CN114509480A - 人工智能生物传感器在检测5-o-甲基维斯阿米醇苷抗过敏活性中的应用 - Google Patents
人工智能生物传感器在检测5-o-甲基维斯阿米醇苷抗过敏活性中的应用 Download PDFInfo
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Abstract
本发明提供一种人工智能生物传感器在检测5‑O‑甲基维斯阿米醇苷抗过敏活性中的应用，所述人工智能生物传感器为以AlGaAs/GaAs HEMT器件为换能器，以修饰在HEMT器件上的Syk蛋白为识别原件，自主构建的Syk‑AlGaAs/GaAs HEMT生物传感器，检测浓度低至pM级，检测范围涵盖五个数量级，具有高灵敏度和强专属性，为抗过敏活性成分的辨识提供了技术支撑；进一步通过斑马鱼体内活性实验，发现了5‑O‑甲基维斯阿米醇苷具有显著的抗过敏活性。该发明形成一套基于人工智能生物传感技术的抗过敏活性成分筛选方法。
Description
技术领域
本发明属于中药领域，具体涉及一种人工智能生物传感器在检测5-O-甲基维斯阿米醇苷抗过敏活性中的应用。
背景技术
过敏反应，又称Ⅰ型超敏反应，是异常免疫反应的结果，会引发炎症，血管舒张，神经过敏等。据统计全世界约有3亿人患有哮喘，超过5亿人患有变应性鼻炎，湿疹及特异性皮炎患者约有4亿人。IgE介导的肥大细胞脱颗粒是诱发AR的重要过程之一。Src家族激酶(Srcfamily kinase，SFKs)Syk、Lyn、Fyn等参与调控该过程的起始信号。研究表明抑制Syk激酶活性可以完全阻断肥大细胞或嗜碱粒细胞的脱颗粒。Lee等发现槐花通过抑制肥大细胞中Syk和LAT的磷酸化，进一步抑制Akt和原型MAP激酶的磷酸化，从而抑制肥大细胞脱颗粒；而日本山茶花的叶提取物则通过抑制Lyn发挥肥大细胞脱颗粒的抑制作用。此外，研究发现Syk可通过激活半胱氨酸蛋白酶caspase-1的多分子复合物，活化炎性小体而导致炎症反应发生。Syk小分子抑制剂的辨识是免疫性疾病的重要突破口。
对于抑制剂的识别与筛选，生物传感器为分子间相互作用的识别提供了前沿技术支撑。高电子迁移率晶体管(High Electron Mobility Transistor,HEMT)作为第三代电化学型器件，具有压电极化和自发极化等电学特性，使其在二元界面上产生高的电子迁移率和二维电子气密度(2DEG)，从而使得对外加应力极其敏感。器件表面条件的状态改变，如结合生物分子，都会改变HEMT通道中2DEG的变化，进而改变电流。因此，HEMT生物传感器以其更高的灵敏度和更强的专属性为小分子抑制剂的辨识提供了关键技术支撑。
发明内容
为了克服现有技术的缺点与不足，提高抗过敏小分子辨识的准确性和灵敏度，开发高效抗过敏药物，本发明的首要目的在于提供一种人工智能生物传感器在检测5-O-甲基维斯阿米醇苷抗过敏活性中的应用。
一种人工智能生物传感器在检测5-O-甲基维斯阿米醇苷抗过敏活性中的应用，其中人工智能生物传感器的制备包括以下步骤：
(1)以AlGaAs/GaAs HEMT半导体材料为载体，采用CHI-660E电化学工作站，测定AlGaAs/GaAs HEMT器件各通道的源极和漏极间的电流电压变化(IDS-VDS)曲线，判断比较各通道性能，选择性能优良的通道作为测试载体；
(2)以AlGaAs/GaAs HEMT半导体材料为载体，将干净的AlGaAs/GaAs HEMT器件置于含巯基试剂3-巯基丙酸(3-MPA)中，于室温下浸泡24小时，在AlGaAs/GaAs HEMT器件表面生成Au-S键，形成自组装单层；
(3)用去离子水洗去AlGaAs/GaAs HEMT器件表面的巯基试剂，加入浓度为20mM碳酰二亚胺盐酸盐(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride，EDC)和浓度为50mM的N-羟基丁二酰亚胺(N-hydroxysuccinimide，NHS)的等体积混合水溶液，活化反应15-30min，生成稳定的胺类活化产物，用于活化羧基；
(4)用磷酸盐缓冲溶液(PBS)清洗AlGaAs/GaAs HEMT器件，加入Syk蛋白的PBS溶液，置4℃冷藏反应2-24小时，即得Syk-AlGaAs/GaAs HEMT生物传感器；
(5)采用XPS技术测定Syk-AlGaAs/GaAs HEMT上修饰Syk抗体前后元素的变化，并采集其IDS-VDS信号。
本发明进一步提供了上述Syk-AlGaAs/GaAs HEMT生物传感器在识别Syk抑制剂中的应用，具体步骤如下：
(1)5-O-甲基维斯阿米醇苷标准品溶液：精密称取5-O-甲基维斯阿米醇苷标准品(分子量为452.45)2.3mg于5mL容量瓶中，用浓度为0.1M的PBS溶液定容，混匀，即得浓度约为1.0mM的5-O-甲基维斯阿米醇苷标准品溶液；按10倍浓度梯度稀释10次，依次得0.1mM至0.1pM 5-O-甲基维斯阿米醇苷标准品溶液，备用；
(2)以制备好的Syk-AlGaAs/GaAs HEMT生物传感器为反应器件，按浓度由低到高(0.1pM至1.0mM)依次向Syk-AlGaAs/GaAs HEMT器件上加入5-O-甲基维斯阿米醇苷溶液，采用探针平台和电化学工作站记录源极-漏极间的电流强度(IDS)，不同浓度下的IDS-VDS信号如图2所示；由图可知，当浓度范围为1.0pM-0.1mM时，IDS信号的绝对值呈规律性下降；
(3)以5-O-甲基维斯阿米醇苷溶液浓度的对数(Lg[Ag])为横坐标，以IDS变化相对值(I-I0)/I0为纵坐标，进行线性拟合，结果表明当浓度范围为1.0pM-0.1mM时，线性关系较好；取1.0pM-10nM五个浓度点进行线性拟合，结果如图3所示，由图可知，线性关系良好，方程为y＝-0.0427x-0.5015,R2＝0.9725；
(4)根据(3)的浓度范围，以5-O-甲基维斯阿米醇苷溶液的浓度([Ag])为横坐标，以浓度([Ag])/电流变化(I-I0,ΔI)为纵坐标，进行线性拟合，得到线性方程为y＝2369.5x+8E-05,R2＝1；根据公式
其中[Ab]为Syk浓度，[Ag]为5-O-甲基维斯阿米醇苷溶液的浓度[C]，K及KA为结合常数，KD为解离常数，ΔI为电流变化值，ΔImax为电流最大变化值。
计算5-O-甲基维斯阿米醇苷与Syk相互作用的解离常数KD，得KD＝3.376×10-8M。
本发明还提供了5-O-甲基维斯阿米醇苷抗过敏活性的验证。
(1)斑马鱼胚胎获取：雌、雄斑马鱼分开喂养，照明14h和黑暗10h交替进行，以人工颗粒状饵料和刚孵出的卤虫无节幼体定时喂养。采卵时取健康性成熟的斑马鱼按雌、雄1:1的比例放入交配缸内，次日9-10时获得受精卵。对受精卵进行消毒和洗涤后移入斑马鱼胚胎培养用水(含5.0mM NaCl，0.17mM KCl，0.4mM CaCl2，0.16mM MgSO4)中，28℃下控光培养。；
(2)药物处理及图像获取：在斑马鱼胚胎发育至3dpf(days post fertilization)时，挑选健康斑马鱼，小心随机吸入24孔板各孔中，每孔10枚胚胎。设定空白对照组、模型组、阳性药组和药物处理组。阳性药组给予20μM布洛芬溶液。单独给药组分别给予62.5、125、250、500和1000μM 5-O-甲基维斯阿米醇苷溶液。空白对照组和模型组给予斑马鱼养殖水。给药24h后，用20μM 5H2O·CuSO4处理模型组、阳性药组和药物处理组。5H2O·CuSO4处理1h后，从各组中随机取6条斑马鱼，用0.22％三卡因麻醉1min，置于体式荧光显微镜(AXIOZoom.V16,ZEISS,Oberkochen,Germany)下拍照。
(3)数据处理：5H2O·CuSO4可引起斑马鱼急性炎症，其特征是巨噬细胞迅速迁移到侧线神经丘。使用Image-Pro Plus软件记录各组斑马鱼巨噬细胞迁移数量。
(4)斑马鱼巨噬细胞迁移数量：斑马鱼巨噬细胞迁移数量以相对于模型组数量的百分比进行归一化。单独给药组结果见图4。由图可知，相比模型组，5-O-甲基维斯阿米醇苷在浓度为125μM和250μM时均具有抑制作用，且在250μM时抑制作用最强，甚至强于阳性对照组，这表明浓度为250μM的5-O-甲基维斯阿米醇苷标准品溶液具有较好的抑制巨噬细胞迁移的抗过敏作用。
本发明的有益效果是：
Syk参与过敏反应及多种肿瘤的炎症反应通路，是重要的疾病治疗靶点之一，其抑制剂的识别成为研究热点。AlGaAs/GaAs HEMT器件以其高灵敏度和强专属性为分子间相互作用的识别提供重要技术支撑。本发明将Syk修饰于AlGaAs/GaAs HEMT器件之上，构建Syk-AlGaAs/GaAs HEMT生物传感器，直接识别Syk抑制剂，检测浓度低至pM级，检测范围涵盖5-10个数量级，具有很高的灵敏度。此外，Syk-AlGaAs/GaAs HEMT生物传感器具有很高的专属性，能够直观反应化合物与Syk相互作用情况，在抑制剂的识别中具有独特优势。而结合斑马鱼体内验证实验，对抑制剂的抗过敏活性进行了验证，形成了一套基于人工智能生物传感技术的抗过敏活性成分筛选方法。
附图说明
图1蛋白修饰XPS测定结果。
图2不同浓度5-O-甲基维斯阿米醇苷与Syk结合的IDS-VDS信号变化。
图3不同浓度5-O-甲基维斯阿米醇苷与Syk结合的线性拟合结果。
图4不同浓度5-O-甲基维斯阿米醇苷对斑马鱼巨噬细胞迁移数量的影响。
图5不同浓度5-O-甲基维斯阿米醇苷干预斑马鱼巨噬细胞迁移情况
具体实施方式
实施例1一种人工智能生物传感器的制备
(1)采用CHI-660E电化学工作站，测定AlGaAs/GaAs HEMT器件各通道的IDS-VDS曲线，判断比较各通道性能。
(2)将干净的AlGaAs/GaAs HEMT器件置于3-MPA水溶液，于室温下浸泡，在AlGaAs/GaAs HEMT器件表面生成Au-S键，形成自组装单层；
(3)用去离子水洗去AlGaAs/GaAs HEMT器件表面的3-MPA，加入20mM EDC和50mMNHS体积比为1:1的混合水溶液，生成稳定的胺类活化产物用于活化羧基；
(4)用PBS清洗AlGaAs/GaAs HEMT器件，加入巨噬细胞游走抑制因子(Syk)抗体，反应完成，即得Syk-AlGaAs/GaAs HEMT器件生物传感器。
(5)采用X射线能谱测定AlGaAs/GaAs HEMT上修饰Syk抗体前后元素的变化，图1展示了HEMT裸片、修饰3-MPA后及修饰MIF后的XPS谱图，其中(a)为三者全扫描结果，(b)、(c)和(d)分别为C1s、N1s和O1s的高分辨XPS谱图。N1s峰的峰高及半峰宽在修饰3-MPA后有所增加，而在蛋白固定后显著增加，从其高分辨谱中分解出两个组分，峰中心位于397.55eV和397.7eV，分别对应于蛋白的中性氨基(NH2)和质子化的氨基(NH3+)。未处理芯片表面的C1s高分辨谱可归属为两个明显的峰，分别在285.0eV(对应于C-H)和294.0(对应于C-O)。碳元素的信号来自于基底的有机物污染。自主装单层形成后，在292.0eV处出现了一个新的羰基(O-C＝O)峰；羰基由NHS/EDC激活，并与蛋白的氨基生成肽键(-CONH-)的形成，C1s位于294.0eV处的高分辨峰被展宽。
实施例2一种人工智能生物传感器在5-O-甲基维斯阿米醇苷抗过敏活性检测中的应用
(1)待测样品溶液的制备：精密称取5-O-甲基维斯阿米醇苷标准品(分子量为452.45)2.3mg于5mL容量瓶中，用浓度为0.1M的PBS溶液定容，混匀，即得浓度约为1.0mM的5-O-甲基维斯阿米醇苷标准品溶液；按10倍浓度梯度稀释10次，依次得0.1mM至0.1pM 5-O-甲基维斯阿米醇苷标准品溶液，备用；
(2)以实施例1制备好的Syk-AlGaAs/GaAs HEMT生物传感器为反应器件，按浓度由低到高(0.1pM至1.0mM)依次向Syk-AlGaAs/GaAs HEMT器件上加入5-O-甲基维斯阿米醇苷标准品溶液，采用探针平台和电化学工作站记录源极-漏极间的电流强度(IDS)，不同浓度下的IDS-VDS信号如图2所示；由图可知，当浓度范围为0.1pM至1.0μM时，IDS信号的绝对值呈规律性下降；
(3)以5-O-甲基维斯阿米醇苷标准品溶液浓度的对数(Lg[Ag])为横坐标，以IDS变化相对值(I-I0)/I0为纵坐标，进行线性拟合，结果表明当浓度范围为0.1pM-1.0μM时，线性关系较好；取0.1pM-1.0μM 8个浓度点进行线性拟合，结果如图3所示，由图可知，线性关系良好，方程为y＝-0.003x-0.0466,R2＝0.9728；
(4)根据(3)的浓度范围，以盐酸西替利嗪溶液的浓度([Ag])为横坐标，以浓度([Ag])/电流变化(I-I0,ΔI)为纵坐标，进行线性拟合，得到线性方程为y＝786.41x+2E-06,R2＝0.9997；根据公式
其中[Ab]为Syk浓度，[Ag]为盐酸西替利嗪溶液的浓度[C]，K及KA为结合常数，KD为解离常数，ΔI为电流变化值，ΔImax为电流最大变化值。
计算5-O-甲基维斯阿米醇苷与Syk相互作用的解离常数KD，得KD＝2.543×10-9M。
实施例3基于斑马鱼巨噬细胞迁移数量的5-O-甲基维斯阿米醇苷抗过敏活性验证
(1)斑马鱼胚胎获取：雌、雄斑马鱼分开喂养，照明14h和黑暗10h交替进行，以人工颗粒状饵料和刚孵出的卤虫无节幼体定时喂养。采卵时取健康性成熟的斑马鱼按雌、雄1:1的比例放入交配缸内，次日9-10时获得受精卵。对受精卵进行消毒和洗涤后移入斑马鱼胚胎培养用水(含5.0mM NaCl，0.17mM KCl，0.4mM CaCl2，0.16mM MgSO4)中，28℃下控光培养。；
(2)药物处理及图像获取：在斑马鱼胚胎发育至3dpf(days post fertilization)时，挑选健康斑马鱼，小心随机吸入24孔板各孔中，每孔10枚胚胎。设定空白对照组、模型组、阳性药组和药物处理组。阳性药组给予20μM布洛芬溶液。单独给药组分别给予62.5、125、250、500和1000μM 5-O-甲基维斯阿米醇苷溶液。空白对照组和模型组给予斑马鱼养殖水。给药24h后，用20μM 5H2O·CuSO4处理模型组、阳性药组和药物处理组。5H2O·CuSO4处理1h后，从各组中随机取6条斑马鱼，用0.22％三卡因麻醉1min，置于体式荧光显微镜(AXIOZoom.V16,ZEISS,Oberkochen,Germany)下拍照。
(3)数据处理：5H2O·CuSO4可引起斑马鱼急性炎症，其特征是巨噬细胞迅速迁移到侧线神经丘。使用Image-Pro Plus软件记录各组斑马鱼巨噬细胞迁移数量。
(4)斑马鱼巨噬细胞迁移数量：斑马鱼巨噬细胞迁移数量以相对于模型组数量的百分比进行归一化。单独给药组结果见图4和图5。由图可知，相比模型组，5-O-甲基维斯阿米醇苷在浓度为125μM和250μM时均具有抑制作用，且在250μM时抑制作用最强，甚至强于阳性对照组，这表明浓度为250μM的5-O-甲基维斯阿米醇苷标准品溶液具有较好的抑制巨噬细胞迁移的抗过敏作用。
Claims (7)
1.人工智能生物传感器在检测5-O-甲基维斯阿米醇苷抗过敏活性中的应用，其特征在于，包括一种人工智能生物传感器，以AlGaAs/GaAs HEMT器件为换能器，以修饰在HEMT器件上的络氨酸激酶Syk蛋白为识别原件，自主构建的Syk-AlGaAs/GaAs HEMT生物传感器。
2.权利要求1中所述的人工智能生物传感器的制备方法，即Syk-AlGaAs/GaAs HEMT生物传感器的制备方法，其特征在于，具体制备步骤如下：
(1)将干净的AlGaAs/GaAs HEMT器件置于含巯基试剂中，于室温下浸泡17小时-24小时，在AlGaAs/GaAs HEMT器件表面生成Au-S键，形成自组装单层；
(2)用去离子水洗去AlGaAs/GaAs HEMT器件表面的巯基试剂，加入羧基活化剂碳酰二亚胺盐酸盐和N-羟基丁二酰亚胺，生成稳定的胺类活化产物用于活化羧基；
(3)用磷酸盐缓冲溶液清洗AlGaAs/GaAs HEMT器件，加入Syk蛋白，置于4℃环境下反应，即得Syk-AlGaAs/GaAs HEMT器件生物传感器。
3.根据权利要求2所述的一种人工智能生物传感器的制备方法，其特征在于，羧基活化剂的活化时间为15-30min，权利要求2步骤(3)中Syk的反应时间为2小时-24小时。
4.由权利要求2-3任一所述的制备方法得到的一种人工智能生物传感器，即Syk-AlGaAs/GaAs HEMT生物传感器。
5.权利要求4所述的一种人工智能生物传感器在制备检测Syk潜在抑制剂中的应用，其特征在于，具体步骤如下：
(1)根据权利要求2-3任一所述方法制备Syk-AlGaAs/GaAs HEMT生物传感器，依次加入浓度由低到高的化合物溶液，采用探针平台和电化学工作站记录源极-漏极间的电流强度；
(2)以化合物浓度的对数为横坐标，以电流变化相对值为纵坐标，进行线性拟合，确定Syk-AlGaAs/GaAs HEMT生物传感器用于检测Syk抑制剂的浓度范围；根据公式计算抑制剂与Syk相互作用强度，判断抑制剂对Syk的潜在抑制作用。
6.根据权利要求5所述的一种人工智能生物传感器在制备检测Syk潜在抑制剂中的应用，其特征在于，权利要求5步骤(1)中化合物的浓度范围为0.1pM–10μM，测量电压范围为0–5V。
7.权利要求1-6任一所述的5-O-甲基维斯阿米醇苷的抗过敏活性。
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