School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 807, Taiwan.
Biosensors (Basel). 2023 May 24;13(6):574. doi: 10.3390/bios13060574.
In this study, a simple, easy and convenient fluorescent sensing system for the detection of the vascular endothelial growth factor (VEGF) based on VEGF aptamers, aptamer-complementary fluorescence-labeled probe and streptavidin magnetic beads was developed in one single tube. The VEGF is the most important biomarker in cancer, and it is investigated that the serum VEGF level varied according to the different types and courses of cancers. Hence, efficient quantification of VEGF is able to improve the accuracy of cancer diagnoses and the precision of disease surveillance. In this research, the VEGF aptamer was designed to be able to bind with the VEGF by forming G-quadruplex secondary structures; then, the magnetic beads would capture the non-binding aptamers due to non-steric interference; and finally, the fluorescence-labeled probes were hybridized with the aptamers captured by the magnetic beads. Therefore, the fluorescent intensity in the supernatant would specifically reflect the present VEGF. After an overall optimization, the optimal conditions for the detection of VEGF were as followed, KCl, 50 μM; pH 7.0; aptamer, 0.1 μM; and magnetic beads, 10 μL (4 μg/μL). The VEGF could be well quantified within a range of 0.2-2.0 ng/mL in plasma, and the calibration curve possessed a good linearity (y = 1.0391x + 0.5471, r = 0.998). The detection limit (LOD) was calculated to be 0.0445 ng/mL according to the formula (LOD = 3.3 × σ/S). The specificity of this method was also investigated under the appearance of many other serum proteins, and the data showed good specificity in this aptasensor-based magnetic sensing system. This strategy provided a simple, sensitive and selective biosensing platform for the detection of serum VEGF. Finally, it was expected that this detection technique can be used to promote more clinical applications.
在这项研究中，开发了一种基于血管内皮生长因子（VEGF）适体、适体互补荧光标记探针和链霉亲和素磁珠的简单、易用、方便的荧光传感系统，可在单个试管中检测 VEGF。VEGF 是癌症中最重要的生物标志物，研究表明，血清 VEGF 水平根据癌症的不同类型和阶段而变化。因此，VEGF 的有效定量能够提高癌症诊断的准确性和疾病监测的精确性。在这项研究中，VEGF 适体被设计为能够通过形成 G-四链体二级结构与 VEGF 结合；然后，由于空间位阻干扰，磁珠会捕获非结合适体；最后，荧光标记探针与磁珠捕获的适体杂交。因此，上清液中的荧光强度将特异性反映当前的 VEGF。经过全面优化，检测 VEGF 的最佳条件如下：KCl，50 μM；pH 值 7.0；适体，0.1 μM；磁珠，10 μL（4 μg/μL）。VEGF 在血浆中的定量范围为 0.2-2.0 ng/mL，校准曲线具有良好的线性关系（y = 1.0391x + 0.5471，r = 0.998）。根据公式（LOD = 3.3 × σ/S）计算检测限（LOD）为 0.0445 ng/mL。在许多其他血清蛋白存在的情况下，还研究了该方法的特异性，结果表明该基于适体的磁传感系统具有良好的特异性。该策略为血清 VEGF 的检测提供了一种简单、灵敏和选择性的生物传感平台。最后，预计该检测技术可用于促进更多的临床应用。
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