组成图示
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传感器类型
综述或非传感器论文
检测对象
槲皮素-3-葡萄糖醛酸苷（quercetin-3-glucuronide, Q3G）、人纤溶酶（plasmin）活性；样品基质：PBS 缓冲液、MCF-7 细胞培养体系
检测原理
在 IAsys plus 共振镜生物传感器中，羧酸盐表面经 EDC/NHS 活化后共价固定人纤溶酶，并用乙醇胺封闭。当 Q3G 加入时，其竞争性结合纤溶酶催化位点，使传感器界面质量/折射率发生变化，引起共振角（arcsec）改变；Q3G 浓度越高，结合量越大，信号随浓度升高并趋于饱和，通过结合/解离时间曲线可计算 kass、kdiss 和 KD。该过程无化学放大，依赖直接结合事件。另以 Chromozym-PL 为底物，纤溶酶催化释放 p-硝基苯胺，410 nm 吸光度反映酶活；Q3G 或提取物竞争占据活性位点，使产物生成速率下降，从而定量抑制。
检测灵敏度
原文未报告LOD、线性范围、灵敏度斜率或相关系数。
效应效果
生物传感器表面在至少50次结合事件中保持稳定性和酶活性，每个实验点为5次重复平均。Q3G与固定纤溶酶呈单指数、非协同结合，KD为175.2±22.8 nM，kass为17692±262 M−1 s−1，kdiss为3.1±0.4 ms−1，平衡KD*为199.2±21.8 nM；分光光度法测得KD为140±15 nM，整体提取物KD为0.34±0.10 mg/L。其他色谱组分对纤溶酶活性影响不显著，提示选择性主要来自Q3G。细胞实验中，Q3G浓度依赖抑制纤溶酶诱导的E-钙黏蛋白切割，并显著延缓MCF-7划痕迁移，效果优于槲皮素和整体提取物，接近aprotinin。作者认为Q3G是设计抗纤溶酶相关肿瘤转移药物的模板。
传感器的构成
- 基底/换能器：IAsys plus 共振镜生物传感器与羧酸盐比色皿（carboxylate cuvettes），提供光学共振信号读出
- 活化层：EDC/NHS 等摩尔混合液，活化羧酸盐表面以共价固定蛋白
- 识别/捕获元件：人纤溶酶（plasmin，0.2 mg/mL，10 mM 醋酸缓冲液 pH 5.5），固定于表面作为结合靶标
- 封闭剂：1 M 乙醇胺（pH 8.5），封闭未反应羧基
- 分析物/配体：槲皮素-3-葡萄糖醛酸苷（Q3G），以不同浓度加入并与固定纤溶酶结合
- 再生/洗脱：PBS（含0.05% Tween 20）及 pH 5.5 CH3COONa 洗涤，用于基线恢复和表面再生
中文摘要
小地榆（Sanguisorba minor）等多种可食用草药长期用于传统医学，其有益作用至少部分归因于多酚成分对多种酶活性的直接调节。本研究采用反相高效液相色谱对乙醇提取物进行表征，并用多级质谱鉴定主要分析物；随后检测整体提取物及主要分离成分对人纤溶酶活性的调节作用，包括对合成底物的酶活测定和在人乳腺癌细胞培养模型中的功能实验。通过分光光度法与生物传感器相结合的方法，获得酶-抑制剂相互作用的动力学和平衡参数。结果显示，槲皮素-3-葡萄糖醛酸苷（quercetin-3-glucuronide, Q3G）是提取物在体外抑制纤溶酶的主要成分，其抑制常数处于高纳摩尔范围；结合生物信息学、酶学和结合分析，该抑制被判定为竞争性抑制。细胞实验进一步表明，Q3G可有效抑制纤溶酶诱导的癌细胞黏附丧失。结果表明，小地榆提取物主要通过Q3G限制纤溶酶介导的肿瘤细胞运动，该葡萄糖醛酸化黄酮可作为设计治疗纤溶酶异常激活相关疾病的新药模板。
英文摘要
BACKGROUND: Sanguisorba minor, as well as several other edible herbs and vegetables, has been used extensively in traditional medicine. The observed beneficial effects can be attributed at least in part to the direct modulation of several enzymatic activities by its polyphenolic constituents.
METHODS: The ethanol extract of Sanguisorba minor was characterized by reversed-phase liquid chromatography, and most relevant analytes were identified by multiple stage mass spectrometry. The whole extract and the most relevant isolated constituents were tested for their ability to modulate the activity of human plasmin both toward a synthetic substrate and in human breast cancer cell culture models. Kinetic and equilibrium parameters were obtained by a concerted spectrophotometric and biosensor-based approach.
RESULTS: Quercetin-3-glucuronide was recognized as the compound mainly responsible for the in vitro plasmin inhibition by S. minor extract, with an inhibition constant in the high nanomolar range; in detail, our approach based on bioinformatic, enzymatic and binding analyses classified the inhibition as competitive. Most interestingly, cell-based assays showed that this flavonoid was effective in suppressing plasmin-induced loss of cancer cell adhesion.
GENERAL SIGNIFICANCE: Our results show that the extract from Sanguisorba minor limits plasmin-mediated tumor cell motility in vitro, mostly due to quercetin-3-glucuronide. This glucuronated flavonoid is a promising template for rational designing of anticancer drugs to be used in the treatment of pathological states involving the unregulated activity of plasmin.