Research On Novel Methods For In Situ Detection Of Cellular Functional Molecules

For the past twenty years, the multidisciplinary interface of biology, chemistry, medical science, physics, materialogy and etc. has created good opportunities for the revolution of analytical methods, and brought brand new ideas for the biological analysis. Cells, especially tumor cells, have always been a hot spot for researchers. Tumor-related cellular functional molecules, such as cell surface glycans and intracellular telomerase, have also kept continuous attraction. Glycans,because of the complexity of the structure, lack effective and sensitive detection methods. In this dissertation, by combining multi-functional nanomaterials, electrochemistry and optical technology, various multi-functionalized nanoprobe were fabricated for the in situ detection of sialic acid (SA). In addition, up to now, most of the methods use cell extract for telomerase activity analysis, thus fail to detect telomerase in situ. Therefore, in this dissertation, several telomerase-responsive nanoprobes were constructed for the in situ detection of intracellular telomerase activity. The establishment of multiple in situ detection strategies for cellular functional molecules provides powerful tools for cancer diagnosis, therapy, drug screening and the medical research of cancer. This dissertation includes the following five parts:1. In Situ Electrochemical Assay of Cell Surface Sialic Acids Featuring Highly Efficient Chemoselective Recognition and a Dual-Functionalized Nanohorn ProbeA novel nanohorn probe was designed through dual functionalization of single-walled carbon nanohorn (SWNH) with 3-aminophenyl boronic acid (APBA) and avidin, featuring highly efficient chemoselective recognition, for the in situ detection of SAs on cell surface. Using BGC-823 cells as the model, sodium periodate (NaIO4) was employed to selectively oxidize the C-7 position of cell surface SAs,and the formed aldehyde group could be targeted by biotin hydrazide (BH), which could be recognized by avidin on SWNH probe.After the combination of the probe and the cells, mannan-conjugated gold nanoparticles (AuNPs) were added, and captured by APBA on the probe. The bound AuNPs could be released from the nanohorn probe by a competition reaction of fructose with APBA to perform the electrochemical detection. This method could be applied for the detection of cells number, and the SA expression level on a single cell. A wide linear response to cells ranging from 5.0×102 to 1.0×106 cells mL-1 with a detection limit of 380 cells mL-1 was obtained, and the average amount of SAs on each cell was calculated to be 1.1×108. This method owns high sensitivity and good repeatability, and could be used for the dynamic monitoring of the expression level of cell surface SAs under the treatment of drugs.2. Fluorescence Off-On Switchable Gold Nanoprobe for In Situ Imaging of Cell Surface Sialic AcidsA fluorescence off-on switchable gold nanoprobe was constructed by functionalization of polysialic acid (PSA)-conjugated AuNPs with fluorescent 3-(dansylamino)phenylboronic acid (DAPB) for the in situ imaging of cell surface SA. Using HeLa cells as the model, cells were firstly incubated with the probe. The DAPB bound on the probe could be removed to the cell surface SA, which "turned on" the SA. This probe could be applied for the in situ imaging and quantitative detection of cell surface SA, and the average amount of SAs on each HeLa cell was calculated to be 7.0×108 This probe could also be used for the dynamic monitoring the expression of cell surface SA in response to SA-related drugs. This strategy provides a potential tool for cancer diagnosis and therapy.3. Switchable Fluorescent Imaging of Intracellular Telomerase Activity Using Telomerase-Responsive Mesoporous Silica NanoparticleAn in situ detection method was developed for the analysis of intracellular telomerase activity. Using mesoporous silica nanoparticle (MSN) as the carrier, a black hole fluorescence quencher is covalently immobilized on the inner walls of the mesopores, while fluorescein was loaded in the mesopores, and a wrapping DNA,O1, was used to seal the MSN through electrostatic adsorption. The fluorescence of fluorescein was quenched by BHQ, while in the presence of telomerase and dNTPs, the designed 01 could be extended, producing telomeric repeated sequence TTAGGG at its 3’end, which was just complementary with its 5’end. The hybridization of the complementary sequences at 5’and 3’ends produced a rigid hairpin-like DNA structure, and led to the detachment of the extended 01 from MSN and the release of the entrapped fluorescein to turn "on" the fluorescence. After incubation of MSN probe with cells, the mesopores could be opened under the effect of telomerase in cytoplasm, and the fluorescence signal was on, which realized the in situ detection of intracellular telomerase activity. The telomerase activity was estimated to be 3.0×10-11,2.0×10-11 and 8.0×10-13 IU for each HeLa, BEL and QSG cells, respectively. This approach exhibits good performance for sensitive in situ tracking of telomerase activity in living cells, and could distinguish various cells with different levels of telomerase activity. It could also be employed for monitoring the change of intracellular telomerase activity in response to telomerase-based drugs. This strategy provides good application prospects for biomedicine and clinical research.4. A Smart Vesicle Kit for In Situ Monitoring of Intracellular Telomerase Activity Using A Telomerase-Responsive ProbeA smart vesicle kit was designed for in situ imaging and detection of cytoplasmic telomerase activity. The vesicle kit contained a telomerase primer (TSP) and a Cy5-tagged molecular beacon (MB) functionalized gold nanoparticle probe, which were encapsulated in liposome for intracellular delivery. After the vesicle kit was transfected into cytoplasm, the released TSP could be extended in the presence of telomerase to produce a telomeric repeated sequence at the 3’end, which was just complementary with the loop of MB assembled on probe surface. Thus the MB was opened upon hybridization to switch the fluorescent state from "off to "on". The fluorescence signal depended on telomerase activity, leading to a novel strategy for in situ imaging and quantitative detection of telomerase activity. The cytoplasmic telomerase activity was estimated to be 3.2×10-11,2.4×10-11 and 8.6×10-13 IU in each HeLa, BEL tumor and QSG normal cell, respectively, demonstrating the capability of this approach to distinguish tumor from normal cells.The proposed method could be employed for dynamic monitoring of the intracellular telomerase activity in response to a telomerase-based drug, suggesting the potential application in discovery and screening of telomerase-targeted anticancer drugs.5. A Robust Probe for Lighting up Intracellular Telomerase via Primer Extension to Open A Nicked Molecular BeaconA nicked molecular beacon (MB) functionalized probe has been designed for in situ imaging and detection of intracellular telomerase activity. The nick separates the MB into two segments:the shorter telomerase primer (TSP) sequence as a part of 5’-end stem, and the longer sequence for forming the loop with one thiol-labeled 3’-end stem. The MB can be opened by substitutional hybridization of telomerase-triggered stem elongation product, which leads to the separation of Cy5 labeled at 5’-end nick from gold nanoparticle (AuNP) as nanocarrier and thus inhibits the energy transfer from Cy5 to AuNP.Upon endocytosis of the probe, the TSP can be extended by intracellular telomerase at its 3’end to produce telomeric repeated sequence, which leads to the inner chain substitution and thus turns on the switch of Cy5 fluorescence. The probe provides a one-step incubation way for quantification and monitoring of the telomerase activity in living cells. The telomerase activity in single HeLa, MCF, BGC, BEL and QSG cell was estimated to be 3.1×10-11,1.4×10-11,2.5×10-11, 2.1×10-11 and 8.4×10-13 IU, respectively. The practicality of the proposed approach for distinguishing tumor from normal cells and monitoring the decrease of telomerase activity during treatment with anti-tumor drug demonstrates its potential in clinical diagnostic and therapeutic monitoring.