CN114958748A - 高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料 - Google Patents
高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料 Download PDFInfo
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Abstract
本申请属于生物材料技术领域,具体涉及一种高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料。所述纳米磁性亲和材料包括硼酸修饰的磁性氮化碳纳米片M‑BCN、亲和素‑生物素化适配体Apt‑Avi,所述亲和素‑生物素化适配体Apt‑Avi由生物素化适配体和亲和素进行预组装制备;所述亲和素‑生物素化适配体通过亲和素的糖基与M‑BCN中硼酸配体的定向连接结合,负载到M‑BCN上。通过将适配体有序排列于材料表面,促进适配体与CTC间的相互作用,从而提高CTC的捕获效率。同时该材料在捕获CTC细胞后,可以通过温和且可逆的释放方式对CTC进行无损释放,提高释放CTC的活性,使其能够进行后续的分析。
Description
技术领域
本申请属于生物材料技术领域,具体涉及一种高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料。
背景技术
循环肿瘤细胞(Circulating tumor cells,CTCs)是从原发肿瘤脱落进入血液循环***的肿瘤细胞,是导致肿瘤转移和复发的重要因素。循环肿瘤细胞不仅携带了肿瘤细胞的全部基因组,并且在癌症病变早期就己存在于外周血循环中,因此,循环肿瘤细胞被作为癌症研究的重要对象和癌症早期诊断的重要标志物。利用高效、准确的分离富集方法从复杂的血液成分中获得高纯度、无损、活性的CTC细胞是进行下游分析的前提。然而,CTC在全血中的数量极少,使它们的分离具有挑战性。因此制备出符合细胞高效捕获和无损释放的亲和材料是目前研究的一个热点。
适配体(Aptamer)是一类具有二级结构的核酸,被称为抗体的化学替代品,由于其可编程设计、成本低和易于修改等优点,常被用于制造细胞捕获亲和材料。除了捕获,从捕获材料上释放CTC仍然是下游分析的一项艰巨挑战。捕获的CTC通常粘附在基质上或被困在微结构中,这阻碍了提取其可靠的生化信息。因此,温和并可控地释放CTC是对其进行深入分析的先决条件。近年来,基于相应的CTC捕获原理,广大科研工作者们致力于开发不同的细胞释放方法。目前基于适配体捕获CTC的释放原理主要通过简单地改变其构象,使其失去亲和力和特异性,从而达到CTC释放的目的,包括热变性(Lab Chip 2012,12,3504)、核酸酶消化(ACS Appl.Mater.Interfaces 2015,7,24001)和互补序列杂交(Nano Res.2018,11,2592)等方法。但这些方法或多或少都会对细胞产生一定损伤。另外,捕获界面上适配体由于修饰方法不当也导致适配体有效性低,捕获效果差。
适配体的细胞捕获界面上,由于适体与细胞捕获界面之间的强相互作用,直接结合往往会导致适配体失活;没有有序排列的适配体的界面也存在链间纠缠和局部过度拥挤的问题,这极大地降低了适配体的效率,从而无法对CTC进行高效捕获。
对于常见的CTC释放方法,当采用热变形的方法释放CTC时,适体的热变性随着温度的升高而发生,脆弱的肿瘤细胞在升高的温度下会受到一定程度的损伤,一些具有高熔点的适体需要相对较高的温度才能进行热变性,因而无法释放出有活力的CTC。采用核酸酶的释放,核酸酶在一定程度上会损害细胞,明显降低细胞活性,且核酸酶降解对化学稳定的适配体无效。基于互补序列杂交的策略提供了一种释放CTC用于下游分析的非破坏性方法。但是这种方法需要复杂的互补序列设计,需要仔细选择合适的反应条件,以及相对长的处理时间。因此,急需开放一种新型的CTC捕获亲和材料,以实现CTC的可控、温和、无损释放。
发明内容
针对现有技术中存在的问题,本发明提出一种纳米磁性亲和材料,通过将适配体有序排列于材料表面,促进适配体与CTC间的相互作用,从而提高CTC的捕获效率。同时该材料在捕获CTC细胞后,可以通过温和且可逆的释放方式对CTC进行无损释放,提高释放CTC的活性,使其能够进行后续的分析。
本发明的具体方案如下:
一种高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料,包括硼酸修饰的磁性氮化碳纳米片(M-BCN)、亲和素-生物素化适配体(Apt-Avi),所述亲和素-生物素化适配体Apt-Avi由生物素化适配体和亲和素进行预组装制备而成。
亲和素-生物素化适配体通过亲和素的糖基与M-BCN中硼酸配体的定向连接结合,负载到M-BCN上。
本发明的纳米磁性亲和材料基于硼酸与糖基之间的作用,将亲和素-生物素化适配体定向连接到硼酸修饰的磁性氮化碳纳米片上,生物素化适配体可以实现CTCs的特异性捕获,通过硼酸的定向偶联作用,有效提高了适配体在材料表面的有序排列,提高了材料对CTC的捕获能力和捕获效率。由于B-N作用,硼酸配体与亲和素中的糖基的顺式二羟基在生理条件下即可形成环状硼酸酯,而在弱酸性介质中,环状硼酸酯发生解离,在果糖存在的条件下,由于竞争作用可进一步加速这种解离作用,基于此实现了弱酸性条件下联合果糖竞争作用对CTCs的无损释放。
优选地,所述生物素化适配体(Bio-Apt)与亲和素(Avidin)进行的预组装,以摩尔比例4:1进行。
所述生物素化适配体(Bio-Apt)为能够靶向CTC细胞或CTC生物标记物的适配体,例如TLS11a(序列:5-bio-TTT TTA CAG CAT CCC CAT GTG AAC AAT CGC ATT GTG ATT GTTACG GTT TCC GCC TCA TGG ACG TGC TG-3),或者适配体SYL3C、MUC1等等。
所述的硼酸修饰的磁性氮化碳纳米片M-BCN以磁性g-C3N4纳米片为基体材料,表面修饰有硼酸配体。用于修饰的硼酸为含有羧基和硼酸基的化合物,优选为羧基苯硼酸,最好为4-羧基苯硼酸,其分子结构中的羧基与石墨相氮化碳(g-C3N4)表面的氨基缩合成酰胺键,硼酸基作为与亲和素中的糖基结合的配体。
上述材料的一种制备方法包括以下步骤:
S1:制备硼酸修饰g-C3N4:在干燥条件下,将羧基苯硼酸溶解在溶剂中,加入酰胺缩合剂,之后加入g-C3N4粉末进行反应;反应结束后进行离心分离,取出离心分离得到的沉淀,洗去溶剂和酰胺缩合剂,冻干后获得硼酸修饰g-C3N4。
该步骤的目的是为了将羧基苯硼酸中的羧基与石墨相氮化碳(g-C3N4)表面的氨基反应缩合成酰胺键,实现g-C3N4的硼酸修饰,羧基苯硼酸与g-C3N4粉末的质量比例优选为(3-4):1。具体的反应条件可根据酰胺缩合剂的种类进行确定。
优选地,溶剂采用N,N-二甲基甲酰胺(DMF),酰胺缩合剂为促进酰胺化反应的缩合剂,优选采用N,N-二异丙基乙胺(DIEA)和2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU),为确保反应充分进行,羧基苯硼酸:DIEA:HATU的摩尔比例可以为1:(1-3):(1-1.5),反应温度约为40℃左右,反应时间15h以上。
酰胺缩合剂还可以采用HBTU(苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯)/DIEA的组合,或者EDC/NHS(碳二亚胺类缩合剂/羟基琥珀酰亚胺)等等,只要能实现促进氨基和羧基的酰胺化反应即可,促进酰胺化的具体反应条件可参考现有技术中的记载。
如果采用上述的DMF溶剂以及DIEA/HATU缩合剂,则一种可用的洗去沉淀表面溶剂和酰胺缩合剂的方法为:先用DMF清洗以去除HATU;再用乙醇清洗以去除DMF;然后用水洗以去除乙醇;再用稀盐酸清洗以去除DIEA;最后用水洗以去除稀盐酸和盐。
S2:将硼酸修饰g-C3N4剥离成硼酸修饰的氮化碳纳米片(BCN)。
该步骤可以采用现有技术中已有的将g-C3N4剥离成纳米片的方法,优选采用超声辅助液体剥离法,具体方法为将硼酸修饰g-C3N4粉末置于水中,以600-800W的超声功率,超声处理16-20h。超声处理后的悬浮液可通过以下方式收集剥离出的BCN:
先对超声处理后的悬浮液进行一次较低转速的离心(4000-6000rpm)分离,收集上清液,此时的上清液即为剥离出的BCN分散液,再将BCN分散液进行一次较高转速的离心(15000rpm以上)分离,收集沉淀,即剥离出的BCN,冻干备用。
S3:将硼酸修饰的氮化碳纳米片BCN制备成硼酸修饰的磁性氮化碳纳米片(M-BCN)。
为便于捕获了CTCs后的纳米材料的分离,将BCN制备成磁性材料,该过程可采用现有技术中制备磁性氮化碳的方法,比如使三价铁盐和二价铁盐在碱性条件下,通过水热反应形成Fe3O4颗粒负载在氮化碳表面上。
下面提供一种将BCN制备成磁性材料M-BCN的具体参考方法:将步骤S2获得的BCN置于水中进行超声分散,待BCN分散均匀后,加入FeCl3·6H2O和FeCl2·4H2O,继续进行超声分散,将混合液转移至反应釜中,调节pH至三价铁和二价铁离子能够发生共沉淀(pH9-10),在温度80-120℃反应1-1.5h,冷却至室温,将分离出的固体洗涤后干燥备用,即为制备成的M-BCN;所述BCN、FeCl3·6H2O和FeCl2·4H2O的质量比例为2:(2-2.5):(0.8-1)。
S4:适配体修饰:将生物素化适配体溶液和亲和素溶液经孵育(0.5-2h)后,相互混合进行预组装,如上文所述,优选以4:1的摩尔比例进行预组装。
之后加入M-BCN,室温条件下反应(可以通过震荡等方式加速反应,反应进行15-30min),即可得到负载了亲和素-生物素化适配体Apt-Avi的M-BCN(Apt-Avi-BCN),也即要制备的纳米磁性亲和材料,经多次洗涤并重悬备用。
此步骤中Apt-Avi和M-BCN的比例能满足Apt-Avi的充分负载即可,具体来说,亲和素-生物素化适配体Apt-Avi与M-BCN的质量比例可采用(1-1.5):1。
所述纳米磁性亲和材料的洗涤可采用PBS-BSA溶液,优选采用1%BSA。BSA可以减少材料的非特异性吸附。
上述纳米磁性亲和材料的使用方法如下:
将含CTC的体液样本加入到所述纳米磁性亲和材料分散体中孵育,对体液中的CTC进行捕获;对进行了CTC捕获后的混合液体进行磁分离,去除磁分离后的上清液,获得含有捕获了CTC的纳米磁性亲和材料沉淀。
在需要释放CTC时,向沉淀中加入溶有果糖的弱酸性(pH6.6-6.8)PBS缓冲液进行孵育,使CTC从纳米磁性亲和材料表面释放,之后进行磁分离,从上清液中得到CTC。
与传统的通过化学共轭方法在捕获界面上随机固定捕获配体不同,本发明的纳米磁性亲和材料中,硼酸与亲和素糖蛋白之间的共价结合具有位点特异性和高度定向性,使适配体排列有序。由于适配体的有序排列、亲和素的局部多价效应和缓冲效应,极大地避免了适配体链间纠缠和局部过度拥挤的问题,使材料对CTC的亲和力大大提高。同时,由于硼酸盐偶联是可逆的,酸性条件下通过果糖的竞争性结合很容易实现温和的细胞释放。
上述纳米磁性亲和材料Apt-Avi-BCN的制备过程,以及其捕获和释放CTC的原理示意图见图1。
本发明的有益效果:
本发明的纳米磁性亲和材料利用硼酸作为生物正交探针,将亲和素有序排列在硼酸修饰的磁性氮化碳纳米片表面,用于生物素化适配体的定向有序组装。适配体的有序取向在很大程度上缓解了链间纠缠和局部过度拥挤的问题。同时,亲和素起到缓冲作用,避免了适配体与界面直接相互作用而导致的失活。亲和素/生物素1:4组装产生的局部多价效应也增强了工程界面的细胞捕获能力。与游离适配体相比,本发明的细胞捕获材料的CTC亲和力提高了约100倍。此外,可逆的硼酸偶联使得被捕获的CTC能在酸性果糖条件下进行温和释放,并且释放后具有较高的细胞活力。本发明的材料界面对全血样本CTC的捕获效率为91.85%,释放效率为92.41%,并且能够从肿瘤患者的外周血中直接分离CTC,具备在癌症早期诊断与治疗的临床潜力。
附图说明
图1:(A)Apt-Avi-BCN材料制备过程。(B)Apt-Avi-BCN界面捕获释放CTC工作原理示意图。(C)Apt-Avi-BCN界面3D结构图。(D)CTC与材料界面结合和释放情况示意图。
图2:(A)全血添加不同数量的HepG2后,Apt-Avi-BCN的捕获和释放效率。(B)Apt-Avi-BCN释放的HepG2活细胞(绿色,AO染色)和死细胞(红色,PI染色)的荧光图像,其中只有箭头标示的两处为死细胞,其余均为活细胞。(C)释放的HepG2细胞培养0-96h后的增殖图像。比例尺:50μm。
图3:(A)肝癌患者血样中分离的WBC和CTC的代表性免疫染色图像。比例尺:20μm。CTC 1:EpCAM阴性;CTC 2:EpCAM阳性;(B)健康志愿者和不同分期的肝癌患者血液样本中CTCs数目。
具体实施方式
下面结合具体实施案例,对本发明的纳米磁性亲和材料及其制备、应用方法进行介绍,但不作为对本发明方法的具体限定。
本实施例中的g-C3N4粉末采用以下方法合成:称取3g二氰二胺于50mL氧化铝坩埚中,盖上盖子并用铝箔纸包裹密封,放入马弗炉中,以3℃min-1的速度升温至600℃并保持2h。待反应物冷却至室温取出,所得淡黄色固体即为g-C3N4,用玛瑙研钵将g-C3N4研磨成均匀固体粉末,备用。
按照以下具体方法制备所述纳米磁性亲和材料:
S1:制备硼酸修饰g-C3N4:在干燥条件下,称取166mg 4-羧基苯硼酸(4-CPBA)至50mL锥形瓶中,沿瓶壁缓慢加入30mL DMF,在40℃下搅拌溶解后,加入0.4mL DIEA和456mgHATU(4-CPBA、DIEA、HATU的摩尔比例约为1:2.3:1.2)保持40℃搅拌反应10min,之后加入50mg制备研磨得到的g-C3N4粉末,在40℃条件下搅拌反应16h。
反应结束后将锥形瓶中样品在6000rpm转速下离心3min,弃去上清液,清洗沉淀:先用DMF清洗以去除HATU;再用乙醇清洗以去除DMF;然后用水洗以去除乙醇;在用稀盐酸洗以去除DIEA;最后用水洗以去除稀盐酸和盐;清洗结束后冻干所得即为硼酸修饰g-C3N4。
S2:用超声辅助液体剥离法将硼酸修饰g-C3N4剥离成硼酸修饰的氮化碳纳米片(BCN):取100mg硼酸修饰的g-C3N4于250mL锥形瓶中,倒入100mL二次水。连续超声16h,超声功率800W。随后将超声所得奶黄色悬浮液在5000rpm转速下离心15min,收集上清,即为BCN分散液。将得到的上清在15000rpm转速下离心10min,收集沉淀,即BCN。最后将沉淀冷冻干燥后称重备用。
S3:硼酸修饰的磁性氮化碳纳米片(M-BCN)的合成:称取0.1g BCN加到锥形瓶中,加入19mL无水乙醇与19ml水的混合液,800W超声5小时,得均匀分散的BCN。再加入0.12gFeCl3·6H2O和0.047g FeCl2·4H2O,超声分散30min。将混合液转移至100mL聚四氟乙烯反应釜内衬中,加入2mL氨水并混合均匀,调节pH至10左右,将反应釜放入烘箱内100℃反应1h。自然冷却至室温,将分离出的固体用无水乙醇和去离子水洗涤,真空干燥箱中60℃干燥12h,取出备用。
S4:适配体修饰:生物素化适配体TLS11a(Bio-Apt,序列:5-bio-TTT TTA CAG CATCCC CAT GTG AAC AAT CGC ATT GTG ATT GTT ACG GTT TCC GCC TCA TGG ACG TGC TG-3)用于特异性靶向。先将250μL的Bio-Apt(13.3μM)与250μL的亲和素溶液(200μg mL-1)在37℃下孵育1小时,以4:1的比例将Bio-Apt与亲和素进行预组装。加入500μL M-BCN(250μgmL-1),室温震荡30min,得到的负载了Bio-Apt的M-BCN(Apt-Avi-BCN)在500μL PBS(1%BSA以减少非特异性吸附)中反复洗涤重悬备用。
上述方法制备的Apt-Avi-BCN纳米磁性亲和材料可用于细胞的捕获与释放:在500μL的待测样本中加入500μL的Apt-Avi-BCN分散液,37℃孵育30min,最后磁分离去除上清液,获得含有捕获了细胞的纳米磁性亲和材料的沉淀。
细胞捕获后,向沉淀加入1ml含60mM果糖的PBS缓冲液(pH=6.8),在37℃温下轻摇30min。进行磁性分离,释放的细胞留在磁性分离后的上清液中,并在显微镜下计数。
采用以下方法测试所制备的Apt-Avi-BCN纳米磁性亲和材料在血液中的CTC细胞捕获性能:将用DAPI预染色的HepG2细胞加标到全血中。然后,将500μL血液加入500μLApt-Avi-BCN分散液中,混合物在37℃下孵育30分钟。然后将上清液中未捕获的细胞通过磁力分离并在荧光显微镜下计数。细胞捕获效率计算如下:
捕获效率=(加入细胞数-未捕获细胞数)/加入细胞数×100%
采用以下方法测试所制备的Apt-Avi-BCN纳米磁性亲和材料在在血液中的释放性能:细胞捕获后,加入1ml含有60mM果糖的PBS缓冲溶液(pH=6.8),在37℃下轻轻摇动孵育30分钟。将释放的细胞从捕获材料中进行磁性分离并计数。细胞释放效率计算如下:
释放效率=释放细胞数/捕获细胞数×100%
释放的CTC的细胞活力分析和体外培养:用活/死染色方法评估释放的HepG2细胞的细胞活力。吖啶橙(AO)可以穿透活细胞,发出绿色荧光,而死细胞被碘化丙啶(PI)染色并显示红色荧光。释放的细胞用AO和PI共同染色,在荧光显微镜下观察,通过计数活细胞和死细胞计算存活率。释放的HepG2细胞在37℃、5%CO2和DMEM(10%FBS和1%抗生素)下进一步培养。用倒置显微镜拍摄某些时间点的明场图像。
图2为上述捕获性能、释放性能、细胞活力分析和体外培养结果。从图2中可以看出,当HepG2细胞数在50~1000之间时,捕获效率>91.85%,释放效率>92.41%(图2A),DAPI/AO染色检测,其中98%存活(图2B)。收集细胞,置于96孔板中培养,24h稳定贴壁,培养96h细胞长满(图2C)。
通过分析18名肝癌患者和17名健康志愿者未经任何预处理的血液样本,进一步评估了上述Apt-Avi-BCN材料的临床应用:未经预处理,将血样(0.5mL)加入到0.5mL的Apt-Avi-BCN分散体中于37℃孵育30min后,磁分离去除上清液,轻轻洗涤Apt-Avi-BCN。将Apt-Avi-BCN上捕获的细胞与含60mM果糖的PBS溶液(pH=6.8)在37℃下轻轻振荡孵育30min后释放,磁分离从上清中得到细胞。
将分离的细胞按进行免疫荧光染色来鉴定CTC:用4%的聚甲醛溶液固定细胞,用3%的山羊血清溶液封闭细胞,然后用DAPI、anti-CK和anti-CD45进行免疫荧光染色。最后,在正置荧光显微镜下观察细胞并拍照。CD45阴性、DAPI和CK阳性(DAPI+/CK+/CD45-)的细胞被识别为CTCs,CD45和DAPI阳性、CK阴性(DAPI+/CK-/CD45+)的细胞被识别为WBC(图3A)。在所有肝癌患者中均发现并分离到CTC,每1ml血液中有5~43个CTC,而在健康供者血液中未发现CTC。同时,捕获的CTC数目不仅在健康供体与患者之间有显著差异,而且在三个阶段的患者之间也有区别(图3B)。
Claims (10)
1.一种高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料,其特征在于,包括硼酸修饰的磁性氮化碳纳米片M-BCN、亲和素-生物素化适配体Apt-Avi,所述亲和素-生物素化适配体Apt-Avi由生物素化适配体和亲和素进行预组装制备;
所述亲和素-生物素化适配体通过亲和素的糖基与M-BCN中硼酸配体的定向连接结合,负载到M-BCN上。
2.根据权利要求1所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料,其特征在于,所述亲和素-生物素化适配体中,生物素化适配体和亲和素以4:1的摩尔比例进行预组装。
3.根据权利要求1所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料,其特征在于,所述硼酸修饰的磁性氮化碳纳米片M-BCN中,基体材料为磁性g-C3N4纳米片,修饰的硼酸为羧基苯硼酸。
4.权利要求1至3中任一项所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的制备方法,其特征在于,包括以下步骤:
S1:制备硼酸修饰g-C3N4:在干燥条件下,将羧基苯硼酸溶解在溶剂中,加入酰胺缩合剂,之后加入g-C3N4粉末进行反应;
反应结束后进行离心分离,取出离心分离得到的沉淀,洗去溶剂和酰胺缩合剂,冻干后获得硼酸修饰g-C3N4;
S2:将硼酸修饰g-C3N4剥离成硼酸修饰的氮化碳纳米片BCN;
S3:将硼酸修饰的氮化碳纳米片BCN制备成硼酸修饰的磁性氮化碳纳米片M-BCN;
S4:适配体修饰:将生物素化适配体溶液和亲和素溶液经孵育后进行预组装,之后加入M-BCN,室温条件下反应,得到负载了亲和素-生物素化适配体Apt-Avi的M-BCN,即要制备的纳米磁性亲和材料,经洗涤后重悬备用。
5.根据权利要求4所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的制备方法,其特征在于,所述步骤S1中,溶剂为N,N-二甲基甲酰胺DMF,酰胺缩合剂为N,N-二异丙基乙胺DIEA和2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯HATU;
羧基苯硼酸:DIEA:HATU的摩尔比例为1:(1-3):(1-1.5);羧基苯硼酸与g-C3N4粉末的质量比例为(3-4):1。
6.根据权利要求5所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的制备方法,其特征在于,所述步骤S1中,洗去溶剂和酰胺缩合剂的方法为:先用DMF清洗以去除HATU;再用乙醇清洗以去除DMF;然后用水洗以去除乙醇;再用稀盐酸洗以去除DIEA;最后用水洗以去除稀盐酸和盐。
7.根据权利要求4所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的制备方法,其特征在于,所述步骤S2中,剥离方法采用超声辅助液体剥离,液体采用水,超声功率为600-800W,超声时间16-20h。
8.根据权利要求4所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的制备方法,其特征在于,所述步骤S3的方法为:将步骤S2获得的BCN置于水中进行超声分散,待BCN分散均匀后,加入FeCl3·6H2O和FeCl2·4H2O,继续进行超声分散,将混合液转移至反应釜中,调节pH,在温度80-120℃反应1-1.5h,冷却至室温,将分离出的固体洗涤后干燥备用;
所述BCN、FeCl3·6H2O和FeCl2·4H2O的质量比例为2:(2-2.5):(0.8-1)。
9.根据权利要求4所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的制备方法,其特征在于,所述步骤S4中,亲和素-生物素化适配体Apt-Avi与M-BCN的质量比例为(1-1.5):1,所述洗涤采用PBS-BSA溶液。
10.权利要求1至3中任一项所述的高效捕获和无损释放循环肿瘤细胞的纳米磁性亲和材料的应用方法,其特征在于,将含CTC的体液样本加入到所述纳米磁性亲和材料分散体中孵育,对CTC进行捕获;
对进行了CTC捕获后的混合液体进行磁分离,去除磁分离后的上清液,加入溶有果糖的弱酸性PBS缓冲液进行孵育,使CTC从纳米磁性亲和材料表面释放,之后进行磁分离,从上清液中得到CTC。
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