Study On Novel Methods For Cytosensing And Detection Of Glycan Expression On Cell Surface

Glycans, as one of the basic components for all eukaryotic cells, participate in many important biological processes including cell adhesion, cell-cell communication, signal transduction, receptor activation, and endocytosis. Different cell types vary in the nature of their surface glycan expression, and a single cell type may change its surface glycan expression profile during cell growth and differentiation processes. The changes of glycan expression on cell surface glycoproteins have been demonstrated to be associated with many diseases, such as inflammation and cancers. Therefore, it is very important to develop specific, highly sensitive, and high-throughput methods to detect glycan expression on cell surfaces for decoding relationship between tumor and glycosylation, understanding the role of glycosylation change in formation and metathesis of malignant tumors, and developing new methods for the treatment of cancer. In this thesis, a series of new cell-based sensors have been developed by combining material science, nanotechnology and chemical biology for in situ, convenient and highly sensitive detection of glycan expression on living cells. In addition, the effect of drugs on the cell surface glycan expression has also been investigated. This dissertation includes the following four parts:1. Electrochemiluminescence of quantum dots for cytosensor and dynamic monitoring of cell surface carbohydrate expressionA novel electrochemiluminescent (ECL) cytosensing strategy for sensitive dynamic monitoring of carbohydrate expression on living cell surfaces was designed by combining the specific recognition of lectin to carbohydrate group with the functionalization of immobilized CdSe quantum dots (QDs), which acted as ECL emitting species. The ECL cytosensor was constructed by covalently binding thioglycolic acid-capped CdSe QDs to chitosan-AuNPs composite modified glassy carbon electrode and then lectin to the QDs. The immobilized functional QDs showed high ECL sensitivity and good stability. The immobilized lectins could efficiently capture cells to the modified electrode surface by the specific binding of cell surface carbohydrates to the lectins, which resulted in the decrease in ECL intensity. The decrease magnitude depended on the number of captured cells, and thus the expression of cell surface carbohydrates. Using human leukemic K562 cells as a model, four lectins specifically related to the surface carbohydrates of K562 cells were covalently bound to QDs immobilized electrodes, respectively. The resulting cytosensors could detect the carbohydrate expression pattern on living cells with identical results with flow cytometric analysis, and be further employed for dynamic monitoring of surface carbohydrate expression change of K562 cells in response to drugs. This strategy provided a promising platform for highly sensitive cytosensing and cell biologic study based on cell surface carbohydrate expression.2. Electrochemiluminescent biosensing of carbohydrate-functionalized CdS nanocomposites for in situ label-free analysis of cell surface carbohydrateA facile electrochemiluminescent (ECL) strategy for in situ label-free monitoring of carbohydrate expression on living cells was designed by integrating the specific recognition of lectin to carbohydrate with a carbohydrate-functionalized CdS nanocomposite. The mercaptopropionic acid-capped CdS quantum dots were firstly immobilized on carbon nanotubes modified electrode and then functionalized with carbohydrate using mannan as a model on the surface. The carbohydrate-functionalized CdS nanocomposite showed high ECL sensitivity and good stability, and could be used for competitive recognition to concanavalin A with the target cells in solution, which led to a change of ECL intensity due to the resistance of concanavalin A. The change depended on both the cell number and the expression level of cell surface carbohydrate. A wide linear response to cells ranging from 2×103 to 1×107 cells mL-1 with a detection limit of 1.2×103 cells mL-1 was obtained. The proposed biosensor could be used to in situ evaluate cell surface glycan, and the average number of mannose moieties on single living BGC cell was detected to be 8.7×107. This sensitive strategy was further used for facile monitoring of dynamic carbohydrate expression on living cells in response to drugs. The proposed method could be further expanded to high-throughput detection with the addition of more specific glycan-lectin pairs to the repertoire.3. Highly sensitive chemiluminescent imaging for visualization of glycan expression on living cells using multifunctional nanoprobesA novel chemiluminescent (CL) imaging method with high sensitivity was developed for in situ monitoring of cell surface carbohydrate expression. The multifunctional nanoprobes were fabricated by assembling biotin-DNA and horseradish peroxidase (HRP) on gold nanoparticles (GNPs). The biotin-DNA could recognize avidin-labeled glycan site on cell surface, and HRP acted as an enzymatic signal amplification molecule for CL imaging. The hydroxyl sites of sialyl and galactosyl groups on cell surfaces were oxidized into aldehydes by periodate and galactose oxidase respectively, followed by aniline-catalyzed hydrazone ligation with a suitable tag. Using the interaction between avidin and biotin, the multifunctional nanoprobes were effectively recognized to the glycan sites on cell surfaces, and then the glycan expression could be monitored by CL imaging with high sensitivity. Using human liver cancer HCCC-9810 cells as a model, this CL imaging strategy could detect HCCC cells ranging from 6×102 to 1×107 cells mL-1 with a low detection limit of 300 cells mL-1. More importantly, this method was further used for screening the cancer cells, and monitoring dynamic carbohydrate expression on living cells. It can be anticipated that this method could be used for clinical diagnosis and treatment of cancer.4. Highly sensitive fluorescent analysis of dynamic glycan expression on living cells using glyconanoparticles and functionalized quantum dotsA double signal amplification strategy was designed for highly sensitive and selective in situ monitoring of carbohydrate on living cells. The double signal amplification included the multiplex sandwich binding of functionalized quantum dots (QDs) to both glycan groups on cell surface and glyconanoparticles and a cadmium cation sensitized fluorescence emission of Rhod-5N. Using sialic acid-phenylboronic acid recognition system as a model, the 3-aminophenylboronic acid functionalized QDs (APBA-QDs) were synthesized by covalently binding APBA to mercaptopropionic acid-capped CdS QDs, and the glyconanoparticles, polysialic acid-stabilized gold nanoparticles (PSA-AuNPs), were prepared by a one-pot procedure. The APBA-QDs firstly recognized the SA groups on BGC-823 human gastric carcinoma (BGC) cells and then the PSA on AuNPs, which were further used to bind more APBA-QDs on cell surface for signal amplification. After the bound QDs were dissolved to release the Cd2+, a Cd2+sensitized fluorescence method was developed for the detection of BGC cells in a linear range from 5.0×102 to 1.0×107 cells mL-1 with a limit of detection down to 210 cells mL-1 (8 cells in 40μL solution) and the dynamic monitoring of SA expression variation on cell surface. The monitoring result was identical with that from flow cytometric analysis. This approach showed high specificity and acceptable reproducibility. This strategy provided a promising platform for highly sensitive cytosensing and cytobiologic studv.