Xiaosheng Ye

Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration

Aptamers have emerged as promising molecular probes for in vivo cancer imaging, but the reported “always-on” aptamer probes remain problematic because of high background and limited contrast. To address this problem, we designed an activatable aptamer probe (AAP) targeting membrane proteins of living cancer cells and achieved contrast-enhanced cancer visualization inside mice. The AAP displayed a quenched fluorescence in its free state and underwent a conformational alteration upon binding to target cancer cells with an activated fluorescence. As proof of concept, in vitro analysis and in vivo imaging of CCRF-CEM cancer cells were performed by using the specific aptamer, sgc8, as a demonstration. It was confirmed that the AAP could be specifically activated by target cancer cells with a dramatic fluorescence enhancement and exhibit improved sensitivity for CCRF-CEM cell analysis with the cell number of 118 detected in 200 μl binding buffer. In vivo studies demonstrated that activated fluorescence signals were obviously achieved in the CCRF-CEM tumor sites in mice. Compared to always-on aptamer probes, the AAP could substantially minimize the background signal originating from nontarget tissues, thus resulting in significantly enhanced image contrast and shortened diagnosis time to 15 min. Furthermore, because of the specific affinity of sgc8 to target cancer cells, the AAP also showed desirable specificity in differentiating CCRF-CEM tumors from Ramos tumors and nontumor areas. The design concept can be widely adapted to other cancer cell-specific aptamer probes for in vivo molecular imaging of cancer.

Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

Poly(Thymine)-Templated Fluorescent Copper Nanoparticles for Ultrasensitive Label-Free Nuclease Assay and Its Inhibitors Screening

Noble-metal fluorescent nanoparticles have attracted considerable interest on account of their excellent properties and potential applicable importance in many fields. Particularly, we recently found that poly(thymine) (poly T) could template the formation of fluorescent copper nanoparticles (CuNPs), offering admirable potential as novel functional biochemical probes. However, exploration of poly T-templated CuNPs for application is still at a very early stage. We report herein for the first example to develop a novel ultrasensitive label-free method for the nuclease (S1 nuclease as a model system) assay, and its inhibitors screening using the poly T-templated fluorescent CuNPs. In this assay, the signal reporter of poly T of 30 mer (T30) kept the original long state in the absence of nuclease, which could effectively template the formation of fluorescent CuNPs. In the presence of nuclease, poly T was digested to mono- or oligonucleotide fragments with decrease of fluorescence. The proposed method was low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. The assay process was very rapid with only 5 min for the formation of fluorescent CuNPs. The capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified. A high sensitivity exhibited with a detectable minimum concentration of 5 × 10(-7) units μL(-1) S1 nuclease, which was about 1-4 orders of magnitude more sensitive than the developed approaches.

Iodide-Responsive Cu–Au Nanoparticle-Based Colorimetric Platform for Ultrasensitive Detection of Target Cancer Cells

Colorimetric analysis is promising in developing facile, fast, and point-of-care cancer diagnosis techniques, but the existing colorimetric cancer cell assays remain problematic because of dissatisfactory sensitivity as well as complex probe design or synthesis. To solve the problem, we here present a novel colorimetric analytical strategy based on iodide-responsive Cu-Au nanoparticles (Cu-Au NPs) combined with the iodide-catalyzed H2O2-TMB (3,3,5,5-tetramethylbenzidine) reaction system. In this strategy, bimetallic Cu-Au NPs prepared with an irregular shape and a diameter of ∼15 nm could chemically absorb iodide, thus indirectly inducing colorimetric signal variation of the H2O2-TMB system. By further utilizing its property of easy biomolecule modification, a versatile colorimetric platform was constructed for detection of any target that could cause the change of Cu-Au NPs concentration via molecular recognition. As proof of concept, an analysis of human leukemia CCRF-CEM cells was performed using aptamer Sgc8c-modified Cu-Au NPs as the colorimetric probe. Results showed that Sgc8c-modified Cu-Au NPs successfully achieved a simple, label-free, cost-effective, visualized, selective, and ultrasensitive detection of cancer cells with a linear range from 50 to 500 cells/mL and a detection limit of 5 cells in 100 μL of binding buffer. Moreover, feasibility was demonstrated for cancer cell analysis in diluted serum samples. The iodide-responsive Cu-Au NP-based colorimetric strategy might not only afford a new design pattern for developing cancer cell assays but also greatly extend the application of the iodide-catalyzed colorimetric system.

A facile graphene oxide-based DNA polymerase assay.

The DNA polymerase assay is fundamental for related molecular biology investigations and drug screenings, however, the commonly used radioactive method is laborious and restricted. Herein, we report a novel, simple and cost-effective fluorometric DNA polymerase detection method by utilizing graphene oxide (GO) as a signal switch. In this strategy, in the absence of DNA polymerase, the fluorophore-labeled template ssDNA could be strongly adsorbed and almost entirely quenched by GO. However, as DNA polymerase exists, the polymerized dsDNA product might lead to a much lower quenching efficiency after addition of GO due to the much weaker interaction of dsDNA with GO than ssDNA, thus resulting in a much higher fluorescence signal detected. As proof of concept, the quantitative DNA polymerase activity assay was performed using the Klenow fragment exo(-) (KF(-)) as a model. It was confirmed that, after optimization of detection conditions, KF(-) activity could be sensitively detected through facile fluorescence measurements, with a detection limit of 0.05 U mL(-1) and a good linear correlation between 0.05-2.5 U mL(-1) (R(2) = 0.9928). In addition, this GO-based method was further inspected to evaluate the inhibitive behaviors of several drugs toward KF(-) activity, the result of which firmly demonstrated its potential application in polymerization-targeted drug screening.

The adenine DNA self-assembly of pH- and near-infrared-responsive gold nanorod vehicles for the chemothermal treatment of cancer cells

Despite the remarkable progress in the construction of nanostructures for drug delivery in cancer therapy, multifunctional nanoplatforms with synergistic advantages over any single-model nanostructures are still encouraging. Herein, a dual pH- and near-infrared (NIR)-responsive nanotherapeutic system for the chemothermal treatment of cancer cells was achieved by using adenine DNA coated gold nanorod vehicles (poly(A)/AuNR). The poly(A)/AuNRs were prepared by self-assembling thiolated poly(A) on the surface of the AuNRs via Au-S bonding. In this system, the poly(A) provided the ability to load coralyne, a model chemotherapeutic drug, through adenine-coralyne-adenine specific binding. Under low-pH or high-temperature conditions, adenine-coralyne-adenine would be unstable, leading to the stimuli-responsive release of coralyne for cancer chemotherapy. The AuNRs showed a high efficiency for the conversion of NIR light into heat, providing the fundamental basis of hyperthermal cancer therapy, and also promoting the triggered release of coralyne from poly(A) upon NIR irradiation. The characteristics of the poly(A)/AuNRs have been investigated by using TEM and UV-vis spectroscopy. It was shown that about 160 copies of poly(A) could be assembled on one AuNR prepared with the dimensions of about 30 nm in length and 10 nm in width, and each poly(A)/AuNR could load about 2500 coralyne molecules. The in vitro studies using human hepatoma SMMC-7721 cells demonstrated that the coralyne loaded poly(A)/AuNRs could be endocytosed and demonstrated an efficient operation in a cellular acidic environment and NIR irradiation, leading to significant cytotoxicity through the excellent chemothermal synergistic effects. We believe that this developed multimode nanostructure will provide potential applications for cancer therapy.

Gold nanorod-seeded synthesis of Au@Ag/Au nanospheres with broad and intense near-infrared absorption for photothermal cancer therapy

As a widely-adopted agent for photothermal therapy (PTT), gold nanorods (Au NRs) remain problematic due to the cytotoxicity derived from cetyltrimethylammonium bromide (CTAB) and the comparatively weak and narrow near-infrared (NIR) absorption band. To address this problem, in this study, we propose a shape-controllable and spectrum-adjustable method of synthesis for Au@Ag/Au nanoparticles (NPs) through first coating a Ag nanolayer on the Au NR seed and a subsequent replacement reaction with HAuCl4 to yield a Ag/Au alloy nanoshell. Results from TEM and UV-vis spectra analysis showed that the thickness of the Ag layers directly determined the shape and size of the NPs, and the formation of Ag/Au nanoshells effectively enhanced the NIR absorbance of the NPs. Remarkably, the optimum Au@Ag/Au nanospheres (NSs) with a diameter of ∼40 nm were revealed to have a broad and intense absorption cross section from 400 to 1100 nm, a ∼4.7 times higher hyperthermic effect than Au NRs, and low dark-cytotoxicity. By using A549 lung cancer as the model, a series of in vitro investigations was performed and demonstrated that Au@Ag/Au NSs could efficaciously kill cancer cells under a 980 nm irradiation. The efficacy of PTT could be further improved by increasing the concentration, incubation time or irradiation time of the NSs. Moreover, a preliminary in vivo study also showed that, after injection into the A549 tumor, Au@Ag/Au NSs could cause an obvious necrosis at the irradiation site. Thus, a novel, promising and highly-effective NIR PTT agent has been developed, which might greatly advance the application of PTT in biomedical research.

Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy.

DNA nanotriangle-scaffolded activatable aptamer probe with ultralow background and robust stability for cancer theranostics

Activatable aptamers have emerged as promising molecular tools for cancer theranostics, but reported monovalent activatable aptamer probes remain problematic due to their unsatisfactory affinity and poor stability. To address this problem, we designed a novel theranostic strategy of DNA nanotriangle-scaffolded multivalent split activatable aptamer probe (NTri-SAAP), which combines advantages of programmable self-assembly, multivalent effect and target-activatable architecture. Methods: NTri-SAAP was assembled by conjugating multiple split activatable aptamer probes (SAAPs) on a planar DNA nanotriangle scaffold (NTri). Leukemia CCRF-CEM cell line was used as the model to investigate its detection, imaging and therapeutic effect both in vitro and in vivo. Binding affinity and stability were evaluated using flow cytometry and nuclease resistance assays. Results: In the free state, NTri-SAAP was stable with quenched signals and loaded doxorubicin, while upon binding to target cells, it underwent a conformation change with fluorescence activation and drug release after internalization. Compared to monovalent SAAP, NTri-SAAP displayed greatly-improved target binding affinity, ultralow nonspecific background and robust stability in harsh conditions, thus affording contrast-enhanced tumor imaging within an extended time window of 8 h. Additionally, NTri-SAAP increased doxorubicin loading capacity by ~5 times, which further realized a high anti-tumor efficacy in vivo with 81.95% inhibition but no obvious body weight loss. Conclusion: These results strongly suggest that the biocompatible NTri-SAAP strategy would provide a promising platform for precise and high-quality theranostics.