Amine-Specific Fluorescent Probe Used For Crosslinking Degree Detection Of Bovine Pericardial In Bioprosthetic Valve Making

OBJECTIVEBiological materials are widely used in the field of cardiovascular surgery, such as porcine aortic valve,bovine pericardium, bovine jugular vein are usually used to make biological heart valve, vascular with valve, patches, and so on. They should be crosslinked before they were used in surgery. Crosslinking of the biological materials involve the binding of amine groups or carboxyl groups, the crosslinking degree determines the effect of the biological material processing. Therefore, the detection of crosslinking degree is an important method to judge the quality of the crosslinked biological material. At prescent, the method of testing crosslinking degree is indirect and old, it can not reflect the crosslinking effect accurately. Here, we use carboxyfluorescein succinimidyl ester (CFSE) to develop a new method to assess crosslinking effect, which quantified the amount of unbound amines. CFSE is used for its specificities that combines with amino groups at neutral PH and emits green fluorescent in488nm excitation optical wavelength.METHODSThe bovine pericardium was divided into four groups:group A, B, C, D. After being decellularized, each group of bovine pericardium were exposed to0.5%glutaraldehyde solution for0,10,30minutes and6hours respectively. Every piece of pericardium was handled with frozen section. And frozen sections were exposed to carboxyfluorescein succinimidyl ester (CFSE), a fluorescent amine-reactive probe. After2hours’mark, the fluorescent intensity for each image can be observed through fluorescence microscope and an average intensity was calculated by Image Analysis Software. Then we can compare the differences of fluorescence intensity by statistical analysis. In order to compare with the new technique, we adopt a traditional assay to assess the crosslinking degree, Protein extraction assay and polyacrylamide gel electrophoresis (PAGE).RESULTSThe amount of uncrosslinked amines was observed decreasing gradually with fixation time, group A> B> C> D. In other words, the fluorescent intensity became weaker with time. And the extractable protein has a gradual decline with the increase of the crosslinking time.CONCLUSIONSOverall, CFSE has an intrinsically faster reaction rate with amino groups, they form a more stable covalent linkage, which can emits green fluorescent in488nm excitation optical wavelength. The new technique is more sensitive and more direct, it can reflect the cross-linking effect of any part or layer you want. Meanwhile, it maybe reveal the mechanisms of other crosslinking methods.