Study On The Detection Of Acute Lymphoblastic Leukemia Cells Based On Aptamers And Quantum Dots

Acute lymphatic leukemia, a haematological malignancy, has a high incidence inhuman populations and particularly accounts for75%of cancers suffered by childrenand adolescents, which is seriously threatening human life and health safety. In recentyears, the appearance of aptamer as "chemical antibodies" brings a new opportunity todevelop acute lymphatic leukemia diagnosis and genotyping techniques. Aptamer notonly has the high affinity and specificity for targets similar to antibodies, but alsoexhibits several advantages like easy modification, widespread targets, and facilesynthesis and so on, thus affording an ideal tumor-targeted recognition molecule. Atthe same time, with the development of nanotechnology, a novel fluorescentnanomaterial--quantum dots (QDs) have received important attentions in the tumordetection and imaging researches due to its predominant advantages including wideabsorption and narrow emission spectrum, strong fluorescence intensity, tunableemission spectra and so on. In this thesis, aiming at the development of novel methodsfor detecting acute lymphatic leukemia cells, two kinds of novel tumor cell detectionprobes were constructed by combining advantages of aptamer as the noveltumor-targeted recognition molecule and excellent optical properties of QDs as thenovel fluorescent marker and a study on the highly specific and sensitive detectionand analysis of CCRF-CEM cells was performed using flow cytometry. The two partsdiscussed in this thesis are as follows:1. Detection of CCRF-CEM cells based on aptamer-functionalized QDsfluorescent probeTo overcome the drawbacks of fluorescenct dye-labeled aptamer for the detectionof acute lymphatic leukemia cells, such as low signal intensity, limited sensitivity andinsufficient photostability, excellent optical properties of near-infrared fluorescentQDs as the novel marker have been utilized, including intensive fluorescenceemission, good photostability and low autofluorescence interference from complexbiological systems. By choosing CCRF-CEM cells as the target and its specificaptamer sgc8c as the target recognition molecule, a novel fluorescent detection probehas been successfully constructed based on the specific interation between avidin andbiotin by modifying biotin-labeled sgc8c onto avidin-coated QDs. Through asystematic characterization using agarose electrophoresis, UV-vis absorption and fluorescence spectroscopy, it was revealed that when the mole ratio of ap tamer to QDswas10:1, majority sgc8c could be conjugated onto the surface of QDs and did notobviously affected QDs’ fluorescence. After the condition optimization of this probefor detecting CCRF-CEM cells using flow cytometry, the specific detection andanalysis of CCRF-CEM cells both in buffer and mouse serum was successfullyachieved. It was shown that on the same condition, the detection sensitivity ofsgc8c-functionalized QDs has been substantially enhanced about seven times bycomparison with the conventional fluorescent dye-labeled probe. This potentiallyaffords a detection method with high sensitivity and specificity for human acutelymphatic leukemia cells.2.Detection of CCRF-CEM cells based on split aptamer and QDs-mediated FRETeffectIn order to solve shortages of the above―always on‖probe in detecting tumorcells like high signal background and limited contrast, the specific aptamer sgc8cagainst CCRF-CEM cells was adopted and splited into two pieces, which were thenrespectively labeled with fluorescenct donor and acceptor. Based on the principle thatthese two pecies could be induced by target to form a certain recognitionconformation and then make the donor-acceptor pair close to initiate FRET, a novelactivatable fluorescent detection probe for CCRF-CEM cells was successfullyconstructed. Firstly by utilizing Cy3-Cy5as the donor-acceptor pair, a systematicoptimization of this probe has been conducted including split sites, probe ratios andconcentrations, reaction time, buffer composition and labeling sites of dyes. It wasrevealed that the probe composed of sgc8c-3a and sgc8c-2b with the mole ratio of1:8,the sgc8c-3a concentration of40nM and dye labeled at the split site, exhibited highaffinity to target tumor cells and the optimized FRET activatable function afterincubation in binding buffer for90min. On this basis, the split aptamer probeintegrated with QDs-Cy5FRET effect was constructed by using QDs to substitute Cy3as the donor. Uusing Ramos cells as the control, flow cytometry assays showed thatthis probe could preserve the high affinity against CCRF-CEM cells, but the FRETsignal was faint and should be improved, which might be due to the stereospecificblockade of QDs.