CN115386371A - 蓝色荧光量子点制备方法及作为谷胱甘肽检测探针的应用 - Google Patents
蓝色荧光量子点制备方法及作为谷胱甘肽检测探针的应用 Download PDFInfo
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Abstract
本发明涉及纳米材料技术领域，尤其涉及一种蓝色荧光量子点制备方法及作为谷胱甘肽检测探针的应用，以L‑胱氨酸(L‑cys)和叶酸(FA)为原料，通过一步水热法合成了能够发蓝光的蓝色荧光碳量子点，对其进行了透射电镜、紫外可见光谱、红外光谱、荧光光谱表征，同时考察了碳量子点的选择性和抗干扰能力，证实所构建的碳量子能够选择性识别谷胱甘肽，其荧光强度在一定范围内与谷胱甘肽浓度呈线性关系。该制备方法路线设计合理，操作简单，重复性好，对设备要求低；制得的蓝色荧光量子点水溶性好，稳定性高，荧光性能优异，能够专一性的用于检测谷胱甘肽。
Description
技术领域
本发明涉及纳米材料技术领域，具体为一种蓝色荧光量子点制备方法及作为谷胱甘肽检测探针的应用。
背景技术
谷胱甘肽(GSH)是一种由L-谷氨酸、L-半胱氨酸和甘氨酸通过肽键缩合构成的三肽。GSH在生物系统中起着关键作用，并具有许多细胞功能，如细胞内信号转导、维持细胞内氧化还原状态、基因调节、解毒和异源代谢等。GSH含有游离的巯基，在清除细胞内的自由基、超氧化物和过氧化物等中起着极其重要的作用。研究发现许多疾病的产生与细胞内GSH水平的变化有关，包括人类免疫缺陷病毒、帕金森、肝损伤、糖尿病、阿尔茨海默病、炎症和心血管疾病等，所以GSH含量的检测对于某些疾病的诊断具有重要意义。目前，已经开发出多种方法来检测GSH或监测细胞内GSH的变化，主要有高效液相色谱法、表面增强拉曼光谱法、质谱法、电化学法、化学发光法、荧光光谱法等，但是这些方法存在选择性差、毒性大、灵敏度低和操作复杂等等问题。
碳量子点作为一种新型的荧光材料被广泛应用于荧光分析和检测中。碳量子点(Carbon quantum dots，CDs)，是一种尺寸小于10nm的、近似球形的碳纳米材料，一般由无定形结构或sp2杂化纳米晶体结构的碳核与其表面丰富的官能团(例如羧基，羟基，醛基，氨基等)组成。近年来，碳量子点在化学和生物传感、光催化、生物成像、药物传递、发光二极管和太阳能电池中受到广泛的关注，与有机荧光团和半导体量子点相比，碳点具有易于制造、良好的稳定性、低的细胞毒性和高的生物相容性。
发明内容
针对现有技术中GSH检测存在选择性差、毒性大、灵敏度低和操作复杂的问题，本发明提供一种蓝色荧光量子点制备方法及作为谷胱甘肽检测探针的应用，该制备方法操作简单，重复性好，原料易得，对设备要求低；制得的蓝色荧光量子点水溶性好，稳定性高，荧光性能优异，能够专一性的用于检测谷胱甘肽。
本发明是通过以下技术方案来实现：
一种蓝色荧光量子点制备方法，以L-胱氨酸和叶酸为原料，采用水热合成法制得发蓝光的蓝色荧光碳量子点，具体步骤如下：
S1，在高温下将L-胱氨酸、叶酸和去离子水混合后反应，之后冷却至室温，获得初始反应物；
S2，利用分散试剂分散初始反应物，之后进行离心，收取上清液进行微孔滤膜过滤、透析和冷冻干燥处理，获得蓝色荧光量子点。
优选的，在S1中，L-胱氨酸、叶酸和去离子水的配比为1g：(1～2)g：10mL。
优选的，在S1中，高温时的温度为180℃，时间为30min。
优选的，在S2中，分散剂为蒸馏水。
优选的，在S2中，微孔滤膜过滤时采用0.22μm注射器滤膜过滤。
优选的，在S2中，透析时采用1000Da的透析袋超纯水中进行，时间为6h。
一种蓝色荧光量子点，由所述的蓝色荧光量子点制备方法所制，所述蓝色荧光量子点的平均粒径为5.7nm，表面电荷为-16.75mV；所述蓝色荧光量子点表面含有亲水性官能团。
一种蓝色荧光量子点作为根据谷胱甘肽检测探针的应用，所述蓝色荧光量子点作为专一性荧光检测探针检测癌细胞内的谷胱甘肽含量，在阳离子、阴离子和有机物单独存在时，仅谷胱甘肽与蓝色荧光量子点发生淬灭。
优选的，所述蓝色荧光量子点对谷胱甘肽的检测浓度区间为200μmol·L-1～8000μmol·L-1。
与现有技术相比，本发明具有以下有益效果：
本发明一种蓝色荧光量子点制备方法采用水热合成法制备蓝色荧光量子点，其合成步骤简单、反应条件较为容易控制且消耗能耗低，可持续规模生产，产物的蓝色荧光量子产率较高，被认为是一种较为经济有效的方法。
本发明制备的量子点克服了传统金属量子点的缺点，具有无毒、高荧光量子产率、水溶性好、生物相容性好、光稳定性好等优点，使得CDs在分析传感、药物递送、生物成像、金属离子检测、光介导治疗和发光二极管等领域得到了广泛的探索。
蓝色荧光量子点具有优良的量子产率，不需要进一步的化学修饰和表面功能化，就可以用于生物体内GSH的荧光检测。
本发明制备的蓝色荧光量子点对GSH的响应非常灵敏，这一特性使CDs能够灵敏检测生物样品中的GSH及其含量变化，为与GSH浓度相关疾病的检测和诊断提供了技术基础。
附图说明
图1为本发明制备的蓝色荧光量子点结构表征相关结果；其中，(A)为CDs透射电镜图；(B)为粒径分布图；(C)为Zeta电位图；
图2为本发明制备的蓝色荧光量子点CDs的红外光谱图；
图3为本发明制备的蓝色荧光量子点CDs的紫外吸收图谱、荧光激发光谱和发射光谱；
图4为本发明制备的蓝色荧光量子点CDs在不同波长激发下的发射图谱；
图5为本发明制备的蓝色荧光量子点CDs在不同阳离子(A)、阴离子(B)和有机物(C)溶液中的荧光强度图；
图6为本发明制备的蓝色荧光量子点CDs在不同浓度GSH中的荧光谱图；
图7为GSH标准溶液的溶液浓度与荧光淬灭率关系曲线。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定。
本发明公开了一种蓝色荧光量子点制备方法，以L-胱氨酸和叶酸为原料，采用水热合成法制得发蓝光的蓝色荧光碳量子点，具体步骤如下：
S1，在高温180℃下将配比为1g：(1～2)g：10mL的L-胱氨酸、叶酸和去离子水混合后反应30min，之后冷却至室温，获得初始反应物；本实施例中配比为0.4g：0.45g：4mL。
S2，利用分散试剂分散初始反应物，之后进行离心，收取上清液进行微孔滤膜过滤、透析和冷冻干燥处理，获得蓝色荧光量子点，蓝色荧光量子点呈深棕色固体粉末状。
其中，分散剂为蒸馏水，离心时的转速为10000rpm，时间20min；微孔滤膜过滤时采用平均孔径为0.22μm注射器滤膜过滤。透析时采用1000Da的透析袋在超纯水中透析为6h，每隔2h换一次超纯水。
一种蓝色荧光量子点，由蓝色荧光量子点制备方法所制。蓝色荧光量子点的相关表征如下：
(1)平均粒径和Zeta电位表征
采用ZEN3600型马尔文动态光散射粒度仪测定CDs平均粒径和Zeta电位，测试温度为25℃。
(2)TEM微观形貌与结构表征
取少量CDs于5mL离心管内，向离心管内加入2mL无水乙醇并超声5min，将样品均匀滴在400目铜网上，采用Tecnai G2F20场发射透射电镜在80KV加速电压下观测样品形貌。
结构参见图1所示，图1中(A)为CDs高分辨透射电子显微镜图，从图中可以看出所制备的CDs呈球形，并且分散性好。通过高分辨透射电镜图及粒径分布图(B)可以看出所制备的CDs平均粒径为5.7nm。(C)为CDs的表面Zeta电位图，可以看出其电位为-16.75mV。
(3)FT-IR表征
取CDs冻干样品，溴化钾压片。采用Tensor27型红外光谱仪测定红外吸收图谱，扫描范围为500～4000cm-1。测试结果如图2所示，其中3319cm-1和3205cm-1的吸收峰可归因于N-H的面内伸缩振动。在1569cm-1和1502cm-1处的特征峰归因于苯环骨架特征峰。在1384cm-1和1333cm-1处的特征峰反映了C-N的伸缩振动。1182cm-1和1102cm-1处的峰归因于C-O的伸缩振动。816cm-1和756cm-1处的特征峰反映了N-H的面外弯曲振动。同时，碳量子点表面含有丰富的羟基、羧基、羰基等亲水性官能团，使其具有良好的水溶性和优异的荧光性能。
(4)蓝色荧光量子点的光学性质
配制pH＝7.4的HCl-Tris缓冲溶液，称取制备的荧光碳点，用超纯水溶解，准确配制1mg/mL的荧光碳点母液。移取400μL上述荧光碳点储存液置于5mL比色管中，加入pH＝7.4的HCl-Tris缓冲溶液稀释至4mL，得到0.1mg/mL荧光碳点储存液，配制两份。一份用紫外可见分光光度计进行吸光度检测；另一份用荧光分光光度计进行荧光检测，得到激发光谱和发射光谱。
使用紫外可见分光光度计和荧光分光光度计分别对CDs样品进行测试。如图3所示，CDs的紫外-可见吸收光谱在276和347nm处出现两个峰。碳点在276nm处的特征吸收峰,归因于到碳量子点核内芳香族sp2杂化碳的π→π*跃迁吸收。在347nm处的特征吸收峰,归因于碳量子点表面羧基中C＝O双键的n→π*跃迁吸收。随后，测定了CDs的荧光性能，如图4，在最佳激发波长(395nm)下,碳量子点465nm处呈现明显的荧光发射峰,且该发射峰的峰位和强度随激发波长的变化发生明显变化。当激发波长从375nm增加到415nm时,荧光发发射峰位发生红移,荧光强度明显增加,之后随发射波长的不断增加,荧光强度反而降低，这种随激发波长依赖的特性被认为是由于碳量子点的尺寸效应或表面发光位点不同所引起。
一种蓝色荧光量子点作为根据谷胱甘肽检测探针的应用，蓝色荧光量子点作为专一性荧光检测探针检测癌细胞内的谷胱甘肽含量，在阳离子、阴离子和有机物单独存在时，仅谷胱甘肽与蓝色荧光量子点发生淬灭。其中，蓝色荧光量子点对谷胱甘肽的检测浓度区间为200μmol·L-1～8000μmol·L-1。
蓝色荧光量子点的专一性检测
(1)离子干扰实验
采用逐级稀释法以PBS(pH＝7.4)缓冲液为溶剂配制浓度为10mmol·L-1的杂离子母液(M+：NH4 +，Mn2+，Ba2+，Ca2+，K+，Zn2+，Mg2+，Na+等；M-：NO3 -、Cl-、SO4 2-、SO32-、CO3 2-、HCO3 -、CH3COO-、HPO4 2-等)。将所配得的杂离子母液进行稀释配得浓度为10mmol·L-1的待测液。向上述待测液中加入3.1所配置的CDs母液，使待测液中CDs的浓度为100μg·mL-1。将所配好的溶液用荧光分光光度计测定其荧光光谱。
(2)有机物干扰实验
采用逐级稀释法以PBS(pH＝7.4)缓冲液为溶剂配制浓度为10mmol·L-1的有机物母液(PPI，SPI，HSA，BSA，OVA，Lys，Arg，Tyr等)。将所配得的有机物母液进行稀释配得浓度为10mmol·L-1的有机物待检测液。向上述待测液中加入3.1所配置的CDs母液，使待测液中CDs的浓度为100μg·mL-1。将所配好的溶液用荧光分光光度计测定其荧光光谱。
为了进一步测定通过不同碳源和氮源所制备的碳量子点仅能够被GSH特异性淬灭。
通过CDs在不同阳离子、阴离子及有机物溶液中荧光强度进行探究。如图5中(A)(B)为不同阳、阴离子溶液中CDs的荧光强度分布图，由图5可以看出，GSH对荧光碳量子点有明显的荧光猝灭作用，而其他阳离子(NH4 +，Mn2+，Ba2+，Ca2+，K+，Zn2+，Mg2+，Na+等)、阴离子(NO3 -、Cl-、SO4 2-、SO32-、CO3 2-、HCO3 -、CH3COO-、HPO4 2-等)和有机物(PPI，SPI，HSA，BSA，OVA，Lys，Arg，Tyr等)对碳量子点荧光强度的猝灭作用影响甚微。因此，本发明所制备的蓝色荧光量子点对GSH的检测具有较强的选择性，且不同阳离子对GSH的荧光猝灭干扰性很小，说明所制备的蓝色荧光量子点不会被杂离子与有机物淬灭，都具有较强的检测专一性。
(3)CDs检测GSH线性关系研究
准确称取上述制备的CDs并将其溶于PBS(pH＝7.4)缓冲液中得到浓度为100μg·mL-1的CDs母液；采用逐级稀释法以PBS(pH＝7.4)缓冲溶液为溶剂依次配制浓度为1mmol·L-1，10mmol·L-1的GSH母液。
以上述所配液体为母液，PBS(pH＝7.4)缓冲液为溶剂分别配制浓度为0，10，20，40，80，100，200，300，500，1000，2000，4000，6000，8000μmol·L-1的GSH待检测液。向上述待测中加入所配制的CDs母液，使待测液中CDs的浓度为100μg·mL-1。将所配好的溶液用荧光分光光度计测定其荧光光谱。
使用荧光分光光度计对CDs样品进行测试的荧光光谱测试，从图6中可以看出，随着GSH浓度的增加，荧光强度呈现出逐渐减弱的趋势。从图7中可以看出，当GSH浓度在200～2000μmol·L-1和2000～8000μmol·L-1范围内，两者呈良好的线性关系，相关系数分别为0.996和0.998，y＝8.958x10-4x+2.076和y＝3.52x10-4x+3.127。
通过拟合方程可以看出所设置的GSH浓度与对应的荧光强度具有指数关系，其检测区间为200～8000μmol·L-1，检测极限为0.027μmol·L-1。
以上所述的仅仅是本发明的较佳实施例，并不用以对本发明的技术方案进行任何限制，本领域技术人员应当理解的是，在不脱离本发明精神和原则的前提下，该技术方案还可以进行若干简单的修改和替换，这些修改和替换也均属于权利要求书所涵盖的保护范围之内。
Claims (9)
1.一种蓝色荧光量子点制备方法，其特征在于，以L-胱氨酸和叶酸为原料，采用水热合成法制得发蓝光的蓝色荧光碳量子点，具体步骤如下：
S1，在高温下将L-胱氨酸、叶酸和去离子水混合后反应，之后冷却至室温，获得初始反应物；
S2，利用分散试剂分散初始反应物，之后进行离心，收取上清液进行微孔滤膜过滤、透析和冷冻干燥处理，获得蓝色荧光量子点。
2.根据权利要求1所述的蓝色荧光量子点制备方法，其特征在于，在S1中，L-胱氨酸、叶酸和去离子水的配比为1g：(1～2)g：10mL。
3.根据权利要求1所述的蓝色荧光量子点制备方法，其特征在于，在S1中，高温时的温度为150～200℃，时间为20～30min。
4.根据权利要求1所述的蓝色荧光量子点制备方法，其特征在于，在S2中，分散剂为蒸馏水。
5.根据权利要求1所述的蓝色荧光量子点制备方法，其特征在于，在S2中，微孔滤膜过滤时采用0.22μm注射器滤膜过滤。
6.根据权利要求1所述的蓝色荧光量子点制备方法，其特征在于，在S2中，透析时采用1000Da的透析袋在超纯水中进行，时间为6h。
7.一种蓝色荧光量子点，由如权利要求1～6所述的蓝色荧光量子点制备方法所制，其特征在于，所述蓝色荧光量子点的平均粒径为4～6nm，表面电荷为-16.75mV；所述蓝色荧光量子点表面含有亲水性官能团。
8.一种如权利要求7所述的蓝色荧光量子点作为根据谷胱甘肽检测探针的应用，其特征在于，所述蓝色荧光量子点作为专一性荧光检测探针检测癌细胞内的谷胱甘肽含量，在阳离子、阴离子和有机物单独存在时，仅谷胱甘肽与蓝色荧光量子点发生淬灭。
9.根据权利要求8所述的蓝色荧光量子点制备方法，其特征在于，所述蓝色荧光量子点对谷胱甘肽的检测浓度区间为200μmol·L-1～8000μmol·L-1。
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| 刘辉;赵永福;韩怀远;刘亚雯;冶保献;: "基于氮掺杂碳纳米颗粒荧光猝灭-恢复方法检测还原型谷胱甘肽", 化学试剂, no. 02, pages 126 - 130 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116023932A (zh) * | 2022-12-09 | 2023-04-28 | 山西大学 | 一种用于谷胱甘肽检测的荧光探针及其制备 |
| CN116023932B (zh) * | 2022-12-09 | 2024-03-12 | 山西大学 | 一种用于谷胱甘肽检测的荧光探针及其制备 |
| CN115948163A (zh) * | 2023-01-03 | 2023-04-11 | 扬州工业职业技术学院 | 一种选择性识别谷胱甘肽的近红外荧光探针 |
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