| In recent years,gold nanopaticles(AuNPs) were widely used in catalysis,optics, elecrics,sulface enhanced Raman scattering,biosensor and emerging field of nanotechnology,due to the special chemical and physical properties of AuNPs. Owing to their optical property,macromolecules such as protein and DNA modified AuNPs are of great interests in the fields of therapeutics,diagnostics and other aspects. Carbohydrate compounds are a kinds of important biological molecules,which can forming glycolipids and glycoproteins,and play essential roles in recognition, immunological activity and other physiological activity.However,research on carbohydrate modified AuNPs was at primary stage,and further studies were needed for developing the manufacturing technique and exploring their potential applications..Heparin is a highly sulfated and negative polysaccharide,which is present on the surface of mast cells.It has numerous important biological activities by interacting with diverse biological important proteins.This work was aimed to preparation of thiolated heparin modified AuNPs and use them to detect ATⅢ.Detailed work includes:(1) preparation of thiolated heparin;(2) preparation of thiolated heparin modified gold nanoparticles;(3) detection of ATⅢ.Heparin with an aldehyde group(HEP-CHO) as reducing end was prepared by nitrous acid degradation method from heparin sodium.The amino group of paminothiophenol (PATP) forms a unstable Schiff base with the aldehyde group of HEP-CHO.Then,thiolated heparin was obtained through reductive amination and sodium cyanoboronhydride as catalyst.The percentage of thiolated heparin was improved by controlling reaction time and solvent.The amount of thiol group on heparin was measured by Ellman's reagent.It was found that 11.13%heparin was thiolated when using dimethyl sulfoxide to acetic acid was 8:1(v/v) as solvent and the reaction time was 6 hours.IR and NMR spectum of HEP-CHO and thiolated heparin indicated the existence of thiol group.Gold nanoparticles were prepared by the sodium citrate reduction method.We discussed the effects of quantity of sodium citrate and pH value of solution on the properties of AuNPs.It was found that the gold nanopaticles with excellent paticle size was formed at a rate of sodium citrate to chloroauric acid was 6:1(mol/mol),and them were stable at the range of pH value was 7.0～11.0.The result of transmission electron microscope showed that gold nanoparticles had homogeneous particle diameter and good dispersion.The average diameter and maximum UV absorbance of gold nanoparticles were about 10 nm and 523 nm.We also discussed the effects of quantity of thiolate heparin on the properties of modified gold nanoparticles,and determined the optimum modified condition.Binding rate of thiolated heparin on gold nanoparticles was from 27%～38%when the concentration of thiolated heparin was 0.125～25 mg/ml in 0.5 hour.The average diameter and maximum UV absorbance of modified gold nanoparticles were about 22 nm and 531 nm.The red shift of the maximum UV absorbance was 8 nm,suggesting the size effect on the maximum UV absorbance. Heparin was end-point immobilized on the gold nanoparticles by forming the Au-S bonds between the thiol group of thiolate heparin and Au atom.This immobilized method made gold nanopaticles stable.Finally we discussed the detection sensitivity of thiolated heparin modified gold nanoparticles on detecting ATⅢ.ATⅢwas detectable when the content was 86.2 nmol/l.The maximum UV absorbance of solution was 534 nm and that of solution without ATⅢwas 533 nm.The red shift was 1 nm.Moreover,the extent of red shift of solution was larger when the more ATⅢwas added.Meanwhile,we detectedα-fetoprotein by using gold nanoparticles labeling antibody A4C11 and A14A11.Binding percentage of A4C11 and A14A11 on gold nanoparticles were 70.39%and 92.29%in 6 hours respectively,α-fetoprotein was detectable when the concentration ofα-fetoprotein was 17.2 pmol.The maximum UV absorbance of soution were 533 nm, and that of solution withoutα-fetoprotein were 531 nm.The red shift was 2 nm.It was showed that the detection sensitivity of detectingα-fetoprotein was higher. Furthermore,we dicussed about the different detection mechanisms of those two detection systems.
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