility of this sensitive and specific assay with a tunable dynamic range was demonstrated to be consistent even in human serum samples.

Sensitive Discrimination and Detection of Prion Disease-Associated Isoform with a Dual-Aptamer Strategy by Developing a Sandwich Structure of Magnetic Microparticles and Quantum Dots

The major challenge of prion disease diagnosis at the presymptomatic stage is how to sensitively or selectively discriminate and detect the minute quantity of disease-associated prion protein isoform (PrP(Res)) in complex biological systems such as serum and brain homogenate. In this contribution, we developed a dual-aptamer strategy by taking the advantages of aptamers, the excellent separation ability of magnetic microparticles (MMPs), and the high fluorescence emission features of quantum dots (QDs). Two aptamers (Apt1 and Apt2), which can recognize their two corresponding distinct epitopes of prion proteins (PrP), were coupled to the surfaces of MMPs and QDs, respectively, to make MMPs-Apt1 and QDs-Apt2 ready at first, which then could be coassociated together through the specific recognitions of the two aptamers with their two corresponding distinct epitopes of PrP, forming a sandwich structure of MMPs-Apt1-PrP-Apt2-QDs and displaying the strong fluorescence of QDs. Owing to the different binding affinities of the two aptamers with PrP(Res) and cellular prion protein (PrP(C)), both of which have distinct denaturing detergent resistance, our dual-aptamer strategy could be applied to discriminate PrP(Res) and PrP(C) successfully in serum. Further identifications showed that the present dual-aptamer assay could be successfully applied to the detection of PrP in 0.01% brain homogenate, about 1000-fold lower than that of commonly applied antibody-mediated assays, which can detect PrP just in 10% brain homogenate, indicating that the present designed dual-aptamer assay is highly sensitive and adequate for clinical diagnosis without isolation of target protein prior to assay.

Graphene oxide as an efficient signal-to-background enhancer for DNA detection with a long range resonance energy transfer strategy

A long range resonance energy transfer (LrRET) strategy for label-free and sensitive DNA detection is outlined by introducing graphene oxide (GO) as an efficient signal-to-background enhancer, giving a limit of determination (3σ) of 0.31 nM.

Hydrogen-bond-mediated in situ fabrication of AgNPs/agar/PAN electrospun nanofibers as reproducible SERS substrates.

Reproducibility in surface enhanced Raman scattering (SERS) measurements is a challenge. This work developed a facile way to make highly dispersed uniform silver nanoparticles (AgNPs) loaded in the agar/polyacrylonitrile (PAN) nanofibers by the coupling the electrospinning technology from metal complex-containing polymer solution and in situ photoreductive technique. Agar, as hydrophilic component, was introduced into the electrospinning solution considering that its abundant hydroxyl group sites could greatly improve the contents of silver ions in the polymers because of the rich silver ion chelated with the hydroxyl group, whereas hydrophilic agar was integrated with hydrophobic PAN by -OH···N≡C- hydrogen bonds as a bridge. Meanwhile, the in situ photoreductive reaction was made under different light irradiations such as desk lamp, 365 nm UV-lamp, and 254 nm UV-lamp. High yield of stable AgNPs with highly uniform and dispersion are available in the agar/PAN nanofibers after the in situ photoreductive reaction, supplying the possibility of reproducible SERS signals. To identify that concept of proof, a facile approach for the determination of malachite green (MG) in three environmental practical samples was demonstrated by using the composite nanofibrous material irradiated by 365 nm UV-lamp, giving the minimum detection concentration of MG as low as 0.1 μmol/L with a good linear response ranging from 0.1-100 μmol/L (R(2) = 0.9960).

Ultra-sensitive detection of prion protein with a long range resonance energy transfer strategy

An ultra-sensitive detection strategy for prion protein is proposed based on the long range resonance energy transfer (LrRET) from quantum dots (QDs) to the surface of gold nanoparticles (AuNPs), in which process energy donor-acceptor separation distance ranges from 9 to 22 nm.

Sensitive detection of prion protein through long range resonance energy transfer between graphene oxide and molecular aptamer beacon

The traditional molecular aptamer beacon (MAB) is designed by combining an aptamer to a molecular beacon, and its two terminals are labelled with a fluorescent moiety (donor) and quenching moiety (acceptor), respectively. However, it usually has a high background because of the low energy transfer efficiency between the donor and the acceptor. In order to overcome these drawbacks, we have developed a novel MAB with just one fluorescently labelled end, which acts as the donor, and graphene oxide (GO) introduced as the acceptor for target detection by employing long range resonance energy transfer (LrRET) as the signal-transduction mechanism from GO to MAB. To test the validity of the designed MAB system, cellular prion protein (PrPC) has been used as the model target. It was found that the fluorescence of the designed MAB is completely quenched by GO, supplying a very low background. Conversely, the quenched fluorescence is recovered significantly with the addition of PrPC, so that PrPC can be detected over a wide range of 10.2–78.8 μg mL−1 with a detection limit as low as 0.309 μg mL−1 and with high selectivity. This GO-based MAB approach is a successful application of LrRET for the detection of PrPC, with advantages such as low costs, high quenching efficiency and good specificity, and it opens up new opportunities for the sensitive detection of biorecognition events.

Metal-enhanced fluorescence of nano-core–shell structure used for sensitive detection of prion protein with a dual-aptamer strategy

Metal-enhanced fluorescence (MEF) as a newly recognized technology is widespread throughout biological research. The use of fluorophore-metal interactions is recognized to be able to alleviate some of fluorophore photophysical constraints, favorably increase both the fluorophore emission intensity and photostability. In this contribution, we developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the presence of prion protein, core-shell Ag@SiO2, which are functionalized covalently by single stranded aptamer (Apt1) of prions and Cyanine 3 (Cy3) decorated the other aptamer (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of the pariticles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission rates of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of 0.05-0.30 nM, which is successfully applied to the detection of PrP in mice brain homogenates.

Electrostatic Assemblies of Well-Dispersed AgNPs on the Surface of Electrospun Nanofibers as Highly Active SERS Substrates for Wide-Range pH Sensing

Surface-enhanced Raman scattering (SERS) has shown high promise in analysis and bioanalysis, wherein noble metal nanoparticles (NMNPs) such as silver nanoparticles were employed as substrates because of their strong localized surface plasmon resonance (LSPR) properties. However, SERS-based pH sensing was restricted because of the aggregation of NMNPs in acidic medium or biosamples with high ionic strength. Herein, by using the electrostatic interaction as a driving force, AgNPs are assembled on the surface of ethylene imine polymer (PEI)/poly(vinyl alcohol) (PVA) electrospun nanofibers, which are then applied as highly sensitive and reproducible SERS substrate with an enhancement factor (EF) of 10(7)-10(8). When p-aminothiophenol (p-ATP) is used as an indicator with its b2 mode, a good and wide linear response to pH ranging from 2.56 to 11.20 could be available, and the as-prepared nanocomposite fibers then could be fabricated as excellent pH sensors in complicated biological samples such as urine, considering that the pH of urine could reflect the acid-base status of a person. This work not only emerges a cost-effective, direct, and convenient approach to homogeneously decorate AgNPs on the surface of polymer nanofibers but also supplies a route for preparing other noble metal nanofibrous sensing membranes.

An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity

Graphene oxide (GO) has been proven as an outstanding fluorescence anisotropy (FA) amplifier. Yet the traditional GO amplified FA assays lack high sensitivity because of the 1:1 binding ratio between target and dye-modified probe. Herein, we report a new target-catalyzed hairpin assembly (CHA), an enzyme-free DNA circuit, assisted GO amplified FA strategy for microRNA-21 (miRNA-21) detection. In the presence of miRNA-21, the CHA was initiated and plenty of H1-H2 duplexes were produced continuously. The obtained H1-H2 duplex could induce the formation of a H1-H2-probe DNA (pDNA) complex by the toehold-mediated strand exchange reaction, which led the dye-modified pDNA to leave away from the GO surface, resulting in a decreased FA of the system. By monitoring the decrease of FA, miRNA-21 could be detected in the range of 0-16 nM. The limit of detection (LOD, 3σ) was 47 pM, which was 194 times lower than that without CHA. In addition, the selectivity of this method has also been enhanced greatly as compared to that without CHA. Our method has great potential to be applied for detecting different types of targets and monitoring diverse molecular interactions by adapting the corresponding nucleotide sequence.