Dynamic Single Molecular Rulers: Toward Quantitative Detection of MicroRNA-21 in Living Cells.

Innovative techniques to measure microRNA (miRNA) in vivo could greatly improve the fundamental understanding of complex cellular processes. Herein, we report a novel method for real-time, quantitative miRNA detection inside living cells based on core-satellite plasmon rulers (PRs). This approach allows for the statistical analysis of single hybridization event caused by target miRNA. We investigated hundreds of satellite leaving events and found that the distribution of the time range for one strand displacement event is miRNA concentration-dependent, which obeyed Poisson statistics. Probing several such PRs under dark-field microscopy would provide precise determination of miRNA in vitro and in living cells, without photobleaching or blinking of the fluorophores. We believe the simple and practical approach on the basis of dynamic PRs with single-molecule sensitivity combined with statistical analysis hold promising potential to visualize native nucleic acids with short sequence and low-abundance.

Multi-segmented CdS-Au nanorods for electrochemiluminescence bioanalysis.

In this study, we have developed a programmable electrochemiluminescence (ECL) system based on multi-segmented CdS-Au nanorod arrays with a sequential and highly tunable structure. The nanorod arrays were synthesized by an electrodeposition method using anodic aluminum oxide (AAO) as the template in which the Au and CdS segments were alternately electrodeposited. Compared to pure CdS nanorod arrays, multi-segmented CdS-Au nanorod arrays have showed a better ECL performance, which can be attributed to two factors: the favorable electron transfer and the surface plasma resonance (SPR) effect of the Au segment. On the one hand, we demonstrated that the Au segment can increase the charge transfer rate of CdS, which is beneficial for the ECL process because the generation of the radical state needs to accept electrons and then generate the radical state. On the other hand, the SPR of Au plasmon-induced local electromagnetic field enhancement can increase the radiative decay rate of CdS which makes the ECL process more efficient and lead to a higher ECL intensity. And also, an ECL sensor with multi-segmented CdS-Au nanorod arrays was constructed to detect prostate protein antigen (PSA). This study provides some basis for designing high-performance ECL emission materials and the construction of biosensors.

Exploration of the Kinetics of Toehold-Mediated Strand Displacement via Plasmon Rulers.

DNA/RNA strand displacement is one of the most fundamental reactions in DNA and RNA circuits and nanomachines. In this work, we reported an exploration of the dynamic process of the toehold-mediated strand displacement via core-satellite plasmon rulers at the single-molecule level. Applying plasmon rulers with unlimited lifetime, single-strand displacement triggered by the invader that resulted in stepwise leaving of satellite from the core was continuously monitored by changes of scattering signal for hours. The kinetics of strand displacement in vitro with three different toehold lengths have been investigated. Also, the study revealed the difference in the kinetics of strand displacement between DNA/RNA and DNA/DNA duplexes. For the kinetics study in vivo, influence from the surrounding medium has been evaluated using both phosphate buffer and cell lysate. Applying core-satellite plasmon rulers with high signal/noise ratio, kinetics study in living cells proceeded for the first time, which was not possible by conventional methods with a fluorescent reporter. The plasmon rulers, which are flexible, easily constructed, and robust, have proven to be effective tools in exploring the dynamical behaviors of biochemical reactions in vivo.

Plasmon-Enhanced Electrochemiluminescence for Nucleic Acid Detection Based on Gold Nanodendrites.

Gold nanodendrites (Au NDs) exhibit extremely strong electromagnetic field located around multiple tip branches due to a plasmon coupling effect. In this work, a novel LSPR-enhanced ECL emission from CdTe nanocrystals (NCs) by Au NDs for the detection of nucleic acid is reported. This system is composed of a thin film of CdTe NCs on glassy carbon electrode (GCE) as anodic ECL emitter and Au NDs as plasmon enhancer. DNA tetrahedron embedded with a stem-loop hairpin structure on one edge was applied as a switch to regulate the distance between CdTe NCs and Au NDs. At original state, the hairpin structure was closed and DNA tetrahedron played in a relaxed state on CdTe NCs film. The ECL emission of CdTe NCs was quenched by proximal Au NDs due to Förster resonance energy transfer (FRET), which was defined as the "turn-off" mode. After the complementary hybridization with target DNA, the hairpin structure changed to a rodlike configuration, resulting in an increased distance between CdTe NCs and Au NDs, and a significant enhancement of ECL induced by LSPR of Au NDs, which was defined as a "turn-on" mode. Along with the asymmetric modification method, a controllable and versatile pathway for modifying nanomaterials, the ECL sensor performed well with great stability and repeatability for nucleic acid detection in the range from 1.0 to 500 fM. Considering the high sensitivity and selectivity in the serum sample assay, this proposed method indicates a great potential for bioassay application.

Ultrasensitive MicroRNA Assay via Surface Plasmon Resonance Responses of Au@Ag Nanorods Etching.

Quantification of trace serum circulate microRNAs is extremely important in clinical diagnosis but remains a great challenge. Herein we developed an ultrasensitive platform for microRNA 141 (miR-141) detection based on a silver coated gold nanorods (Au@Ag NRs) etching process accompanied by surface plasmon resonance (SPR) shift. Both SPR absorption and scattering responses were monitored. Combined amplification cascades of catalyzed hairpin assembly (CHA) and hybridization chain reaction (HCR) with the sensitive SPR responses of plasmonic Au@Ag NRs, the proposed bioassay exhibited ultrahigh sensitivity toward miRNA-141 with dynamic range from 5.0 × 10-17 M to 1.0 × 10-11 M. With target concentration higher than 1.0 × 10-13 M, the color of the solution changed obviously that could be observed with naked eyes. Under dark-field microscopy observation of individual particle, a limit of detection down to 50 aM could be achieved. Owing to the superior sensitivity and selectivity, the proposed method was applied to detecting trace microRNA in serum. Similar SPR assays could be developed simply by redesigning the switching aptamer for the detections of other microRNAs or targets such as small molecule, DNA, or protein. Considering the convenient operation, good performance and simple observation modes of this method, it may have great potential in trace bioanalysis for clinical applications.

DNA tetrahedral scaffolds-based platform for the construction of electrochemiluminescence biosensor.

Proximal metallic nanoparticles (NPs) could quench the electrochemiluminescence (ECL) emission of semiconductor quantum dots (QDs) due to Förster energy transfer (FRET), but at a certain distance, the coupling of light-emission with surface plasmon resonance (SPR) result in enhanced ECL. Thus, the modification strategies and distances control between QDs and metallic NPs are critical for the ECL intensity of QDs. In this strategy, a SPR enhanced ECL sensor based on DNA tetrahedral scaffolds modified platform was reported for the detection of telomerase activity. Due to the rigid three-dimensional structure, DNA tetrahedral scaffolds grafting on the electrode surface could accurately modulate the distance between CdS QDs and luminol labelled gold nanoparticles (L-Au NPs), meanwhile provide an enhanced spatial dimension and accessibility for the assembly of multiple L-Au NPs. The ECL intensities of both CdS QDs (-1.25V vs. SCE) and luminol (+0.33V vs. SCE) gradually increased along with the formation of multiple L-Au NPs at the vertex of DNA tetrahedral scaffolds induced by telomerase, bringing in a dual-potential ECL analysis. The proposed method showed high sensitivity for the identification of telomerase and was successfully applied for the differentiation of cancer cells from normal cells. This work suggests that DNA tetrahedral scaffolds could serve as an excellent choice for the construction of SPR-ECL system.

Distance mediated electrochemiluminescence enhancement of CdS thin films induced by the plasmon coupling of gold nanoparticle dimers.

Gold nanoparticle dimers assembled on the surface of CdS QD thin films served as nano-antennas to mediate the distance-dependent plasmon enhanced electrochemiluminescence of QDs.

Dual-Wavelength Electrochemiluminescence Ratiometry Based on Resonance Energy Transfer between Au Nanoparticles Functionalized g-C3N4 Nanosheet and Ru(bpy)3(2+) for microRNA Detection.

Here, a dual-wavelength ratiometric electrochemiluminescence (ECL) approach is reported based on resonance energy transfer (RET) from graphite-like carbon nitride nanosheet (g-C3N4 NS) to Ru(bpy)3(2+) for sensitive detection of microRNA (miRNA). In this approach, Au nanoparticles (Au NPs) functionalized g-C3N4 NS nanohybrid (Au-g-C3N4 NH) coated on glassy carbon electrode (GCE) could exhibit strong and stable ECL emissions with emission peak centered at 460 nm. The ECL emission at such wavelength matched well with the absorption peak of Ru(bpy)3(2+) as well as impeccably stimulating the emission of Ru(bpy)3(2+) at the wavelength of 620 nm, producing ECL-RET with high efficiency. Thus, based on the ECL signals quenching at 460 nm and increasing at 620 nm, a dual-wavelength ratiometric ECL-RET system was achieved. This system was then utilized for determination of target miRNA. With the attachment of thiol-modified molecular beacon on Au-g-C3N4 NH, target miRNA hybridized with the molecular beacon to form a DNA-RNA duplex. The obtained DNA-RNA duplex could be cleaved by duplex-specific nuclease to release target miRNA which would take part in the next cycle for further hybridization. Finally, the introducing of Ru(bpy)3(2+) was through the probe DNA-Ru(bpy)3(2+) complementary with the rest single-strand DNA on electrode. By measuring the ratio of ECL(460 nm)/ECL(620 nm), we could accurately quantify the concentration of miRNA-21 in a wide range from 1.0 fM to 1.0 nM. This work provides an important reference for the study of dual-wavelength ECL ratiometry and also exhibits potential capability in the detection of nucleic acids.

Elevated expression of Foxp3 in tumor-infiltrating Treg cells suppresses T-cell proliferation and contributes to gastric cancer progression in a COX-2-dependent manner.

The transcription factor Foxp3 plays a key role in CD4(+)CD25(+) regulatory T (Treg) cell function. A correlation has been shown between survival and the frequency of tumor-infiltrating Foxp3-positive Treg cells in cancer patients. However, few studies have characterized the regulation of Foxp3 expression and function in Treg cells, which are known to comprise distinct subsets, with different roles in the complex tumor microenvironment. Here, we show that significantly more Foxp3-positive Treg cells accumulated in gastric tumors. In addition, we found increased expression of Foxp3 protein per cell in tumor-infiltrating Treg cells. Moreover, elevated Foxp3 expression in tumor-infiltrating Treg cells was associated with the TNM stage in gastric cancer patients. Importantly, further investigation within the tumor microenvironment showed that expression of Foxp3 in Treg cells correlated with expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Furthermore, Treg cells with higher levels of Foxp3 were able to suppress the proliferation of autologous CD4(+)CD25(-) T cells. The suppression of the effector T-cell response was reversed by COX inhibitors and PGE(2) receptor-specific antagonists. Our data demonstrate a mechanism by which tumor-infiltrating Treg cells with increased Foxp3 expression can mediate immune suppression via COX-2/PGE(2) production in the gastric cancer microenvironment. Thus, we provide new insights into overcoming regulatory T-cell activity, which may be beneficial for the treatment of human gastric cancer.

CD4(+)CD25(+)CD127(low/-) regulatory T cells express Foxp3 and suppress effector T cell proliferation and contribute to gastric cancers progression.

Increased populations of regulatory T cells (Tregs) impair anti-tumor immunity. Recently, the transcription factor Foxp3 has been reported to play a key role in CD4(+)CD25(+) regulatory T cell function and represents a specific marker for these cells. However, Foxp3 is a nuclear protein and is of limited value in the isolation of Tregs, which is a major reason that many functionally relevant aspects of Treg cells are still unknown. Here, we have characterized CD4(+)CD25(+)CD127(low/)- as the surface marker of regulatory T cells in gastric cancer. 88.1-96.1%of CD25(+)CD127(low/-) T cells expressed Foxp3, the frequency of CD4(+)CD25(+)CD127(low/-) regulatory T cells in the peripheral blood of gastric cancer patients was significantly higher than that in healthy controls. Increased CD4(+)CD25(+)CD127(low/-) regulatory T cells were also present in the tumor microenvironment, such as those found in the ascites fluid, tumor tissue or adjacent lymph nodes. Particularly those Treg cells associated with the TNM stage. In addition, we found that CD4(+)CD25(+)CD127(low/-) Tregs suppressed effector T cell proliferation and also correlated to advanced stage of gastric cancer. Thus, CD4(+)CD25(+)CD127(low/-) can be used as a selective biomarker to enrich human Treg cells and also to perform functional in vitro assays in gastric cancer.

[The effects of tumor antigen gene OVA66 on biological characteristics of tumor cells].

AIM: To explore the effects of tumor antigen gene OVA66 on the biological characteristics of tumor cells. METHODS: The eukaryotic expression vector pcDNA3.1-mock and recombined vector pcDNA3.1-OVA66 were transfected into hepatoma cell line SMMC-7721 by LipofectAMINETM 2000. After stable selection and clone proliferation of cells, the expression of OVA66 gene and protein was detected by RT-PCR, Western blot and immunocytochemical (ICC) staining. The cell growth cycle was analyzed by FACS. The ultrastructure was observed under electron microscope. The experiments in cell migration and invasion were performed in vitro. The OVA66-associated genes were analyzed by genechip technique after gene transfection. RESULTS: OVA66 was highly expressed in 7721 cells transfected with pcDNA3.1-OVA66 at mRNA level. Western blot result demonstrated that OVA66 protein content was notably increased compared with that in control cells. Electron microscope and FACS results indicated that OVA66 gene promoted the growth and proliferation of tumor cells. In vitro, the experiments in cell migration and invasion showed that this gene enhanced migration and invasion of tumor cells. The result of genechip demonstrated that the over- expression of OVA66 gene increased the expression of plasminogen activator inhibitor-1 gene (PAI-1), suggesting that it had some effects on tumor invasion and metastasis. CONCLUSION: Tumor antigen gene OVA66 and it's protein demonstrate a series of biological characteristics of tumor cells, which can promote the proliferation, migration and invasion of tumor cells.

[Identification of a novel HLA-A2-restrictive CTL epitope of an ovary cancer-associated antigen OVA66].

AIM: To identify a novel HLA-A2-restrictive CTL epitope of an ovary cancer-associated antigen OVA66. METHODS: Dendritic cells (DCs), induced from peripheral blood mononuclear cells(PBMCs) by cytokines, were confirmed by morphological observation and FACS. Mature DCs were pulsed with each of the two synthesized peptides which were selected as possible CTL epitopes by software analysis. The pulsed DCs were used to stimulate autologous CD8+ T cells from an HLA-A2+ healthy donor. One week later, the peptides-pulsed autologous PBMCs were used to stimulate the CD8+ T cells for another 3 times at weekly intervals. The stimulated CD8+ T cells were used as CTLs. The cytotoxicity of CTLs to target cells was detected by lactate dehydrogenase (LDH) release assay and the number of T cells secreting antigen-specific IFN-gamma in CTLs was analyzed by enzyme-linked immunospot assay (ELISPOT). RESULTS: The results of morphology observation and FACS indicated that mature DCs were induced from PBMCs. Of the two peptides, peptide L235(FLPDHINIV) induced peptide-specific CD8+ T cells that lysed HLA-A2+ T2 cells pulsed with L235 and OVA66+/HLA-A2+ SW480 cells. Compared with control peptide, L235 increased the number of IFN-gamma producing T cells. CONCLUSION: This novel OVA66-derived CTL epitope L235 can induce HLA-A2-restrictive CTL response, which lays the foundation for preparation of tumor-specific peptide vaccine.