CN116519935A - 一种用于肿瘤标志物cyfra21-1 sers检测的免疫生物芯片及其制备方法 - Google Patents
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Abstract
一种用于肿瘤标志物CYFRA21‑1SERS检测的免疫生物芯片及其制备方法,该芯片包括位于上层的SERS探针和位于下层的免疫基底;制备方法为:采用MB标记AuNRs@Ag,活化后再修饰上CYFRA21‑1A抗体得到SERS探针;在Fe3O4@GO基底上修饰CYFRA21‑1B抗体,然后识别不同浓度的CYFRA21‑1得到Fe3O4@GO免疫基底;将Fe3O4@GO免疫基底浸入到SERS探针中,室温下孵育,用PBS清洗掉未连接的SERS探针,得到免疫生物芯片。该芯片特异性强,能对基底上吸附的CYFRA21‑1特异性识别,可应用于CYFRA21‑1的高效灵敏检测,提升SERS的灵敏度和选择性。
Description
技术领域
本发明属于肿瘤检测技术领域,具体涉及一种用于肿瘤标志物CYFRA21-1 SERS检测的免疫生物芯片及其制备方法。
背景技术
癌症在早期往往是无症状的,生物样本中肿瘤标志物的浓度往往极低。现有的电化学传感器仍然不够灵敏,无法进行早期诊断。有必要设计和开发高灵敏度的生物传感材料和设备,以早期发现肿瘤标志物,并识别癌前病变和转移前恶性肿瘤。
CYFRA21-1目前被认为是一种主要用于检测肺癌的肿瘤标志物,尤其对非小细胞肺癌(non-small cell lung cancer,NSCLC)的诊断具有重要价值。如果肺部存在不清晰的环形阴影,同时血清CYFRA21-1浓度大于30ng/ml,原发性支气管肺癌的可能性非常高。各类非小细胞肺癌阳性检出率为70%~85%。CYFRA21-1的血清浓度水平高低与肿瘤临床分期正相关,也可作为肺癌手术和放化疗后追踪早期复发的有效指标。CYFRA21-1的血清高浓度水平提示疾病处于进展期和预后不良。质量成功的标志是CYFRA21-1的血清浓度迅速下降,反之则表示病灶未完全清除。血清水平下降后又升高,则提示疾病复发。但是,CYFRA21-1阴性也不能排除存在肺癌的可能。CYFRA21-1对各型肺癌诊断的敏感性依次为:鳞癌>腺癌>大细胞癌>小细胞癌。另外,CYFRA21-1对其他肿瘤,如侵袭性膀胱癌、头颈部、乳腺、宫颈、消化道肿瘤均有一定的阳性率。
目前用于检测CYFRA21-1的传统方法主要有酶联免疫吸附法、流动荧光法、荧光法、表面等离子体共振分析法、伏安法和免疫放射测定法,这些方法由于存在设备成本高、线性范围窄等问题限制了它们的应用。
表面增强拉曼散射(SERS)是一种振动光谱技术,已被证明是检测疾病生物标志物最敏感的分析方法之一。SERS是基于贵金属银或金的增强拉曼光谱。与传统的拉曼光谱不同,它具有灵敏度高、化学特异性强、拉曼光谱带窄等特点。它能将拉曼信号增强到104-107幅值,对单分子检测灵敏度高。SERS已被证明是一种超灵敏且应用广泛的检测方法。由于SERS的独特性能,SERS已被许多研究者用于肿瘤标志物的超灵敏检测,特别是用于蛋白质和基因的检测。目前,还未见有用于肿瘤标志物CYFRA21-1 SERS检测的文献报道。
发明内容
本发明的目的在于提供一种用于肿瘤标志物CYFRA21-1 SERS检测的免疫生物芯片及其制备方法,该方法制备得到的芯片特异性强,能对Fe3O4@GO免疫基底上吸附的CYFRA21-1特异性识别,能排除其他与CYFRA21-1结构相似物质的干扰,可应用于CYFRA21-1的高效灵敏检测,提升SERS的灵敏度和选择性。
为实现上述目的,本发明提供了一种用于肿瘤标志物CYFRA21-1 SERS检测的免疫生物芯片,该芯片包括位于上层的SERS探针和位于下层的免疫基底,所述SERS探针是标记MB和修饰CYFRA21-1A抗体的AuNRs@Ag,所述免疫基底是表面修饰CYFRA21-1B抗体和CYFRA21-1的Fe3O4@GO免疫基底。
本发明还提供一种上述用于肿瘤标志物CYFRA21-1 SERS检测的免疫生物芯片的制备方法,包括如下步骤:
(1)SERS探针的制备:采用MB标记AuNRs@Ag,活化后再修饰上CYFRA21-1A抗体得到SERS探针;
(2)Fe3O4@GO免疫基底的制备:在Fe3O4@GO基底上修饰CYFRA21-1B抗体,然后识别不同浓度的CYFRA21-1,得到Fe3O4@GO免疫基底;
(3)免疫生物芯片的制备:将步骤(2)获得的Fe3O4@GO免疫基底浸入到步骤(1)得到的SERS探针中,室温下孵育2h,接着用浓度为0.01M的PBS清洗掉未连接的SERS探针,得到用于肿瘤标志物CYFRA21-1 SERS检测的免疫生物芯片。
进一步的,步骤(1)的具体步骤如下:
(1-1)将0.1mL浓度为0.01M的MB溶液与0.9mL浓度为2.36mg/mL的AuNRs@Ag溶液混合,以250rpm的转速旋转2h,然后以8000rpm的转速离心10min并洗涤数次后分散于1mL浓度为0.01M的PBS溶液中,得到MB标记的AuNRs@Ag纳米棒分散液;
(1-2)将50μL浓度为100mM的EDC和80μL浓度为100mM的NHS加入到1mL MB标记的AuNRs@Ag纳米棒分散液中,在室温下低速摇晃30min,然后用0.01M的PBS洗涤两次,再分散到1mL浓度为0.01M的PBS中,得到活化后的MB标记的AuNRs@Ag纳米棒分散液;
(1-3)向活化后的MB标记的AuNRs@Ag纳米棒分散液中加入100μL浓度为100μg/mL的抗CYFRA21-1A,于4℃下孵育12h,再加入50μL、浓度为3%的BSA作为阻断剂,于4℃下孵育6h,离心后分散在1mL浓度为0.01M的PBS中,得到SERS探针。
进一步的,所述AuNRs@Ag的制备过程如下:
a、将19.7μL浓度为0.01M的HAuCl4、30.3μL去离子水和2mL浓度为0.1M的CTAB混合均匀得到混合溶液,然后在0℃下快速将0.12mL新配制的浓度为0.378mg/mL的硼氢化钠溶液注入上述混合溶液中,混合溶液很快变成棕色,再将混合物溶液在超声水浴箱上涡旋2min,搅拌均匀后,置于24-25℃中2h,制备得到种液;
b、将盛有40mL浓度为36.45mg/mL的CTAC的50mL烧瓶放入30℃油浴中,转速为1200rpm,然后依次向烧瓶中加入400μL浓度为0.01M的AgNO3、788μL浓度为0.01M的HAuCl4、1.212mL的去离子水、0.8mL浓度为1.0M的HCl和0.32mL浓度为0.1M的AA,制备得到生长液;
c、将96μL种液注入到步骤b所配好的生长液中,搅拌30s,生长2h,将其倒入离心管后以8000rpm速度离心10分钟,弃上清后,用去离子水以相同的转速和时间离心清洗2~3次,最后一次离心后得到AuNRs,将其用超声波器分散备用得到AuNRs溶液;
d、向步骤c制备的AuNRs溶液中加入0.08M的CTAC溶液,以8000rpm速度离心10分钟,去上清后再以相同方式离心去上清1次,最后分散于12ml 0.08M的CTAC溶液中,然后加入0.4mL浓度为0.01M的AgNO3和0.32mL浓度为0.1M的AA,用超声波仪充分混合2min,溶液在65℃水浴中孵育4.5h后,以8000转/分离心20min,用去离子水洗涤2次,分散于3mL水中,调整终浓度为2.36mg/mL,得到AuNRs@Ag溶液。
进一步的,步骤(2)的具体步骤如下:
将100μL浓度为5mg/mL的磁性氧化石墨烯分散体分散在1mL浓度为0.01M的PBS溶液中,再加入100μL浓度为100μg/mL的CYFRA21-1B抗体,在4℃下孵育12h以上;再加入50μL、浓度为3%的BSA作为阻断剂,于4℃下孵育6h,最后磁分离收集免疫Fe3O4@GO底物重悬于1mL浓度为0.01M的PBS中;取100μL免疫Fe3O4@GO底物滴入2mL离心管中,然后在离心管中加入40μL不同浓度的CYFRA21-1标准溶液,在室温孵育3h后,将混合物磁分离并用PBS清洗后,得到Fe3O4@GO免疫基底。
与现有技术相比,本发明具有以下优点:
(1)本发明制备得到的芯片特异性强,能对Fe3O4@GO免疫基底上吸附的CYFRA21-1特异性识别,能排除其他与CYFRA21-1结构相似物质的干扰,可应用于CYFRA21-1的高效灵敏检测,提升SERS的灵敏度和选择性。
(2)本发明建立了一种高灵敏度、高选择性检测CYFRA21-1的方法,该方法能够满足方法验证过程中对灵敏性、特异性等方面的要求。目前临床对CYFRA21-1检测方法主要为化学发光法,其检测浓度仅到ng级,而本发明的检测浓度达到了pg级,提高了好几个数量级,这大大改善了现有方法灵敏度低的缺陷。
(3)在本发明中,选择了拉曼信号较高的金银纳米棒做探针,选用磁性氧化石墨烯做磁性基底,拉曼峰强度与CYFRA21-1浓度的对数之间的线性关系更广,本方法在1pg/ml-10ng/ml范围内,CYFRA21-1具有良好的线性关系。
附图说明
图1为本发明检测原理示意图;
图2为实施例一中AuNRs纳米棒和AuNRs@Ag的SEM图;(A)AuNRs纳米棒;(B)AuNRs@Ag;
图3为实施例一中相同浓度下MB分别在AuNRs和AuNRs@Ag上的拉曼光谱图;
图4为实施例一中AuNRs和AuNRs@Ag的紫外可见光谱图;
图5为实施例一中不同MB浓度下AuNRs@Ag纳米探针的拉曼光谱图;
图6为实施例一中Fe3O4@GO免疫基底的TEM图,(A)、刻度为100nm,(B)、刻度为200nm;
图7为实施例一中不同体积(10~100μL)特异性抗体的拉曼光谱图;
图8为实施例一中不同用量(25~400μL)探针的拉曼光谱图;
图9为实施例二中基于SERS双抗体系对CYFRA21-1抗原的特异性试验结果示意图;
图10为实施例三中CYFRA21-1在不同浓度下的SERS光谱图;
图11为实施例三中CYFRA21-1(1pg mL-1-10ng mL-1)在448cm-1峰值处的浓度Lg值与SERS强度的标准曲线;
图12为实施例四中不同稀释倍数下人血清的SERS光谱图;
图13为实施例四中不同稀释倍数下人血清的SERS信号与CYFRA21-1浓度的线性关系图。
具体实施方式
以下结合附图和具体实施例对本发明作进一步详细说明。
除非特别说明,本发明采用的试剂、方法和设备均为本技术领域常规试剂、方法和设备。
以下实施例中:
CYFRA21-1A抗体为捕获抗体,浓度为4.1mg/ml;CYFRA21-1B抗体为检测抗体,浓度为8.9mg/ml,为霍尔姆斯公司生产。
CYFRA21-1、CA199、CA125、NSE、CEA均来自于徐州医科大学附属医院东院检验科的定标液。
磁性氧化石墨烯分散体购买于江苏先丰纳米材料科技有限公司。
实施例一
一种用于肿瘤标志物CYFRA21-1 SERS检测的免疫生物芯片的制备方法,包括如下步骤:
(1)SERS探针的制备:采用MB标记AuNRs@Ag,活化后再修饰上CYFRA21-1A抗体得到SERS探针;
具体制备过程为:
(1-1)将0.1mL浓度为0.01M的MB溶液与0.9mL浓度为2.36mg/mL的AuNRs@Ag溶液混合,以250rpm的转速旋转2h,然后以8000rpm的转速离心10min并洗涤数次后分散于1mL浓度为0.01M的PBS溶液中,得到MB标记的AuNRs@Ag纳米棒分散液;
(1-2)将50μL浓度为100mM的EDC和80μL浓度为100mM的NHS加入到1mL MB标记的AuNRs@Ag纳米棒分散液中,在室温下低速摇晃30min,然后用0.01M的PBS洗涤两次,再分散到1mL浓度为0.01M的PBS中,得到活化后的MB标记的AuNRs@Ag纳米棒分散液;
(1-3)向活化后的MB标记的AuNRs@Ag纳米棒分散液中加入100μL浓度为100μg/mL的抗CYFRA21-1A,于4℃下孵育12h,再加入50μL、浓度为3%(即将3g的牛血清白蛋白完全溶解于100mL的PBS溶液中)的BSA作为阻断剂,以防止抗原的非特异性吸收,于4℃下孵育6h,离心后分散在1mL浓度为0.01M的PBS中,得到SERS探针。
所述AuNRs@Ag溶液的制备过程为:
a、将19.7μL浓度为0.01M的HAuCl4、30.3μL去离子水和2mL浓度为0.1M的CTAB混合均匀得到混合溶液,然后在0℃下快速将0.12mL新配制的浓度为0.378mg/mL的硼氢化钠溶液注入上述混合溶液中,混合溶液很快变成棕色,再将混合物溶液在超声水浴箱上涡旋2min,搅拌均匀后,置于24-25℃中2h,制备得到种液;
b、将盛有40mL浓度为36.45mg/mL的CTAC的50mL烧瓶放入30℃油浴中,转速为1200rpm,然后依次向烧瓶中加入400μL浓度为0.01M的AgNO3、788μL浓度为0.01M的HAuCl4、1.212mL的去离子水、0.8mL浓度为1.0M的HCl和0.32mL浓度为0.1M的AA(抗坏血酸),制备得到生长液;
c、将96μL种液注入到步骤b所配好的生长液中,搅拌30s,生长2h,将其倒入离心管后以8000rpm速度离心10分钟,弃上清后,用去离子水以相同的转速和时间离心清洗2~3次,最后一次离心后留下1ml的AuNRs,将其用超声波器分散备用得到AuNRs溶液;
d、配置适量的0.08M的CTAC溶液,将其加入步骤c制备的AuNRs溶液中,以8000rpm速度离心10分钟,去上清后再以相同方式离心去上清液1次,最后分散于12ml 0.08M的CTAC溶液中,然后加入0.4mL浓度为0.01M的AgNO3和0.32mL浓度为0.1M的AA(抗坏血酸),用超声波仪充分混合2min,溶液在65℃水浴中孵育4.5h后,以8000rpm离心20min,用去离子水洗涤2次,分散于3mL水中,调整终浓度为2.36mg/mL,得到AuNRs@Ag溶液。
双金属纳米棒的表征分析如下:
从图2A和图2B可以清楚地观察到金纳米棒表面覆盖了一层银。当AuNRs@Ag和AuNRs浓度相同时,以9:1比例测量的AuNRs@Ag和10-2M MB的拉曼强度明显高于原始AuNRs(图3)。双金属纳米棒的SERS效应比单金属纳米棒强得多,这是由于双金属纳米棒两端存在极强的电磁场(图3)。区间间耦合的电磁增强在内部间隙产生了更多的“热点”,从而诱导出更强的SERS信号,Nikoobakht等人将这一现象归因于棒两端的避雷针效应。可以推断,Au和Ag之间的协同效应对增强有很大的贡献,两种纳米探针的紫外-可见光谱(图4)表明,双金属纳米棒AuNRs@Ag在900nm左右有一个金纳米棒特异性峰,在650nm左右有一个银纳米棒明显峰。这些结果表明AuNRs@Ag的构建是成功的。
所述MB浓度的优化过程为:利用制备好的AuNRs@Ag纳米棒进一步优化MB的浓度,将不同浓度的MB溶液(10-9M-10-1M)以9:1的比例加入到AuNRs@Ag溶液中,测量拉曼强度。从图5中可以看出,当MB浓度降低到10-9M时,仍然可以看到拉曼信号,表明Au@Ag NRs有很明显的拉曼强度。随着MB浓度的降低,拉曼强度逐渐降低。MB浓度为10-2M时拉曼强度最大;但随着MB浓度的增加,拉曼强度变化不明显。因此,为了获得较强的拉曼信号,选择10-2M作为后续SERS检测的最佳浓度。随后,为了进一步量化SERS活性,计算了AuNRs@Ag的拉曼增强因子(EF)。通过比较AuNRs@Ag作为探针时MB(448cm-1)的SERS强度和不使用探针时MB(448cm-1)在10-3mol/L时的拉曼强度来估计EF。MB的EFs计算范围为1.34×10~2.15×107,说明探针对低浓度分子的检测灵敏度较高。
(2)Fe3O4@GO免疫基底的制备:
将100μL浓度为5mg/mL的磁性氧化石墨烯分散体分散在1mL浓度为0.01M的PBS溶液中,再加入100μL浓度为100μg/mL的CYFRA21-1B抗体,在4℃下孵育12h以上;再加入50μL、浓度为3%(即将3g的牛血清白蛋白完全溶解于100mL的PBS溶液中)的BSA作为阻断剂,于4℃下孵育6h,最后磁分离收集免疫Fe3O4@GO底物重悬于1mL浓度为0.01M的PBS中;
取100μL免疫Fe3O4@GO底物滴入2mL离心管中,然后在离心管中加入40μL不同浓度的CYFRA21-1标准溶液,在室温孵育3h后,将混合物磁分离并用PBS清洗以去除未结合的抗原后,得到Fe3O4@GO免疫基底。从图6中可以看出,灰色薄片的层状结构为氧化石墨烯,支撑在其上的黑色颗粒为Fe3O4,说明Fe3O4@GO免疫基底成功制备。
通过进一步优化,确定了免疫Fe3O4@GO底物的最佳用量和免疫条件的优化。图7和图8分别为添加到SERS平台的不同浓度的免疫Fe3O4@GO底物和抗体偶联免疫探针的拉曼光谱。随着免疫Fe3O4@GO底物量从10μL增加到100μL,拉曼信号逐渐增强。拉曼信号强度的增强可能是由于抗原-抗体结合。加入50μL和100μL免疫Fe3O4@GO底物,拉曼信号差异不大(图7)。同样,随着探针用量从25μL增加到400μL,拉曼强度逐渐增加(图8);然而,当探针用量从200μL增加到400μL时,拉曼信号并没有发生变化,这可能与衬底或探针的饱和有关。基于此,衬底和探针的最佳体积分别为100μL和200μL。
(3)免疫生物芯片的制备:将步骤(2)获得的100μLFe3O4@GO免疫基底浸入到步骤(1)得到的200μL SERS探针中,室温下孵育2h,接着用浓度为0.01M的PBS清洗掉未连接的SERS探针,最后将沉淀分散在200μL去离子水中,得到用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片用于SERS检测,检测原理如图1所示。
实施例二
SERS免疫分析的特异性测试
通过在SERS免疫分析平台上添加不同抗原来检测SERS免疫分析的特异性。在免疫测定反应体系中加入40μL CYFRA21-1(100pg/mL)、CA199(281U/mL)、CA125(281U/mL)、NSE(50.5ng/mL)、CEA(100ng/mL)时(图9),只有CYFRA21-1蛋白的拉曼信号较强,而其他抗原蛋白的拉曼信号都很弱,说明免疫测定体系对CYFRA21-1具有特异性。
实施例三
SERS免疫分析的线性
采用不同浓度的CYFRA21-1对所提出的基于SERS的免疫分析法进行检测。不同浓度CYFRA21-1的SERS免疫分析反应的SERS光谱具有标记分子MB的3个强拉曼峰(448cm-1、1441cm-1和1626cm-1)(图10)。利用MB在448cm-1处的SERS强度对靶抗原进行定量评估(图10)。在没有CYFRA21-1的情况下,没有检测到明显的SERS信号,说明洗涤过程可以去除几乎所有未反应的探针。随着靶抗原浓度的增加,SERS信号增强。此外,随着抗原的稀释,SERS强度降低。当CYFRA21-1的浓度降低到1pg mL-1时,MB在448cm-1处的SERS峰值变得非常微弱。CYFRA21-1(1pg mL-1-10ng mL-1)在448cm-1峰值处的浓度lg值与SERS强度的标准曲线如图11所示。相应的线性方程为int=5657[Lg[CYFRA21-1]]+5055(相关系数R2=0.9916,N=3)。根据3σ规则(σ为标准差),估计10个空白样品的检出限为0.8943pg mL-1。这些结果表明,基于SERS的夹心免疫分析法具有良好的线性关系。
实施例四
人血清样本中SERS免疫分析的验证
为验证该免疫分析方法的潜在诊断价值,采用本发明所提出的SERS方法和化学发光法对3份临床血清样本进行了CYFRA21-1检测。采用SERS免疫法测定CYFRA21-1的浓度与化学发光法测定的浓度吻合较好,且在临床可接受的范围内,变异系数(CV)可接受。将其中一份血清样品用PBS溶液分别稀释至10倍、100倍和1000倍,检测SERS信号(图12和表1),SERS信号与CYFRA21-1浓度呈线性关系(图13和表1)。通过标准线性方程计算血清中目标蛋白的浓度(表1)。在人血清样本中,CYFRA21-1的SERS免疫测定最低检出量为6.68pg mL-1。因此,基于SERS的检测技术具有应用于临床检测在人类血清中CYFRA21-1的潜力。
表1计算稀释血清样品中的蛋白质含量
实施例五
方法比较:SERS免疫分析法与其他CYFRA21-1检测方法的比较
表2.SERS免疫分析法与其他CYFRA21-1检测方法的比较。
如表2所示,与这些传统方法相比,表面增强拉曼光谱(SERS)具有很多显著的优势,具有更高选择性、高灵敏度等优良性能。
Claims (5)
1.一种用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片,其特征在于,该芯片包括位于上层的SERS探针和位于下层的免疫基底,所述SERS探针是标记MB和修饰CYFRA21-1A抗体的AuNRs@Ag,所述免疫基底是表面修饰CYFRA21-1B抗体和CYFRA21-1的Fe3O4@GO免疫基底。
2.一种如权利要求1所述的用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片的制备方法,其特征在于,包括如下步骤:
(1)SERS探针的制备:采用MB标记AuNRs@Ag,活化后再修饰上CYFRA21-1A抗体得到SERS探针;
(2)Fe3O4@GO免疫基底的制备:在Fe3O4@GO基底上修饰CYFRA21-1B抗体,然后识别不同浓度的CYFRA21-1,得到Fe3O4@GO免疫基底;
(3)免疫生物芯片的制备:将步骤(2)获得的Fe3O4@GO免疫基底浸入到步骤(1)得到的SERS探针中,室温下孵育2h,接着用浓度为0.01M的PBS清洗掉未连接的SERS探针,得到用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片。
3.根据权利要求2所述的一种用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片的制备方法,其特征在于,步骤(1)的具体步骤如下:
(1-1)将0.1mL浓度为0.01M的MB溶液与0.9mL浓度为2.36mg/mL的AuNRs@Ag溶液混合,以250rpm的转速旋转2h,然后以8000rpm的转速离心10min并洗涤数次后分散于1mL浓度为0.01M的PBS溶液中,得到MB标记的AuNRs@Ag纳米棒分散液;
(1-2)将50μL浓度为100mM的EDC和80μL浓度为100mM的NHS加入到1mL MB标记的AuNRs@Ag纳米棒分散液中,在室温下低速摇晃30min,然后用0.01M的PBS洗涤两次,再分散到1mL浓度为0.01M的PBS中,得到活化后的MB标记的AuNRs@Ag纳米棒分散液;
(1-3)向活化后的MB标记的AuNRs@Ag纳米棒分散液中加入100μL浓度为100μg/mL的抗CYFRA21-1A,于4℃下孵育12h,再加入50μL、浓度为3%的BSA作为阻断剂,于4℃下孵育6h,离心后分散在1mL浓度为0.01M的PBS中,得到SERS探针。
4.根据权利要求2或3所述的一种用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片的制备方法,其特征在于,所述AuNRs@Ag的制备过程如下:
a、将19.7μL浓度为0.01M的HAuCl4、30.3μL去离子水和2mL浓度为0.1M的CTAB混合均匀得到混合溶液,然后在0℃下快速将0.12mL新配制的浓度为0.378mg/mL的硼氢化钠溶液注入上述混合溶液中,混合溶液很快变成棕色,再将混合物溶液在超声水浴箱上涡旋2min,搅拌均匀后,置于24-25℃中2h,制备得到种液;
b、将盛有40mL浓度为36.45mg/mL的CTAC的50mL烧瓶放入30℃油浴中,转速为1200rpm,然后依次向烧瓶中加入400μL浓度为0.01M的AgNO3、788μL浓度为0.01M的HAuCl4、1.212mL的去离子水、0.8mL浓度为1.0M的HCl和0.32mL浓度为0.1M的AA,制备得到生长液;
c、将96μL种液注入到步骤b所配好的生长液中,搅拌30s,生长2h,将其倒入离心管后以8000rpm速度离心10分钟,弃上清后,用去离子水以相同的转速和时间离心清洗2~3次,最后一次离心后得到AuNRs,将其用超声波器分散备用得到AuNRs溶液;
d、向步骤c制备的AuNRs溶液中加入0.08M的CTAC溶液,以8000rpm速度离心10分钟,去上清后再以相同方式离心去上清1次,最后分散于12ml0.08M的CTAC溶液中,然后加入0.4mL浓度为0.01M的AgNO3和0.32mL浓度为0.1M的AA,用超声波仪充分混合2min,溶液在65℃水浴中孵育4.5h后,以8000rpm离心20min,用去离子水洗涤2次,分散于3mL水中,调整终浓度为2.36mg/mL,得到AuNRs@Ag溶液。
5.根据权利要求2所述的一种用于肿瘤标志物CYFRA21-1SERS检测的免疫生物芯片的制备方法,其特征在于,步骤(2)的具体步骤如下:
将100μL浓度为5mg/mL的磁性氧化石墨烯分散体分散在1mL浓度为0.01M的PBS溶液中,再加入100μL浓度为100μg/mL的CYFRA21-1B抗体,在4℃下孵育12h以上;再加入50μL、浓度为3%的BSA作为阻断剂,于4℃下孵育6h,最后磁分离收集免疫Fe3O4@GO底物重悬于1mL浓度为0.01M的PBS中;取100μL免疫Fe3O4@GO底物滴入2mL离心管中,然后在离心管中加入40μL不同浓度的CYFRA21-1标准溶液,在室温孵育3h后,将混合物磁分离并用PBS清洗后,得到Fe3O4@GO免疫基底。
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