Xiaohai Yang

Pyrene‐Excimer Probes Based on the Hybridization Chain Reaction for the Detection of Nucleic Acids in Complex Biological Fluids

China Scholarship Council (CSC); ACS; US NIH; China NSFC[20805038]; National Basic Research Program of China[2007CB935603, 2010CB732402]; China National Grand Program on Key Infectious Disease[2009ZX10004-312]; Key Project of Natural Science Foundation of China[90606003]; International Science & Technology Cooperation Program of China[2010DFB30300]; Hunan Provincial Natural Science Foundation of China[10JJ7002]

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.

Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction

Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 × 10−15 M.

Functionalized silica nanoparticles: a platform for fluorescence imaging at the cell and small animal levels.

Going in vivo, including living cells and the whole body, is very important for gaining a better understanding of the mystery of life and requires specialized imaging techniques. The diversity, composition, and temporal-spatial variation of life activities from cells to the whole body require the analysis techniques to be fast-response, noninvasive, highly sensitive, and stable, in situ and in real-time. Functionalized nanoparticle-based fluorescence imaging techniques have the potential to meet such needs through real-time and noninvasive visualization of biological events in vivo. Functionalized silica nanoparticles (SiNPs) doped with fluorescent dyes appear to be an ideal and flexible platform for developing fluorescence imaging techniques used in living cells and the whole body. We can select and incorporate different dyes inside the silica matrix either noncovalently or covalently. These form the functionalized hybrid SiNPs, which support multiplex labeling and ratiometric sensing in living systems. Since the silica matrix protects dyes from outside quenching and degrading factors, this enhances the photostability and biocompatibility of the SiNP-based probes. This makes them ideal for real-time and long-time tracking. One nanoparticle can encapsulate large numbers of dye molecules, which amplifies their optical signal and temporal-spatial resolution response. Integrating fluorescent dye-doped SiNPs with targeting ligands using various surface modification techniques can greatly improve selective recognition. Along with the endocytosis, functionalized SiNPs can be efficiently internalized into cells for noninvasive localization, assessment, and monitoring. These unique characteristics of functionalized SiNPs substantially support their applications in fluorescence imaging in vivo. In this Account, we summarize our efforts to develop functionalized dye-doped SiNPs for fluorescence imaging at the cell and small animal levels. We first discuss how to design and construct various functionalized dye-doped SiNPs. Then we describe their properties and imaging applications in cell surface receptor recognition, intracellular labeling, tracking, sensing, and controlled release. Additionally, we have demonstrated the promising application of dye-doped SiNPs as contrast imaging agents for in vivo fluorescence imaging in small animals. We expect these functionalized dye-doped SiNPs to open new opportunities for biological and medical research and applications.

Aptamer-based analysis of angiogenin by fluorescence anisotropy

Recognition and monitoring proteins in real time and in homogeneous solution has always been a difficult task. Here, we introduce a signal transduction strategy for quick protein recognition and real-time quantitative analysis in homogeneous solutions based on a high-affinity aptamer for protein angiogenin (Ang). The method takes advantage of the sensitive anisotropy signal change of fluorophore-labelled aptamer upon protein/aptamer binding. When the labelled aptamer is bound with its target protein Ang, the increased molecular weight causes the rotational motion of the fluorophore attached to the complex to become much slower. Therefore, increasing the amount of Ang results in a raised anisotropy value of the Ang/aptamer. By monitoring the anisotropy change, we are able to detect the binding events between the aptamer and Ang, and measure Ang concentration quantitatively in homogeneous solutions. This assay is highly selective, with a detection limit of 1 nM of Ang. The dissociation constant of the Ang/aptamer binding is determined in the nanomolar range and changes with increasing salt concentration. One can also use our assay to compare the binding affinities of different ligands for the target molecule. Ang in serum samples of malignant lung cancer was also detected. Efficient protein detection using aptamer-based fluorescence anisotropy measurements is expected to find wide applications in protein monitoring, cancer diagnosis, drug screening and other fields.

Graphene oxide–gold nanoparticles hybrids-based surface plasmon resonance for sensitive detection of microRNA

In this study, a simple and sensitive surface plasmon resonance (SPR) biosensor for miRNA detection was developed using graphene oxide-gold nanoparticles (GO-AuNPs) hybrids as signal amplification element. Taking advantage of the GO-AuNPs hybrids and their enhanced performance in SPR biosensors, the detection of miRNA was carried out in only two steps. Firstly, the thiolated capture DNA probe with a short complete complementary sequence was immobilized on the Au film surface to recognize the part sequence of target miRNA. Subsequently, the assistant DNA-linked GO-AuNPs hybrids were employed to bind the other section of the target. It was found that the developed SPR biosensor was able to achieve a detection limit as low as 1 fM. Moreover, the method showed excellent ability to discriminate differences among miRNA-200 family members. Notably, human miRNA from cancer cells could also be detected, and the results were in excellent agreement with the ones obtained using qRT-PCR. On the basis of these findings, we believe that this method has great potential for quantitative detection of miRNA in biomedical research and early clinical diagnostics.

Screening of DNA Aptamers against Myoglobin Using a Positive and Negative Selection Units Integrated Microfluidic Chip and Its Biosensing Application

An aptamer screening method using a positive and negative selection units integrated microfluidic chip was introduced. Here, myoglobin (Myo), one of the early markers to increase after acute myocardial infarction, was used as the model. After 7-round selection, the aptamers, which exhibited dissociation constants (K(d)) in the nanomolar range (from 4.93 to 6.38 nM), were successfully obtained using a positive and negative selection units integrated microfluidic chip. The aptamer with the highest affinity (K(d) = 4.93 nM) was then used for the fabrication of a label-free supersandwich electrochemical biosensor for Myo detection based on target-induced aptamer displacement. The detection limit of this aptamer-based electrochemical biosensor was 10 pM, which was significantly lower than that of those previous antibody-based biosensors for Myo detection. This work may not only develop a strategy for screening aptamer but also offer promising alternatives to the traditional analytical and immunological methods for Myo detection.

Real-time imaging of protein internalization using aptamer conjugates.

Angiogenin is a potent angiogenic factor that is known to play an important role in tumor angiogenesis. In this paper, we investigate the cellular internalization of angiogenin conjugated with its highly specific aptamer. By using fluorophore-labeled aptamer and confocal laser scanning microscopy, we have developed a novel and simple method by which to visualize the real-time process of angiogenin internalization. Specifically, when aptamer-angiogenin conjugates were added into cell cultures, conjugates could be selectively bound to HUVE cells (human umbilical vein endothelial cells) and MCF-7 cells (human breast cancer cells). Nuclear staining and Z-axis scanning studies demonstrated that the aptamer-angiogenin conjugates were internalized to intracellular organelles, and dynamic confocal imaging studies indicated that the conjugates were quickly internalized. These results provide the first evidence that a fluorophore-labeled aptamer can be used as a fluorescent probe to visualize the spatiotemporal process of protein internalization in real time.

Exciton Energy Transfer-Based Fluorescent Sensing through Aptamer-Programmed Self-Assembly of Quantum Dots

A novel exciton energy transfer-based ultrasensitive fluorescent sensing strategy for the detection of biological small molecules and protein has been established through split aptamer-programmed self-assembly of quantum dots (QDs). The signal is produced from exciton energy transfer of the self-assembled QDs. The recognition is accomplished using an aptamer sensor scaffold designed with two split fragment sequences, which specifically bind to the model analytes. The extent of particle assembly, induced by the analyte-triggered self-assembly of QDs, leads to an exciton energy transfer effect between interparticles, giving a readily detectable fluorescent quenching and red shift of the emission peak, which enables us to quantitate the target in dual signal modes. The application of the technique is well demonstrated using two representative split aptamer-based model systems for the detection of adenosine and thrombin. The sensitivity of this exciton energy transfer-based fluorescent sensing is much better than that of plasmonic coupling-based colorimetric methods. Limit of detections (LODs) down to 12 nM and 15 pM can be achieved for adenosine and thrombin, respectively. The sensing strategy is proposed as a general platform for robust and specific aptamer-target analysis which could be further developed to monitor a wide range of target analytes. The concept and methodology developed in this work shows a good promise in the study of molecular binding events in the biological and medical applications.

Surface plasmon resonance biosensor for sensitive detection of microRNA and cancer cell using multiple signal amplification strategy

A sensitive and versatile surface plasmon resonance (SPR) biosensor was proposed for the detection of microRNA (miRNA) and cancer cell based on multiple signal amplification strategy. Thiol-modified hairpin probe, including a sequence complementary to the target miRNA, was first immobilized on the Au film. In the presence of target miRNA, the stem-loop structure of hairpin probe was unfolded, and then DNA-linked Au nanoparticles (AuNPs) were hybridized with the terminus of the unfolded hairpin probe. Subsequently, DNA-linked AuNPs initiated the formation of DNA supersandwich structure through the addition of two report DNA sequences. Owing to the electronic coupling between localized plasmon of the AuNPs and the surface plasmon wave, as well as the enhancement of the refractive index of the medium over the Au film induced by DNA supersandwich structure, the SPR response was significantly enhanced. Next, numerous positively charged silver nanoparticles (AgNPs) were absorbed onto the long-range DNA surpersandwich equably, resulting in a further increase of SPR response. Due to the enzyme-free multiple signal amplification strategy, as low as ca. 0.6 fM miRNA-21 could be detected. In addition, this biosensor showed high selectivity toward single-base mismatch. More importantly, this SPR biosensor was also used for cancer cell detection coupled with the cell-specific aptamer modified magnetic nanoparticles. Given that the biosensor avoided enzyme introduction, the limitation of the enzyme was overcome. The versatile biosensor has great potential for the broad applications in the field of clinical analysis.