Study On The Analysis Of Estrogen Receptor Using The Microfluidic-chip Immunoelectrophoresis

The increase expression level of estrogen receptors （Estrogen Receptor, ER） dueto the long-term over-stimulation of estrogen was an important reason to breast cancer.Interaction between ER and estrogen and participate in the incidence of breastcancer’s development plays an important role in the development of breast cancer.The expression level of ER was closely related to the diagnosis, treatment andprognosis of breast cancer. ER alpha is the main form of ER in breast cancer cells.The traditional methods such as immunohistochemical can only measure the relativelevel of ERα, but not the quantitative level.In this paper, a versatile programmable eight-path-electrode power supply （PEPS）and a laser-induced fluorescence detector （LIFD）-based microfluidic system fordetecting ERα of the human breast cancer MCF-7cells was provided. The advantagesof this method were less of samples, simple, fast, and easy to implement. Fivechapters were included as follows:In the chapter one, the structure and the significance of ERα, the concepts of themicrofluidic-chip immunoelectrophoresis, the fabrication and modification methodsof the chip as well as the detection methods were recommended.In the chapter two, a PEPS and LIFD-based microfluidic system for detecting ERαof the human breast cancer MCF-7cells was provided by using a home-synthesizedfluorescent probe （anti-ERα-FITC）. On a simple cross microchip, including sampleinjection, electrophoresis separation and detection of the reaction products ofanti-ERα-FITC, the quantitative analysis of ERα was automatically carried out usingthe electrokinetic pinched injection and laser-induced fluorescence detection （LIFD）.The standard curve of the concentration of the sample with the change of the peak area of the anti-ERα-FITC before and after was established. y=9.226+0.176x, R²=0.9857.The linear range of ERα was1.880×10^-92.268×10^-7mol/L. The lowest detectionlimit was9.400×10^-10mol/L.In the chapter three, a PEPS and LIFD-based microfluidic system for detectingERα of the human breast cancer MCF-7cells homogenate was provided by using ahome-synthesized fluorescent probe （anti-ERα-FITC）. According to the standardcurve, the ERα in the MCF-7cell homogenates was2.857×10^-7mol/L with the celldensity of1.450×10⁷cells/mL.In the chapter four, the feasibility of the PEPS and LIFD-based microfluidicsystem for detecting ERα of the human breast cancer MCF-7cells homogenate wasprovided by using a home-synthesized fluorescent probe （anti-ERα-FITC） was provedby ELISA. The standard curve linear equation, y=-0.03737+0.01037x, R²=0.9915,was establish by ELISA. The linear range of ERα was9.400×10^-101.880×10^-7mol/L.The lowest detection limit was9.400×10^-11mol/L. According to the standard curve,the ERα in the MCF-7cell homogenates was1.183×10^-7mol/L with the cell densityof1.450×10⁷cells/mL, with the same as the LIFD-based microfluidic system.In the chapter five, the preliminary exploratory experiment of the analysis anddetectsion of mRNA of ERα in single MCF-7cell by using mRNA probes wasestablish to determine, the cell density of104cells/mL, the detection excitationwavelength with532nm.