组成图示
点击图片查看大图 · 依据论文自动绘制
传感器类型
荧光生物传感器
检测对象
钾离子（K+）；样品基质：5 mM Tris-HCl缓冲液（含20 mM NaCl、2 mM MgCl2），面向血清/生物流体
检测原理
该传感器采用适配体-互补探针均相荧光检测策略。未修饰血栓结合DNA适配体与两端标记吡rene的部分互补寡核苷酸先杂交形成异源双链，此时两个吡rene距离较远，仅产生弱单体荧光。加入K+后，K+与适配体结合并诱导其形成椅型G-四链体结构，使互补寡核苷酸从适配体上被竞争置换释放。释放的探针通过自身5'和3'端互补序列折叠成发卡结构，使两端吡rene分子靠近并形成激基复合物，在485 nm处产生显著荧光增强。K+浓度越高，被置换释放的探针越多，激基复合物荧光强度越大，从而实现定量检测。该策略属于signal-on检测，背景低，且吡rene激基复合物寿命较长，有利于时间分辨测量。
检测灵敏度
LOD: 4.0 × 10^-4 M；线性范围: 6.0 × 10^-4–2.0 × 10^-2 M；灵敏度斜率: +11.6% per 1 mM K+ ions
效应效果
在5 min孵育下，方法对K+检出限为0.4 mM，低于血清K+正常范围3.5–5.3 mM，灵敏度良好。选择性方面，在Na+、Mg2+、Ca2+和NH4+存在下，仅K+引起明显激基复合物荧光增强；Na+浓度超过100 mM时才出现微弱信号，且2 mM KCl在100 mM NaCl存在下仍可获得与无Na+时相近的荧光响应，表明抗Na+干扰能力强。作者指出，吡rene激基复合物荧光寿命约40 ns，明显长于生物背景<5 ns，可通过时间分辨荧光消除背景，因此该策略有望用于血清和活细胞中K+检测。论文未报告RSD、实际样品回收率或与ELISA/HPLC/qPCR的定量对比。
传感器的构成
- 检测体系：溶液相均相体系，无需固相基底或电极，反应在5 mM Tris-HCl（pH 7.2）缓冲液中进行
- 识别元件：未修饰血栓结合DNA适配体 d(GGT TGG TGT GGT TGG)，特异性结合K+并形成椅型G-四链体
- 信号探针：两端标记吡rene（pyrene）的部分互补寡核苷酸 d(TGG TGT CAA CCA CAC CA)，与适配体杂交后被K+置换并自折叠产生激基复合物荧光
- 反应缓冲液：5 mM Tris-HCl（pH 7.2）含20 mM NaCl和2 mM MgCl2，维持杂交、适配体折叠及离子环境
- 荧光读出装置：Hitachi F-4500荧光分光光度计，激发340 nm、发射485 nm，监测吡rene激基复合物荧光强度
中文摘要
本文开发了一种基于适配体和吡rene标记荧光探针的钾离子（K+）灵敏生物传感器。以未修饰的血栓结合DNA适配体作为分子识别元件，将一段与适配体部分互补的寡核苷酸两端标记吡rene，用于转导K+与适配体的结合事件。当体系中存在K+时，K+诱导适配体形成椅型四链体结构，使互补寡核苷酸从适配体上被置换释放；释放的互补寡核苷酸通过自折叠形成发卡结构，使两端吡rene分子靠近并产生激基复合物荧光。相反，在无K+时仅观察到吡rene单体发射。在最优条件下，吡rene相对荧光强度与K+浓度在6.0×10^-4至2.0×10^-2 mol/L范围内呈线性关系，检出限为4.0×10^-4 mol/L。此外，该方法在生物流体中常见的Na+、NH4+、Mg2+和Ca2+存在下仍能以高选择性检测K+。由于操作简便、灵敏度高且特异性好，该检测策略在生物环境中具有广阔应用潜力。
英文摘要
A novel and sensitive biosensor based on aptamer and pyrene-labeled fluorescent probes for the determination of K+ was developed. The aptamer was used as a molecular recognition element and a partially complementary oligonucleotide with the aptamer was labeled by pyrene moieties at both ends to transduce the binding event of K+ with aptamer. In the presence of K+, the complementary oligonucleotides were displaced from aptamers, which was accompanied by excimer fluorescence of pyrenes because the self-hairpin structure of the complementary oligonucleotide brought pyrene moieties into close proximity. However, it gave only monomer emission in the absence of K+. Under optimum conditions, the relative fluorescence intensity of pyrene was proportional to the concentration of K+ in the range of 6.0 x 10(-4) to 2.0 x 10(-2) M. A detection limit of 4.0 x 10(-4) M was achieved. Moreover, this method was able to detect K+ with high selectivity in the presence of Na+, NH4+, Mg2+, and Ca2+ ions of biological fluids. In brief, the assay may have great potential applications, especially in a biological environment because of its simplicity, sensitivity, and specificity.