CN108562745A - 功能化氧化石墨烯氮化碳溴化银纳米材料及用途 - Google Patents
功能化氧化石墨烯氮化碳溴化银纳米材料及用途 Download PDFInfo
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Abstract
本发明涉及医学中肿瘤标志物检测领域,具体是制备氧化石墨烯‑氮化碳‑溴化银纳米复合物材料,利用其可见光激活效应构建比色免疫分析方法,实现对前列腺抗原的检测。合成功能化氧化石墨烯‑氮化碳‑溴化银(GO‑C3N4‑AgBr)纳米复合物材料,步骤制备氧化石墨烯‑氮化碳(GO‑C3N4)复合物;将上述制备好的GO‑C3N4复合物进行羧基化处理;制备氧化石墨烯‑氮化碳‑溴化银(GO‑C3N4‑AgBr)纳米复合物材料;对GO‑C3N4‑AgBr进行功能化修饰。本发明还涉及利用制备的功能化氧化石墨烯‑氮化碳‑溴化银(GO‑C3N4‑AgBr)纳米复合物材料构建免疫复合物的方法及用途。
Description
技术领域
本发明涉及医学中肿瘤标志物检测领域,具体是制备氧化石墨烯-氮化碳-溴化银纳米复合物材料,利用其可见光激活效应构建比色免疫分析方法,实现对前列腺抗原的检测。
背景技术
比色分析方法是一种灵敏度和准确度均很高的分析方法,只需裸眼观察就可进行半定量分析,结合紫外可见分光光度计能实现微量甚至痕量检测。由于其价格低廉、设备小型化、操作简便等优点,比色分析方法被广泛应用于环境、医学、食品检测等领域(X. Zhou,J. Nie, and B. Du, ACS Appl. Mater. Interfaces, 2017, 9 (24), 20913–20921; C.Dong, Z. Wang, Y. Zhang, X. Ma, M. Iqbal, L. Miao, Z. Zhou, Z. Shen, A. Wu,ACS Sens., 2017, 2 (8), 1152–1159)。其中肿瘤标记物是肿瘤细胞产生的,含量远高于正常细胞的特异性物质(W. Chen, R. Zheng, P. Baade, S. Zhang, H. Zeng, F. Bray,A. Jemal, X. Yu, J. He, Ca. Cancer J. Clin. 66 (2016) 115–132.)。监测肿瘤标志物有助于提高肿瘤患者的诊断率,监测肿瘤早期转移,并可为患者根治性手术后是否进行辅助化疗、疗效判断、复发转移等具有一定的指导意义,对患者的病情预后判断有着至关重要的临床意义。基于抗原-抗体特异性反应的免疫分析法是检测肿瘤标志物最为常用的方法。结合比色和免疫分析方法优点的比色免疫分析方法是目前检测肿瘤标志物的研究热点之一。
天然酶的结构容易发生变化、在生物体内含量很低、储存条件比较苛刻等因素大大限制了其实际应用。由于纳米材料模拟酶对酸、碱、温度具有较好的稳定性且催化活性较高,已成为生命分析化学等相关领域的研究热点之一。纳米材料模拟酶在比色传感、生物传感、降解环境污染物、电化学传感等方面已显示出诱人的应用前景 (Dutta, S., Ray, C.,Mallick, S., Sarkar, S., Sahoo, R., Negishi, Y., Pal, T., 2015. J. Phys.Chem. C. 119, 23790-23800. Qin, W., Su, L., Yang, C., Ma, Y., Zhang, H.,Chen, X., 2014. J. Agric. Food. Chem. 62, 5827-5834. Zhao, H., Dong, Y.,Jiang, P., Wang, G., Zhang, J., 2015. ACS Appl. Mater. Interfaces. 7, 6451-6461.)。从实际应用的角度考虑,探寻具有高催化活性的、稳定的、可重复利用的模拟酶纳米材料尤为重要。
近几年,一些光催化纳米材料在太阳光激发下也被发现具有过氧化物模拟酶活性,如石墨烯,TiO2, AgI等 (Wang, G., Xu, X., Qiu, L., Dong, Y., Li, Z., Zhang,C., 2014a. ACS Appl. Mater. Interfaces. 6, 6434−6442. Wang, G., Xu, X., Wu,X., Cao, G., Dong, Y., Li, Z. J., 2014b. J. Phys. Chem. C. 118, 28109-28117.Wang, G., Jin, L., Dong, Y., Wu, X., Li, Z., 2015. Biosens. Bioelectron. 64,523-529.)。本文选择制备氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr复合纳米材料,并用其作为标记物构建比色免疫分析方法,用于前列腺抗原的检测。纳米材料的制备方法简单、快速、成本低,以此构建的比色免疫分析方法具有较好的选择性、稳定性和重现性。
发明内容
本发明所要解决的技术问题是:如何提供一种可见光激活的比色免疫分析方法,以GO-C3N4-AgBr复合纳米材料作为标记物在常规条件下实现对前列腺抗原的检测,并具有较低的检测限,且能用于实际血清样品的检测。
本发明所采用的技术方案是:
合成功能化氧化石墨烯-氮化碳-溴化银(GO-C3N4-AgBr)纳米复合物材料,按照如下的步骤进行
步骤一、制备氧化石墨烯-氮化碳(GO-C3N4)复合物,将尿素加入到氧化石墨烯分散溶液中,该混合液再80℃下持续搅拌3个小时,之后将其转移至烘箱,在70℃下放置过夜,最后将该产品在550℃下煅烧2.5个小时,获得的样品即为GO-C3N4;
步骤二、将上述制备好的GO-C3N4复合物进行羧基化处理,将GO-C3N4样品分散在浓硝酸溶液中,在125 ℃下回流24个小时,之后将其冷却至室温,离心清洗得到羧基化的GO-C3N4复合物;
步骤三、制备氧化石墨烯-氮化碳-溴化银(GO-C3N4-AgBr)纳米复合物材料,将上述羧基化的GO-C3N4复合物超声分散在二次水中,之后加入硝酸银溶液,避光搅拌1小时,随后逐滴加入溴化钠溶液继续搅拌2小时,通过离心、清洗和干燥得到GO-C3N4-AgBr样品;
步骤四、对GO-C3N4-AgBr进行功能化修饰,将上述制备好的GO-C3N4-AgBr超声分散在二次水中,同时该水溶液中含有N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺磺酸钠盐(NHS),该混合溶液在室温下搅拌30分钟,之后加入前列腺抗体(Ab),在4℃下缓慢震荡过夜,离心清洗得到抗体功能化的GO-C3N4-AgBr,记为Ab-GO-C3N4-AgBr。
作为一种优选方式:步骤一中尿素的量为0.6-0.9克;氧化石墨烯溶液的量为100-150毫升,浓度为0.15毫克/毫升;步骤二中GO-C3N4的量为1-1.5克;浓硝酸溶液的量为100-150毫升,浓度为5摩尔;步骤三中二次水的量为10-15毫升;硝酸银的量为5-7.5毫升,浓度为0.5毫摩尔;溴化钠的量为10-15毫升,浓度为0.5毫摩尔;步骤四中二次水的量为1-1.5毫升; EDC的浓度为100-150毫摩尔;NHS的浓度为100-150毫摩尔;前列腺抗体的量为500-750微升。
利用制备的功能化氧化石墨烯-氮化碳-溴化银(GO-C3N4-AgBr)纳米复合物材料构建免疫复合物的方法:在室温下,将磁珠与前列腺抗体孵育过夜,记为MB/Ab,清洗后再孵育不同浓度的前列腺抗原(PSA)30分钟,记为MB/Ab/PSA,清洗之后孵育权利1制备好的Ab-GO-C3N4-AgBr纳米共轭复合物30分钟,记为MB/Ab/PSA/Ab-GO-C3N4-AgBr。
作为一种优选方式:磁珠的量为1-1.5毫升,浓度为5毫克/毫升;前列腺抗体的量为500-750微升;不同浓度的前列腺抗原是指0纳克/毫升、0.5纳克/毫升、1纳克/毫升、2纳克/毫升、4纳克/毫升、10纳克/毫升、15纳克/毫升、25纳克/毫升。
可见光激活构建好的免疫复合物(MB/Ab/PSA/Ab-GO-C3N4-AgBr),用于比色免疫检测前列腺抗原的方法,将MB/Ab/PSA/Ab-GO-C3N4-AgBr免疫复合物分散在醋酸缓冲溶液中,加入四甲基联苯胺溶液,利用氙灯(λ ≥ 400)照射5分钟,通过紫外分光光度计检测TMB的紫外吸收来定量前列腺抗原的浓度。
作为一种优选方式:醋酸缓冲液的量为2-3.6毫升,pH为4;TMB的量为100-180微升,浓度为12毫摩尔/升;;通过紫外分光光度计检测TMB的紫外吸收来定量前列腺抗原的浓度是指前列腺抗原浓度与吸光度成线性相关性,对应的线性方程为y = 0.42 log(x) +0.63,其中,x是前列腺抗原的浓度,单位是纳克/毫升,y是检测的吸光度。
本发明的有益效果是:本发明方法制备功能化氧化石墨烯-氮化碳-溴化银(GO-C3N4-AgBr)纳米复合物材料,利用其可被可见激发的效应构建比色免疫分析方法对前列腺抗原进行检测。此比色免疫分析方法具有较好的灵敏度,并且检测速度快,准确率高,检测前列腺抗原有较好的专一性。
附图说明
图1是GO-C3N4-AgBr扫描电子显微镜(SEM)图;
图2是GO-C3N4-AgBr X-射线衍射(XRD)图;
图3(A)比色免疫传感器的构建示意图;(B)光激活显色的机理图;
图4是前列腺抗原的浓度与TMB吸光度的线性图。
具体实施方式
可见光激活的比色免疫分析方法,按照如下的步骤进行
步骤一、制备氧化石墨烯-氮化碳(GO-C3N4)复合物,将0.6-0.9克尿素加入到100-150毫升氧化石墨烯分散溶液中(0.15毫克每毫升),该混合液再80℃下持续搅拌3个小时,之后将其转移至烘箱,在70℃下放置过夜,最后,将该产品在550℃下煅烧2.5个小时,获得的样品即为GO-C3N4。
步骤二、将上述制备好的GO-C3N4复合物进行羧基化处理,将1-1.5克 GO-C3N4样品分散在100-150毫升浓硝酸溶液中(5M),在125℃下回流24个小时,之后将其冷却至室温,离心清洗得到羧基化的GO-C3N4复合物。
步骤三、制备氧化石墨烯-氮化碳-溴化银(GO-C3N4-AgBr)纳米复合物材料,将上述羧基化的GO-C3N4复合物超声分散在10-15毫升二次水中,之后加入5-7.5毫升硝酸银(0.5mM),避光搅拌1小时,随后逐滴加入10-15毫升溴化钠溶液(0.5 mM)继续搅拌2小时,通过离心、清洗和干燥得到GO-C3N4-AgBr样品;其扫描电镜图和X-射线衍射图如图1和图2所示,同时其光催化显色的机理图如图3A所示。
步骤四、对GO-C3N4-AgBr进行功能化修饰,将上述制备好的GO-C3N4-AgBr超声分散在1-1.5毫升二次水中,同时该水溶液中含有N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(100 mM)和N-羟基琥珀酰亚胺磺酸钠盐(100 mM),该混合溶液在室温下搅拌30分钟,之后加入500-750微升前列腺抗体(Ab),在4℃下缓慢震荡过夜,离心清洗得到抗体功能化的GO-C3N4-AgBr,记为Ab-GO-C3N4-AgBr。
利用制备的功能化氧化石墨烯-氮化碳-溴化银(GO-C3N4-AgBr)纳米复合物材料构建免疫复合物的方法,示意图如图3A所示,在室温下,将1-1.5毫升磁珠(5毫克/毫升)与500-750微升前列腺抗体孵育过夜,记为MB/Ab,清洗后再分别孵育浓度为0纳克/毫升、0.5纳克/毫升、1纳克/毫升、2纳克/毫升、4纳克/毫升、10纳克/毫升、15纳克/毫升、25纳克/毫升的前列腺抗原(PSA)30分钟,记为MB/Ab/PSA,清洗之后孵育上述制备好的Ab-GO-C3N4-AgBr纳米共轭复合物30分钟,记为MB/Ab/PSA/Ab-GO-C3N4-AgBr。将MB/Ab/PSA/Ab-GO-C3N4-AgBr免疫复合物分散在2-3.6a 毫升pH为4的醋酸缓冲溶液中,加入100-180a微升四甲基联苯胺溶液(12毫摩尔/升),利用氙灯(λ ≥ 400)照射5分钟,通过紫外分光光度计检测TMB的紫外吸收来定量前列腺抗原的浓度。
通过紫外分光光度计检测TMB的紫外吸收来定量氯霉素的浓度是指,将免疫传感器在氙灯下照射5分钟后,在200-800波长范围内进行紫外光谱扫描,当前列腺抗原的浓度在0纳克/毫升时,观察到空白缓冲液的紫外吸收峰为0.40,当前列腺抗原的浓度在0.5纳克/毫升时,得到的紫外吸收峰开始大于0.40,前列腺抗原的浓度检测范围是0.5纳克/毫升到25纳克/毫升,如图4,在此范围内,前列腺抗原的浓度与紫外吸收成线性相关性,其线性相关系数平方是0.987,对应的线性方程为y = 0.42 log(x) + 0.63 (R2 = 0.987, n =14)其中,x是前列腺抗原的浓度,单位是纳克/毫升,y是紫外吸光度。其最低检测限为0.5纳克/毫升(信噪比为3),与其它检测方法相比,构建的比色免疫传感器具有较低的检测限和较宽的检测范围(R是线性相关系数,R2是线性相关系数的平方,n代表的是实验次数)。
前列腺抗原浓度与吸光度的对应关系如下表所示:
x(纳克/毫升) | 0 | 0.5 | 1 | 2 | 4 | 10 | 15 | 25 |
y(Abs) | 0.40 | 0.48 | 0.67 | 0.78 | 0.89 | 0.98 | 1.11 | 1.27 |
实际样品分析
用构建的比色免疫传感器检测胎牛血清中添加的前列腺抗原,添加的前列腺抗原浓度分别为1纳克/毫升、5纳克/毫升、10纳克/毫升,由线性相关方程y = 0.42 log(x) + 0.63计算得到血清中的前列腺抗原的浓度分别为1.10纳克/毫升、4.89纳克/毫升、10.75纳克/毫升,回收率为110%、97%、107%,证明该传感器可以用来检测实际血清样品中的前列腺抗原浓度。
专一性分析
将制得的比色免疫传感器分别在空白缓冲溶液、10纳克/毫升的不同干扰物质(甲胎蛋白,癌胚抗原,免疫球蛋白,Ca2+,Mg2+, Zn2+,Fe2+,H2O2,葡萄糖)的缓冲溶液中孵育30分钟后,用二次水充分洗涤,然后检测,构建的比色传感器与上述九种干扰物质作用后,测得的吸光度值与空白组比较(0.40)相差不大(< 2%)。相反,当构建的比色传感器与0.5纳克/毫升的氯霉素作用时,吸光度值变化显著,为0.48。说明由GO-C3N4-AgBr构建的可见光激发比色免疫分析方法对检测前列腺抗原有较好的专一性。
Claims (6)
1.一种功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料制备方法,其特征在于:按照如下的步骤进行
步骤一、制备氧化石墨烯-氮化碳GO-C3N4复合物,将尿素加入到氧化石墨烯分散溶液中,在80℃下持续搅拌3个小时,在70℃下放置6-12小时,在550 oC 下煅烧2.5个小时,获得氧化石墨烯-氮化碳GO-C3N4复合物;
步骤二、将上述制备好的氧化石墨烯-氮化碳GO-C3N4复合物进行羧基化处理,将氧化石墨烯-氮化碳GO-C3N4复合物分散在浓硝酸溶液中,在125℃下加热反应24个小时,之后将其冷却至室温,离心清洗得到羧基化的GO-C3N4复合物;
步骤三、制备氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料,将步骤二制备的羧基化的GO-C3N4复合物超声分散在二次水中,之后加入硝酸银溶液,避光搅拌1小时,随后逐滴加入溴化钠溶液继续敞口搅拌2小时,通过离心、清洗和干燥得到石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料;
步骤四、对石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料进行功能化修饰,将制备好的石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料超声分散在二次水中,同时在该水溶液中添加N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺磺酸钠盐(NHS),在室温下搅拌30分钟,之后加入前列腺抗体(Ab),在4℃下缓慢震荡6-12小时,离心清洗得到功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料。
2.根据权利要求1所述的一种功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料制备方法,其特征在于:步骤一中尿素的量为0.6-0.9克;氧化石墨烯溶液的量为100-150毫升,浓度为0.15毫克/毫升;步骤二中氧化石墨烯-氮化碳GO-C3N4复合物量为1-1.5克;浓硝酸溶液的量为100-150毫升,浓度为5摩尔/升;步骤三中二次水的量为10-15毫升;硝酸银的量为5-7.5毫升,浓度为0.5毫摩尔/升;溴化钠的量为10-15毫升,浓度为0.5毫摩尔/升;羧基化的GO-C3N4复合物的量为1-1.5克;步骤四中二次水的量为1-1.5毫升;EDC的浓度为100-150毫摩尔/升;NHS的浓度为100-150毫摩尔/升;前列腺抗体的量为500-750微升;石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料的量为1-1.5克。
3.利用功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料构建免疫复合物,其特征在于:在室温下,将磁珠与前列腺抗体孵育6-12小时,记为MB/Ab,清洗后再孵育不同浓度的前列腺抗原(PSA)30分钟,记为MB/Ab/PSA,清洗之后孵育功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料溶液30分钟,记为MB/Ab/PSA/Ab-GO-C3N4-AgBr。
4.根据权利要求3利用功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料构建免疫复合物,其特征在于:磁珠的量为1-1.5毫升,浓度为5毫克/毫升;前列腺抗体的量为500-750微升;不同浓度的前列腺抗原是指0纳克/毫升、0.5纳克/毫升、1纳克/毫升、2纳克/毫升、4纳克/毫升、10纳克/毫升、15纳克/毫升、25纳克/毫升;功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物溶液的量为500-750微升,浓度为1毫克/升。
5.功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料构建免疫复合物的用途,其特征在于:MB/Ab/PSA/Ab-GO-C3N4-AgBr分散在醋酸缓冲溶液中,加入四甲基联苯胺溶液,利用氙灯(λ ≥ 400)照射5分钟,通过紫外分光光度计检测TMB的紫外吸收来定量前列腺抗原的浓度。
6.根据权利要求5所述的功能化氧化石墨烯-氮化碳-溴化银GO-C3N4-AgBr纳米复合物材料构建免疫复合物的用途,其特征在于:醋酸缓冲液的量为2-3.6毫升,pH为4;TMB的量为100-180微升,浓度为12毫摩尔/升;通过紫外分光光度计检测TMB的紫外吸收来定量前列腺抗原的浓度是指前列腺抗原浓度与吸光度成线性相关性,对应的线性方程为y = 0.42 log(x) + 0.63,其中,x是前列腺抗原的浓度,单位是纳克/毫升,y是检测的吸光度。
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Application publication date: 20180921 |