CN116904190B - 一种可以同时检测六价铬和抗坏血酸的荧光探针及其制备方法与应用 - Google Patents
一种可以同时检测六价铬和抗坏血酸的荧光探针及其制备方法与应用 Download PDFInfo
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Abstract
本申请公开了一种可以同时检测六价铬和抗坏血酸的荧光探针及其制备方法与应用,可以同时检测六价铬和抗坏血酸的荧光探针的制备方法包括以下步骤:(1)称取阿胶粉溶于去离子水,涡旋并超声使其充分溶解,得到混合溶液;(2)将混合溶液置于高压反应釜中,加热反应,得到阿胶碳量子点溶液;(3)阿胶碳量子点溶液冷却至室温,转移到离心管中,离心,得到阿胶碳量子点溶液上清液;(4)取阿胶碳量子点溶液上清液,过滤,在真空下冷冻干燥得到阿胶碳量子点固体粉末;(5)取适量阿胶碳量子点固体粉末溶于超纯水,得到所述荧光探针,保存备用。本申请制备过程简单,不使用有毒化学试剂,对环境友好无污染,成本低廉且荧光强度和量子产率高。
Description
技术领域
本申请涉及一种可以同时检测六价铬和抗坏血酸的荧光探针及其制备方法与应用,属于荧光纳米材料及食品与环境科学技术领域。
背景技术
六价铬(Cr(VI))是一种广泛使用的工业材料,多用于冶金、化工、铸铁、皮革、印刷等方面,但六价铬被人体过量吸收可能致癌,还会引发诸多其他的健康问题,被列为一类致癌物。随着工业的不断发展,六价铬在水中的排放量也日益增多,可能会对土壤中农作物等植物的生长造成一定的危害,若对水质处理不当而被人体摄入后,会在人体内不断积累,从而对人体的呼吸系统,神经免疫系统以及胃肠道造成极大的危害,对皮肤黏膜也具有强烈的刺激作用。因此,开发一种高效灵敏的,用于检测六价铬的方法至关重要。
抗坏血酸(AA),是一种水溶性维生素,具有很强的还原能力,多存在于新鲜水果和蔬菜中,临床上可以参与机体的多种反应,是人体许多代谢途径中关键的辅酶因子。抗坏血酸可以治疗和预防感冒,预防缺铁性贫血,提高人体的免疫力。此外还能够缓解一些重金属中毒,防治坏血病,还有清除羟基和预防肿瘤的作用。由于人体无法合成,因此只能从水果和蔬菜等食物以及医药品中获取。因此督促我们寻找并建立一种更加准确,灵敏的检测抗坏血酸含量的方法。
传统的Cr(VI)和AA的检测方法有很多种,包括原子吸收光谱法、色谱法、电化学法、比色法、毛细管电泳法,化学发光法和荧光光谱法等,而其中,荧光法因其高灵敏度,设备简单,操作方便等优势受到了广泛的关注。然而,目前的荧光碳量子点荧光探针(CDs)的制备方法包括水热法、微波法、激光灼烧法等等,这些方法需要消耗大量能量才能合成碳量子点,存在能耗高,成本高且不环保的问题。
发明内容
为了解决上述问题,提供了一种可以同时检测六价铬和抗坏血酸的荧光探针的制备方法,通过采用绿色环保的阿胶粉作为原料,制备可以同时检测六价铬和抗坏血酸的荧光探针,制备过程简单,不使用有毒、复杂的化学试剂,对环境友好无污染,成本低廉且荧光强度和量子产率高。
根据本申请的一个方面,提供了一种可以同时检测六价铬和抗坏血酸的荧光探针的制备方法,包括以下步骤:
(1)称取阿胶粉溶于去离子水,涡旋并超声使其充分溶解,得到混合溶液;
(2)将步骤(1)得到的混合溶液置于高压反应釜中,加热反应,得到阿胶碳量子点溶液;
(3)将步骤(2)得到的阿胶碳量子点溶液冷却至室温,转移到离心管中,离心,得到阿胶碳量子点溶液上清液;
(4)取步骤(3)得到的阿胶碳量子点溶液上清液,过滤,在真空下冷冻干燥得到阿胶碳量子点固体粉末;
(5)取适量阿胶碳量子点固体粉末溶于超纯水,得到所述荧光探针,保存备用。
可选地,步骤(1)中,所述阿胶粉与去离子水的用量比为(0.5~0.75)g:(10~15)mL。
具体地,在阿胶粉与去离子水在此用量比范围以内,所制备的荧光探针具有较高的荧光强度和荧光量子产率。
可选地,步骤(2)中,反应温度为160~200℃,反应时间为5~12h。
具体地,步骤(2)中,反应温度可以为160℃、170℃、180℃、190℃、200℃;反应时间可以为5h、6h、7h、8h、9h、10h、11h、12h。在此反应温度和反应时间内,所制备的荧光探针具有较高的荧光强度和荧光量子产率。
可选地,步骤(3)中离心为用离心机以4000~4200r/m的速度离心10~15min。
具体地,步骤(3)中离心以去除大的颗粒杂质。
可选地,步骤(2)中,高压反应釜为聚四氟乙烯内胆高压反应釜;步骤(4)中过滤采用0.22μm滤膜。
具体地,通过滤膜过滤以去除细小的颗粒杂质。
可选地,所述冷冻干燥的温度为-70~-80℃,冷冻干燥的时间为24~48h。
可选地,所述荧光探针的浓度为20~50mg/mL。
具体地,浓度过低会导致用量过大,浓度过高会使得待测物消耗过多,二者均会增加实际检测中的误差。
根据本申请的另一方面,提供了一种可以同时检测六价铬和抗坏血酸的荧光探针,采用上述制备方法制得。
根据本申请的又一方面,还提供了上述荧光探针在检测水环境中的六价铬含量方面的应用。
具体地,测定方法包括以下步骤:
(1)量取200μL浓度为20~50mg/mL荧光探针溶液与400μL Cr(VI)溶液(1-400μM)于5mL离心管中混合,并向其中添加PBS缓冲液(pH=9)将溶液体积定容至3mL;
(2)将步骤(1)中得到的混合溶液于室温下涡旋2min使其充分反应后,记录其荧光强度。
根据本申请的又一方面,还提供了上述荧光探针在检测新鲜水果和维生素C片中的抗坏血酸含量方面的应用。
具体地,测定方法包括以下步骤:
(1)量取200μL浓度为20~50mg/mL荧光探针溶液和400μL的Cr(VI)溶液(400μM)在5mL离心管中混合,室温下涡旋2min使CDs的荧光强度猝灭至一定程度,从而形成CDs/Cr(VI)荧光探针;
(2)向步骤(1)中形成的CDs/Cr(VI)荧光探针中加入1mL的AA溶液(1-400μM)混合,并用PBS缓冲液(pH=9)定容体积至3mL;
(3)将步骤(2)中的到的混合液在室温下涡旋2min使其充分反应后,记录其荧光强度。
本申请的有益效果包括但不限于:
1.本申请通过采用绿色环保的阿胶粉作为原料,制备可以同时检测六价铬和抗坏血酸的荧光探针,制备过程简单,不使用有毒、复杂的化学试剂,对环境友好无污染,成本低廉且荧光强度和量子产率高。
2.本申请提供的荧光探针,合成的阿胶碳量子点表面含有丰富的-OH和-NH等含氧官能团,因此在水溶液中表现出良好的分散性,颗粒间没有明显的聚集,可在实际检测中被作为一种高效的荧光传感器。
3.本申请提供的荧光探针对Cr(VI)和AA的检测都表现出较好的选择性和灵敏度,几乎不会受到其他离子的干扰,在一定范围内与二者均呈线性关系,因此可以实现在水环境和食品中对Cr(VI)和AA的含量检测,具有十分广阔的应用前景。
4.本申请提供的荧光探针在高盐浓度下基本保持稳定,且在较宽的pH值范围内(4-9)也具有较好的荧光效果,同时具有较强的光漂白性,在紫外灯下持续照射90min荧光强度也不会发生明显的变化。
5.本申请提供的荧光探针检测时间短,操作简单,反应可在2min内迅速完成,且在室温下操作即可。
附图说明
此处所说明的附图用来提供对本申请的进一步理解,构成本申请的一部分,本申请的示意性实施例及其说明用于解释本申请,并不构成对本申请的不当限定。在附图中:
图1为本申请实施例1涉及的CDs的透射电子显微镜以及晶格间距分析图;
图2为本申请实施例1涉及的CDs的粒径分布图;
图3为本申请实施例1涉及的CDs的X射线衍射分析图;
图4为本申请实施例1涉及的CDs的红外光谱图;
图5为本申请实施例1涉及的CDs的X射线光电子能谱图;
图6为本申请实施例1涉及的CDs的合成条件优化图;
图7为本申请实施例1涉及的CDs在不同浓度盐溶液下的荧光光谱图(A)以及在最佳激发波长下用紫外灯持续照射90min的抗光漂白性荧光光谱图(B);
图8为本申请实施例涉及的CDs,CDs/Cr(VI),CDs/Cr(VI)/AA的紫外吸收光谱图和最佳荧光激发与发射图谱;
图9为本申请实施例1涉及的CDs在不同激发波长下(320-510nm)的荧光光谱图;
图10为本申请实施例3涉及的CDs对Cr(VI)和AA的检测条件优化图;
图11为本申请实施例3涉及的Cr(VI)的紫外吸收光谱图和最佳荧光激发与发射图谱;
图12为本申请实施例涉及的CDs,CDs/Cr(VI),CDs/Cr(VI)/AA的荧光寿命图谱;
图13为本申请实施例2涉及的CDs的荧光强度与不同浓度Cr(VI)的荧光发射光谱图;
图14为本申请实施例2涉及的不同浓度的Cr(VI)与CDs荧光强度的线性关系图;
图15为本申请实施例3涉及的CDs/Cr(VI)的荧光强度与不同浓度AA的荧光发射光谱图;
图16为本申请实施例3涉及的不同浓度的AA与CDs/Cr(VI)荧光强度的线性关系图;
图17为本申请实施例3涉及的不同金属阳离子和阴离子对CDs溶液荧光强度影响的对比图;
图18为本申请涉及的可以同时检测六价铬和抗坏血酸的荧光探针的制备及检测原理示意图。
具体实施方式
下面结合实施例详述本申请,但本申请并不局限于这些实施例。
除非另行定义,文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。本发明所使用的试剂或原料均可通过常规途径购买获得,如无特殊说明,本发明所使用的试剂或原料均按照本领域常规方式使用或者按照产品说明书使用。此外,任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。本专利中所述的较佳实施方法与材料仅作示范之用。
具体地,本申请中采用Tecnai G2 F30透射电镜(TEM)(美国FEI公司)分析CDs的形貌;X射线光电子能谱仪(XPS)(美国Thermo Fisher公司)对CDs的元素组成和含量进行了表征;D8-Focus X射线衍射仪(XRD)(德国Brook公司)分析了CDs的X射线衍射(XRD)谱。紫外可见吸收光谱(UV-Vis)在UV-5500PC分光光度计(中国上海元析仪器有限公司)上记录,傅里叶变换红外光谱(FT-IR)采用Nicolet iN10光谱仪(美国Thermo Fisher公司)进行记录,研究CDs的官能团信息。F97荧光分光光度计(中国上海棱光技术有限公司)用来记录荧光强度,最佳激发和发射波长为375/457nm,狭缝宽度均设置为10nm;荧光分光光度计的参数可设置为:扫描速度(1000nm/min),激发带宽(10nm),发射带宽(10nm),增益(中,650V)。
实施例1荧光探针的制备
(1)精密称量0.5g阿胶粉溶于10mL去离子水中,涡旋并超声使其充分溶解,得到混合溶液;
(2)将得到的混合溶液置于聚四氟乙烯内胆高压反应釜中,于200℃烘箱中反应12h,得到阿胶碳量子点溶液;
(3)阿胶碳量子点溶液冷却至室温后,转移到50ml离心管中用离心机以4200r/m的速度离心15min以除去一些大的固体杂质;
(4)取阿胶碳量子点溶液上清液并用0.22μm的滤膜进行过滤,滤除小的颗粒杂质,然后于-80℃真空冷冻干燥48h,即得阿胶碳量子点固体粉末。
(5)取适量粉末溶于超纯水中得到浓度为20mg/mL的荧光探针,并于4℃
下保存备用。
如图1所示,实施例1制备的CDs呈球形,具有良好的分散性和高结晶度。从图1右上角的插图也可以看出碳点晶格条纹的间距为0.24nm,与石墨烯的晶面间距相似。
如图2所示,实施例1制备的CDs的粒径主要分布在10nm范围内,平均粒径为6.15nm。
如图3所示,实施例1制备的CDs的XRD分析图谱,可以看出在20-30°之间有一个明显的较宽衍射峰,在2θ=22.9°的位置存在一个强衍射峰,与石墨晶格间距(002)相符合,表明实施例1制备的CDs含有石墨类似结构。
如图4所示,实施例1制备的CDs的红外光谱图,发现在3444.07cm-1处的吸收带强而宽,可能归因于O-H和N-H单键的伸缩振动。除此之外,1633.88cm-1处的红外吸收峰是由羰基C=O的伸缩振动引起的,1409.73cm-1的吸收带则来自C-O单键和C-NH的弯曲振动。在1139.74cm-1左右的小的特征峰可能来自于C-O-C的拉伸振动或C-OH的伸缩振动。因此推测结构中可能存在酰胺的结构,同时也证实了碳点表面含有大量亲水的氨基和羟基。
如图5所示,图5(A)是以291.7、404.7和535.8eV为中心的三个特征峰可分别归因于C1s、N 1s和O1s的结合能,表明实施例1制备的CDs中含C、N、O元素。图5(B)是C1s的高分辨率谱图,在284.7、285.9和287.9eV处呈现三个拟合峰,分别属于C-C/C=C、C-O和C=O,表明实施例1制备的CDs中有不饱和C=C双键的形成。而图5(C)中,530.6eV、531.0eV和532.8eV处的三个拟合峰分别对应于C-OH、C=O和-OH三个含氧官能团,证实了羰基的存在。图5(D)的N1s光谱在399.6eV和400.8eV处有两个吸收峰,分别代表C-N-C/C-NH和-NH键,上述结果也与FT-IR红外光谱的结果良好吻合。
如图6所示,图6(A)可以看出在160-200℃范围内,随着温度的增高,荧光强度逐渐增大,并在200℃时达到最大值。图6(B)则显示了不同合成时间的影响,发现荧光强度会随着时间的延长逐渐增加,而在反应9h之后基本无变化。因此我们选择最佳条件为在200℃的条件下反应9h。
如图7所示,本申请实施例1涉及的CDs在不同浓度盐溶液下的荧光光谱图7(A)显示出本申请制备的荧光探针在0.1-2mol·L-1盐溶液下均能保持稳定;以及图7(B)所示,在最佳激发波长下用紫外灯持续照射90min的抗光漂白性荧光强度几乎无变化。
如图9所示,为本申请实施例1涉及的CDs在不同激发波长下(320-510nm)的荧光光谱图。
实施例2Cr(VI)的测定
取实施例1中的CDs溶液200μL,向其中加入400μL不同浓度(1-400μM)的Cr(VI)溶液,用PBS缓冲液(pH=9)将溶液体积定容至3mL,室温下涡旋2min使其充分反应,测量并记录混合液的荧光强度。
如图13所示,Cr(VI)浓度在1-400μM范围内时,CDs的荧光强度会随着Cr(VI)浓度的增加而降低。
如图14所示,将Cr(VI)浓度与荧光强度进行线性拟合,结果发现荧光强度与Cr(VI)浓度在1-400μM范围内呈现良好的线性关系,回归方程为y=4.758[Cr(VI)]+4634,R2为0.9932,且根据公式3σ/k可以计算得出检测限低至0.08μM,可以用于实际样品中Cr(VI)的测定。
对于河水和自来水中Cr(VI)的测定,首先进行适当的预处理。将河水离心,用0.22μm的微孔滤膜进行过滤;自来水则煮沸15min以除去一些干扰物质,然后将二者用PBS缓冲液(pH=9)稀释100倍。将得到的实际样品溶液与CDs混合,室温下反应2min,测试其荧光强度。将该数值代入到上述得到的线性回归方程中,即可计算出实际样品中Cr(VI)的浓度。
实施例3AA的测定
将200μL实施例1中的CDs溶液与400μL浓度为400μM的Cr(VI)溶液混合,室温下涡旋2min使荧光强度猝灭至一定程度,从而形成CDs/Cr(VI)荧光探针。然后向其中加入1mL的AA溶液(1-400μM),并用PBS缓冲液(pH=9)定容体积至3mL,室温下涡旋2min使其充分反应,测量并记录荧光强度。
如图8所示为本申请实施例涉及的CDs,CDs/Cr(VI),CDs/Cr(VI)/AA的紫外吸收光谱图和最佳荧光激发与发射图谱,左上角的插图为CDs在日光与紫外灯下的溶液颜色对比图。
如图10所示,图10(A)优化了反应时间的影响,可以看出在加入Cr(VI)反应2min左右后,碳点的荧光强度发生了显著的猝灭,且在之后的20min内荧光强度基本保持不变,说明碳点与Cr(VI)反应迅速。在此体系中加入AA后,发现荧光强度可在2min内发生一定程度的恢复,且在之后的20min内也没有发生明显的变化,因此选择2min作为最佳反应时间。
图10(B)中,CDs/Cr(VI)以及CDs/Cr(VI)/AA体系在20-80℃范围内,荧光强度均没有发生明显的变化,说明对温度不敏感,因此我们选择在室温下进行反应。
此外,我们还研究了pH值的影响,如图10(C)所示,在pH值为9时,Cr(VI)对碳点的猝灭程度达到最低,而在CDs/Cr(VI)体系中加入AA后,发现荧光强度在pH值为4-10之间即可发生一定程度的恢复,因此我们选择PBS缓冲液(pH=9)来对溶液的酸碱度进行调节。
如图11所示,可以明显的看到Cr(VI)的吸收光谱与CDs的激发和发射光谱发生了重叠,说明二者之间可能存在内部过滤效应(IFE),从而诱导了猝灭现象的发生。
此外,还研究了CDs和CDs/Cr(VI)体系的荧光寿命以进一步探讨其猝灭机理。从图12可以看出,CDs在加入Cr(VI)前后,荧光寿命从6.43ns变为6.90ns,仅发生了微小的变化,呈现出静态猝灭的规律。而当AA加入到CDs/Cr(VI)体系中时,由于AA的强还原性,又可以通过将Cr(VI)变为Cr(III)而使荧光部分恢复。因此推断CDs和Cr(VI)之间的荧光猝灭是通过内滤效应的静态猝灭机制引起的。
从图15中可以看出,当AA浓度在0-400μM范围内时,CDs/Cr(VI)的荧光强度会随着抗坏血酸浓度的增加而降低。
将AA浓度与荧光强度进行线性拟合,从图16中可以发现荧光强度与AA浓度在0-400μM范围内具备良好的线性关系,回归方程为y=3.829[AA]+2683,R2为0.9984,检测限经计算可低至0.14μM,适用于实际样品中AA的测定。
我们对新鲜水果和维生素C片中的AA进行检测。首先将柠檬和橙子去皮切片,葡萄去皮,置于榨汁机中分别搅碎成汁。取10片维C片,用研钵将其研磨成均匀的粉末。称取0.2g粉末溶于20mL纯净水中,超声10min使其溶解。将上述得到的果汁和维生素C片溶液分别用离心机以4200r/m的速度离心20min,取上清液,用0.22μm的微孔滤膜进行过滤以除去其中的不溶物。测定前用PBS缓冲液(pH=9)稀释500倍,然后与CDs溶液混合,室温下反应2min并测试其荧光强度。将得到的数值代入到上述的线性回归方程中,即可计算出新鲜水果和维生素C片中AA的浓度。
如图17所示,还研究了不同金属阳离子(Zn2+,Ba2+,Al3+,Cd2+,Co2+,Cu2+,Mg2+,Mn2+,K+,Na+,Fe2+,Fe3+)和阴离子(HPO4 2-,H2PO4 -,CO3 2-,HCO3 -,SO4 2-,NO2 -,NO3 -)对碳点荧光强度的影响。首先将浓度为1M的各种阳离子和阴离子分别添加到碳点溶液中,发现在375nm最佳激发波长下,只有在Cr(VI)存在时,荧光强度才发生了显著的荧光猝灭。然而从图中可以看出,Fe3+也具有一定的猝灭效果,但效果不如Cr(VI)灵敏,说明该荧光探针对Cr(VI)的选择性较好。为了避免实际测定过程中Fe3+可能存在的影响,使测定结果更加准确,我们向其中添加掩蔽剂焦磷酸或NaF,发现都能够能够很好的将其掩蔽。
如图18所示为本申请涉及的可以同时检测六价铬和抗坏血酸的荧光探针的制备及检测原理示意图。
以上所述,仅为本申请的实施例而已,本申请的保护范围并不受这些具体实施例的限制,而是由本申请的权利要求书来确定。对于本领域技术人员来说,本申请可以有各种更改和变化。凡在本申请的技术思想和原理之内所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
Claims (10)
1.一种检测六价铬和抗坏血酸的荧光探针的制备方法,其特征在于,包括以下步骤:
(1)称取阿胶粉溶于去离子水,涡旋并超声使其充分溶解,得到混合溶液;
(2)将步骤(1)得到的混合溶液置于高压反应釜中,加热反应,得到阿胶碳量子点溶液;
(3)将步骤(2)得到的阿胶碳量子点溶液冷却至室温,转移到离心管中,离心,得到阿胶碳量子点溶液上清液;
(4)取步骤(3)得到的阿胶碳量子点溶液上清液,过滤,在真空下冷冻干燥得到阿胶碳量子点固体粉末;
(5)取适量阿胶碳量子点固体粉末溶于超纯水,得到所述荧光探针,保存备用。
2.根据权利要求1所述的制备方法,其特征在于,步骤(1)中,所述阿胶粉与去离子水的用量比为(0.5~0.75)g:(10~15)mL。
3.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,反应温度为160~200℃,反应时间为5~12h。
4.根据权利要求1所述的制备方法,其特征在于,步骤(3)中离心为用离心机以4000~4200r/m的速度离心10~15min。
5.根据权利要求1所述的制备方法,其特征在于,步骤(2)中,高压反应釜为聚四氟乙烯内胆高压反应釜;步骤(4)中过滤采用0.22μm滤膜。
6.根据权利要求1所述的制备方法,其特征在于,所述冷冻干燥的温度为-70~-80℃,冷冻干燥的时间为24~48h。
7.根据权利要求1所述的制备方法,其特征在于,所述荧光探针的浓度为20~50mg/mL。
8.一种检测六价铬和抗坏血酸的荧光探针,其特征在于,采用权利要求1~7任一项所述制备方法制得。
9.权利要求8所述的荧光探针在检测水环境中的六价铬含量方面的应用。
10.权利要求8所述的荧光探针在检测新鲜水果和维生素C片中的抗坏血酸含量方面的应用。
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