组成图示
示意图生成中
传感器类型
表面等离子共振(SPR)生物传感器
检测对象
生物素标记蛋白（biotinylated proteins, BP）；样品基质：HeLa 细胞膜样品/细胞裂解液（HBS 稀释）
检测原理
细胞表面蛋白先经 Biotin-(AC5)2-OSu 生物素化，使质膜蛋白共价携带生物素。将含生物素化蛋白（BP）的膜样品注入亲和素固定化 SPR 表面后，BP 通过生物素–亲和素高亲和作用特异性结合；随后注入 2 mol/L guanidine-HCl 和 0.5% Triton X-100 洗涤液去除非特异性结合组分。结合量增加使传感器表面质量/折射率变化，引起 SPR 共振条件改变，Biacore 1000 以共振单位（RU）读出净结合量。由于重复注入–再生导致表面结合能力逐渐衰减，方法在每组测定末端注入 1 unit/mL BP 测定衰减比，并按算术衰减模型校正 RU，再经校准标准曲线定量 BP 滴度。该过程无需参考表面。
检测灵敏度
标准曲线范围: 0–2.0 unit ml^-1 BP；相关系数 = 0.998（校准后标准曲线）；相关系数 = 0.937（校准前标准曲线）；第四组校准后相关系数 = 0.91
效应效果
方法具有良好选择性：MOLT-4 细胞生物素化后荧光仅位于细胞周边，Tween 20 预处理则胞内也出现荧光，说明生物素化主要限于质膜；生物素化 BSA/OVA 经洗涤后 RU 不变，证实 avidin–biotin 结合不被洗脱。校准后，前三组验证样品计算值与理论值之比由 0.92–1.04（SD 0.09–0.23）改善至 1.00–1.01（SD 0.04–0.09）；第四组比值 >1.5、SD >0.5，不可用。未校准标准曲线第四组斜率衰减至第一组的 1/8，校准后相关系数仅 0.91。BP 滴度与氨基肽酶 N 活性的膜纯化比无显著差异（p > 0.01）。方法无需参考表面，可节省一半样品体积和时间，但芯片寿命有限，可通过再固定 avidin 回收。
传感器的构成
- 基底/换能器：CM5 传感器芯片（提供羧基用于固定）
- 活化层：EDC/NHS（N-乙基-N'-(3-二乙氨基丙基)碳二亚胺/N-羟基琥珀酰亚胺）活化羧基形成活性酯
- 识别元件：avidin（亲和素）固定于活化表面，特异性结合生物素化蛋白
- 封闭剂：1 mol/L ethanolamine-HCl（乙醇胺盐酸盐）封闭游离酯基
- 信号标记物：biotinylated proteins（BP，生物素标记蛋白），其生物素与 avidin 结合产生 SPR 信号
- 洗涤液：2 mol/L guanidine-HCl + 0.5% Triton X-100，去除非特异性结合组分
- 再生剂：100 mmol/L NaOH，用于表面再生
- 运行缓冲液：HBS（HEPES-buffered saline，10 mM HEPES、150 mM NaCl、1 mM EDTA、0.005% Tween 20，pH 7.4）
中文摘要
动物细胞质膜蛋白可作为质膜纯化程度的指标。本文提出一种基于表面等离子共振（SPR）生物传感器测定质膜蛋白的简便方法。用 5-[5-(N-琥珀酰亚胺氧羰基)戊基酰胺]己基-D-生物素酰胺对 MOLT-4 细胞进行生物素化后，仅在细胞周边区域观察到生物素化蛋白；HeLa 细胞表面蛋白同样可被生物素化。将 HeLa 细胞膜样品注入亲和素固定化 SPR 表面，随后用洗涤液去除非特异性结合组分，并在洗涤后直接根据绝对共振单位（RU）测定生物素化蛋白（BP）含量，该方法无需参考表面。由于重复注入导致表面结合量逐渐衰减，引入基于每次注入衰减比的 RU 校准方法，使校准后 BP 滴度与理论估算值的相关性显著提高。同一传感器表面可成功完成三轮 BP 测定。在膜组分纯化过程中，由 BP 滴度判断的纯化程度与膜组分中氨基肽酶 N 活性升高程度一致，表明 BP 滴度可作为质膜纯化的指标。
英文摘要
Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-D: -biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.