Kayran Yasin Ugur, Ozkan-Ariksoysal Dilsat, Topkaya Seda Nur, Kaymaz Burcin Tezcanli, Can Buket Kosova
Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Izmir. Turkey.
Department of Medicinal Biology, Faculty of Medicine, Ege University, Izmir. Turkey.
Comb Chem High Throughput Screen. 2017;20(1):56-63. doi: 10.2174/1386207320666170103154226.
Surface Plasmon Resonance (SPR) based biosensor system was developed for the detection of Delta F508 (ΔF508del) Cystic Fibrosis (CF) mutation in both synthetic and real samples.
In order to provide an effective hybridization between probe and the Polymerase Chain Reaction (PCR) amplicons (target), streptavidin was bound to the surface and biotin-tag probe was sent to the streptavidin-coated surface. For the target preparation, blood samples were collected from the patients who suffer from CF. Following the DNA isolation; samples were amplified with PCR with biotin-tag. Before sending the biotin-tag PCR amplicons onto the modified surface, amplicons were also interacted with the helper oligonucleotides to prevent re-annealing of the denatured DNA strands. This kind of 'multiple surface binding' method helps increasing the sensitivity of the detection.
The limit of detection (S/N= 3) was calculated as 12.24 pico-mole/ml for PCR-like synthetic long target sequence and 13x105 molecules for real samples in less than half an hour.
Using the both biotin-tag probe and the helper oligonucleotides together, hybridization was achieved much more efficiently than traditional denaturation protocols for real samples and biotinfree hybridization detection. To the best of our knowledge, the procedure described in this study is one of the simplest, rapid and sensitive methods for CF mutation detection with SPR based biosensor system in real samples.
开发基于表面等离子体共振（SPR）的生物传感器系统，用于检测合成样品和实际样品中的ΔF508del囊性纤维化（CF）突变。
为了使探针与聚合酶链反应（PCR）扩增子（靶标）有效杂交，将链霉亲和素结合到表面，并将生物素标记的探针送至链霉亲和素包被的表面。为了制备靶标，从患有CF的患者身上采集血样。DNA分离后，样品用生物素标记的引物进行PCR扩增。在将生物素标记的PCR扩增子送至修饰表面之前，扩增子还与辅助寡核苷酸相互作用，以防止变性DNA链重新退火。这种“多表面结合”方法有助于提高检测灵敏度。
对于类似PCR的合成长靶序列，检测限（S/N = 3）计算为12.24皮摩尔/毫升，对于实际样品，在不到半小时内检测限为1.3×10⁵个分子。
同时使用生物素标记的探针和辅助寡核苷酸，与传统的实际样品变性方案和无生物素杂交检测相比，杂交效率更高。据我们所知，本研究中描述的方法是基于SPR的生物传感器系统在实际样品中检测CF突变的最简单、快速且灵敏的方法之一。
