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
三硝基甲苯（TNT，trinitrotoluene）；样品基质：水溶液/PBS缓冲液（文中亦讨论地雷土壤残留蒸气浓缩后检测）
检测原理
该传感器基于间接竞争免疫SPR检测。TNP-β-ala-OVA通过EDC/NHS胺偶联固定在CM5芯片羧甲基葡聚糖基质上，作为固相抗原。兔抗TNP多克隆抗体与固相抗原结合后，使金膜表面质量与折射率增加，SPR共振角/入射角发生偏移Δθ0。当样品中存在游离TNT时，TNT与抗体竞争结合，使抗体结合到固相抗原的量减少，Δθ降低；以100×(Δθ/Δθ0)表示抑制率，TNT浓度越高，角移越小。随后注入抗兔IgG二抗，与已结合的一抗结合，增加表面质量，放大角移并降低检出限。
检测灵敏度
LOD: 3 × 10−11 g/ml (30 ppt)；LOD: 1 × 10−11 g/ml (10 ppt)；线性范围: 3 × 10−11 to 3 × 10−7 g/ml；r = 0.915
效应效果
该SPR免疫传感器对TNT具有较高选择性：与二硝基芳香化合物及N-(2,4,6-TNP)-烷基胺交叉反应低于1%或无抑制，但对TNP-aha和TNA交叉反应分别高于13911%和1470%。4 ℃下在10−11 g/ml TNT可产生约15%抑制，抗体在50 ℃下未降解。各浓度点RSD（n=3）小于4%；重复20次免疫反应后信号下降约10%，可用NaOH/甘氨酸-HCl再生，但作者认为现场检测宜用一次性芯片。加入抗兔IgG后角移增强约40%，检出限由3×10−11 g/ml降至1×10−11 g/ml。作者认为便携SPR适合地雷现场检测，土壤残留蒸气约0.1 ppt需浓缩100倍。
传感器的构成
- 基底/换能器：金膜玻璃片（Au-coated glass slide）嵌于塑料支撑平台，作为SPR光学换能基底。
- 修饰层：羧甲基葡聚糖（CM-dextran）共价结合于Au表面，增加结合容量并降低非特异吸附。
- 化学偶联层：EDC/NHS胺偶联形成NHS酯，将TNP-β-ala-OVA共价固定；乙醇胺盐酸盐（ethanolamine HCl）封闭未反应酯基。
- 固相识别/捕获层：TNP-β-ala-OVA固定于葡聚糖基质，作为固相抗原捕获抗体。
- 液相识别元件：兔抗TNP多克隆抗体（anti-TNP Ab，rabbit IgG）与固相抗原结合，受TNT竞争抑制。
- 信号放大标记：抗兔IgG抗体（anti-rabbit IgG）结合已结合抗体，增加表面质量并放大SPR角移。
- 读出系统：Biacore J-2000 SPR生物传感器，监测入射角/共振角变化（Δθ）。
中文摘要
本研究在兔体内制备了针对三硝基苯基（TNP）衍生物的多克隆抗体，并将其用于表面等离子共振（SPR）生物传感器检测三硝基甲苯（TNT）。将TNP-孔介孔蓝血蛋白（TNP-KLH）偶联物免疫兔，经Protein G纯化血清获得抗TNP多克隆抗体。作者系统评价了该抗体对多种硝基芳香化合物的交叉反应性和亲和力，并考察了抗TNP抗体与TNT亲和力的温度依赖性。TNT定量基于间接竞争免疫分析原理：TNP-β-丙氨酸-卵白蛋白（TNP-β-ala-OVA）通过胺偶联固定在金表面葡聚糖基质上，当溶液中存在游离TNT时，TNT抑制TNP-β-ala-OVA与抗TNP抗体的免疫反应，使入射角偏移降低。该免疫分析在3×10−11至3×10−7 g/ml范围内对TNT表现出良好灵敏度。为进一步提高灵敏度，使用抗兔IgG抗体进行信号放大；在游离TNT与抗TNP抗体混合液流过芯片后注入抗兔IgG并测量入射角偏移，观察到信号增强，检出限改善至1×10−11 g/ml。
英文摘要
A polyclonal antibody against trinitrophenyl (TNP) derivatives was raised in rabbit, and the antibody was applied to detection of trinitrotoluene (TNT) using a surface plasmon resonance (SPR) biosensor. TNP-keyhole limpet hemocyanine (TNP-KLH) conjugate was injected into a rabbit, and a polyclonal anti-TNP antibody was realized after purification of the sera using protein G. Aspects of the anti-TNP antibody against various nitroaromatic compounds, such as cross-reactivities and affinities, were characterized. The temperature dependence of the affinity between the anti-TNP antibody and TNT was also evaluated. The quantification of TNT was based on the principle of indirect competitive immunoassay, in which the immunoreaction between the TNP-beta-alanine-ovalbumin (TNP-beta-ala-OVA) and anti-TNP antibody was inhibited in the presence of free TNT in solution. TNP-beta-ala-OVA was immobilized to the dextran matrix on the Au surface by amine coupling. The addition of a mixture of free TNT to the anti-TNP antibody was found to decrease the incidence angle shift due to the inhibitory effect of TNT. The immunoassay exhibited excellent sensitivity for the detection of TNT in the concentration range of 3x10(-11) to 3x10(-7)g/ml. To increase the sensitivity of the sensor, anti-rabbit IgG antibody was used. After flowing the mixture of free TNT and anti-TNP antibody, anti-rabbit IgG antibody was injected, and the incidence angle shift was measured. Amplification of the signal was observed and the detection limit was improved to 1x10(-11)g/ml.