CN115212861B - 一种基于表面增强拉曼散射信号的编码微球及应用 - Google Patents
一种基于表面增强拉曼散射信号的编码微球及应用 Download PDFInfo
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Abstract
本发明公开了一种基于表面增强拉曼散射信号的编码微球及应用,所述编码微球包括具有表面增强拉曼散射效应壳层的磁性聚合物微球和拉曼编码分子,其中具有表面增强拉曼散射效应的壳层不仅可以负载拉曼编码分子而且可以显著增强拉曼编码信号;还公开了所述拉曼编码微球结合增强拉曼报告分子载体在一种或多种目标物检测中的应用。本发明采用的具有表面增强拉曼散射效应壳层的微球显著增强了编码分子的拉曼信号的强度,解决了拉曼信号在指纹区峰过于复杂容易重叠、在静默区信号本征强度太低而难以进行高通量编码的问题,使得拉曼信号编码能力显著提升,有望成为继荧光信号编码之后的另一有力的编码手段,为液相芯片体外检测技术提供了新的发展方向。
Description
技术领域
本发明涉及体外检测领域,具体地,涉及一种基于表面增强拉曼散射信号的编码微球及应用。
背景技术
液相芯片是重要的体外多因子检测技术之一。其技术核心在于利用荧光、发光寿命、光散射、图形等信号构建编码库的编码微球。每一种编码微球代表一种待测物,因此编码微球的数量直接影响液相芯片的多因子检测能力。目前通用的荧光编码微球库理论上可以实现100多种待测物的同时检测,有效地提高了体外检测的效率和准确性,在疾病的早期筛查、精准诊断和治疗预后中发挥了重要作用。
然而基于荧光编码微球的液相芯片平台还存在一些问题。荧光信号本身存在光谱重叠、浓度猝灭和能量转移等问题,这限制了编码数量的进一步增加,且不利于编码库的精确设计和预测。而且荧光信号易受到温度、pH等环境因素的影响,为了实现信号长期稳定性需要较为复杂的保护手段。同时不同的荧光分子一般对应不同的激发光和发射谱带,这使得检测设备的光路设计复杂且成本增加。基于荧光报告信号的液相芯片平台也难以实现待测物的超灵敏检测。因此发展基于新的信号形式的液相芯片平台十分必要。
拉曼信号是一种反映分子振动转动信息的散射光信号,由分子的结构决定,峰位稳定,不存在能量转移和浓度猝灭,谱峰窄且尖锐,能被同一激发波长激发,在侧向层析检测、平板阵列等体外检测平台中都已取得了很好的应用。但是拉曼信号用于液相芯片却仍然存在一些瓶颈亟待突破。首先指纹区的拉曼峰多且密集,选择可以明确彼此区分且互不干扰的峰用于编码难度较大;其次虽然随着在静默区具有独特信号峰的拉曼分子的发展,在静默区可以选择一系列彼此互不干扰的拉曼峰进行编码,但是拉曼信号的本征强度很低,也无法实现信号峰的强度编码。目前很难构建超过20因子的拉曼编码库,这极大限制了拉曼液相芯片体系的发展。
因此,体外检测领域的研究者致力于开发一种能够实现静默区拉曼信号有效增强的编码微球,通过静默区拉曼信号在波长和强度两个维度的编码,构建编码能力强大的拉曼微球编码库,以便获得高通量、超灵敏的拉曼液相芯片平台,为疾病的早期筛查、准确诊断和治疗预后提供有效手段。
发明内容
针对现有技术中的缺陷,本发明的目的是提供一种基于表面增强拉曼散射信号的编码微球及应用。
本发明的目的是通过以下方案实现的:
本发明的第一方面提供一种基于表面增强拉曼散射信号的编码微球,其特征在于,包括具有表面增强拉曼散射效应壳层的磁性聚合物微球和拉曼编码分子。
优选的,所述微球的表面增强拉曼散射效应壳层包括:贵金属银的表面增强壳层、贵金属金的表面增强壳层、贵金属金银的复合增强壳层、金属铜的表面增强壳层及其他具有明显表面增强拉曼散射效应的金属和半导体壳层中的一种或多种。
优选的,所述拉曼编码分子包括:4,4’-联吡啶、罗丹明6G、异硫氰酸孔雀石绿、尼罗河蓝A、结晶紫、亚甲基蓝、4-巯基苯甲酸、2,3,5,6-四氟-4-巯基苯甲酸、5,5’-二硫双歧-(2-硝基苯甲酸)、2-萘硫醇、苯硫醇、4-羟基苯硫醇、4-硝基苯硫醇、4-溴苯硫醇、4-氯苯硫醇、2-氯苯硫醇、4-异丙基苯硫醇、3,4-二氯苯硫醇、4-巯基吡啶、4-巯基甲苯、4-巯基苯甲腈、4-氟噻吩、4-氨基苯硫酚、4-甲氧基硫酚、4-巯基苯乙炔、4,4’-(1,2-乙炔基)双[苯硫醇]、4-(2-(三甲基硅基)乙炔基)苯硫醇和各类聚炔衍生物中的一种或多种。
优选的,所述贵金属银的表面增强壳层微球和贵金属金银的复合增强壳层微球可以通过以下制备方法制备得到:
步骤一、利用SPG膜乳化-溶剂挥发法在室温下制备四氧化三铁纳米粒子掺杂的磁性聚合物微球;
步骤二、将制备的所述磁性聚合物微球置于室温碱性环境中进行表面的多巴胺自氧化聚合形成聚多巴胺层包覆,得到磁性聚多巴胺壳层微球;
步骤三、室温下,在制备的所述磁性聚多巴胺壳层微球表面原位还原银氨离子生长银纳米粒子壳层,得到银纳米粒子壳层微球;
步骤四、室温下,将制备的所述银纳米粒子壳层微球表面通过种子诱导二次还原硝酸银和氯金酸,分别得到二次生长银纳米粒子复合壳层微球和二次生长银-金纳米粒子复合壳层微球。
步骤五、室温下,在制备的银纳米粒子壳层微球、二次生长银纳米粒子复合壳层微球和二次生长银-金纳米粒子复合壳层微球表面静电吸附拉曼编码分子,得到表面增强拉曼散射信号的编码微球。优选的,所述步骤一中四氧化三铁的掺杂量为1%~10%。
优选的,所述步骤二中多巴胺溶液浓度为0.1~4mg/mL,多巴胺溶液体积为5~20mL。。
优选的,所述步骤三中银氨离子浓度为5~25mg/mL,银氨溶液体积为5~20mL。
优选的,所述制备方法中二次生长使用的硝酸银浓度为8.5×10-3mg/mL~8.5×10-2mg/mL、硝酸银水溶液体积为0.1~2mL;氯金酸浓度为1.70×10-2mg/mL~0.17mg/mL、氯金酸水溶液体积为0.1~2mL。
优选的,所述制备方法中编码分子的用量为每个微球0~1×10-6mol。
本发明的第二方面提供一种基于拉曼静默区的表面增强拉曼散射信号的编码微球的应用,所述编码微球可用于一种或多种蛋白目标物的超灵敏检测。
与现有技术相比,本发明具有如下的有益效果:
(1)本发明制备的基于表面增强拉曼散射信号的编码微球采用贵金属金银壳层微球作为拉曼增强基底,制备方法简便可靠,可以大批量制备表面贵金属壳层包覆均匀完全、粒径均一性好的贵金属微球,而且微球贵金属壳层表面可以进一步调控,可以实现非常显著的表面增强拉曼散射效果,比颗粒增强策略提高了2个数量级,从而能够完成对静默区拉曼分子在强度维度的编码,所构建的拉曼编码库编码能力强大,在静默区范围内使用10个拉曼峰进行5强度编码就可以实现近1000万个编码子,而相对来说,荧光编码受限于光谱重叠和能量转移,能够实现的编码子小于2000个;
(2)本发明提供的基于微球的拉曼编码信号表面增强策略,使得拉曼信号也可用于液相芯片平台的编码库构建,有效克服了现行荧光编码策略的不足,而且可以清晰分辨的拉曼编码还有利于辅助开发拉曼流式细胞仪;
(3)本发明制备的静默区表面增强拉曼编码微球可以作为载体,结合表面标记报告拉曼分子的金纳米花,微球贵金属壳层和金纳米花的突触纳米结构可以共同增强报告拉曼信号,从而实现对蛋白待测物的多因子超灵敏检测,这对基于液相芯片的体外多因子检测技术的进一步发展有重要意义。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更明显:
图1是实施例1制备的银纳米粒子壳层微球的SEM图片;
图2是实施例2制备的二次生长银纳米粒子复合壳层微球的SEM图片;
图3是实施例3制备的二次生长银-金纳米粒子复合壳层微球的SEM图片;
图4是本实施例3中制备的拉曼静默区的表面增强拉曼散射信号的编码库;
图5是实施例1中制备的所述4-巯基苯甲腈编码银纳米粒子微球作为载体,利用液相芯片检测技术得到的归一化后的免疫检测标准曲线;
图6是实施例2中制备的所述4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银纳米粒子复合壳层微球作为载体,利用液相芯片检测技术得到的归一化后的免疫检测标准曲线;
图7是实施例3中制备的所述4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银-金纳米粒子复合壳层微球作为载体,利用液相芯片检测技术得到的归一化后的免疫检测标准曲线;
图8是文献报道的使用溶胀吸附法、溶胀异相聚集法、层层组装法图和种子吸附生长法图制备得到的贵金属壳层微球的SEM图片。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明,但不以任何形式限制本发明。应当指出的是,对本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变化和改进。这些都属于本发明的保护范围。
实施例1
本实施例所制备的静默区表面增强拉曼散射信号编码微球是表面标记4-巯基苯甲腈的银纳米粒子壳层微球,具体制备步骤为:
步骤1、使用SPG膜乳化-溶剂挥发法制备2.5%四氧化三铁纳米粒子掺杂的苯乙烯-马来酸酐共聚物的磁性微球;
步骤2、将所述步骤1得到的20mg磁性微球充分分散于20mL Tris缓冲液(pH=8.5)中,再加入20mL浓度为1mg/mL的多巴胺的Tris缓冲液(pH=8.5)溶液,室温搅拌3小时,纯水磁分离清洗3遍,重新分散于纯水中;
步骤3、将所述步骤2得到的1.5×108个聚多巴胺层磁性微球分散于10mL 10mg/mL的银氨离子水溶液中,室温搅拌1小时,纯水磁分离清洗3遍并真空干燥。
步骤4、将所述步骤3得到的5×104个银纳米粒子壳层微球重新分散于50μL纯水中,加入50μL 5×10-3M的4-巯基苯甲腈溶液,震荡孵育1小时,乙醇磁分离清洗3遍,得到4-巯基苯甲腈编码的银纳米粒子壳层微球。
如图1所示,在扫描电子显微镜下可观察到所制备的银纳米粒子壳层编码微球表面均匀且包覆完全,微球的粒径均一性、形貌一致性好,借助贵金属壳层的表面增强拉曼散射效应可以显著增强静默区拉曼信号,从而能够实现拉曼信号的强度编码。
实施例2
本实施例所制备的静默区表面增强拉曼散射信号编码微球是表面标记4-(2-(三甲基硅基)乙炔基)苯硫醇的二次生长银纳米粒子复合壳层微球,具体制备步骤为:
步骤1、使用SPG膜乳化-溶剂挥发法制备2.5%四氧化三铁纳米粒子掺杂的苯乙烯-马来酸酐共聚物的磁性微球;
步骤2、将所述步骤1得到的20mg磁性微球充分分散于20mL Tris缓冲液(pH=8.5)中,再加入20mL浓度为1mg/mL的多巴胺的Tris缓冲液(pH=8.5)溶液,室温搅拌3小时,纯水磁分离清洗3遍,重新分散于纯水中;
步骤3、将所述步骤2得到的1.5×108个聚多巴胺层磁性微球分散于10mL 10mg/mL的银氨离子水溶液中,室温搅拌1小时,纯水磁分离清洗3遍并真空干燥。
步骤4、将所述步骤3得到的106个银纳米粒子壳层微球重新分散于50μL纯水中,加入1mL 0.15mM的硝酸银水溶液和0.5mL 0.45mM的抗坏血酸的水溶液,震荡孵育1小时,纯水磁分离清洗3遍,得到二次生长银纳米粒子复合壳层微球。
步骤5、将所述步骤3得到的5×104个二次生长银纳米粒子复合壳层微球重新分散于50μL纯水中,加入50μL 5×10-3M的4-(2-(三甲基硅基)乙炔基)苯硫醇溶液,震荡孵育1小时,乙醇磁分离清洗3遍,得到4-(2-(三甲基硅基)乙炔基)苯硫醇编码的二次生长银纳米粒子复合壳层微球。
如图2所示,在扫描电子显微镜下可观察到以所制备的银纳米粒子壳层编码微球为种子二次生长银纳米粒子,所制得的银纳米粒子复合壳层均一性好、包覆完全,同样可用于实现拉曼信号的贵金属壳层表面增强散射。
实施例3
本实施例所制备的静默区表面增强拉曼散射信号编码微球是表面标记4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇的二次生长银-金纳米粒子复合壳层微球,具体制备步骤为:
步骤1、使用SPG膜乳化-溶剂挥发法制备2.5%四氧化三铁纳米粒子掺杂的苯乙烯-马来酸酐共聚物的磁性微球;
步骤2、将所述步骤1得到的20mg磁性微球充分分散于20mL Tris缓冲液(pH=8.5)中,再加入20mL浓度为1mg/mL的多巴胺的Tris缓冲液(pH=8.5)溶液,室温搅拌3小时,纯水磁分离清洗3遍,重新分散于纯水中;
步骤3、将所述步骤2得到的1.5×108个聚多巴胺层磁性微球分散于10mL 10mg/mL的银氨离子水溶液中,室温搅拌1小时,纯水磁分离清洗3遍并真空干燥。
步骤4、将所述步骤3得到的106个银纳米粒子壳层微球重新分散于50μL纯水中,加入1mL 0.3mM的氯金酸水溶液和0.5mL 0.45mM的盐酸羟胺的水溶液,震荡孵育1小时,纯水磁分离清洗3遍,得到二次生长银-金纳米粒子复合壳层微球。
步骤5、将所述步骤3得到的5×104个二次生长银-金纳米粒子复合壳层微球重新分散于50μL纯水中,加入25μL 5×10-3M的4-(2-(三甲基硅基)乙炔基)苯硫醇溶液和25μL 5×10-3M的4-巯基苯甲腈溶液,震荡孵育1小时,乙醇磁分离清洗3遍,得到4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇二元编码的二次生长银-金纳米粒子复合壳层微球。
如图3所示,在扫描电子显微镜下可观察到以所制备的银纳米粒子壳层编码微球为种子二次生长金纳米粒子,所制得的银-金纳米粒子复合壳层均一性好、包覆完全,同样可用于实现拉曼信号的贵金属壳层表面增强散射。
如图4所示,使用所制备的贵金属纳米粒子壳层微球可以构建基于4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇的25因子的编码库。
实施例4
本实施例是将实施例1中制备的所述4-巯基苯甲腈编码银纳米粒子微球作为载体,利用液相芯片检测技术,实现对肿瘤标志物癌胚抗原(CEA)的含量的定量检测。具体步骤为:
步骤1、将实施例1中制备的4-巯基苯甲腈编码银纳米粒子微球通过静电吸附在表面偶联癌胚抗原的单克隆捕获抗体;
步骤2、在1.5毫升离心管中分别加入上述包被有癌胚抗原的单克隆捕获抗体的4-巯基苯甲腈编码银纳米粒子微球,然后加入病人血清,室温振荡孵育1h,通过磁分离清洗,除去血清中未捕获的癌胚抗原;
步骤3、然后向离心管中分别加入生物素标记的单克隆检测抗体和链霉亲和素标记的金纳米花2-萘硫酚报告分子载体,室温振荡孵育30min,通过磁分离清洗,除去过量的生物素标记的检测抗体和金纳米花;
步骤4、用拉曼共聚焦显微镜读取数据,得到病人血清中癌胚抗原的含量。
如图5所示为归一化后的免疫检测标准曲线。
实施例5
本实施例是将实施例2中制备的所述4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银纳米粒子复合壳层微球作为载体,利用液相芯片检测技术,实现对肿瘤标志物甲胎蛋白(AFP)的含量的定量检测。具体步骤为:
步骤1、将实施例2中制备的4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银纳米粒子复合壳层微球通过静电吸附在表面偶联甲胎蛋白的单克隆捕获抗体;
步骤2、在1.5毫升离心管中分别加入上述包被有甲胎蛋白的单克隆捕获抗体的4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银纳米粒子复合壳层微球,然后加入病人血清,室温振荡孵育1h,通过磁分离清洗,除去血清中未捕获的甲胎蛋白;
步骤3、然后向离心管中分别加入生物素标记的单克隆检测抗体和链霉亲和素标记的金纳米花2-萘硫酚报告分子载体,室温振荡孵育30min,通过磁分离清洗,除去过量的生物素标记的检测抗体和金纳米花;
步骤4、用拉曼共聚焦显微镜读取数据,得到病人血清中甲胎蛋白的含量。
如图6所示为归一化后的免疫检测标准曲线。
实施例6
本实施例是将实施例3中制备的所述4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银-金纳米粒子复合壳层微球作为载体,利用液相芯片检测技术,实现对肿瘤标志物癌蛋白125(CA125)的含量的定量检测。具体步骤为:
步骤1、将实施例3中制备的4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银-金纳米粒子复合壳层微球通过静电吸附在表面偶联癌蛋白125的单克隆捕获抗体;
步骤2、在1.5毫升离心管中分别加入上述包被有癌蛋白125的单克隆捕获抗体的4-巯基苯甲腈和4-(2-(三甲基硅基)乙炔基)苯硫醇编码二次生长银-金纳米粒子复合壳层微球,然后加入病人血清,室温振荡孵育1h,通过磁分离清洗,除去血清中未捕获的癌蛋白125;
步骤3、然后向离心管中分别加入生物素标记的单克隆检测抗体和链霉亲和素标记的金纳米花2-萘硫酚报告分子载体,室温振荡孵育30min,通过磁分离清洗,除去过量的生物素标记的检测抗体和金纳米花;
步骤4、用拉曼共聚焦显微镜读取数据,得到病人血清中癌蛋白125的含量。
如图7所示为归一化后的免疫检测标准曲线。
对比例1
本对比例是将实施例1中制备的处于2个不同编码位置的所述4-巯基苯甲腈编码银纳米粒子微球作为载体,利用液相芯片检测技术,实现对肿瘤标志物癌胚抗原(CEA)和甲胎蛋白(AFP)的含量的联合定量检测并与文献报道的基于量子点编码荧光微球的液相芯片对CEA和AFP的联合定量检测结果进行对比。对比结果如表1所示,具体步骤为:
步骤1、将实施例1中制备的处于2个不同编码位置的4-巯基苯甲腈编码银纳米粒子微球通过静电吸附在表面分别偶联癌胚抗原的单克隆捕获抗体和甲胎蛋白的单克隆捕获抗体。量子点荧光微球通过羧基分别共价偶联癌胚抗原的单克隆捕获抗体和甲胎蛋白的单克隆捕获抗体;
步骤2、在1.5毫升离心管中分别加入上述包被有癌胚抗原的单克隆捕获抗体和包被有甲胎蛋白的单克隆捕获抗体的4-巯基苯甲腈编码银纳米粒子微球,对比管中加入上述包被有癌胚抗原的单克隆捕获抗体和包被有甲胎蛋白的单克隆捕获抗体的量子点荧光微球,然后分别加入病人血清,室温振荡孵育1h,通过磁分离清洗,除去血清中未捕获的癌胚抗原和甲胎蛋白;
步骤3、然后向离心管中分别加入生物素标记的癌胚抗原单克隆检测抗体、生物素标记的甲胎蛋白单克隆检测抗体和链霉亲和素标记的金纳米花2-萘硫酚报告分子载体,对比管中加入生物素标记的癌胚抗原单克隆检测抗体、生物素标记的甲胎蛋白单克隆检测抗体和链霉亲和素标记的PE荧光报告分子,室温振荡孵育30min,通过磁分离清洗,除去过量的生物素标记的检测抗体、金纳米花和PE报告分子;
步骤4、拉曼编码微球使用拉曼共聚焦显微镜读取数据,量子点荧光编码微球使用流式细胞仪读取数据,分别得到病人血清中癌胚抗原和甲胎蛋白的含量。
表1拉曼散射信号增强微码编球与荧光微球对CEA和AFP的联合定量检测结果
[1]Y.Leng,W.Wu,L.Li,K.Lin,K.Sun,X.Chen,and W.Li,Magnetic/FluorescentBarcodes Based on Cadmium-Free Near-Infrared-Emitting Quantum Dots forMultiplexed Detection,Adv.Funct.Mater.,2016,26,7581-7589.
如表1所示,所构建的基于表面增强拉曼散射的银纳米粒子表面编码微球-金纳米花拉曼报告分子的拉曼液相芯片平台的检测灵敏度比基于量子点荧光编码微球-荧光染料报告分子的荧光液相芯片平台提高了2个数量级,这表明所构建的拉曼液相芯片不仅能够解决荧光信号编码数量受限和编码位置不可控的问题,还能够实现更加灵敏的检测性能。
对比例2
如图8所示为文献报道的使用溶胀吸附法图8A(Tian Q,Yu X,Zhang L,etal.Monodisperse raspberry-l ike multihollow polymer/Ag nanocompositemicrospheres for rapid catalytic degradation of methylene blue[J].Journal ofColloid&Interface Science,2017,491:294-304.)、溶胀异相聚集法图8B(Lee J H,Mahmoud M A,Sitterle V B,et al.Highly Scattering,Surface-Enhanced RamanScattering-Active,Metal Nanoparticle-Coated Polymers Prepared via CombinedSwell ingHeteroaggregation[J].Chemi stry of Materials,2009,21(23):5654-5663.)、层层组装法图8C(Zhang S,Xu Z,Guo J,et al.Layer-by-Layer Assembly ofPolystyrene/Ag for a Highly Reproducible SERS Substrate and Its Use for theDetection of Food Contaminants[J].polymers,2021,13:3270.)和种子吸附生长法图8D(Chen K,Chou L,Song F,et al.Fabrication of metal nanoshell quantum-dotbarcodes for biomolecular detection[J].Nano Today,2013,8(3):228–234.)制备得到的贵金属微球SEM图片。
与图1、图2、图3对比可见,所提出的膜乳化-PDA原位还原生长法所制备的金属壳层微球表面包覆更完全而且具有更好的形貌均一性。
综上所述,本发明使用多巴胺层进行原位吸附和还原生长,不用再额外引入还原剂和吸附剂,可以快速在微球表面生长一层包覆完整的银纳米粒子,利用二次生长还能进一步调控表面金属组成和形貌,不仅制备步骤简单、快速高效,使用的试剂安全无毒,而且相比于其他制备方法,制备的金属壳层微球表面包覆更完整且具有更好的均一性,表面增强拉曼散射效果更加显著,有效解决了拉曼信号在液相芯片体系中难以实现强度编码的问题,扩展了液相芯片编码库的范围,有利于实现超灵敏的拉曼液相芯片体外检测。
以上对本发明的具体实施例进行了描述。需要理解的是,本发明并不局限于上述特定实施方式,本领域技术人员可以在权利要求的范围内做出各种变化或修改,这并不影响本发明的实质内容。在不冲突的情况下,本申请的实施例和实施例中的特征可以任意相互组合。
Claims (7)
1.一种基于表面增强拉曼散射信号的编码微球的应用,其特征在于,所述编码微球可用于一种或多种蛋白目标物的超灵敏检测;
所述编码微球包括具有表面增强拉曼散射效应壳层的磁性聚合物微球和拉曼编码分子;
所述具有表面增强拉曼散射效应壳层的磁性聚合物微球为贵金属银的表面增强壳层微球和可以进一步调控的二次生长银纳米粒子复合壳层微球和二次生长银-金纳米粒子复合壳层微球时,通过以下制备方法制备得到:
步骤一、利用SPG膜乳化-溶剂挥发法制备四氧化三铁纳米粒子掺杂的磁性聚合物微球;
步骤二、将制备的所述磁性聚合物微球置于碱性环境中进行表面的多巴胺自氧化聚合形成聚多巴胺层包覆,得到磁性聚多巴胺壳层微球;
步骤三、在制备的所述磁性聚多巴胺壳层微球表面原位还原银氨离子生长银纳米粒子壳层,得到银纳米粒子壳层微球;
步骤四、将制备的所述银纳米粒子壳层微球表面通过种子诱导二次还原硝酸银和氯金酸,分别得到二次生长银纳米粒子复合壳层微球和二次生长银-金纳米粒子复合壳层微球;
步骤五、在制备的银纳米粒子壳层微球、二次生长银纳米粒子复合壳层微球和二次生长银-金纳米粒子复合壳层微球表面静电吸附拉曼编码分子,得到表面增强拉曼散射信号的编码微球。
2.根据权利要求1所述的应用,其特征在于,所述拉曼编码分子包括:4,4’-联吡啶、罗丹明6G、异硫氰酸孔雀石绿、尼罗河蓝A、结晶紫、亚甲基蓝、4-巯基苯甲酸、2,3,5,6-四氟-4-巯基苯甲酸、5,5’-二硫双歧-(2-硝基苯甲酸)、 2-萘硫醇、苯硫醇、4-羟基苯硫醇、4-硝基苯硫醇、4-溴苯硫醇、4-氯苯硫醇、2-氯苯硫醇、4-异丙基苯硫醇、3,4-二氯苯硫醇、4-巯基吡啶、4-巯基甲苯、4-巯基苯甲腈、4-氟噻吩、4-氨基苯硫酚、4-甲氧基硫酚、4-巯基苯乙炔、4,4’-(1,2-乙炔基)双[苯硫醇]、4-(2-(三甲基硅基)乙炔基)苯硫醇、聚炔衍生物中的一种或多种。
3.根据权利要求1所述的应用,其特征在于,所述步骤一中四氧化三铁的掺杂量为1%~10%。
4.根据权利要求1所述的应用,其特征在于,所述步骤二中多巴胺溶液浓度为0.1~4mg/mL,多巴胺溶液体积为5~20 mL。
5.根据权利要求1所述的应用,其特征在于,所述步骤三中银氨离子浓度为5~25 mg/mL,银氨溶液体积为5~20 mL。
6.根据权利要求1所述的应用,其特征在于,所述步骤四中硝酸银浓度为8.5 × 10-3mg/mL~8.5 × 10-2 mg/mL、硝酸银水溶液体积为0.1~2 mL;氯金酸浓度为1.70 × 10-2mg/mL~0.17 mg/mL、氯金酸水溶液体积为0.1~2 mL。
7.根据权利要求1所述的应用,其特征在于,所述步骤五中编码分子的用量为每个微球0~1×10-6mol。
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