Design And Synthesis Of Magnetic Composite System Based On Nano Materials And The Applications In Biology And Medical Science

The main reason for the death of cancer patients is the spread and metastasis of thecirculating tumor cells (CTCs) through the way such as blood circulation. The detection of CTCsmay help to study the mechanism of tumor metastasis, evaluate the therapeutic effect, andmonitor the metastasis or recurrence of cancer.Two kinds of new methods were established to achieve the detection of CTCs: a capturemethod based on a size amplified immune magnetic microbeads, and a detection method basedon the photothermal effect of graphene. Based on the traditional membrane filtration andimmune-magnetic separation, the efficient capture and detection of CTCs were realized by thedirect microscopic counting and the thermal contrast detection based on the photothermal effectof graphene, respectively. These two methods had the same incubation and capture procedure butdifferent detection mode, the combination of these two methods allowed us to detect all sampleswith the wide range of cancer cell numbers. The whole strategy had a series of advantages suchas rapid, simple, sensitive, and so on. The paper mainly consisted of three parts as follows:In chapter one, the capture and detection of CTCs, nano materials and their photothermaleffects, and the brief introduction of the magnetic microbeads and polydimethylsiloxane weresummarized. The photothermal effects of nanogold/graphene and their research progress, and theresearch progress of the capture and detection of CTCs was emphasized.In chapter two, a novel capture and detection method of CTCs based on the size amplifiedimmune magnetic microbeads strategy was designed and constructed. The high captureefficiency and capture purity were simultaneously achieved by combined use of sizeamplification-membrane filtration and immune-magnetic separation. The magnetic microbeadswere modified with anti-EpCAM antibodies. The immune-magnetic microbeads wouldspecifically recognize the EpCAM antigens on the surface of MCF-7cells. Following incubation,membrane filtration and magnetic separation were operated to achieve the capture process. The membrane-retained MCF-7cells were observed and counted under an optical microscope tocalculate the capture efficiency and purity. The concentration of anti-EpCAM antibodies andincubation time were optimized. At the same time, the size distribution curves of MCF-7cells,leukocytes and size-amplified MCF-7cells were constructed. The optimal concentration ofanti-EpCAM antibodies was1.5μg·mL-1, and the optimal incubation time was1h. Under thisbest conditions, high purity (>98%) and capture efficiency (>78%), rapid (<2hours) andsimple detection of CTCs were realized. At the same time, this rapid detection method was alsoapplied to the detection of CTCs in human blood, the minimum detection limit could reach5cells/1mL. This method was suitable for the detection of CTCs in blood samples with a smallnumber of CTCs.In chapter three, a strategy based on the photothermal effect of the graphene functionalizedon the magnetic microbeads was established. The high capture efficiency and sensitive detectionof CTCs were respectively realized by the combined use of membrane filtration andimmune-magnetic separation in chapter two and the thermal contrast detection. The grapheneoxides (GOs) were modified with anti-EpCAM antibodies, and the magnetic microbeads weremodified with anti-IgG antibodies. The GOs were mixed with the magnetic microbeads, and thenthe magnetic microbeads were surrounded by GOs through the anti-EpCAM-anti-IgG interaction.This GO-magnetic microbead complex could specifically recognize the EpCAM antigens on thesurface of MCF-7cells. The cell capture and thermal contrast detection could be simultaneouslyachieved by using magnetic microbeads and the photothermal effect of GOs, respectively.Following incubation, the membrane filtration and immune-magnetic separation were operated, alaser pen was then used to irradiate the GOs to produce temperature variation on the cell-retainedmembrane. The numbers of cells were determined by an infrared thermometer, according to thestandard curve of the cell number and the temperature variation. The concentration ofanti-EpCAM antibodies/anti-IgG antibodies and incubation time were optimized. At the sametime, the standard curve of the cell number and the temperature variation was established. Theoptimal concentrations of anti-EpCAM antibodies and anti-IgG antibodies were1.0μg·mL-1, andthe optimal incubation time was1h. Under this best conditions, high sensitivity (150cells),rapid (<1.5hours) and simple detection of CTCs were realized. This rapid detection method wasalso applied to the detection of CTCs in human blood, the recovery ratio of89.54%was obtained. This strategy was suited to the detection of CTCs in blood samples with a relatively largenumber of CTCs.