CN116969445A - 一种对pH敏感的长波碳点的制备方法及应用 - Google Patents
一种对pH敏感的长波碳点的制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种对pH敏感的长波碳点的制备方法及其在荧光墨水中的应用，属于碳纳米材料技术领域。本发明通过室温下取中性红和L‑半胱氨酸于去离子水中超声溶解；然后进行加热反应，自然冷却至室温后过滤，再通过透析膜透析得到碳点水溶液，冷冻干燥得到L‑CDs。本发明制备得到的L‑CDs对pH与次氯酸根非标记检测灵敏度高、成本低、检测过程简单，对生物样品的危害性小，更加安全可靠。该L‑CDs还可成成功用于细胞内成像。
Description
技术领域
本发明属于碳纳米材料技术领域，具体涉及一种对pH敏感的长波碳点的制备方法及其在荧光墨水中的应用。
背景技术
细胞内pH的稳定在信号转导、细胞增殖、细胞凋亡等多种代谢生理过程中起着重要作用。细胞内pH值异常可导致癌症、慢性肾病、关节炎多种病理症状的发生。健康细胞的正常pH值为7.2～7.4，而许多继发性疾病的病理组织中常发生pH的不平衡和波动。迄今为止，多种技术被用于细胞内pH的检测，如微电极、核磁共振和吸收光谱。然而，传统方法存在着成本高、采样过程复杂、操作复杂等难以克服的缺点。荧光检测具有灵敏度高、成本低、检测过程简单等优点，基于CDs的荧光检测策略因其低毒、低成本、高生物相容性和快速检测而受到广泛关注。因此，开发一种对pH敏感的CDs的制备方法来监测细胞内pH值的变化具有重要意义。
近年来，假冒已成为一个日趋严重的社会问题，因此采用高科技解决方案来预防和制止假冒行为至关重要。现有的多种防伪技术中，荧光防伪因具有强防伪能力，且识别快捷、操作简便而被广泛使用。而防止假冒的常用荧光材料主要是无机磷光体、量子点、有机荧光染料等。然而，无机磷光体合成复杂和原料昂贵，量子点包括对人类和环境造成不良影响的重金属，而有机染料的热稳定性、光稳定性和化学稳定性和抗光漂白性较差。因此，制备光稳定性好、毒性低、抗光漂白性好的荧光防伪材料对荧光防伪技术的发展有重大意义。碳量子点由于其良好的生物相容性、环境友好性、低成本和优异的抗光漂白性等优点，可以用作数据加密和储存中的高质量新型荧光油墨。碳量子点制备的发光油墨可以封装在喷墨打印机或注入钢笔、中性笔中或用毛笔直接蘸墨，在不含荧光剂的纸、纺织品、皮革、玻璃等上面打印或书写字体或图案，同时也可以结合二维码、条形码等实现双重加密。且碳量子点的水溶性优异，用碳量子点制备的防伪油墨下墨流畅，不会造成堵塞，因此开发一种具有优异性能的碳量子点，对荧光防伪技术的发展有重大意义。
发明内容
针对上述问题，本发明提供了一种对pH敏感的长波碳点的制备方法及应用。
为了达到上述目的，本发明采用了下列技术方案：
一种对pH敏感的长波碳点的制备方法，包括如下步骤：室温下取中性红和L-半胱氨酸于去离子水中超声溶解；然后进行加热反应，自然冷却至室温后过滤，再通过透析膜透析得到碳点水溶液，冷冻干燥得到L-CDs。
进一步，中性红和L-半胱氨酸的质量比为5.9：11.8。
进一步，所述超声溶解的时间是3min～5min。
进一步，所述加热反应的加热温度为160℃～220℃，加热时间为4h～8h。
进一步，去离子水的添加量为20mL。
进一步，所述过滤是通过0.22μm的滤膜过滤。
进一步，所述透析膜透析是用500～1000Da的透析膜透析2～3d。
进一步，将溶解后的溶液装入特氟龙内衬，再密封入不锈钢高压水热反应釜，进行加热反应。
一种如上述制备方法制得的长波碳点以及在荧光墨水中的应用。
与现有技术相比本发明具有以下优点：
1.本发明操作步骤简单，通过简单的一步水热法即可得具有长波发射的碳点。
2.本发明制备得到的L-CDs对pH与次氯酸根非标记检测灵敏度高、成本低、检测过程简单，对生物样品的危害性小，更加安全可靠。该L-CDs还可成成功用于细胞内成像。
3.本发明制备得到的L-CDs具有优异的光稳定性和化学稳定性，基于L-CDs制备的荧光墨水能够响应pH变化，在防伪领域具有良好的应用潜力。
附图说明
图1(a)为L-CDs的TEM与HRTEM图；(b)为L-CDs的尺寸分布图；(c)为L-CDs的AFM图；
图2为L-CDs的FTIR图；
图3为L-CDs的(a)XPS全谱(b)C 1s、(c)N 1s、(d)O 1s和(e)S2p的高分辨率元素谱；
图4(a)为L-CDs紫外可见吸收光谱、激发光谱与发射光谱；(b)为L-CDs在不同激发光下的发射光谱；
图5(a)为不同pH下L-CDs溶液的荧光强度；(b)为不同KCl浓度下L-CDs溶液的荧光强度；(c)氙灯照射不同时间后L-CDs的荧光强度；
图6(a)为L-CDs的pH滴定曲线；(b)L-CDs的为荧光强度与pH的Boltzmann非线性拟合及pH为(c)4.2-5.8、(d)5.8-7.4的线性拟合；
图7为不同pH下L-CDs溶液的Zeta电位；
图8(a)为L-CDs的次氯酸根滴定曲线；(b)为L-CDs的荧光淬灭程度与次氯酸根浓度的非线性拟合与(c)线性拟合；(d)L-CDs的选择性检测；
图9(a)为含与不含次氯酸根的L-CDs溶液的荧光寿命曲线；(b)含与不含次氯酸根的L-CDs溶液与次氯酸根溶液的紫外可见吸收光谱；
图10为Hela细胞存活率与L-CDs浓度之间的关系；
图11(a)为在pH为4、7、9时L-CDs对Hela细胞的共聚焦成像；(b)为L-CDs与L-CDs/ClO-对Hela细胞的共聚焦成像；
图12为分别在(上图)日光、(下图)365nm紫外线下，(a)使用0.26mg/mLL-CDs溶液和(b)0.26mg/mLL-CDs溶液(PH＝10)和(c)0.26mg/mLL-CDs溶液(PH＝2)在滤纸上手写“山西大学SXU”的相关照片。
具体实施方式
实施例1
一种用于pH与次氯酸根非标记检测的长波碳点的制备方法，包括如下步骤：
在20mL去离子水中加入5.9mg NR和11.8mg L-cys，溶液超声3min使前体溶解，将溶液装入50mL特氟龙内衬，密封入不锈钢高压水热反应釜，置于烘箱中，在180℃下加热4h。反应结束待所得溶液自然冷却至室温后用0.22μm滤膜过滤，后用500～1000Da透析膜透析3d，再经冷冻干燥得到L-CDs粉末。罗丹明B为参照物，L-CDs的相对量子产率为2.53％。
实施例2
本发明实施例1制备的L-CDs的TEM图像、尺寸分布和AFM图像表征如图1所示。该碳点显示出规则性球状形态特征，呈单分散排布，L-CDs纳米颗粒分散均匀，层间晶格间距为0.21nm，表明存在石墨烯结构；平均粒径为2.43nm；AFM图表征了L-CDs纳米颗粒的高度。
实施例3
本发明实施例1制备的L-CDs的红外光谱(FTIR)表征如图2所示。该碳点在614、1101、1405、1630、1711、3210和3306cm-1处分别观察到7个特征峰，证实了L-CDs表面存在O-H、N-H、C-O、C-N、C＝C、C＝N、C＝O、C-H化学键的存在。
实施例4
本发明实施例1制备的L-CDs的X射线光电子能谱(XPS)表征如图3所示。在195、282、396和528eV处可以观察到四个明显的结合能峰，分别对应S2p、C 1s、N 1s和O1s四种元素，表明该碳点由C、N、O、S四种元素组成。图3b、3c、3d和3e分别为C1s、N 1s、O 1s和S2p的高分辨率XPS光谱。C 1s元素谱可分为284.8eV下的C-C/C＝C、285.3eV下的C＝O和287.25eV下的C-O三个分峰；在N1s元素谱中，398.25eV、398.85eV和400.65eV三个分峰分别对应吡啶N、吡咯N和石墨烯N；在O1s元素谱中，在398.85eV和400.65eV分别得到C-O和C＝O两个分峰；在S2p元素谱中，可以得到两个分峰(167.65eV的S2p 1/2和168.85eV的S2p 3/2)。元素分析结果表明，S被成功掺杂于L-CDs上，L-CDs表面存在各种含氮、含氧、含硫基团。
实施例5
本发明实施例1制备的L-CDs的紫外可见吸收光谱、激发光谱与发射光谱如图4a所示，L-CDs在不同激发光下的发射光谱如图4b所示。该L-CDs在272nm和531nm处可分别观察到两个吸收峰，分别归因于C＝C键的π→π*跃迁和C＝O/C-N键的n→π*跃迁，最大激发与发射波长分别为520nm，587nm。图4b表示随着激发波长的增加，L-CDs的荧光呈现出略微的红移。
实施例6
本发明实施例1制备的L-CDs不同pH、盐浓度环境中的荧光强度及在氙灯照射下的稳定性如图5所示；该L-CDs的荧光强度随pH的上升而逐渐淬灭，表明该L-CDs可对pH的变化产生荧光响应(图5a)。在不同浓度的KCl溶液(0-4mol/L)中，L-CDs的荧光强度保持相对稳定，表明L-CDs对溶液盐浓度有良好的耐受性(图5b)。用氙灯连续照射L-CDs，持续60min，结果表明该L-CDs有较好的光稳定性(图5c)。
实施例7
本发明实施例1制备的L-CDs对pH的非标记检测如图6所示。首先以硼酸、磷酸、冰乙酸制备了一系列pH为2-10、间隔区间为0.2的BR缓冲溶液。向2mL不同pH的BR缓冲液中加入60μL的L-CDs溶液(0.26mg/mL)，测量溶液的荧光光谱，观察pH值与L-CDs溶液最大荧光强度之间的关系。如图6(a)所示，当pH逐渐上升时，L-CDs的荧光强度明显减弱。采用Boltzmann方程对L-CDs的荧光强度与pH间的关系进行非线性拟合(图6b)，得到了两段线性范围，分别对应4.2-5.8与5.8-7.4，图6(c)为pH在4.2-5.8间时的线性拟合结果，图6(d)为pH在5.8-7.4间的线性拟合，结果表明L-CDs具有监测pH变化的能力。
实施例8
本发明实施例1制备的L-CDs对pH的非标记检测的响应机制如图7所示，在不同pH下L-CDs溶液的Zeta电位在-23.4mV-23.6mV之间变化，pH逐渐升高时L-CDs的Zeta电位随之降低，这种变化在pH为2-5时尤其明显，据此推测L-CDs对pH的响应机制是L-CDs表面含氮官能团在不同pH环境中的质子化和去质子化。
实施例9
本发明实施例1制备的L-CDs对次氯酸根的非标记检测如图8所示。首先将不同量的次氯酸根溶液加入60μL的L-CDs溶液(0.26mg/mL)中，并用去离子水稀释至2mL，得到一系列不同次氯酸根浓度(0-300μM)的待测溶液，测量其荧光光谱，在λex/λem为520/587nm处进行记录。如图8a所示，随着次氯酸根浓度从0μmol/L到300μmol/L逐渐增加，L-CDs溶液的荧光强度呈现显著降低的趋势。图8b为采用Boltzmann方程对淬灭程度与次氯酸根浓度的非线性拟合结果。图8c显示了线性拟合方程，线性范围为10-162.5μmol/L，检出限为1.021μmol/L。随后检测了L-CDs对不同离子的选择性(图8d)，将60μL L-CDs溶液用去离子水稀释至2mL，加入不同的离子并检测荧光强度变化，结果表明，次氯酸根能引起L-CDs明显的荧光淬灭，这种淬灭对其他离子的存在具有较高的耐受性，说明L-CDs对次氯酸根的检测具有良好的选择性。
实施例10
本发明实施例1制备的L-CDs对次氯酸根的响应机制如图9所示，图9(a)为L-CDs溶液的荧光寿命与含有ClO-(浓度为0.5mmol/L)的L-CDs溶液的荧光寿命，两者之间未观察到显著性变化。图9(b)中在L-CDs溶液中加入次氯酸根后，在272nm处的吸收峰出现一定程度红移且吸光度略下降，在531nm的吸收峰蓝移且吸光度明显降低，这表明L-CDs与次氯酸根发生作用导致L-CDs表面化学结构发生了变化，进而导致了光学性质的改变。结合荧光寿命和UV/Vis吸收光谱的检测结果，推断L-CDs对次氯酸根的荧光响应机制为静态猝灭。
实施例11
本发明实施例1制备的L-CDs对Hela细胞的毒性测试如图10所示，本发明使用标准MTT比色法检测L-CDs在Hela细胞中的细胞毒性，在96孔板中接种Hela细胞后将其置于温度37℃、CO2体积分数为5％的培养箱中，培养1d后吸弃培养液，加入不同浓度L-CDs的培养基，再培养1d后加入10μL浓度为5mg/mL的MTT溶液培养4h，去除上清液后加入150μL二甲基亚砜，振荡10min后使用酶联免疫检测仪测定490nm吸光度。由图10可见，随着L-CDs浓度的增加，细胞的存活率逐渐降低，但各浓度组的细胞存活率均能维持在较高水平(高于80％)。由此推测，L-CDs的细胞毒性较低，在生物监测和细胞成像方面具有应用潜力。
实施例12
本发明实施例1制备的L-CDs对Hela细胞的细胞成像如图11所示，首先将HeLa细胞置于15mm的玻璃皿中，在37℃下孵育24h。当细胞孵育完成后，用PBS(pH＝7.4)清洗细胞三次以去除杂质，用不同pH值(4,7,9)的PBS溶液孵育2h后用L-CDs(0.26mg/mL)处理HeLa细胞2h，小心丢弃培养基，加入含次氯酸根的新鲜培养基培养30min后，用PBS溶液冲洗处理过的细胞，进行共聚焦成像观察。图11(a)为不同pH处理后的细胞图像，结果表明，随着pH值的增加，可明显观察到Hela细胞的荧光强度逐渐降低。图11b表明在次氯酸根的作用下Hela细胞相对于只经过L-CDs处理的细胞，荧光发生了明显淬灭。以上结果表明，L-CDs具有对细胞内pH和次氯酸根浓度的成像和监测能力，在生物监测领域具有一定应用潜力。
实施例13
本发明实施例1制备的L-CDs在防伪中的应用如图12所示，由L-CDs制成的荧光油墨应用于防伪书写。具体操作为将0.26mg/ml的L-CDs溶液制备的荧光油墨封装到钢笔的墨囊中，在纸上进行手写。如图12a所示，用L-CDs油墨在纸上写的“山西大学SXU”在日光下几乎不可见，在365nm紫外光照射发出橙色荧光。本发明基于上述实施例7中L-CDs对pH的非标记检测，制作了不同pH的0.26mg/ml的L-CDs溶液，并将其按上述步骤在纸上书写。如图12b所示，当pH＝10时，该碳点溶液书写“山西大学SXU”在日光下完全不可见，且在紫外光照射下所发出的荧光颜色变浅，强度变弱；而将其溶液pH调到2时，如图12c所示，在365nm紫外光照射下又发出橙色荧光，且荧光强度比之前增加，这也对应于制备的L-CD其荧光能够响应pH变化。表明该L-CDs作为隐形荧光油墨在防伪方面具有良好的应用潜力。
综上所述，本发明使用中性红和L-半胱氨酸通过一步水热法成功合成了发黄光的L-CDs。该L-CDs对pH和次氯酸根浓度有特异性荧光响应，当环境pH值在4.2～7.4之间时，L-CDs的荧光强度与pH值之间表现出良好的线性关系。次氯酸根可导致L-CDs溶液的荧光发生明显淬灭，线性范围为10-162.5μM，检出限为1.021μM，根据紫外可见吸收光谱和荧光寿命检测结果，响应机制为静态淬灭。细胞毒性实验结果显示L-CDs具有较低的细胞毒性和良好的生物相容性，随后使用L-CDs对Hela细胞进行细胞成像实验，结果表明L-CDs可用于细胞内的荧光成像，且能对细胞内的pH变化与次氯酸根浓度实现荧光成像监测。这意味着L-CDs具有潜在的生物监测领域应用价值，从而进一步丰富了长波长L-CDs的理论及其在荧光监测、生物传感方面的应用。
本发明说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。尽管上面对本发明说明性的具体实施方式进行了描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
Claims (10)
1.一种对pH敏感的长波碳点的制备方法，其特征在于：包括如下步骤：室温下取中性红和L-半胱氨酸于去离子水中超声溶解；然后进行加热反应，自然冷却至室温后过滤，再通过透析膜透析得到碳点水溶液，冷冻干燥得到L-CDs。
2.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：中性红和L-半胱氨酸的质量比为5.9：11.8。
3.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：所述超声溶解的时间是3min～5min。
4.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：所述加热反应的加热温度为160℃～220℃，加热时间为4h～8h。
5.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：去离子水的添加量为20mL。
6.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：所述过滤是通过0.22μm的滤膜过滤。
7.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：所述透析膜透析是用500～1000Da的透析膜透析2～3d。
8.根据权利要求1所述一种对pH敏感的长波碳点的制备方法，其特征在于：将溶解后的溶液装入特氟龙内衬，再密封入不锈钢高压水热反应釜，进行加热反应。
9.一种如权利要求1～8任意一项所述制备方法制得的长波碳点。
10.一种如权利要求1～8任意一项所述制备方法制得的长波碳点在不同pH值下荧光墨水中的应用。
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