New Fluorescence Methods For Drug Screening And Apoptosis Detection

Cancer, also called malignant tumor, is a disease damaging human being health. The main therapy of tumors is treatment of chemical drugs, which can induce the death of tumor cell. Screening safe and effective antitumor drugs from thousands of compounds is a hotspot for tumor treatment research. The tumor cell death includes apoptosis and necrosis pathway, and apoptosis pathway is play important roles in many biological processes. Detection of drug-induced apoptotic tumor cells could be used to evaluate therapeutic effects and prognosis, which is very important in treatment of cancer. At present, many methods are used for antitumor drug screening and detection of apoptosis in the clinic. To develop high sensitivity, versatility, small samples requirement for drug screening and cell apoptosis detection is still imminent. Fluorescence correlation spectroscopy(FCS) is well suitable for this requirements as a single molecule detection technique.In this dissertation we presents certain new methods for drug screening and apoptosis cell detecting by FCS, flow cytometry and fluorescence images detection using organic fluorescent dyes and quantum dots(QDs) as labeling probes. The main work in the following aspects:(1) A sensitive and microscale method for drug screening is described using single molecule spectroscopy FCS. The principle of this method is mainly based on the competition of candidate drugs to the fluorescent probe-target complexes and the excellent capacity of FCS for sensitively distinguishing the free fluorescent probes and the fluorescent probe-target complexes in solution. Firstly, the screening of protein kinase inhibitors was used as a model, we established the theoretical model of monitoring binding process of fluorescent probes and targets, and the competition of candidate drugs to the fluorescent probe-target complexes by FCS. And then, the dasatinib derivatives were synthesized and labeled with fluorescent dye Alexa 488, the binding and dissociation processes of Alexa 488-dasatinib and ABL1 were systematically investigated. The dissociation constant and dissociation rate for Alexa 488-dasatinib-ABL1 complex were determined, the values were 40 nM and 1.8 × 10-3 S-1 respectively. Finally, the established method was used to screen candidate drugs. The dissociation constants of ABL1 kinase to six known drugs for treating chronic myeloid leukemia(CML) were evaluated and the results obtained are well consistent with the reported values, the fitting correlation coefficient R2 value is greater than 0.986 and the fitting errors are less than 0.07. And, a homemade chip with micro-wells was successfully utilized in FCS measurements as the carrier of samples, and the sample requirements were only 1~2 μL in this case, the R2 values are greater than 0.979 and the fitting errors are less than 0.12. Our results demonstrated that the drug screening method described here is sensitive, universal, and small sample and reagent requirement, and can be used to evaluate the affinity between candidate compounds and target without labeled candidate compounds, kinase and specific substrate. We believe that this method will become a high throughput plat-form for screening of small molecule drugs.(2) A novel strategy for sensitive detection of drug-induced apoptosis was proposed by FCS. FCS can be used for sensitive and selective assay of DNA fragmentation from drug-induced apoptotic cells without separation. We firstly developed a highly sensitive FCS method for characterization of DNA fragments using SYBR Green I as fluorescent DNA-intercalating dye, the R2 value is greater than 0.972 and the fitting error is less than 0.1. And then, we established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin(LDM). FCS method established was used to directly detect diffusion behavior of the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data, the R2 values are greater than 0.914 and the fitting errors are less than 0.15. The characteristic diffusion time of DNA fragments was used asan important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of LDM concentration, which implied that DNA fragmentation occurred in LDM induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gelelectrophoresis.The method described here is simple and sensitive, reproducible. And more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nanoliter level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.(3) A new method for in situ detecting drug-induced apoptosis was developed using fluorescence imaging, flowcytometry and FCS technique. The principle of this method is based on QDs labeled AnnexinV recognition of apoptotic cells. Annexin V was linked with different surface modified QDs firstly, and the QDs-Annexin V complexes was characterized the by FCS and capillary electrophoresis after purified by gel column chromatography. Then, we studied on the nonspecific adsorption of cells to different surface modified QDs, and found that there was a little nonspecific adsorption of QDs modified with amino groups in detection apoptosis and cell imaging. The results of cell imaging and flow cytometric analysis were consistent with apoptosis detection test kit. Furthermore, QDs fluorescent probe was incubated with apoptotic PANC-1 cells, and was detected by the fluorescence correlation spectroscopy-confocal laser scanning microscopy(FCS-CLSM) system. The diffusion coefficients of QDs on cells with different states are from 0.032 μm2/s to 0.115 μm2/s. QDs fluorescent probe can be detected the cell apoptosis sensitively and accurately.