| Development and application of single cell genomics, transcriptomics, proteomics, and metabolomics have become the major way to find the chemicals basis of life and advance our knowledge of molecular mechanism to a deeper and clearer extent. Single cell level analysis is more advantageous to our understanding of microenvironment of the body. Conventional cell research methods can obscure the cell-to-cell heterogeneity and cause misunderstanding of functions of cell population. However, direct analysis of individual cells can overcome the inherent limitations of investigating cellular biology on large and heterogeneous cell populations and reveal the important biological information covered by a mean result, which is very helpful for the early diagnosis and prevention against some diseases. Moreover, for the smallest units of organisms and function relatively independent unicellular material composition analysis will help to clarify various biological processes at the single cell level. However, because of the intricacy and minute volumes of most cells, analysis at single cell level poses significant sensitivity demands. Thus, in order to accurately, in situ and directly analyse single cell, this paper have made some exploring researches as follows:1. Cell is an extremely complex system, so it has serious matrix effects during mass spectrometry (MS) analysis. Moreover, the amount of chemicals in cell is very low, which would greatly reduce the accuracy and reliability of analysis. To decrease or control the matrix effects, we explored a novel ambient ionization approach in which porous material, such as filter paper, was placed on the surface of a piezoelectric ceramic disk to carry liquid and solid samples. Moreover, matrix effects (including ion suppression effect) are greatly reduced in this novel ambient ionization approach. This method, termed as paper assisted ultrasonic spray ionization (PAUSI) allows spray and ionization directly from complicated matrixes in both liquid and solid phases, which enables direct MS analysts of analytes from bovine serum with NaCl 150g/L; viscous samples such as olive oil and toothpaste and biological fluid, for instance cell culture fluid without any sample preparation. With facilitation of porous material (c.a. filter paper), fine droplets carrying analyte were generated via the micro-channel of the porous material without any sample preparation. The gas of cell release is nearly immediate readout of how cells react to environmental influences. However, the gas is very difficult to measure because of high humidity of the released gas. A humidity independent mass spectrometric method was developed for rapid analysis of gas phase chemicals. Raw gas phase chemicals released from plant at different physiological stages were directly identified in real time using this method2. Cell is the minimum unit to maintain the biological activity. To ensure better life activity, the structural of the molecules in cell is very diversity. In order for a single cell technology to be useful for (systems) biology research, it is important that the technology can either measure a wide range of metabolites such that the technology could be used for phenotypic screening on the single cell level or can measure and quantify fewer metabolites in a targeted manner. A new method which has capacity for the simultaneous analysis of nonpolar and polar compounds was developed. A novel ambient hybrid ionization technique termed plasma enhanced paper assisted ultrasonic spray ionization (PEPAUSI) was used. This technique combines features of plasma-and spray-type ionization but with the unique capability of enabling the simultaneous and direct detection of molecules within a broader range of polarities and molecular weights. It integrates the benefits and circumvents some of the limitations of plasma-type (molecular weight limited) and spray-type (polarity range limited) ionization techniques. Furthermore, matrix effects, especially ion suppression effect, are greatly overcome/alleviated in this approach.3. The dynamics of chemicals in cell is very rapid. To more complete coverage of the metabolome, better and faster identification of metabolites, and nondestructive measurement are needed. Plasma enhanced paper assisted ultrasonic spray ionization mass spectrometry (PEPAUSI-MS) has been introduced as a powerful tool for monitoring reaction process and intermediates in situ real-time. An inferred reaction route for the conversion of Nip to Amp using NaBH4 was gived, based on the recognized intermediates via PEPAUSI-MS. Interestingly, isotope detailed analysis shows the hydrogen atoms of the amine group of the product are from water molecules, not come from BH4". Moreover, some fresh obtained insights include the intermediates have been found and confirmed, this catalytic process can be slightly spontaneous without catalysts, not completely stopped, and it is a reversible reaction, not totally irreversible one-way reaction. All the results implied that PEPAUSI-MS has the capacity for monitoring the composition and concentration changes of complex system in situ real-time4. An ESI-MS method based on induced nanoESI-MS for single cell analysis was developed. Single cell capillary microsampling was performed by a motorized micromanipulator mounted on a universal stand next to an upright microscope. A micro-pipette holder with a new nanoelectrospray tip was attached to the micromanipulator for the cells. Then the intracellular fluid was sucked from inner single cell into the nanoelectrospray emitter. Then the emitter was subjected to induced nanoESI MS, in detail, a home-made power supply to provide 3 kV AC voltage with a frequency of 200 Hz on the electrode outside the spray emitter with induced nanoESI. The metabolites of single HEK-293 cell haves been detected using this method. Moreover, the changes of metabolites that from injured HEK-293 cell treated by different carbon tetrachloride concentration has been measured using this method, and found that there was a strong correlation between the CCl4 concentration and the amount of intracellular creatinine, which was consistent with the clinical diagnose of kidney injury using the concentration of creatinine in blood. To achieve both chemical profiling and electrophysiology assay, patch clamp electrophysiology and ESI/MS were combined in present study. We discovered coexistance of Glu and GABA in main body of both excitatory and inhibitory nerouns. The metabolism of Glu and GABA in various brain area and at various ages were also investigated. It might potentially benefit study in life sciences, including cell biology, pharmacology, pathology, and toxicology at single cellular level. |
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