Roque A C A, Bispo S, Pinheiro A R N, Antunes J M A, Gonçalves D, Ferreira H A
REQUIMTE/CQFB, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
J Mol Recognit. 2009 Mar-Apr;22(2):77-82. doi: 10.1002/jmr.913.
Magnetic particles (MNPs) offer attractive possibilities in biotechnology. MNPs can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide (Fe3O4) MNPs with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat IgG), bovine serum albumin (BSA) as blocking agent, and a complementary antibody labelled with FITC (anti-goat IgG). The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated MNPs, a 5% (w/v) concentration of BSA in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.
磁性颗粒（MNPs）在生物技术领域展现出诱人的潜力。MNPs能够接近目标生物实体，因为其可控尺寸范围从几纳米到几十纳米，并且其表面可以进行修饰，以增加对所需分子的亲和力和特异性。此外，它们可以通过外部磁场梯度进行操控。在这项工作中，对具有不同包被剂的氧化铁（Fe3O4）MNPs进行了研究，特别是关于抗体在表面附着的策略（共价吸附和物理吸附），以及封闭缓冲液组成和孵育时间对所观察到的特异性和非特异性相互作用的影响。所考虑的生物模型系统由包被抗体（山羊IgG）、作为封闭剂的牛血清白蛋白（BSA）以及用异硫氰酸荧光素（FITC）标记的互补抗体（抗山羊IgG）组成。通过荧光显微镜观察抗体结合情况，并对信号强度进行定量。在评估测定效率时，考虑了阴性和阳性对照的平均灰度值之比以及阳性对照中可达到的最大强度。与蛋白质吸附相比，包被抗体的共价固定更为成功。对于共价固定，二氧化硅包被的MNPs、封闭缓冲液中5%（w/v）浓度的BSA以及1小时的孵育时间在测定灵敏度方面产生了最佳结果。然而，当孵育时间为10分钟进行测定时，荧光信号降低了44%，但测定特异性得以保持。
