ICP-MS Techniques And Their Application In Cancer Cells Analysis

Cancer is one of the most serious diseases that cause death worldwide. The presence of CTCs in the peripheral blood has been regarded as an important cancer biomarker. The detection of CTCs especially at low concentration has a drastic effect on the accurate early diagnosis of cancer. Recently, element-tagged ICP-MS-based immunoassay techniques have gained much attention for cancer cell detection. ICP-MS is one of the most sensitive techniques providing a wide dynamic range, low detection limits, low matrix effects and element specificity. Recently, ICP-MS technique has become one of the most versatile and sensitive complementary tools in the emerging field of life-science related research. To take full advantages of element-tagged ICP-MS-based immunoassay techniques for cell analysis, considerable attention has been paid to the labeling tags. Inorganic nanoparticles are employed to improve the tag signal intensity of cancer cells detection, high sensitivity can be easily obtained because of large quantities of detectable atoms in each nanoparticle tag. It is well known that individual cells differ from each other in many aspects even under identical environmental condition. In consideration of average data can mask the underlying information, except for the bulk analysis, it is necessary to study individual cell for better understanding of cellular heterogeneity. Time-resolved ICP-MS technique emerges as a powerful tool for single cell analysis. The spike signal obtained for each element is corresponding to the presence of a cell and the spike signal intensity can be used for the determination of the metal quality of the cell.Disseminated cells circulate in the blood at extremely low concentrations, making the detection of low-frequency cancer cells difficult. To achieve acceptable detection sensitivity, appropriate sample preparation prior to ICP-MS detection is required for the specific identification, isolation and enrichment of target cells from the heterogeneous suspension. Currently, immunomagnetic separation is routinely used for target cells separation from a blood sample.Unlike conventional fluorescence-based cytometry, mass cytometry is a destructive technique. It can replace fluorescence cytometry in multiplex cell analysis, but can not perform cell sorting.The design and synthesis of novel multifunctional probes, in which cellular biomarkers label with fluorescent agents and element tags, are greatly expected to perform a more comprehensive multiplex assay of cancer cells, achieving more accurate early diagnosis of cancers.The aim of this dissertation is to develop immunomagnetic separation technique combined with element-tagged ICP-MS-based immunoassay method for cancer cells analysis using nanoparticles as a label probe; to design a trifunctional probe (IgG-Cy3-conjugated UCNPs) for two ways of visualization of CTCs and combination with ICP-MS determination; to explore time-resolved ICP-MS technique for single cell analysis. The major contents of this dissertation are described as follows:(1) A new method for detecting tumor cells at low levels based on coupling of element-tagged immunoassay with ICP-MS detection has been developed In this method, PbS NPs are functionalized to bond covalently with the anti-EpCAM antibody, which is specific to the highly expressed EpCAM antigen on the surface of HepG2 cells, but not on HeLa cells. The interference of HeLa cells on detection of HepG2 cells was investigated. A series of conditions influencing the immunoassay were carefully optimized, including the incubation time, the concentration of the labeling probe and the elution conditions. Under the optimized conditions, the linear range of 800-40000 and the limit of detection of 282 HepG2 cells were obtained, and the relative standard deviation for seven replicate determinations of HepG2 cells was 5.0%(3000 HepG2 cells).(2) An efficient, specific and sensitive immunoassay protocol for detection of tumor cells by using inductively coupled plasma mass spectrometry (ICP-MS) with two probes has been developed. Magnetic nanobeads modified with anti-CD3 were used as capture probes for efficient and fast magnetic separation of Jurkat T cells from a mixture of cells, and gold nanoparticles (Au NPs) conjugated with anti-CD2 were used as detection probes for ICP-MS measurement.The interference of A549 and 97L cells (CD2/CD3-negative cells) on the capturing and detection of Jurkat T cells was investigated. The conditions for this immunoassay were carefully optimized, including the incubation time and temperature, the concentration of the labeling probe, and the elution conditions. Under the optimized conditions, the linear range of 300-30000 and the limit of detection of 86 Jurkat T cells were obtained, and the relative standard deviation for seven replicate detections of Jurkat T cells was 5.2% (3000 Jurkat T cells).(3) A new method of UCNPs-Ab2-Cy3 nanoprobe for two ways of visualization of tumors and combined with ICP-MS for the detection of cancer cells has been developed. This concept of the all-in-one probe facilitates the selective labeling of targeted biomarkers and thus a subsequent multi technology for cancer cells using various detection techniques, offering more comprehensive information over a single assay. In this work, EpC AM-positive HepG2 cells were used as a model and EpCAM-negative Vero cells as the control group. The tirfunctional probe consisted of Ab2 unit to catch cancer cells via targeting anti-EpCAM which has reacted with EpCAM antigen overexpressed on cellular surface, Cy3 moiety for fluorescence imaging, UCNPs for upconversion luminescence imaging under near-infrared irradiation and subsequent ICP-MS determination of Yb in UCNPs(NaYF4:Yb3+, Er3+). Under the optimized conditions, Cy3 confocal images excited at 561 nm and upconversion luminescence images excited at 980 nm were observed, and ICP-MS determination of Yb was obtained.(4) A new method of time-resolved ICP-DRC-MS for the determination of Mg contents in single Jurkat T cell for the first time has been developed. Time-resolved ICP-MS technique for single cell analysis can be considered as a special case of single-particle analysis in which intact biological cells are introduced directly into ICP for measurement. Cells are directly sprayed as single-cell aerosols into torch to produce a plume of metal atoms through the way of vaporizing and ionization of its atomic constituents, and then quantified by mass detector. The spike signal obtained is corresponding to the presence of a cell, and the spike signal intensity is the metal quality in a single cell. DRC was used to reduce spectral interferences to improve the analytical performance.92% He as collision gas and 8% H2 as reaction gas were selected to reduce spectral interferences. The factors were optimized in detail. The distribution of Mg spike intensity of Jurkat T cell was approximately log-normal. The number of spikes was proportional to the cell number density. Under the optimized conditions, Mg quality in 20 individual cells is ranging from 0.09 to 0.29 fg cell-1. Thus, the average Mg content for these 20 cells was 0.18 fg cell-1. Because of the cellular heterogeneity, Mg contents in the individual Jurkat T cell were not equal even though they were the same cell population. The average Mg content was 0.94 fg cell-1 and 0.48 fg cell-1 respectively determined by acid digestion and cell rupture of Jurkat T cell suspensions (number density was 1.7 × 106 cell mL-1).(5) A new method to provide a single cell flow has been developed by using a symmetric cross-over microfluidic chip based on the symmetric hydrodynamic focusing effect. This is achieved by controlling the width of focused stream to the same order magnitude of the size of cell on a cross-over microchannel device. The hydrodynamic focusing experiments were accomplished on a cross-over microchannel device using sheath flows from the two side channels and cell suspensions for the sample flow from the inlet channel. Optimization of the experimental conditions, including sheath flow reagent, the microchannel width in a symmetric cross-over chip and the velocity ratio of sheath flow to sample flow to make sure the width of the focused stream reduced to the same order of magnitude as the cell size. Under the optimized conditions, a single cell flow could be obtained.