CN114384049B - 基于破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法 - Google Patents
基于破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法 Download PDFInfo
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Abstract
本发明公开一种基于破伤风人免疫球蛋白‑金簇为荧光探针的破伤风抗原测定方法,其特征是基于破伤风人免疫球蛋白‑金簇的荧光和免疫原性的双功能特性,一方面,金簇具有荧光特性,另一方面它能够与破伤风抗原结合,利用金簇在检测线与控制线荧光强度比值的变化,直接用于破伤风抗原的测定。测定体系的荧光强度比值变化值∆(T/C)与破伤风抗原浓度在0.4~6.0μg/mL的对数呈线性关系,检测限为0.03μg/mL。本发明所构建的方法操作简便、检测时间短、特异性高、灵敏度好等优点,可用于破伤风抗原的含量测定。
Description
技术领域
本发明涉及以破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法,属于生物化学及纳米技术领域。
背景技术
破伤风是由破伤风梭状芽孢杆菌分泌的神经毒素而引起的具有急性感染性、中毒性的疾病,它具有潜伏期短、起病急、病情严重、病死率高等特点。破伤风神经毒素由两个二硫化物连接亚基组成,重链(100000 道尔顿)含有结合和易位结构域,轻链(50000 道尔顿)含有锌依赖的蛋白酶结构域。破伤风抗原的检测方法与破伤风疫苗生产的安全性关系密切,目前为止,破伤风抗原均使用豚鼠或小鼠进行生物制剂的风险评估,其在伦理和经济方面的可接受性较低。因此,寻找一种新的破伤风抗原的检测技术在医学诊断及疫苗研发等方面具有重要的意义。
金纳米团簇(AuNCs)具有良好的光稳定性、较大的斯托克位移和毒性低等特点,优于其他金属纳米团簇和量子点,正因为如此,它已经成为一种新型的荧光探针,并广泛应用于光学传感、生物成像和体外生物测定。在过去的几年里,各种生物分子(如蛋白质、多肽和氨基酸)已经被广泛用作稳定金核的生物模板。生物分子介导的金纳米团簇对生物体具有较低的毒性和良好的生物相容性,有望成为生命科学和环境研究的新型分析工具。研究人员已经发现许多不同的蛋白质,包括人免疫球蛋白、胃蛋白酶、辣根过氧化物酶、胰岛素、溶菌酶和胰蛋白酶等,均可以作为制备金纳米团簇的生物配体,此外,这些金纳米团簇可以保留生物分子的固有功能(如催化活性、识别能力,免疫原性等)。以破伤风人免疫球蛋白为生物模板,能构建具有红色荧光的破伤风人免疫球蛋白-金簇,该金簇同时保持荧光和免疫原性的双功能特性,并能够很好的应用于破伤风抗原的检测。因此在生物传感器和疫苗研发等领域研究中具有巨大的应用前景。
本发明以破伤风人免疫球蛋白为生物模板,构建了破伤风人免疫球蛋白-金簇荧光探针,并利用此探针建立了一种测定破伤风抗原的新方法。
发明内容
本发明的目的是提供一种利用破伤风人免疫球蛋白-金簇为荧光探针,建立一种测定破伤风抗原的新方法。
为了实现上述目的,本发明采用以下技术方案:
一种基于破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法,其特征是基于破伤风人免疫球蛋白-金簇的荧光和免疫原性的双功能特性,一方面,金簇具有荧光特性,另一方面它能够与破伤风抗原结合,利用金簇在检测线与控制线荧光强度比值的变化,直接用于破伤风抗原的测定。
2、所使用的破伤风人免疫球蛋白-金簇采用破伤风人免疫球蛋白直接还原氯金酸的方法制备:将浓度为17.8~142 mg/mL 的破伤风人免疫球蛋白溶液和浓度为1.5~3.5mol/L的氢氧化钠溶液加入到浓度为0.5~7.5 mmol/L的氯金酸溶液中,混匀,置于25~60°C水浴恒温反应15~150分钟,反应结束后用截留分子量为3500的透析袋对反应液进行透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。
3、所使用的破伤风人免疫球蛋白-金簇采用破伤风人免疫球蛋白直接还原氯金酸的方法制备:将浓度为2.5 moL/L的NaOH溶液与浓度为2.5 mmoL/L氯金酸依次加入到为59.3 mg/mL的人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到人免疫球蛋白-金簇荧光材料水溶液。
4、所使用的破伤风人免疫球蛋白-金簇荧光材料水溶液为浅色,紫外-可见光谱在280 nm处出现蛋白质的特征吸收峰,在紫外灯照射下产生强烈的红色荧光,最大激发波长和发射波长分别为351 nm和619 nm,量子产率为13%,荧光寿命为7.8 μs。
5、所述的破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法,其特征是利用破伤风人免疫球蛋白-金簇在Chemiscope series 5600成像仪上检测线和控制线的荧光强度比值的变化值∆(T/C)判断破伤风抗原的含量,所使用的激发波长为365 nm。
所述的一种破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法,其特征是所破伤风抗原测定的线性范围为0.4~6.0 μg/mL,最低检测限为0.03 μg/mL。
为了实现上述目的,本发明采用具体技术方案如下:
(一)破伤风人免疫球蛋白-金簇荧光材料的制备
以下过程中使用的所有玻璃器皿均经过王水浸泡,并用双蒸水彻底清洗,晾干。破伤风人免疫球蛋白-金簇荧光材料的制备过程如下:将0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理。纯化后的破伤风人免疫球蛋白-金簇溶液置于冰箱4℃避光保存。
(二)破伤风抗原的检测方法
利用免疫层析试纸条对破伤风抗原进行测定:免疫层析试纸条由样品垫、结合垫、吸水垫和硝酸纤维素膜组成。将浓度为100 µg/mL的破伤风人免疫球蛋白-金簇1µL加入49µL双蒸水、2 μL 浓度为5% (w/w) Triton X-100、2 μL浓度为 5% (w/w) PEG400、4 μL 浓度为 5% (w/w) 蔗糖,1 μL 浓度为1 mol/L HEPES缓冲液和2.5 μL (w/w) 乙二醇,然后将荧光抗体溶液滴加在结合垫中,真空干燥1 h。接下来将硝酸纤维素膜裁剪为4 mm宽度,并将浓度为1.5 µg/mL的破伤风抗毒素和浓度为0.5 µg/mL的羊抗人IgG抗体分别划线于检测线和控制线。将硝酸纤维素膜真空干燥15 min,然后将吸水垫、结合垫和样品垫依次组装在硝酸纤维素膜上,并安装在4 mm塑料卡盒上。检测时,将破伤风抗原(6 μL)加入 20 mmoL/LHEPES(54 μL)和 5% (w/w) PEG 400(40 μL)溶液中,然后将其滴加于样品垫上,10 min后,在荧光成像仪Chemiscope series 5600上拍照,并采用Image J 1.51软件分析检测线和控制线荧光强度比值(T/C),通过检测样品与空白样品荧光强度变化值∆(T/C)标准曲线进行破伤风抗原的测定。
本发明的优点:
(1)本发明以破伤风人免疫球蛋白为模板原位合成金纳米团簇荧光材料。制备方法绿色环保,操作简便快捷,重现性好。
(2)本发明所制备的破伤风人免疫球蛋白-金簇具有强烈的红色荧光(最大发射波长为619 nm),量子产率高(13%),长荧光寿命(7.8 μs),及较大的斯托克斯位移(107 nm)等特点。
(3)本发明所制备的破伤风人免疫球蛋白-金簇仍保持抗体的免疫原性,对破伤风抗原的亲和力为2.27×10-8moL/L。
(4)本发明所构建的方法特异性好、检测过程简便、快速。
附图说明
图1为破伤风人免疫球蛋白-金簇荧光纳米材料分别在可见光(A)和紫外灯下(B)的外观对照图。
图2为破伤风人免疫球蛋白-金簇荧光纳米材料的紫外-可见吸收光谱图。
图3为破伤风人免疫球蛋白-金簇荧光纳米材料的荧光光谱图。
图4为破伤风人免疫球蛋白浓度对金簇荧光强度的影响图。
图5为氢氧化钠浓度对金簇荧光强度的影响图。
图6为反应时间对金簇荧光强度的影响图。
图7为反应温度对金簇荧光强度的影响图。
图8为金簇荧光纳米材料的荧光寿命图。
图9为金簇荧光纳米材料的X射线光电子能谱图。图中:A为金的4f图,B为硫的2p图。
图10为金簇荧光纳米材料与破伤风抗原的共振荧光光谱图。图中:A为破伤风人免疫球蛋白-金簇,B为破伤风抗原,C为破伤风人免疫球蛋白-金簇+破伤风抗原。
图11为金簇荧光纳米材料与破伤风抗原的原子力显微镜图。图中:A为破伤风人免疫球蛋白-金簇,B为破伤风人免疫球蛋白-金簇+破伤风抗原。
图12为金簇荧光纳米材料对不同浓度破伤风抗原的表面等离子共振图。
图13为基于破伤风人免疫球蛋白-金簇检测破伤风抗原的方法示意图。
图14为检测体系检测线与控制线的荧光强度比值变化值∆(T/C)与破伤风抗原浓度对数的线性关系图。
图15为不同抗原对检测体系荧光强度的影响图,编号a~e分别为破伤风抗原、脊髓灰质炎病毒抗原、卡介苗抗原、流行性脑脊髓膜炎抗原,乙型脑膜炎抗原。破伤风抗原的浓度为2 μg/mL,其余抗原的浓度均为20 μg/mL。
具体实施方式
实例1:
将0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。所得到的荧光纳米材料溶液在可见光下为浅色(见图1中的A),紫外灯照射下产生强烈的红色荧光(见图1中的B),紫外-可见光谱在280 nm处出现蛋白质的特征吸收峰(见图2),最大激发波长和发射波长分别为351 nm和619 nm(见图3),量子产率为13%。
实例2:
将0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为17.8~142 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。如图4所示,溶液在619 nm处的荧光强度值(F619)在破伤风人免疫球蛋白溶液浓度为59.3 mg/mL时达到最大。
实例3:
将浓度为0.2 mL浓度为1.5~3.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。如图5所示,溶液在619 nm处的荧光强度值(F619)在氢氧化钠溶液浓度为2.5 moL/L时达到最大。
实例4:
将0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育15~150分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。如图6所示,溶液在619 nm处的荧光强度值(F619)在在反应时间为30分钟时达到最大。
实例5:
将0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于25~60℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。如图7所示,溶液在619 nm处的荧光强度值(F619)在在反应温度为37 ℃时达到最大。
实例6:
将0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。将所得的溶液进行荧光寿命测定,测得金簇的荧光寿命值为7.8 μs(见图8)。
实例7:
0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。将所得溶液冷冻干燥后得到粉末,取所得粉末进行X射线光电子能谱测定,在84.9 eV和88.8 eV处出现金的4f峰,在163.5 eV和164.4 eV处出现硫的2p峰(见图9中的A为金的4f图,B为硫的2p图)。
实例8:
0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。将所得溶液进行原子力显微镜测试,测得破伤风人免疫球蛋白-金簇高度约为2 nm,而将破伤风人免疫球蛋白-金簇和破伤风抗原溶液混合后,测得高度约为5 nm,增加了3 nm,说明二者发生了相互作用(见图10中的A为破伤风人免疫球蛋白-金簇的原子力显微镜图,B为破伤风抗原的原子力显微镜图,C为破伤风人免疫球蛋白-金簇 + 破伤风抗原的原子力显微镜图)。
实例9:
0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。将所得溶液进行共振荧光测试,当溶液中只含有破伤风人免疫球蛋白-金簇(0.2 µmol/L)或破伤风抗原(0.3 µmol/L)时,二者的共振荧光强度均处于较低水平。当破伤风人免疫球蛋白-金簇(0.2 µmol/L)和破伤风抗原(0.3 µmol/L)共存时,溶液的共振荧光强度急剧升高,说明二者发生相互作用,导致粒径增大。(见图11中的A为破伤风人免疫球蛋白-金簇的共振荧光光谱图(0.2 µmol/L),B为破伤风人免疫球蛋白-金簇(0.2 µmol/L)+ 破伤风抗原(0.3 µmol/L)的共振荧光光谱图)。
实例10:
0.2 mL浓度为2.5 moL/L的NaOH溶液与2 mL浓度为2.5 mmoL/L氯金酸依次加入到2 mL浓度为59.3 mg/mL的破伤风人免疫球蛋白溶液中,混匀后置于37℃恒温水浴槽中孵育30分钟。待反应结束后用分子量为3500的透析袋对反应液透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液。将所得溶液进行表面等离子共振测试,将破伤风人免疫球蛋白-金簇固定在CM5表面等离子共振芯片上,当破伤风抗原的浓度由3.125 nmol/L逐渐递增至200 nmol/L时,仪器形成不同强度的表面等离子共振曲线,通过软件Biacore T100计算出亲和力常数为2.27×10-8mol/L。(见图12中为破伤风人免疫球蛋白-金簇对不同浓度破伤风抗原的表面等离子共振图:a-f分别代表浓度为3.125,6.25,12.5,25,50,200 nmol/L的破伤风抗原)。
实例11:
取1µL实例1所制得的破伤风人免疫球蛋白-金簇荧光材料水溶液(100 µg/mL)加入缓冲液中(内含49 µL双蒸水、2 μL 浓度为5% (w/w) Triton X-100、2 μL浓度为 5% (w/w) PEG400、4 μL 浓度为 5% (w/w) 蔗糖,1 μL 浓度为1 mol/L HEPES缓冲液和2.5 μL(w/w) 乙二醇),并滴加在结合垫中,真空干燥1 h。然后将6 µL不同浓度的破伤风抗原溶液加入缓冲液中(内含54 μL浓度为20 mmoL/LHEPES缓冲液和 40 μL 浓度为5% (w/w) PEG400),并将其滴加于样品垫上,10 min后,在荧光成像仪Chemiscopeseries 5600上拍照,并采用Image J 1.51软件分析检测线和控制线荧光强度比值(T/C),通过检测样品与空白样品荧光强度变化值∆(T/C)标准曲线进行破伤风抗原的浓度测定。随着破伤风抗原浓度的增大,∆(T/C)逐渐增大。如图13所示,在0.4~6.0 μg/mL范围内∆(T/C)与破伤风抗原浓度的对数呈线性关系,检测限为0.03 μg/mL。
实例12:
取1µL实例1所制得的破伤风人免疫球蛋白-金簇荧光材料水溶液(100 µg/mL)加入缓冲液中(内含49 µL双蒸水、2 μL 浓度为5% (w/w) Triton X-100、2 μL浓度为 5% (w/w) PEG400、4 μL 浓度为 5% (w/w) 蔗糖,1 μL 浓度为1 mol/L HEPES缓冲液和2.5 μL(w/w) 乙二醇),并滴加在结合垫中,真空干燥1 h。然后将6 µL破伤风抗原溶液、脊髓灰质炎病毒抗原、卡介苗抗原、流行性脑脊髓膜炎抗原和乙型脑膜炎抗原加入缓冲液中(内含54μL浓度为20 mmoL/LHEPES缓冲液和 40 μL 浓度为5% (w/w) PEG 400),并将其滴加于样品垫上,10 min后,在荧光成像仪Chemiscopeseries 5600上拍照,并采用Image J 1.51软件分析检测线和控制线荧光强度比值(T/C),通过检测样品与空白样品荧光强度变化值∆(T/C)进行破伤风抗原的特异性进行测定。如图13所示,破伤风抗原的∆(T/C)荧光强度值约为1.6,明显高于其他样品。这说明该方法检测破伤风抗原具有很强的特异性。
Claims (2)
1.一种基于破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法,其特征是基于破伤风人免疫球蛋白-金簇的荧光和免疫原性的双功能特性,金簇具有荧光特性,它能够与破伤风抗原结合,利用金簇在检测线与控制线荧光强度比值的变化,直接用于破伤风抗原的测定;
所使用的破伤风人免疫球蛋白-金簇采用破伤风人免疫球蛋白直接还原氯金酸的方法制备:将浓度为17.8~142 mg/mL 的破伤风人免疫球蛋白溶液和浓度为1.5~3.5 mol/L的氢氧化钠溶液加入到浓度为0.5~7.5 mmol/L的氯金酸溶液中,混匀,置于25~60°C水浴恒温反应15~150分钟,反应结束后用截留分子量为3500的透析袋对反应液进行透析纯化处理,得到破伤风人免疫球蛋白-金簇荧光材料水溶液;
利用破伤风人免疫球蛋白-金簇在荧光成像仪上检测线和控制线的荧光强度比值的变化值∆(T/C)判断破伤风抗原的含量,所使用的激发波长为365 nm;
所使用的破伤风人免疫球蛋白-金簇荧光材料水溶液为浅色,紫外-可见光谱在280 nm处出现蛋白质的特征吸收峰,在紫外灯照射下产生强烈的红色荧光,最大激发波长和发射波长分别为351 nm和619 nm,量子产率为13%,荧光寿命为7.8 μs。
2.根据权利要求1所述的一种破伤风人免疫球蛋白-金簇为荧光探针的破伤风抗原测定方法,其特征是破伤风抗原测定的线性范围为0.4~6.0 μg/mL,最低检测限为0.03 μg/mL。
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