Abstract: Objective To detect the cross-linking degree of bioprosthetic valve with carboxyl fluorescein succinimide ester(CFSE), which can combine with amino groups specifically and irreversibly and emits green fluorescent in 488 nm excitation optical wavelength. Methods Decellularized bovine pericardium was soaked in 0.5% glutaraldehyde solution and divided into four groups:blank control group, 10 minutes group, 30 minutes group and 6 hours group. Each specimen was made from frozen section respectively and immersed in the solution of CFSE, as a fluorescent amine-reactive probe. The fluorescence image of every frozen section can be observed by fluorescence microscopy and average fluorescence intensity of each image was measured with image analysis software. At the same time, the protein which was extracted from each group's bovine pericardium was tested by SDS-PAGE to compare with the amino immune fluorescent probe technology. Results The number of cross-linking amino reduced gradually with increased cross-linking time, as reflected in the light intensity of the groups, control group > 10 minutes group>30 minutes group>6 hours group,with remarkable statistics significance among groups (P<0.01). Conclusion Compared with the SDS-PAGE method, amino immune fluorescent probe technology is more intuitive, sensitive, and it can reflect the crosslinking effect of valves organization at any part and any layer. The amino immunefluorescent probe technology may provide a new theoretical basis and train of thought for degree detection of artificial biological valve crosslinking.

Key words: Bioprosthetic valve, Degree of cross-linking, Carboxyl fluorescein succinimide ester, Immunofluorescence probe