Ping Ping Hu

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.

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.

Aptamer-mediated nanoparticle-based protein labeling platform for intracellular imaging and tracking endocytosis dynamics.

Although nanoparticles have been widely used as optical contrasts for cell imaging, the complicated prefunctionalized steps and low labeling efficiency of nanoprobes greatly inhibit their applications in cellular protein imaging. In this study, we developed a novel and general strategy that employs an aptamer not only as a recognizer for protein recognition but also as a linker for nanoreporter targeting to specifically label membrane proteins of interest and track their endocytic pathway. With this strategy, three kinds of nanoparticles, including gold nanoparticles, silver nanoparticles, and quantum dots (QDs), have been successfully targeted to the membrane proteins of interest, such as nucleolin or prion protein (PrP(C)). The following investigations on the subcellular distribution with fluorescent immunocolocalization assay indicated that PrP(C)-aptamer-QD complexes most likely internalized into cytoplasm through a classical clathrin-dependent/receptor-mediated pathway. Further single-particle tracking and trajectory analysis demonstrated that PrP(C)-aptamer-QD complexes exhibited a complex dynamic process, which involved three types of movements, including membrane diffusion, vesicle transportation, and confined diffusion, and all types of these movements were associated with distinct phases of PrP(C) endocytosis. Compared with traditional multilayer methods, our proposed aptamer-mediated strategy is simple in procedure, avoiding any complicated probe premodification and purification. In particular, the new double-color labeling strategy is unique and significant due to its superior advantages of targeting two signal reporters simultaneously in a single protein using only one aptamer. What is more important, we have constructed a general and versatile aptamer-mediated protein labeling nanoplatform that has shown great promise for future biomedical labeling and intracellular protein dynamic analysis.

Highly Photoluminescent Molybdenum Oxide Quantum Dots: One-Pot Synthesis and Application in 2,4,6-Trinitrotoluene Determination.

As a well-studied transition-metal semiconductor material, MoOx has a wider band gap than molybdenum disulfide (MoS2), and its property varies dramatically for the existence of several different allotropes and suboxide phases of molybdenum oxides (MoOx, x < 3). In this manuscript, a one-pot method possessing the advantages of one pot, easily prepared, rapid, and environmentally friendly, has been developed for facile synthesis of highly photoluminescent MoOx quantum dots (MoOx QDs), in which commercial molybdenum disulfide (MoS2) powder and hydrogen peroxide (H2O2) are employed as the precursor and oxidant, respectively. The obtained MoOx QDs can be further utilized as an efficient photoluminescent probe, and a new turn-off sensor is developed for 2,4,6-trinitrotoluene (TNT) determination based on the fact that the photoluminescence of MoOx QDs can be quenched by the Meisenheimer complexes formed in the strong alkali solution through the inner filter effect (IFE). Under the optimal conditions, the decreased photoluminescence of MoOx QDs shows a good linear relationship to the concentration of TNT ranging from 0.5 to 240.0 μM, and the limit of detection was 0.12 μM (3σ/k). With the present turn-off sensor, TNT in river water samples can be rapidly and selectively detected without tedious sample pretreatment processes.

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.

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.

Label-Free Detection of Prion Protein with Its DNA Aptamer through the Formation of T-Hg2+-T Configuration

Though rapid tests were developed for mass screening of prion diseases in the last century, bovine spongiform encephalopathy (BSE) was still epidemic in some European countries. The main reason is that the sensitivity of such tests is insufficient for detecting animals that are incubating with prion diseases at the presymptomatic stage. Driven by this, in this contribution, we developed a novel sensitive label-free method taking advantage of DNA aptamer for prion proteins (PrP) detection through the formation of T-Hg(2+)-T configuration. In the presence of Hg(2+) ions, double-strand structures formed due to the strong binding affinity of Hg(2+) ions to the T bases of DNA aptamer, which dramatically enhanced the fluorescence of Syber Green I, a double-strand indicator. With the addition of prion protein, however, the specific interaction between prion protein and its aptamer forced the destruction of the double-strand structures, and thus the fluorescence of Syber Green I decreased. It was found that there is a linear relationship between the decreased fluorescence intensities and prion protein concentration ranging from 13.0 to 156.0 nmol/L. Compared with other methods, the method presented here holds the advantages of being label-free, rapid, highly sensitive, and selective, which shows great promise for clinical application.

Carbon nanotube–DNA hybrid used for activity monitoring and inhibitor screening of nuclease

Carbon nanotubes (CNTs) can efficiently quench the fluorescence of the adsorbed fluorophores and nonconvalently interact with soft single-stranded DNA (ssDNA). Upon disruption of CNTs-fluorescent oligonucleotides hybrid by nuclease S1, fluorescence turn-on was observed. Using this strategy, a platform based on fluorescence signal for monitoring the activity of nuclease with advantages of high sensitivity and commonality was established, and a linear relationship between initial cleavage reaction rate and nuclease S1 concentration is found in the range of 0.6-8.0 U mL(-1) with a detection limit of 0.08 U mL(-1). Furthermore, by taking pyrophosphate as an example, we use the assay to evaluate the prohibition effect on nuclease, and the extent of fluorescence recovery decreased linearly with increasing the concentration of pyrophosphate in the range of 0.2-1.4 mM, implying that the cleavage reaction by nuclease S1 was prohibited, and therefore this fluorescence assay can also be conveniently utilized for inhibitor screening of nuclease.

Coomassie brilliant blue R-250 as a new surface-enhanced Raman scattering probe for prion protein through a dual-aptamer mechanism

Surface-enhanced Raman scattering (SERS) spectra, which can provide large information about trace amount of chemical and biological species have been widely performed as a well-established tool in complex biological system. In this work, coomassie brilliant blue (R-250) with high affinity to proteins and high Raman activity was employed as a Raman reporter to probe prion protein (PrP) through a dual-aptamer mechanism, and thus an original strategy for PrP determination was proposed, which showed great potential to turn on the SERS response through specific recognition of anti-prion aptamers towards the target protein. Aptamers (Apt1 and Apt 2) recognizing distinct epitopes of PrP with high affinity were first conjugated to Ag@Si NPs, and Ag@Si-PrP/R-250-Ag@Si conjugates were obtained in the presence of PrP/R-250, inducing dramatically enhanced Raman signal. SERS responses enhanced with increasing amount of PrP and a linear equation of ISERS=6729.7+3091.2 cPrP was obtained in the range of 3.0-12.0×10(-9)M with the determination coefficient of 0.988. The proposed strategy is simple, rapid, and high specificity to probe protein-aptamer recognition in the solution.

A Visual Dual-Aptamer Logic Gate for Sensitive Discrimination of Prion Diseases-Associated Isoform with Reusable Magnetic Microparticles and Fluorescence Quantum Dots

Molecular logic gates, which have attracted increasing research interest and are crucial for the development of molecular-scale computers, simplify the results of measurements and detections, leaving the diagnosis of disease either “yes” or “no”. Prion diseases are a group of fatal neurodegenerative disorders that happen in human and animals. The main problem with a diagnosis of prion diseases is how to sensitively and selectively discriminate and detection of the minute amount of PrPRes in biological samples. Our previous work had demonstrated that dual-aptamer strategy could achieve highly sensitive and selective discrimination and detection of prion protein (cellular prion protein, PrPC, and the diseases associated isoform, PrPRes) in serum and brain. Inspired by the advantages of molecular logic gate, we further conceived a new concept for dual-aptamer logic gate that responds to two chemical input signals (PrPC or PrPRes and Gdn-HCl) and generates a change in fluorescence intensity as the output signal. It was found that PrPRes performs the “OR” logic operation while PrPC performs “XOR” logic operation when they get through the gate consisted of aptamer modified reusable magnetic microparticles (MMPs-Apt1) and quantum dots (QDs-Apt2). The dual-aptamer logic gate simplifies the discrimination results of PrPRes, leaving the detection of PrPRes either “yes” or “no”. The development of OR logic gate based on dual-aptamer strategy and two chemical input signals (PrPRes and Gdn-HCl) is an important step toward the design of prion diseases diagnosis and therapy systems.