| Aptamers, well known as "chemical antibodies", possess several advantages including high binding affinity and specificity, easy and reproducible synthesis, long-term chemical stability, and flexible modifications. They are single-stranded oligonucleotides, which are generated from an in vitro method known as SELEX (systematic evolution of ligands by exponential enrichment) and can bind to variety of targets. With the improvement of cell-SELEX technologies, an increasing amount of specific aptamers for tumor cells are obtained, making aptamers ideal candidates as molecular probes for use in early diagnosis and treatment of cancers. However, improving the detection sensitivity is still one of the most critical problems for early diagnosis of cancers. As we known, tyramine signal amplification (TSA) technology with superior signal amplification ability has been widely used in the fields of in situ hybridization, enzyme-linked immunosorbent assay, and flow cytometry techenology, etc. In this thesis, a series of simple and universal cancer cell detection methods have been developed to improve the efficiency of cell recognition, by using lung cancer A549cells as a model, aptamer probes as units for specific recognition, and TSA as the signal amplification technique. The main researches are summarized as follows.1. Aptamer/TSA-based solid phase analysis for identification of A549cellsWith the great potential of aptamer as "diagnostic antibody" in cancer cells detection, aptamer/TSA-based solid phase analysis technique with adherent A549cells as targets was developed for sensitive cancer detection. TSA method is a catalytic-deposition based signal amplification method with geometric amplification ability especially in solid-phase immunoassay. Combining the high specificity and affinity of aptamers and superior signal enhancement capability of TSA, the method has been developed to take in vitro and primary cultured A549cells as targets for the effective identification in buffer and serum, respectively. The approach is simple, specific, and results-intuitive with about110-fold signal enhancement compared with that of traditional fluorescent dye-labeled aptamer probes. Meanwhile, Aptamer/TSA solid phase analysis has a strong anti-interference ability to small physiological changes of A549cells, showing superior enviromental-friendly suitability, which will play a very important role in the clinical diagnosis of lung adenocarcinoma in the future.2. Aptamer/TSA based solid phase analysis with qantum dots as reporter probes for identification of the A549cellsThe signal groups of TSA technology are mainly traditional fluorescent dye molecules, which normally show weak fluorescence and easy photobleaching. As an excellent fluorescent nanomaterials, quantum dots (QDs), have possessed high fluorescence brightness, and anti-photobleaching ability. Thus, aptamer/TSA solid phase analysis with QDs as reporter probes was developed for specific recognition and imaging of A549cells. Compared with the quantum dots linked aptamer/non-TSA labeling methods, the proposed approach was enhanced about2.5times. It has demonstrated a weak sensitivity comparing with that of Strepavidin-FITC as a reporter probe (signal enhanced about110-fold), but showed obvious advantages in anti-photobleaching and stability.3. Aptamer/TSA based homogeneous analysis for identification and highly sensitive detection of A549cells.In order to expand applications of the aptamer/TSA method and to achieve quantitative analysis of cancer cells, an aptamer/TSA homogeneous analysis combined with flow cytometry has been developed for specific recognition and highly sensitive detection of suspension A549cells in buffer and in serum, the detection limit of which were100and125cells, respectively. Compared with the fluorescence microscopy methods, it has shorten the test time, increased the detection efficiency, and confirmed the operability of aptamer/TSA homogeneous analysis method in suspension cancer cells, which is expected to develop into a general method for a variety of tumor cells with rapid and highly sensitive detection. |
PDF Full Text Request