Study On Fast Analysis Of Small Molecules In Single Cells

Cellular analysis is an interdisciplinary frontier in fields of analytical chemistry, biology, and medicine. With the development of modern biology, researchers realize that the same type of cells in the culture medium or in the body are heterogeneous. So the "mean" information obtained by tradational method from cell population is no longer sufficient to meet our needs. In view of single cell analysis providing more accurate and comprehensive information on physiological state and process of cells, high content analysis at single cell levels is imperative. High-throughput analysis of single cells has been achieved by optical imaging techniques that could be combined with a variety of other techniques to achieve comprehensive, dynamic, visual analysis and monitor. Although more researchers are devoted to developing new single-cell imaging techniques, new materials and probes for single cell analysis, the further developpment of single cell analysis for the future clinic test is still urgent. This thesis focuses on the development of novel and rapid single-cell optical analysis technology, new optical probe and their application in cell imaging to realize the rapid analysis of biological small molecules in single cells. The main works are described as follows:1. Fast Analysis of Intracellular Glucose at Single Cells using Electrochemiluminescence ImagingBased on the electroluminescence (ECL) phenomenon of luminol-HkO2 system and ECL imaging system built in our group, analysis of intracellular small biological molecules (glucose) at single cells was acheived for the first time. The cell-cized microewells were fabricated on the ITO surface, and then Au NPs were deposited to amplify the ECL signal for the visualization of H2O2 at low concentration. The optimized ECL imaging system showed that hydrogen peroxide as low as 5?M was visible. The broken of cellular membrane in the presence of Triton X-100 released intracellular glucose into the microwells and reacted with the glucose oxidise to generate hydrogen peroxide, which induced luminol luminescene under positive potential. To achieve the fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using CCD. More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that the intracellular glucose was detected at single cells. The starvation of cells to decrease the intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at single cells were observed, which revealed the high cellular heterogeneity in intracellular glucose at the first time. This electrochemiluminescence assay developed will be potentially applied for the fast analysis of more intracellular molecular in single cells to elucidate the cellular heterogeneity.2. g-C3N4 nanosheet modified microwell array with enhanced electrochemiluminescence for total analysis of cholesterol at single cellsg-C3N4 nanosheet modified microwell array providing enhanced electrochemiluminescence (ECL) and better visible sensitivity(500 nM) was prepared to simultaneously analyze total (membrane and intracellular) cholesterol at single cells. To achieve single cell cholesterol analysis, the individual cells cultured at microwell array was exposed to cholesterol oxidase generating hydrogen peroxide for luminescence analysis of membrane cholesterol; and then, treated with triton X-100, cholesterol esterase and cholesterol oxidase to produce hydrogen peroxide from intracellular cholesterol for luminescence determination. The observation of the luminescence spots at microwells in these two steps confirmed the co-detection of membrane and intracellular cholesterol at single cells. The inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) resulted in less intracellular cholesterol storage (less luminescence) and more membrane cholesterol (more luminescence). The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor membrane and intracellular cholesterol pools at different cellular states, which should offer more information for the study of cholesterol-related pathways at single cells.3. A near-infrared fluorescent aza-bodipy probe for dual-wavelength detection of hydrogen peroxide in living cellsThe realization of near-infrared detection for intracellular hydrogen peroxide is significant for in vivo fluorescence analysis. However, the excitation/emission wavelength of present fluorscence probes is concentrated in visible region. Owing to the high fluorescence intensity, good photostability, narrow emission band and near-infrared emission of aza-bodipy, boronic acid functionalized aza-borondipyrromethene dye (azaBDPBA) was designed and synthesized in our group. Boronic acid groups as electron-deficient group on aza-bodipy react with H2O2 to form electron-rich phenolic group, leading to the red-shift of emission wavelength. Since the the emission of near infrared region offered low-level background interference from cells, and the good selectivity for H2O2, the prepared azaBDPBA fluorescent probe was loaded into the cell to indicate the alteration of intracellular hydrogen peroxide during the biological process. The dual-wavelength emission of this probe guaranteed the accuracy of measurement for intracellular H2O2. Therefore, the intracellular H2O2 was first imaged in near infrared region with dual-wavelength detection of fluorescence probe.