CN107674675A - 检测用叶酸受体靶向碳量子点turn‑on探针的制备方法及其产品和应用 - Google Patents
检测用叶酸受体靶向碳量子点turn‑on探针的制备方法及其产品和应用 Download PDFInfo
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Abstract
本发明公开了一种检测用叶酸受体靶向碳量子点turn‑on探针的制备方法,其特征在于,通过柠檬酸与乙二胺复合制备得到的碳量子点(Cdots),加入硅酸乙酯(TEOS)和碳量子点后制备出特异荧光发光的介孔硅包裹碳量子点(MSN@Cdots)化合物。此化合物特异性结合富有叶酸受体表达的细胞,原叶酸堵塞的荧光因叶酸特异性的释放而打开碳量子荧光发光开关,标示肺部肿瘤特异性标志物中的指示物叶酸受体是否存在,本发明是材料学技术、荧光技术及热疗技术的交叉应用。可用生物分子检测及细胞成像等领域。
Description
技术领域
本发明是生物材料的制备和应用,具体是一种检测用叶酸受体靶向碳量子点turn-on探针的制备方法及其产品和应用。由叶酸和3-氨丙基三乙氧基硅烷(APTES)合成特异性模板,加入硅酸乙酯(TEOS)和碳量子点后制备出特异荧光发光的介孔硅包裹碳量子点(MSN@Cdot)化合物。此化合物特异性结合富有叶酸受体表达的细胞,原叶酸堵塞的荧光因叶酸特异性的释放而打开碳量子荧光发光开关,标示肺部肿瘤特异性标志物的存在,本发明是材料学技术、荧光技术及热疗技术的交叉应用。
背景技术
在中国,环境污染等众多因素诱导下的肺癌发病率和死亡率呈持续走高态势,严重威胁人类健康。早期诊断的肺癌患者其5年生存率可达80%,而局部晚期的肺癌患者缺乏有效的预警监测,失去了手术机会[1]。因此,随着免疫学和分子生物学的发展,肿瘤标志物在癌症的普查、诊断、判断预后和转化,评价疗效和随诊等方面占有重要地位,因此,实现肿瘤细胞标志物的特异性识别在肺癌早期诊断、治疗及研究方面具有举足轻重的临床意义。肺癌标志物通常存在于患者的组织、血液和体液中,进行标志物检测具有高效性、损伤小、标本易获得的特点[2]。常见的肺癌相关标志物有:癌胚抗原(carcinoembryonicantigen)、神经元特异性稀醇化酶(neuron specific enolase)、细胞角蛋白19片段抗原(CYFRA21-1)、胃泌素释放肽前体(ProGRP)、糖链抗原类(Carbohydrate)、跨膜糖蛋白(CD44)及叶酸受体(folate receptor)等[3-7]。
近年来,纳米荧光探针被应用于生化过程研究、疾病标志物诊断和有害化合物检测。单一信号检测和单一的靶向识别易产生假阳性结果。为解决为题,荧光turn-on探针相关研究受到了极大关注,将功能化量子点探针与荧光成像技术结合,利用特异性识别能力的小分子和核酸适配体的识别功能和量子点特异的光学性质作为传感信号,将量子点本身优越的荧光特性和功能化试剂的特异性识别结合构建具有光稳定性好、特异性强的荧光探针,应用以实现肺癌标志物的灵敏性识别和细胞荧光成像研究[8]。碳量子点作为一种新兴材料,受到了广泛的关注。由于其具有尺寸相关荧光性质及热疗作用,具有很大的潜力应用于纳米荧光材料领域。碳量子点的传统合成方法可分为自上而下合成法(包括激光销蚀法、电化学法、弧光放电法)和自下而上合成法(包括化学氧化法、模板法、微波法和热分解法等)[9-10]。本发明以叶酸为功能化试剂,介孔硅材料(MSNs)包覆碳量子点(Cdots)为传感信号的荧光“turn-on”探针,用于肺癌细胞与正常细胞的识别与区分。MSNs@Cdots 探针提供的背景信号干扰少,对于叶酸受体(FR)的荧光“turn-on”响应是基于肺癌细胞内外酸性环境的改变和细胞内FR与FA的高亲和力作用,导致MSNs@Cdots从探针中释放而使荧光信号得到恢复。
参考文献:
[1] 杨玲,李连第。中国肺癌死亡趋势分析及发病、死亡的估计与预测。中国肺癌杂志。2005第8卷。
[2] Mohammad Hasanzadeh, Nasrin Shadjou. Nanomaterials for use inimmunosensing of carcinoembryonic antigen: Recent advance. 2017, 86, 185-205
[3] Xing Wang, Yanying Wang, Xiaoxue Ye. Sensing platform for neuronspecific enolase basedon molecularly imprinted polymerized ionic liquids inbetween gold nanoarrays. Biosensors and Bioelectronics, 2018, 99, 34-39.
[4] Niels Reinmuh, Burkhard Brandt. Prognostic impact of Cyfra21-1 andother serum markers in completely resectednon-small cell lung cancer. LungCancer, 2002, 3, 265-270.
[5]Thomas Muley, Xiaotong Zhang. MA11.09 Progastrin-Releasing Peptide(ProGRP ) to Rule out Progressive Disease in Patients with Small Cell LungCarcinoma. Journal of Thoracic Oncology, 2017, 12, 407-408.
[6]Evgenia Karousou, Suniti Misra. Roles and targeting of the HAS/hyaluronan/CD44 molecular system in cancer. Matrix Biology, 2017, 59, 3-22.
[7] Maria Ines Nunez, Carmen Behrens. High Expression of Folate ReceptorAlpha in Lung Cancer Correlates with Adenocarcinoma Histology and Mutation.Journal of Thoracic Oncology, 2012, 7, 833-840.
[8] Wang X,Qu K,Xu B. Microwave assisted one-step green synthesis ofcell-permeable multicolor photoluminescent carbon dots without surfacepassivation reagents.Journal of Material Chemistry,2011,21, 2445-2450.
[9] Bourlinos A B,Stassinopoulos A.Surface functionalized carbogenicquantum dots.Small,2008,4, 455-458.
[10] Peng H,Travas-Sejdic.Simple aqueous solution route to luminescentcarbogenic dots from carbohydrates.Chemical Materials,2009,21, 5563-5565.。
发明内容
为克服现有技术的不足,本发明目的在于:提供一种检测用叶酸受体靶向碳量子点turn-on探针的制备方法。
本发明的再一目的在于:提供上述方法制得的产品。
本发明的又一目的在于:提供上述叶酸受体靶向碳量子点turn-on探针的应用。
一种检测用叶酸受体靶向碳量子点turn-on探针的制备方法,通过柠檬酸与乙二胺复合制备得到的碳量子点(Cdots),包括如下步骤:
A、水热制备碳量子点,分离纯化后得到碳量子点溶液I;
B、叶酸和3-氨丙基三乙氧基硅烷(APTES)合成特异性模板,加入硅酸乙酯(TEOS)和碳量子点溶液制备特异荧光发光的介孔硅包裹碳量子点MSN@Cdots化合物,洗去模板分子后得到介孔硅包覆的碳量子点溶液;
C、制备叶酸溶液功能化MSN@Cdots化合物,即使用叶酸溶液充盈介孔孔道,计算吸附量后得到所述叶酸受体靶向碳量子点turn-on探针。
本发明公开了一种由叶酸和3-氨丙基三乙氧基硅烷(APTES)合成特异性模板,加入硅酸乙酯(TEOS)和碳量子点后制备出特异荧光发光的介孔硅包裹碳量子点(MSN@Cdots)化合物。此化合物特异性结合富有叶酸受体表达的细胞,原叶酸堵塞的荧光因叶酸特异性的释放而打开碳量子荧光发光开关。
所述量子点为粒径为5~15nm的碳量子点。
步骤B具体为:加入叶酸(模板分子)和APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点加入到上述溶液中随后加入TEOS(交联剂)和氨水,反应产物经反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在,将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
步骤C具体为:MSN@Cdots分散在一定体积的叶酸溶液(PBS,PH值7.0),得到叶酸饱和的MSN@Cdots荧光探针。
向所得固体产物MSN@Cdots中加入待检测的细胞液,根据荧光信号是否发光进行细胞膜表面是否具有叶酸受体蛋白进行判断。
本发明提供一种检测用叶酸受体靶向碳量子点turn-on探针,根据上述任一所述方法制备得到。
本发明还提供一种叶酸受体靶向碳量子点turn-on探针在检测叶酸受体靶向碳量子点的应用。碳量子点的生物相容性良好,在体内使用的安全性好,可以应用于细胞及组织荧光成像。用于标示肺部肿瘤特异性标志物中的指示物叶酸受体是否存在,本发明是材料学技术、荧光技术及热疗技术的交叉应用。可用于肿瘤治疗、生物分子检测及细胞成像等领域。
步骤A所述量子点为粒径为5~15nm的碳量子点。使用的量子点尺寸过小,可能导致量子点的过分弥散,从而影响探针的诊断效率;使用量子点尺寸过大时,则导致所制备量子点荧光效率及热疗效果降低,而造成效果下降等。所述水热制备碳量子点为水溶性的碳量子点,具体为以不同前体在180℃水热反应12h后分离纯化得到。
步骤B具体为:在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 10ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100 反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在。将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
步骤C具体为:50mg的MSN@Cdots分散在一定体积的叶酸溶液(PBS,PH值7.0),在摇床上混合,得到叶酸饱和的MSN@Cdots荧光探针。通过紫外可见分光法在285nm波长测定上层清液的剩余叶酸量。MSN@Cdots对于叶酸的吸附量(Q, m•g-1)。
空隙中的叶酸遮蔽了碳量子点的荧光发光;加入人体组织以后,叶酸靶向A549细胞的叶酸受体,从MSN孔道中漏出,荧光信号得到回复,由光学turn-off转变为turn-on状态。
本发明解决的技术问题是MSN@Cdots的合成制备及特异性荧光响应的问题,提供一种具有光学稳定性好、生物相容性高、具有良好热疗效果和荧光造影能力的量子点的制备方法, 同时工艺简单、材料转化率高、易重复的优点。
本发明优越性在于:该方法原料简单易得,水热法及MSN包覆法操作简单,探针响应速度灵敏,易保存且易于商业化。碳点的生物相容性良好,在体内使用的安全性好,可以应用于细胞及组织荧光成像。此化合物特异性结合富有叶酸受体表达的细胞,原叶酸堵塞的荧光因叶酸特异性的释放而打开碳量子荧光发光开关,标示肺部肿瘤特异性标志物中的指示物叶酸受体是否存在,本发明是材料学技术、荧光技术及热疗技术的交叉应用。可用生物分子检测及细胞成像等领域。
附图说明
图1为实施例的制备方法流程,其中图1a、b为分别为叶酸吸附前、后荧光打开、关闭示意图;
图2为实施例制备所得介孔硅包裹碳量子点(MSN@Cdots)的TEM照片;
图3为实施例所得介孔硅包裹碳量子点(MSN@Cdots)荧光光谱;
图4为叶酸饱和前后的MSN@Cdots荧光光谱;
图5为MSN@Cdots对细胞的荧光显影照片,其中,5a为对HEK293人胚肾细胞及5b为对Hela细胞荧光显影照片。
具体实施方式
以下通过具体的实施例对本发明的技术方案作进一步描述。以下的实施例是对本发明的进一步说明,而不限制本发明的范围。
实施例1
制备介孔硅包覆的碳量子点(MSN@Cdots)化合物:
将甘氨酸0.1g溶于10ml乙醇中避光于常温下搅拌,直至固体全部溶解,得到均匀的溶液,置于20ml四氟乙烯反应釜中置于180摄氏度烘箱中放置12个小时。冷却至室温后,将所得到的溶液缓慢滴加入硅胶柱中静置分离,通过紫外灯确定所得到的碳点位置,收集所得到的碳点溶液,将得到的碳点溶液再次旋蒸,去除其中的有机溶剂后,加入去离子水,即得到甘氨酸缩合的碳量子点溶液,将溶液冷冻干燥后得到碳量子点固体,重配置碳量子去离子水浓度为0.0004g/mL测得荧光产率为57.8%;
在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 5ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100 反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在,制备的介孔硅包覆的碳量子点(MSN@Cdots);
将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。附图2为实施例制备所得MSN@Cdots的TEM照片,制备的探针弥散分布,尺寸大小在40-80nm范围。
实施例2
将甘氨酸0.1g溶于10ml乙醇中避光于常温下搅拌,直至固体全部溶解,得到均匀的溶液,置于20ml四氟乙烯反应釜中置于180摄氏度烘箱中放置12个小时。冷却至室温后,将所得到的溶液缓慢滴加入硅胶柱中静置分离,通过紫外灯确定所得到的碳点位置,收集所得到的碳点溶液,将得到的碳点溶液再次旋蒸,去除其中的有机溶剂后,加入去离子水,即得到甘氨酸缩合的碳量子点溶液,将溶液冷冻干燥后得到碳量子点固体,重配置碳量子去离子水浓度为0.0004g/mL测得荧光产率为57.8%;在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 10ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在。将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。附图2为实施例制备所得MSN@Cdots的TEM照片,制备的探针弥散分布,尺寸大小在40-80nm范围。
实施例3
将甘氨酸0.1g溶于10ml乙醇中避光于常温下搅拌,直至固体全部溶解,得到均匀的溶液,置于20ml四氟乙烯反应釜中置于180摄氏度烘箱中放置12个小时。冷却至室温后,将所得到的溶液缓慢滴加入硅胶柱中静置分离,通过紫外灯确定所得到的碳点位置,收集所得到的碳点溶液,将得到的碳点溶液再次旋蒸,去除其中的有机溶剂后,加入去离子水,即得到甘氨酸缩合的碳量子点溶液,将溶液冷冻干燥后得到碳量子点固体,重配置碳量子去离子水浓度为0.0004g/mL测得荧光产率为57.8%;在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 15ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在。将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
实施例4
将甘氨酸0.1g溶于10ml乙醇中避光于常温下搅拌,直至固体全部溶解,得到均匀的溶液,置于20ml四氟乙烯反应釜中置于180摄氏度烘箱中放置12个小时。冷却至室温后,将所得到的溶液缓慢滴加入硅胶柱中静置分离,通过紫外灯确定所得到的碳点位置,收集所得到的碳点溶液,将得到的碳点溶液再次旋蒸,去除其中的有机溶剂后,加入去离子水,即得到甘氨酸缩合的碳量子点溶液,将溶液冷冻干燥后得到碳量子点固体,重配置碳量子去离子水浓度为0.0004g/mL测得荧光产率为57.8%;在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 15ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在。将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
实施例5
将甘氨酸0.1g溶于10ml乙醇中避光于常温下搅拌,直至固体全部溶解,得到均匀的溶液,置于20ml四氟乙烯反应釜中置于180摄氏度烘箱中放置12个小时。冷却至室温后,将所得到的溶液缓慢滴加入硅胶柱中静置分离,通过紫外灯确定所得到的碳点位置,收集所得到的碳点溶液,将得到的碳点溶液再次旋蒸,去除其中的有机溶剂后,加入去离子水,即得到甘氨酸缩合的碳量子点溶液,将溶液冷冻干燥后得到碳量子点固体,重配置碳量子去离子水浓度为0.0004g/mL测得荧光产率为57.8%;在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 15ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在。将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
实施例6
将甘氨酸0.1g溶于10ml乙醇中避光于常温下搅拌,直至固体全部溶解,得到均匀的溶液,置于20ml四氟乙烯反应釜中置于180摄氏度烘箱中放置12个小时。冷却至室温后,将所得到的溶液缓慢滴加入硅胶柱中静置分离,通过紫外灯确定所得到的碳点位置,收集所得到的碳点溶液,将得到的碳点溶液再次旋蒸,去除其中的有机溶剂后,加入去离子水,即得到甘氨酸缩合的碳量子点溶液,将溶液冷冻干燥后得到碳量子点固体,重配置碳量子去离子水浓度为0.0004g/mL测得荧光产率为57.8%;在50ml锥型瓶中,加入10mg叶酸(模板分子)和60uL APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点 15ml加入到上述溶液中,搅拌30min。随后加入80uLTEOS(交联剂)和5uL氨水(28%,W/v),搅拌8 hours。反应产物经10000rpm离心5min, 0.5% Triton x-100反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在。将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
介孔硅包覆的碳量子点(MSN@Cdots)的荧光光谱,如附图3所示:分别以360nm,380nm, 400nm, 420nm和440nm波长激发介孔硅碳量子点,发现以380nm激发时,得到最强的发射波长为475nm。并以此作为为开关检测的标准强度
介孔硅包覆的碳量子点纳米粒子(MSN@Cdots)的荧光开关效果:将实施例1至6中制得的介孔硅包覆的碳量子点置于分光皿中,用0.01M的叶酸去离子水溶液进行饱和,随后以10000rpm离心收集叶酸饱和的介孔硅包覆的碳量子点(FA-MSN@Cdots)组分;将组分以0.0004g/ml分散于去离子水中,对比前后在380nm激发下的荧光光谱,可以发现叶酸吸附进入介孔硅孔道后,强度极速下降,几乎可以忽略不计。如图1 a、b的叶酸吸附前、后荧光打开关闭示意图所示。图4为叶酸饱和前后的MSN@Cdots荧光光谱。
(MSN@Cdots)对于叶酸标志物靶向细胞(Hela细胞)和非靶向普通细胞(HEK293细胞)的显影观察对比:
将HEK293人胚肾细胞和Hela脑胶质瘤细胞,以2×104 个细胞/ 孔的浓度接种于平底四分皿中( 每孔100μL 培基),加入10μL 不同浓度的碳量子点材料混合入血清培基中在37℃、5%的CO2 条件下孵育2小时;共孵育后加入多聚甲醛进行细胞固定;附图5a、b为碳量子点显影所得的HEK293人胚肾细胞、Hela细胞荧光显微照片,可以观测到HEK293的照片没有任何荧光信号,而Hela细胞在MSN@Cdots发射的荧光照射下清晰的反应出细胞轮廓。
Claims (7)
1.一种检测用叶酸受体靶向碳量子点turn-on探针的制备方法,其特征在于,通过柠檬酸与乙二胺复合制备得到碳量子点(Cdots),包括如下步骤:
A、水热制备碳量子点,分离纯化后得到碳量子点溶液I;
B、叶酸和3-氨丙基三乙氧基硅烷(APTES)合成特异性模板,加入硅酸乙酯(TEOS)和碳量子点溶液I制备特异荧光发光的介孔硅包裹碳量子点MSN@Cdots化合物,洗去模板分子后得到介孔硅包覆的碳量子点溶液;
C、制备叶酸溶液功能化MSN@Cdots化合物,即使用叶酸溶液充盈介孔孔道,计算吸附量后得到所述叶酸受体靶向碳量子点turn-on探针。
2.如权利要求1所述检测用叶酸受体靶向碳量子点turn-on探针的制备方法,其特征在于,所述量子点为粒径为5~15nm的碳量子点。
3.如权利要求1所述检测用叶酸受体靶向碳量子点turn-on探针的制备方法,其特征在于,步骤B具体为:加入叶酸(模板分子)和APTES(功能性单体),模板分子叶酸与APTES通过氢键和静电作用形成模板-单体复合物,将制备的碳量子点加入到上述溶液中随后加入TEOS(交联剂)和氨水,反应产物经反复清洗去除模板,直到紫外可见分光光度法检测没有模板分子存在,将所制备的介孔硅包覆的碳量子点(MSN@Cdots)分散于缓冲溶液,然后储存在4oC保存。
4.如权利要求1所述的肺癌检测用叶酸受体靶向碳量子点turn-on探针的制备方法,其特征在于,步骤C具体为:MSN@Cdots分散在一定体积的叶酸溶液(PBS,PH值7.0),得到叶酸饱和的MSN@Cdots荧光探针。
5.如权利要求1所述检测用叶酸受体靶向碳量子点turn-on探针的制备方法,其特征在于,向所得固体产物MSN@Cdots中加入待检测的细胞液,根据荧光信号是否发光进行细胞膜表面是否具有叶酸受体蛋白进行判断。
6.一种检测用叶酸受体靶向碳量子点turn-on探针,其特征在于根据权利要求1-5任一所述方法制备得到。
7.根据权利要求6所述叶酸受体靶向碳量子点turn-on探针在检测叶酸受体靶向碳量子点的应用。
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