Huiting Lu

Highly Sensitive Multiple microRNA Detection Based on Fluorescence Quenching of Graphene Oxide and Isothermal Strand-Displacement Polymerase Reaction

A simple, highly sensitive, and selective multiple microRNA (miRNA) detection method based on the graphene oxide (GO) fluorescence quenching and isothermal strand-displacement polymerase reaction (ISDPR) was proposed. The capability to discriminate ssDNA and double-stranded nucleic acid structure coupled with the extraordinary fluorescence quenching of GO on multiple organic dye allows the proposed strategy to simultaneously and selectively detect several miRNA labeled with different dyes in the same solution, while the ISDPR amplification endows the detection method with high sensitivity. The strong interaction between ssDNA and GO led to the fluorescent ssDNA probe exhibiting minimal background fluorescence. Upon the recognition of specific target miRNA, an ISDPR was triggered to produce numerous massive specific DNA-miRNA duplex helixes, and a strong emission was observed due to the weak interaction between the DNA-miRNA duplex helix and GO. A miRNA biosensor down to 2.1 fM with a linear range of 4 orders of magnitude was obtained. Furthermore, the large planar surface of GO allows simultaneous quenching of several DNA probes with different dyes and produces a multiple biosensing platform with high sensitivity and selectivity, which has promising application in profiling the pattern of miRNA expression and biomedical research.

Trace and Label-Free MicroRNA Detection Using Oligonucleotide Encapsulated Silver Nanoclusters as Probes

A simple, sensitive, and label-free method for microRNA (miRNA) biosensing was described using oligonucleotide encapsulated silver nanoclusters (Ag-NCs) as effective electrochemical probes. The functional oligonucleotide probe integrates both recognition sequence for hybridization and template sequence for in situ synthesis of Ag-NCs, which appears to possess exceptional metal mimic enzyme properties for catalyzing H(2)O(2) reduction. The miRNA assay employs gold electrodes to immobilize the molecular beacon (MB) probe. After the MB probe subsequently hybridizes with the target and functional probe, the oligonucleotide encapsulated Ag-NCs are brought to the electrode surface and produce a detection signal, in response to H(2)O(2) reduction. An electrochemical miRNA biosensor down to 67 fM with a linear range of 5 orders of magnitude was obtained. Meanwhile, the MB probe allows the biosensor to detect the target with high selectivity. The Ag-NCs-based approach provides a novel avenue to detect miRNA with high sensitivity and selectivity while avoiding laborious label and signal amplification. It is convinced that rational introduction of signal amplification strategy to the Ag-NCs-based bioanalysis can further improve the sensitivity. To our best knowledge, this is the first application of the electrocatalytic activity of Ag-NCs in bioanalysis, which would be attractive for genetic analysis and clinic biomedical application.

Functionalized Graphene Oxide Mediated Adriamycin Delivery and miR-21 Gene Silencing to Overcome Tumor Multidrug Resistance In Vitro

Multidrug resistance (MDR) is a major impediment to successful cancer chemotherapy. Co-delivery of novel MDR-reversing agents and anticancer drugs to cancer cells holds great promise for cancer treatment. MicroRNA-21 (miR-21) overexpression is associated with the development and progression of MDR in breast cancer, and it is emerging as a novel and promising MDR-reversing target. In this study, a multifunctional nanocomplex, composed of polyethylenimine (PEI)/poly(sodium 4-styrenesulfonates) (PSS)/graphene oxide (GO) and termed PPG, was prepared using the layer-by-layer assembly method to evaluate the reversal effects of PPG as a carrier for adriamycin (ADR) along with miR-21 targeted siRNA (anti-miR-21) in cancer drug resistance. ADR was firstly loaded onto the PPG surface (PPGADR) by physical mixing and anti-miR-21 was sequentially loaded onto PPGADR through electric absorption to form anti-miR-21PPGADR. Cell experiments showed that PPG significantly enhanced the accumulation of ADR in MCF-7/ADR cells (an ADR resistant breast cancer cell line) and exhibited much higher cytotoxicity than free ADR, suggesting that PPG could effectively reverse ADR resistance of MCF-7/ADR. Furthermore, the enhanced therapeutic efficacy of PPG could be correlated with effective silencing of miR-21 and with increased accumulation of ADR in drug-resistant tumor cells. The endocytosis study confirmed that PPG could effectively carry drug molecules into cells via the caveolae and clathrin-mediated endocytosis pathways. These results suggest that this PPG could be a potential and efficient non-viral vector for reversing MDR, and the strategy of combining anticancer drugs with miRNA therapy to overcome MDR could be an attractive approach in cancer treatment.

Fluorescent MoS2 Quantum Dots: Ultrasonic Preparation, Up-Conversion and Down-Conversion Bioimaging, and Photodynamic Therapy

Small size molybdenum disulfide (MoS2) quantum dots (QDs) with desired optical properties were controllably synthesized by using tetrabutylammonium-assisted ultrasonication of multilayered MoS2 powder via OH-mediated chain-like Mo-S bond cleavage mode. The tunable up-bottom approach of precise fabrication of MoS2 QDs finally enables detailed experimental investigations of their optical properties. The synthesized MoS2 QDs present good down-conversion photoluminescence behaviors and exhibit remarkable up-conversion photoluminescence for bioimaging. The mechanism of the emerging photoluminescence was investigated. Furthermore, superior (1)O2 production ability of MoS2 QDs to commercial photosensitizer PpIX was demonstrated, which has great potential application for photodynamic therapy. These early affording results of tunable synthesis of MoS2 QDs with desired photo properties can lead to application in fields of biomedical and optoelectronics.

Highly Sensitive and Selective MicroRNA Detection Based on DNA-Bio-Bar-Code and Enzyme-Assisted Strand Cycle Exponential Signal Amplification

Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

Label-free and ultrasensitive microRNA detection based on novel molecular beacon binding readout and target recycling amplification

A label-free and high-sensitive microRNA (miRNA) detection approach by coupling a metal ion-meditated conformational molecular beacon (MB), using novel fluorescent Ag nanocluster (AgNCs) as fluorophore, with endonuclease-assisted target recycling amplification was developed. The assay comprised an Hg(2+) ion-meditated conformational MB probe and an assistant probe that do not hybridize with each other at a specific temperature and can be annealed to each other in the presence of the target to form a Y-shape junction structure and released Hg(2+). The target-MB hybridization event with the help of assistant probe can readily be read out based on the efficient fluorescence quenching of AgNCs by released Hg(2+), while the Y-shape junction structure consisting of the probe MB, assistant probe and target miRNA could be recognized by the endonuclease Nt.BbvCI. The MB probe was then effectively cleaved by the endonuclease, and the regenerated assistant probe and the target further attended another cleavage cycle to implement the signal amplification. The competition displacing interaction between the target and the Hg(2+) endows the biosensor with high sequence discrimination capability, while the high signal-to-noise ratio and target recycling amplification allows the biosensor to detect the target with high sensitivity. Under the optimal conditions, the concentration of target miRNA could be conveniently read out with a linear range from 10 pM to 1 fM. The proposed approach, avoiding any laborious label, possessing high sensitivity and selectivity, provided significant potential applications in future clinical analysis.

Highly sensitive and selective chemiluminescent imaging for DNA detection by ligation-mediated rolling circle amplified synthesis of DNAzyme

A highly sensitive DNA biosensing method down to sub-femtomolar level with excellent selectivity was proposed by designing an amplified synthesis of horseradish peroxidase mimicking DNAzyme and introducing the amplified DNAzyme to chemiluminescent (CL) imaging. The amplified synthesis was achieved by combining a target DNA related ligase reaction with rolling circle amplification (RCA), which produced thousands of repeated sequences to bind hemin and form a mass of horseradish peroxidase-mimicing DNAzyme units. The amplification strategy greatly enhanced the CL emission of the luminol-H(2)O(2) system. The genotyping method displayed highly specific biochemistry in allele discrimination. The novel CL imaging strategy based on ligation-mediated RCA synthesis of DNAzyme showed high fidelity in discriminating single-base mismatch and efficiently facilitated signal amplification for sensitive target DNA detection. It could detect DNA ranging from 1×10(-15) M to 1×10(-11) M with a detection limit of 0.26 fM. The proposed approach provided a robust, cost-efficient, highly sensitive and specific platform for genetic target analysis in bioanalysis and clinic biomedical application.

Multifunctional Poly(l-lactide)–Polyethylene Glycol-Grafted Graphene Quantum Dots for Intracellular MicroRNA Imaging and Combined Specific-Gene-Targeting Agents Delivery for Improved Therapeutics

Photoluminescent (PL) graphene quantum dots (GQDs) with large surface area and superior mechanical flexibility exhibit fascinating optical and electronic properties and possess great promising applications in biomedical engineering. Here, a multifunctional nanocomposite of poly(l-lactide) (PLA) and polyethylene glycol (PEG)-grafted GQDs (f-GQDs) was proposed for simultaneous intracellular microRNAs (miRNAs) imaging analysis and combined gene delivery for enhanced therapeutic efficiency. The functionalization of GQDs with PEG and PLA imparts the nanocomposite with super physiological stability and stable photoluminescence over a broad pH range, which is vital for cell imaging. Cell experiments demonstrate the f-GQDs excellent biocompatibility, lower cytotoxicity, and protective properties. Using the HeLa cell as a model, we found the f-GQDs effectively delivered a miRNA probe for intracellular miRNA imaging analysis and regulation. Notably, the large surface of GQDs was capable of simultaneous adsorption of agents targeting miRNA-21 and survivin, respectively. The combined conjugation of miRNA-21-targeting and survivin-targeting agents induced better inhibition of cancer cell growth and more apoptosis of cancer cells, compared with conjugation of agents targeting miRNA-21 or survivin alone. These findings highlight the promise of the highly versatile multifunctional nanocomposite in biomedical application of intracellular molecules analysis and clinical gene therapeutics.

Fabricating Pt/Sn–In2O3 Nanoflower with Advanced Oxygen Reduction Reaction Performance for High-Sensitivity MicroRNA Electrochemical Detection

Herein, an efficient electrochemical tracer with advanced oxygen reduction reaction (ORR) performance was designed by controllably decorating platinum (Pt) (diameter, 1 nm) on the surface of compositionally tunable tin-doped indium oxide nanoparticle (Sn-In2O3) (diameter, 25 nm), and using the Pt/Sn-In2O3 as electrochemical tracer and interfacial term hairpin capture probe, a facile and ultrasensitive microRNA (miRNA) detection strategy was developed. The morphology and composition of the generated Pt/Sn-In2O3 NPs were comprehensively characterized by spectroscopic and microscopic measurements, indicating numerous Pt uniformly anchored on the surface of Sn-In2O3. The interaction between Pt and surface Sn as well as high Pt(111) exposure resulted in the excellent electrochemical catalytic ability and stability of the Pt/Sn-In2O3 ORR. As proof-of-principle, using streptavidin (SA) functionalized Pt/Sn-In2O3 (SA/Pt/Sn-In2O3) as electrochemical tracer to amplify the detectable signal and a interfacial term hairpin probe for target capture probe, a miRNA biosensor with a linear range from 5 pM to 0.5 fM and limit of detection (LOD) down to 1.92 fM was developed. Meanwhile, the inherent selectivity of the term hairpin capture probe endowed the biosensor with good base discrimination ability. The good feasibility for real sample detection was also demonstrated. The work paves a new avenue to fabricate and design high-effective electrocatalytic tracer, which have great promise in new bioanalytical applications.

Peroxidase-like Fe3O4 nanocomposite for activatable reactive oxygen species generation and cancer theranostics

Photodynamic therapy (PDT) that utilizes apoptosis induced by reactive oxygen species (ROS) has received extensive attention in practical cancer therapy. However, the hypoxic microenvironment of solid tumors significantly limits the efficacy of therapy. Approaches that overcome the barriers to PDT in hypoxic conditions by simultaneously producing ROS exogenously and improving the oxygenation of tumors have never been studied. Herein, an activatable ROS platform was designed that uses the high reactivity of peroxidase-like Fe3O4 toward endogenous hydrogen peroxide (H2O2) to concurrently generate ˙OH as a therapeutic agent and provide O2 for oxygen-dependent PDT. Multifunctional chitosan-encapsulated Fe3O4 nanoparticles modified with CuS and porphyrin (FCCP NPs) were fabricated to achieve multimodal imaging and synergetic therapy. The FCCP NPs possess enhanced intrinsic peroxidase mimetic activity to produce ROS and O2 from endogenous H2O2. Multimodal imaging in vivo, including photoacoustic imaging (PAI), magnetic resonance imaging (MRI), photoluminescence imaging (PLI), and photothermal imaging (PTI), exploits the tumor-targeting property of FCCP NPs upon intravenous injection. It can induce cancer cell death with remarkable efficiency both in vitro and in vivo via synergetic treatment with PDT and photothermal therapy (PTT). This study demonstrates the promise of the activatable generation of ROS and O2 for PDT with nanotechnology to overcome a current deficiency in cancer therapies.