Development And Application Of Multi-fluorescence Microsensors For Early Embryo Temperature And PH Measurement

Since the beginning of the 21st century,early embryos have become the main research objects of cutting-edge biomedical technologies such as cloning,assisted reproduction,and gene repair.However,early embryos are extremely fragile in vitro.Various micromanipulation techniques currently applied to early embryos will affect them to varying degrees,and this effect is often ignored by people.In the past,the quality of early embryos was basically judged by researchers through morphological observation.This method is greatly influenced by the subjective experience of researchers and cannot be quantified.Temperature and pH are extremely important physiology for any living cell.As a living system,the internal environment of the embryo can be maintained relatively stable through self-regulation.Therefore,the quality of the embryo can be quantified by monitoring physiological indicators such as temperature and pH.This provides a new method for grasping the status of embryonic development in real time and assessing the quality of embryos.Therefore,how to safely and accurately measure the temperature and pH in early embryos is of great significance.Aiming at the difficult problem of measuring the internal environmental parameters of early embryos,this subject was inspired by the existing fluorescent probe method for cell parameter measurement and further improved it,and proposed a multi-fluorescence microsensor based on polystyrene microspheres.The newly designed multi-fluorescence microsensor not only inherits the advantages of traditional fluorescent probes such as good targeting,high sensitivity and fast response speed,but also overcomes the shortcomings of traditional fluorescent probes such as cytotoxic side effects,easy leakage,and poor stability.The designed multi-fluorescence microsensor is used with a laser confocal fluorescence microscope with a live cell workstation to complete the monitoring of the whole process of early embryonic development.First,the broad application of fluorescent probes in biological microscopic imaging and sensing is briefly described,the current bottlenecks restricting the development of fluorescent probe methods are summarized,and the improvement of traditional fluorescent probe methods by researchers is listed.Second,the basic principle of fluorescence sensor is introduced and the best improvement scheme is given according to the application scene of this subject.Aminopolystyrene microspheres are used as the carrier of the sensor,the fluorescent probe rhodamine B is filled in the microspheres by the expansion method for temperature measurement,and the microspheres are bonded by chemical bonds.A layer of fluorescein isothiocyanate was modified on the surface of the microspheres for pH measurement.Third,the performance of the multi-fluorescence microsensor was measured,including the measurement of the sensor’s working spectrum,the calibration of temperature sensing,the calibration and temperature compensation of pH sensing,the determination of anti-ion concentration interference,the determination of stability and reversibility.Finally,the mouse oocytes and sperm were successfully prepared and in vitro fertilization was completed,and the early embryos of the experimental subjects were obtained.First,the multi-fluorescent microsensors were implanted into early embryos by piezoelectric ultrasonic microinjection technology,and then the sensors were proved to have low cytotoxicity through control experiments.Finally,the temperature and pH of the microenvironment inside and outside the early embryos were measured.The experimental data show that early embryos show a certain self-sustaining ability after leaving the culture environment,and their temperature is always 4～5℃ higher than the culture medium,but finally,with the distortion and death of early embryos gradually falling to room temperature,the pH in embryos also decreased significantly in the process of distortion.During the normal development of early embryos,the temperature in the embryo will decrease periodically during division,and the decrease range will weaken with the increase of the number of cells in the embryo.At the same time,the small-scale fluctuation of external ambient temperature will not cause the change of temperature in the embryo.Within 4h after changing the medium,the pH in early embryos gradually increased to the level consistent with the medium.