Research On The Detection Of Protein Using Gold Nanoparticle Probes

Carc ingembryonic antigen(CEA) is one of the acid glycoprotein,many research works have been reported abroad.It is said that the CEA level of lung cancer patient is much higher that healthy people,and the positive rate is also the highest.So,the CEA detection is very useful for enhancing the sensitivity for lung cancer's early diagnose. As the concentration of CEA is very low in the serum of the patient,so developing a protein detection method with high sensitivity is very important.With the development of nanotechnology,nanoparticle appears a high potential in the disease detection field because of its special optical and electrical properties,good stability and surface effect,as well as the special biological compatibility.Many protein detection methods using nanoparticle have been reported,however,all these methods needs a long time to operate and are very complicated,the detection appliances are also very expensive,only several labs use them.A easier nanoparticle method,which can use common detection appliance and with high sensitivity,needs to be developed.For this purpose,a novel protein detection method using the gold nanoparticle(AuNP) has been developed.In this method,the micromagnetic particles(MMPs) functionalized with capture antibody,CEA antigen,as well as AuNP probes immobilized with detecting antibody and SA-HRP are used to form a sandwich structures.The mixture is given a further incubation with TMB solution.Then the absorbance value was measured at 450nm.The finished work in this research is as following:1 The mark and representation of the magnetic probe2 The marking method research of the gold nanoparticle probes3 The research on the protein detection method with AuNP probesConclusion: 1 From the fluorescent detection result,the CEA antibody is marked to the MMPs, and it is shown from the immunity detection that the antibodies marked to the MMPs are still actived.2 From the protein stability detection experiment,the minimum quantity of AuNPs marked protein is 0.6μl.The sensitivity of this method is depended on the number of the marked enzyme on them.So the quantity of CEA and SA-HRP needs to be optimized.From the experience, the ideal ratio of detecting antibody CEA(V):SA-HRP(V) is 0.5μl:5μl/100μl AuNPs solution.A high positive A value can be obtained in this condition.The choice of resuspension of AuNP probes also has a key effect on the stability.From the experience, The solution with 50mM Tris(pH7.8,1.25%saccharose,0.2%BSA,0.05%PEG, 0.05%PVP,0.15%Tween20) can maintain the stability,and is the optimal solution.3 The optimization for the protein detection method with AuNP:the incubation solution in the first step is 10 mM PB(PH7.4,1%BSA,5%sucrose,1%PEG, 1%PVP,0.3%Tween20),the incubation solution in the second step is 10mM PBS (PH7.4,0.1%BSA,0.05%Tween20,AuNP probes is 3μ1.The incubation time for the first step is 1h,and 45min for the second step.By this method to draw the CEA caliberation curve,it is found that there is good linear relationship for the CEA concentration in 250pg/ml-25ng/ml,and R~2 equals 0.991.From the sensitivity experiment,it is found that the lowest detection sensitivity is 25pg/mL,however,the detection sensitivity for the traditional ELISA method is only 1.65ng/mL,the detection sensitivity for RIA is 1μg/ml,so this research has gotten the target for high sensitivity protein detection through common optical appliances.