Technical Research Of Har Gabur Fluorescent Carbon Dots-GPC-3 In Early Diagnosis Of Hepatocellular Carcinoma

Objective: In order to improve the early diagnosis rate of liver cancer,it is necessary to couple the Har Gabur fluorescent carbon dots and GPC-3 antibody to identify the single cancer cell of liver cancer.Methods: 1)Preparation of Har Gabur fluorescent carbon dots: weigh 1g of Har Gabur of Mongolian medicine and put it into the simulated gastric juice,place the Beaker with gastric juice in an oil pot of 37 degrees and stir it under a magnetic stirrer for 12 hours,and then collect the gastric juice,centrifugally collect the supernatant by a bench type low-temperature high-speed centrifuge and store it in a refrigerator of 4 degrees,wash its sediment with distilled water twice,and then put it into the prepared simulated intestinal juice again in the same 37 degree oil pot,stir under the magnetic stirrer for 12 hours,then centrifugate again to collect the supernatant and store it in a 4 degree refrigerator.Put the supernatant of the two times into the dialysis membrane and put it in the beaker with distilled water for 48 hours,during which,change the distilled water in the beaker twice.After 48 hours,the two kinds of liquid in the dialysis membrane were mixed together,excited in the microwave oven for 5 minutes,and then put into the freeze-drying machine to collect the freeze-drying powder,which was stored in a 4-degree refrigerator away from light,and characterized by infrared spectrometer and fluorescence spectrophotometer.2)The Har Gabur fluorescent carbon dots was coupled with GPC-3 antibody to construct a complex coupling: 60 μ L acetic acid buffer（0.05 mol/L PH=4.2）and 120 μ L sodium periodate solution（1.5mg/mL PH=4.2）were added into 40 μ L GPC-3 monoclonal antibody solution and keep stirring for 2 hours under the condition of 4-degree dark,add the Har Gabur fluorescent carbon dots solution（5mg /mL）in the antibody solution after 2 hours,and keep stirring for 20 hours under the condition of 4-degree dark,add 20 μ L sodium borohydride solution（2mg/mL PH=7.1）in the composite solution after 20 hours,and keep stirring for 2 hours under the condition of 4-degree dark,After ultrafiltration,the Har Gabur fluorescent carbon dots-GPC-3 was collected and characterized by infrared spectrometer and fluorescence spectrophotometer.3)The liver cancer cells were imaged with Har Gabur fluorescent carbon dots and Har Gabur fluorescent carbon dots-GPC-3（HCDs-GPC-3）: when Hep G2 cells were in good condition,they were cultured in a confocal culture dish for 24 hours and divided into blank group,Har Gabur fluorescent carbon dots group and HCDs-GPC-3 group for 2 hours.After that,the culture solution was sucked out,and the fixed cells were stored at 4 degrees away from light.The cells were examined with confocal microscope to see the binding site with carbon dots and HCDs-GPC-3 and take photos.Results: 1)Under the fluorescence microscope,the Har Gabur fluorescent carbon dots can emit blue,red and green colors.Its emission wavelength reaches its peak at 420 nm.The fluorescence emission peak is wide and symmetrical,showing the maximum fluorescence intensity.The results show that the absorption peak of 3467/cm^-1 is consistent with the characteristic peak of O-H group,and the absorption peak of 1636/cm^-1 is consistent with the characteristic peak of-NH2,indicating that the surface of the Har Gabur fluorescent carbon dots has the functional groups-NH2 and-COOH.2)The blue shift of the wavelength corresponding to the maximum peak value was found by comparing the fluorescence emission patterns of the carbon dots of the couplet and the Har Gabur fluorescent carbon dots.The results showed that the coupling of GPC-3 antibody had an effect on the fluorescence of the Har Gabur fluorescent carbon dots.FTIR of Har Gabur fluorescent carbon dots and HCDs-GPC-3 shows that there is a different absorption peak in the infrared spectrum of the HCDsGPC-3 compared with the Gabur fluorescent carbon dots FTIR spectrum.The peak at 1439/cm^-1 is attributed to the stretching vibration of the amide bond（N-C）.The existence of this characteristic peak indicates the formation of the amide bond（CONH）and the successful synthesis of the coupling.3)In the experiment of liver cancer cells,Har Gabur fluorescent carbon dots and HCDs-GPC-3 were respectively administered to the cells.Through the observation of inverted fluorescence microscope and confocal microscope,it was found that the Har Gabur fluorescent carbon dots could enter the cells smoothly and emit blue fluorescence under the microscope,while the HCDs-GPC-3 could be found to concentrate on the surface of the cell membrane after comparing the Har Gabur fluorescent carbon dots.It does not enter into the cell,indicating that GPC-3 antibody has a very good coupling effect with the fluorescent carbon spot of Har Gabur fluorescent carbon dots,and the antibody also has a good retention of its targeting,which is very important for the early diagnosis of liver cancer.Conclution: 1)By the method of sodium periodate oxidation,the Har Gabur fluorescent carbon dots was successfully coupled with GPC-3 antibody,and a targeted compound coupling（HCDs-GPC-3）was prepared.2)The complex coupling of the Har Gabur fluorescent carbon dots and GPC-3 antibody can specifically identify the hepatoma cells,which is of great significance for the early diagnosis of hepatoma.