采用共沉淀法合成平均粒径为18nm的Fe2O3磁性纳米颗粒，并用具有双功能基团的二巯基丁二酸(HOOC-CH(SH)-CH(SH)-COOH，DMSA)进行表面修饰。透射电子显微镜(TEM, JEOL, JEM-200EX,)，X衍射(XRD, Rigaku, D/Max-RA, λ=1.5405×10-10m, CuK)，磁共振磁强计(VSM，Lakeshore 7407)、Zeta电位分析仪(BECKMAN, Delsa 440SX)等对其进行表征，结果表明Fe2O3纳米颗粒近于球形，立方反尖晶石结构，最大饱和磁化强度为67.6emu/g ， 在pH值为3-11范围内，具有很高的表面电位从而使颗粒不易聚集。
碳化二亚胺(EDC, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) 活化磁性颗粒表面的羧基，使其与抗体表面的羟基形成酰胺键，将两者以化学键结合。采用酶联免疫分析(ELISA)方法，对其表面结合的抗体进行活性分析，结果表明抗体与磁性颗粒结合后仍然保持其活性，同时用未结合抗体的磁性颗粒作对比，发现磁性颗粒本身对心肌肌钙蛋白I没有非特异吸附现象。
Fe2O3 nanoparticles are synthesized by chemical coprecipitation with the average particle size of 18nm, and were chelated with meso-2,3-dimercaptosuccinic acid (HOOC-CH(SH)-CH(SH)-COOH) or DMSA, which fomed strong complexes with the surface layer of the nanoparticles. The particle size and morphology of the samples were determined by transmission electronic microscopy (TEM, JEOL, JEM-200EX,). Powder X-ray diffraction (XRD, Rigaku, D/Max-RA, λ=1.5405×10-10m, CuK) and electronic diffraction ( ED, JEOL, JEM-200EX) were used to determine the crystal structure of the samples. Surface charge measurements were performed with a Zeta Potential Analyzer (BECKMAN, Delsa 440SX). The magnetic measurements were carried out with a Vibrating Sample Magnetometer（VSM，Lakeshore 7407). The results indicate that most of the particles are quasi-spherical and with an average diameter of 18nm and inverse cubic structure. The value of Ms, 67.6 emu/g, at ambient temperature, is slightly smaller than that of bulk maghemite (75.0 emu/g), the particles have high negative charges at the pH range (3-11), which can form strong electrostatic repulsion to protect the particles from congregating.
EDC is a carboxyl and amine-reactive cross-linker, which reacts with a carboxyl group and amino group to form an amide bond. To demonstrate the activity of the maghemite nanoparticles conjugated with antibody (IMMNPS), cardiac troponin I (cTnI) antigen was used as target for detection by an indirect ELISA method. The results show that the antibody remains its biological activity. The blank particles and the boiled sample almost have no effect on the OD values compared with the sample, which can be considered that the blank particles or the boiled sample have neither specific recognition capacity nor non- non-specific adsorption.
Figure 1: The preparation scheme of the anti-human cTnI antibodies and DMSA-coated MNPs
Figure 2:The effect of the non-specific adsorption of the DMSA-coated MNPs and the boiled IMNPs on OD values, and the activity of the IMNPs.
Figure 3: The activity curve of the IMNPs with different amount of anti-human cTnI antibodies.
Figure 4: The stability curves of the IMNPs with different amount of anti-human cTnI antibodies.
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