Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques
Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques
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DOI：
10.1016/j.apsusc.2013.11.040
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发表时间：
2014-01
影响因子：
6.7
通讯作者：
K. Awsiuk;J. Rysz;P. Petrou;A. Budkowski;A. Bernasik;S. Kakabakos;M. Marzec;I. Raptis
中科院分区：
文献类型：
--
作者：
K. Awsiuk;J. Rysz;P. Petrou;A. Budkowski;A. Bernasik;S. Kakabakos;M. Marzec;I. Raptis
为了在（3-氨基丙基）三乙氧基硅烷修饰的sio2上有效固定寡核苷酸探针，采用原子力显微镜、角分辨x射线光电子能谱和飞行时间二次离子质谱对链霉亲和素-生物素体系的四种方法进行了比较。第一种方法包括：吸附生物素化的牛血清白蛋白，用BSA阻断自由表面位点，结合链亲和素和生物素化的寡核苷酸（b-oligo）。最后的步骤是交换在第二程序与固定预制链亲和素- b寡聚偶联物。第三种方法包括链霉亲和素吸附、BSA阻断和b-寡核苷酸结合。最后，链霉亲和素- b-寡聚物在第四种方法中直接固定。ARXPS测定的生物分子表面覆盖率与平均AFM高度一致，与Si+离子强度呈反相关。与第三种方法（0.58（±0.20）mg/m2）和第四种方法（0.41（±0.11）mg/m2）的表面密度较低相比，在第一种方法的最后步骤（2.45（±0.38）mg/m2）和第二种方法（1.31（±0.22）mg/m2）中获得了更高的生物分子覆盖率。磷原子浓度反映了寡核苷酸固定的有效性。次级离子强度，寡核苷酸，链亲和素，牛血清白蛋白和蛋白质的特征，允许额外的洞察层的组成。这些测量结果验证了ARXPS结果，并显示了前两种固定方法在链霉亲和素和表面寡核苷酸密度方面的优势。
To immobilize effectively oligonucleotide probes on SiO2modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m2) and second (1.31(±0.22) mg/m2) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m2) and fourth (0.41(±0.11) mg/m2) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.