Yuyu Tan

Proximity-dependent protein detection based on enzyme-assisted fluorescence signal amplification.

In this paper, we develop a sensitive fluorescence method for protein detection based on proximity extension and enzyme-assisted signal amplification. In this novel method, pairs of proximity probes are designed, and the recognition elements are integrated into the proximity probes. Then proteins are detected by transforming aptamer or antibody-protein binding signals into DNA detection based on proximity effect. In addition, nick sites are introduced into the proximity probes to amplify the detectable signal. As proof of concept, detection of human α-thrombin and human IgG are demonstrated in this study. The aptamers and antibodies are coupled in the proximity probes as recognition elements for human α-thrombin and human IgG respectively. In the presence of target protein, aptamer or antibody-protein binding signals are transformed into detectable signals by the proximity effect, and can be further amplified by enzyme-assisted strand displacement. The above mentioned strategies consequently bring the limit of detection (LOD) to as low as 1 pM for human α-thrombin and 6 pM for human IgG. Furthermore, this method might be extended to sensitive detection of other proteins by changing recognition elements of proximity probes.

Amplified electrochemical DNA sensor using peroxidase-like DNAzyme.

A novel electrochemical DNA sensor was developed here by using peroxidase-like G-quadruplex-based DNAzyme as a biocatalytic label. A hairpin structure including the G-quadruplex-based DNAzyme in a caged configuration and the target DNA probe were immobilized on Au-electrode surface. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated on the electrode surface, triggering the electrochemical oxidization of hydroquinone by H(2)O(2), which provide a quantitative measure for the detection of the target DNA. The DNA target was analyzed with a detection limit of 0.6 nM. This method is simple and easy to design without direct conjugation of redox-active element.

DNA aptamer-based surface plasmon resonance sensing of human C-reactive protein

Human C-reactive protein (CRP) is a powerful predictor and risk factor of inflammation and cardiovascular disease. We have obtained high-affinity DNA aptamers selected by an immobilization-free screening method, and the lowest Kd value of the aptamers is 3.9 nM. The affinity and specificity of the aptamers have been further investigated using aptamer-based SPR analysis, which will be of benefit for constructing DNA aptamer-based sensors for CRP detection.

A fluorescent aptasensor for sensitive detection of human hepatocellular carcinoma SMMC-7721 cells based on graphene oxide

In this work, we reported a simple, rapid and sensitive fluorescence method for the direct detection of cancer cells using a graphene oxide (GO)-based aptasensor. This method took advantage of the exceptional quenching capability of GO for dye-labelled aptamers, and an aptamer was employed as the molecular recognition element. As proof of concept, an aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for human liver cancer cell line SMMC-7721 with high specificity and high affinity, which was applied in this method. Samples with the target cells show high fluorescence intensity, while non-target samples still showed low fluorescence intensity. This GO-based aptasensor exhibited high sensitivity and specificity with a detection limit of 200 SMMC-7721 cells in 200 μL binding buffer. In addition, our strategy can directly detect target cells without washing and separation. Furthermore, the future design of detectors for other cancer cells only requires changing the recognition element for the target cell. More importantly, flow cytometry was used for the specific recognition of target cells from a mixture of cells in fetal bovine serum to demonstrate the potential application of this method for medical diagnostics.

Whole cell-SELEX aptamers for fluorescence staining of frozen hepatocellular carcinoma tissues

Hepatocellular carcinoma is one of the most common and deadly cancers in the world, and the development of specific molecular probes for hepatocellular carcinoma diagnosis is of great significance. Recently, aptamer ZY1, which showed excellent specificity against SMMC-7721 human hepatocellular carcinoma cells, was selected by our group through cell-SELEX. In this study, aptamer ZY1 was labelled with Cy5 to serve as a fluorescence probe, and its efficacy in fluorescence staining of frozen SMMC-7721 xenograft tissues was tested. The results indicated that the aptamer not only showed high specificity in identifying SMMC-7721 carcinoma, but also exhibited high efficiency in differentiating liver carcinomas of different subtypes. Moreover, the time required for tissue staining was as short as 10 min. These results strongly support the potential of the aptamer for future application in cancer diagnosis.