CN113310960B - 硫量子点的合成方法及基于硫量子点测定Fe2+和H2O2的方法 - Google Patents
硫量子点的合成方法及基于硫量子点测定Fe2+和H2O2的方法 Download PDFInfo
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Abstract
本发明一种硫量子点的合成方法及基于硫量子点测定Fe2+和H2O2的方法，解决了现有技术中利用单一信号检测离子浓度总是受到外部因素的影响，如背景干扰和材料特性，导致检测结果误差大等问题。本发明利用比色荧光双信号检测水溶液中的铁(II)和H2O2浓度，可以实现结果的自校正，具有灵敏度高，选择性好，回收率令人满意，相对偏差较低等特点。
Description
技术领域
本发明属于分析检测技术领域，具体涉及基于硫量子点的比色荧光双信号测定食品中的铁(II)和H2O2的方法。
背景技术
铁(Fe)是人体中不可缺少的微量元素，其在生物过程如细胞死亡、细胞代谢、酶催化中有着不可或缺的地位。但是人体内铁含量失衡会引起各种疾病，如铁含量太少会引起贫血，太高会导致帕金森氏病、阿尔茨海默氏病、心肌损伤等疾病。食物和饮用水是人体内铁摄入的主要来源，在人体内活性铁主要以Fe2+形式存在，其含量过多时会由于芬顿反应造成大量活性氧(ROS)积累和脂质过氧化，从而诱导细胞死亡，导致神经退行性疾病产生。因此，能准确、定量地检测人体内Fe2+摄入量对于预防疾病具有重要意义。
H2O2是一种强氧化剂，常常作为漂白剂和消毒剂用于食品、工业、医疗等行业，如印染工业中棉织物漂白、食品和饮料中包装袋的消毒等。然而，人体内H2O2过量会引起氧化应激，从而导致心血管疾病、癌症和神经退行性疾病等疾病。因此有必要研制一种灵敏度高、选择性强的H2O2检测探针。
目前，已有学者报道了对Fe2+和H2O2的各种检测方法，如电化学法、化学发光、光谱法、色谱法等，但是这些方法操作繁琐、成本高、选择性低等缺陷，限制了其实际应用。而荧光法和比色法具有操作简单、响应迅速、灵敏度高、选择性好的优点而被广泛应用。然而大多数报道都基于单信号构建的检测体系，如Chen等设计了Fe2+的比色检测方法基于 EAR–AuNPs线性范围10-500μM，检测线1.5μM。Pezhhan等基于AuNPs的葡萄糖氧化酶的催化活性，用荧光法检测H2O2，线性范围在5-135μM，检测线为3.6μM，然而多种因素会严重影响单信号读取,使得结果和真实值总是有一定差距。荧光法或比色法测定总是受到外部因素的影响，如背景干扰和材料特性。近年来，荧光/比色双信号传感器因其抗干扰能力强而备受关注。
目前，大多数胶体量子点因其含有重金属离子(如CdSe、CdTe等)，使得它们的实际应用被限制。因此，具有溶解度好、毒性低、光致发光稳定等优点的纯元素量子点被提出，如基于Si、C、S的纳米材料。近年来，因S作为丰富的自然资源及其自身的抗菌性，吸引了广大学者的注意力。目前为止，已有SQDs在细胞成像、光催化、LED、传感方面应用的报道。Qiao和他的同事报告了SQD在细胞成像中的应用。将宫颈癌细胞(He La癌细胞) 与SQD孵育后，通过共聚焦激光扫描显微镜(CLSM)对细胞成像，发现有强蓝色荧光在 He La癌细胞的CLSM图像中出现，证实了SQDs极低的细胞毒性。Li等研究了利用SQD 使TiO2纳米粒子敏化以增强其光催化性能。作者发现，SQD与TiO2纳米颗粒的组合可以产生具有高氢放出反应性的复合光催化剂。Wang的小组报告说，第一次使用SQD来制造LED。通过将蓝色发光的SQD和橙色发光的铜纳米晶体(NC)与UV-LED芯片集成在一起，成功制备了白色LED。Tan等人报道了SQDs作荧光传感探针检测Cr(VI)和抗坏血酸(AA)， Cr(VI)可以通过荧光内滤效应(IFE)淬灭SQDs荧光强度，而AA可以将Cr(VI)还原为Cr(III) 从而恢复荧光强度。总之，还没有报道关于同时检测Fe2+和H2O2的SQDs传感探针。
1、Chen文献出处：Chen,X.,Ji,J.,Shi,G.,Xue,Z.,Zhou,X.,Zhao,L.,&Feng,S.(2020). Formononetin in Radix Hedysari extract-mediated green synthesis ofgold nanoparticles for colorimetric detection of ferrous ions in tapwater.Rsc Advances,10(54),32897-32905.
2、Pezhhan文献出处：Pezhhan,H.,Akhond,M.,&Shamsipur,M.(2020).Histidinecapped-gold nanoclusters mediated fluorescence detection of glucose andhydrogen peroxide based on glucose oxidase-mimicking property of goldnanoparticles via an inner filter effect mechanism.Journal of Luminescence,228.
3、Qiao文献出处：Qiao,G.,Liu,L.,Hao,X.,Zheng,J.,Liu,W.,Gao,J.,Zhang,C.C.,&Wang, Q.(2020).Signal transduction from small particles:Sulfur nanodotsfeaturing mercury sensing, cell entry mechanism and in vitro trackingperformance.Chemical Engineering Journal,382, 122907.
4、Li文献出处：Li,S.,Chen,D.,Zheng,F.,Zhou,H.,Jiang,S.,&Wu,Y.(2014).Water-Soluble and Lowly Toxic Sulphur Quantum Dots.Advanced FunctionalMaterials,24(45),7133-7138.
5、Wang文献出处：Wang,H.,Wang,Z.,Xiong,Y.,Kershaw,S.V.,Li,T.,Wang,Y.,Zhai,Y., &Rogach,A.L.(2019).Hydrogen Peroxide Assisted Synthesis of HighlyLuminescent Sulfur Quantum Dots.AngewandteChemie International Edition,58(21),7040-7044.
6、Tan文献出处：Tan,Q.,An,X.,Pan,S.,Liu,H.,&Hu,X.(2021).Hydrogenperoxide assisted synthesis of sulfur quantum dots for the detection ofchromium(VI)and ascorbic acid. Spectrochimicaacta.Part A,Molecular andbiomolecular spectroscopy,247,119122-119122.
发明内容
本发明的目的是针对现有技术的问题而提供一种SQDs的合成方法.
本发明的目的是这样实现的:一种用做荧光探针同时检测Fe+2和H2O2的硫量子点(SQDs) 的合成方法，步骤如下：先将1.4g升华硫、4.0g氢氧化钠、3mL聚乙二醇-200和50ml蒸馏水混合，在70℃下回流4小时得A液；A液与2％的H2O2等体积混合进一步氧化刻蚀得到B液；然后将B液用4MNaOH调pH至7，在转速为3500rpm下离心5min后留下上清液，即得到SQDs。
本发明的另一目的提供一种抗干扰能力强，选择性好，灵敏度高，检阅结果准确的SQDs 的比色荧光双信号测定食品中的Fe2+和H2O2的方法。
本发明的另一目的是这样实现的：一种基于SQDs的荧光双信号测定食品中的Fe2+和 H2O2的方法，按照以下步骤进行：检测水溶液中的铁Fe2+和H2O2浓度的体系中，最佳反应条件的确定；A.SQDs/Fe2+体系分别在不同温度条件下反应一定时间，在450nm和612nm 处分别测定反应液荧光强度和吸光度，确定荧光、比色检测Fe2+最佳反应温度为25℃；B.SQDs/Fe2+体系在最佳反应温度条件下，每隔1min在450nm和612nm处分别测定反应液荧光强度和吸光度，确定荧光、比色检测Fe2+最佳反应时间为4分钟；C.SQDs/Fe2+/H2O2在最佳反应温度条件下，每隔2min在450nm和612nm处分别测定反应液荧光强度和吸光度，确定荧光、比色检测H2O2最佳反应时间为6分钟；SQDs双信号检测Fe2+和H2O2的选择性在最佳反应条件下，加入一定浓度的Fe2+、食品中的干扰物(包括Na+，Ag+，K+，NH4 +， Hg2+，Cu2+，Zn2+，Cd2 +，Ca2+，Mn2+，Mg2+，NO3 -，HCO3 -，CO3 2-和SO4 2-)，然后分别在450nm和612nm处测定反应液荧光强度和吸光度。在25℃条件下，加入一定浓度的Fe2+ 反应4分钟后，加入一定浓度H2O2、食品中的干扰物(包括赖氨酸Lys，甲硫氨酸Met，丝氨酸Ser，天冬氨酸Asn，甘氨酸Gly，谷氨酰胺Gln，精氨酸Arg，Na+，K+，NH4 +，Zn2+， Ca2+，Mn2+，Mg2+和Cu2+)，反应6分钟后，分别在450nm和612nm处测定反应液荧光强度和吸光度；检测Fe2+并建立Fe2+标准曲线，在最佳反应条件下，SQDs加入不同浓度的 Fe2+，在450nm或612nm下测定反应液荧光强度F或紫外吸光度A；以Fe2+浓度为横坐标，荧光强度F或紫外吸光度A为纵坐标，得到Fe2+标准曲线；检测H2O2并建立H2O2标准曲线在最佳反应条件下，加入不同浓度的H2O2，在450nm(或612nm)下测定反应液荧光强度F (或紫外吸光度A)。以H2O2浓度为横坐标，荧光强度F(或紫外吸光度A)为纵坐标，得到H2O2标准曲线(图12-15)。
与现有技术相比，本发明的有益效果是：
(1)本发明提供的SQDs可比色荧光双信号检测水溶液中的Fe2+和H2O2的浓度
(2)本发明提供的双信号检测方法与单一信号检测离子浓度相比，解决了单一信号总是受到外部因素的影响，如背景干扰和材料特性，导致检测结果误差大等问题，本发明双信号测定系统可以实现结果的自校正以及抗干扰能力强，因此比单信号测定结果更准确，本发明具有抗干扰能力强，选择性好，灵敏度高，检测结果准确等特点。
附图说明
图1为反应体系的温度优化
图2为SQDs双信号检测Fe2+的时间优化
图3为SQDs双信号检测H2O2的时间优化
图4为SQDs双信号检测Fe2+荧光选择性
图5为SQDs双信号检测Fe2+紫外光选择性
图6为SQDs双信号检测H2O2荧光选择性
图7为SQDs双信号检测H2O2紫外选择性
图8为SQDs探针荧光法检测Fe2+波普图
图9为SQDs探针荧光法检测Fe2+线性图
图10为SQDs探针比色法检测Fe2+波普图
图11为SQDs探针比色法检测Fe2+线性图
图12为SQDs探针荧光法检测H2O2波普图
图13为SQDs探针荧光法检测H2O2线性图
图14为SQDs探针比色法检测H2O2波普图
图15为SQDs探针比色法检测H2O2线性图
图16为SQDs的HRTEM图
图17为SQDs的粒径分布图
图18为SQDs的光学性质图
图19为SQDs的激发依赖性图
图20为SQDs的XPS谱图
图21为S2p的高分辨谱图
图22为SQDs的FTIR谱图
具体实施方式
下面结合实施例对本发明做详细说明，实施例给出了详细的实施方案和具体操作过程，但本发明的保护范围不限于下述的实施例。
实验药品与仪器设备
PEG-200，升华硫，NaOH，FeSO4等试剂购自成都科龙有限公司。H2O2购自成都金山化学试剂有限公司。荧光分光光度计(日立，F-4500，日本东京)紫外可见分光光度计(上海仪仪仪器有限公司，上海A390)分别用于测量系统的紫外可见光和荧光光谱。SQD的表征参数(形态，尺寸等)是通过高分辨率透射电子显微镜(HRTEM)(日本，JEOL 2100F)获得的。通过ESCALAB 250Xi 光电子能谱仪(Thermo Scientific，美国)测量XPS光谱，以了解元素组成。FTIR 光谱是通过傅立叶变换红外光谱仪(FTIR-8400S，Shimadzu，Japan)获得的以鉴定官能团。
实施例1
硫量子点(SQDs)的合成方法
(1)先将1.4g升华硫、4.0g氢氧化钠、3mL聚乙二醇-200在70℃下回流4小时得A液。
(2)A液与2％的H2O2等体积混合进一步氧化刻蚀得到B液。
(3)然后将B液用NaOH(4M)调pH至7，在转速为3500rpm下离心5min后留下上清液，即得到SQDs。
本方法合成的SQDs呈球形且分散良好，晶格条纹间距为0.28nm。SQDs的平均直径为4.09nm(图16-图17)。从图18可以看出SQDs的光学性质，在370nm的激发下，发出450nm 的蓝色荧光，从紫外可见吸收光谱来看，SQDs在200nm-400nm波段有强的吸收峰，241nm 处的强吸收是由于电子的n→σ*跃迁，304和382nm的吸收带可能是由于S2 2-和S8 2-。SQDs 的激发波长从340nm到420nm变化时，发射波长从450nm到510nm变化(图19)，表明 SQDs因粒径分布不同而具有激发依赖性。用x射线光电子能谱(XPS)揭示了SQDs的元素和化学组成。图20结果表明SQDs主要由C、O、S组成。由S2p的高分辨XPS光谱可知， S2p包含161.7eV、162.8eV、163.5eV、166.0eV、167.9eV、169.1eV五个信号(图21)。其中161.7eV、162.8eV的结合能分别由S2-(2p 2/3)和S2-(2p 1/2)组成，163.5eV处的峰值可以归因于原子硫的存在，166.0eV处的峰值可以被认为是SO3 2-，167.9eV、169.1eV的结合能可以归因于SO4 2-(2p 2/3)和SO4 2-(2p1/2)。结果表明，SQDs表面附着大量的亚硫酸盐与硫酸盐。SQDs的FTIR光谱如图22所示，在3420cm-1和1644cm-1吸收带分别是OH的伸缩振动和H2O的弯曲振动，2917cm-1、1464cm-1、1353cm-1、1117cm-1、938cm-1分别是 PEG的特征峰。1025cm-1处的吸收带是C-O的伸缩振动，670cm-1处的为S-O弯曲振动的吸收带，583cm-1处为S-S的伸缩振动。
实施例2
(1)用SQDs检测Fe2+并建立Fe2+标准曲线
在最佳反应条件下，SQDs中加入一定浓度梯度的Fe2+，反应一定时间，450nm(或612nm) 下测定反应液荧光强度F(或紫外吸光度A)。以Fe2+浓度为横坐标，荧光强度F(或紫外吸光度A)为纵坐标，得到Fe2+标准曲线(图3)。
(2)检测H2O2并建立H2O2标准曲线
在最佳反应条件下，SQDs/Fe2+体系中加入一定浓度梯度的H2O2，反应一定时间，450nm(或 612nm)下测定反应液荧光强度F(或紫外吸光度A)。以H2O2浓度为横坐标，荧光强度F(或紫外吸光度A)为纵坐标，得到H2O2标准曲线
实施例1、2中，最佳反应条件为：温度25℃，Fe2+和H2O2反应时间分别为4分钟和6分钟。
实施例3
为了评估本发明实施例提供的检测Fe2+和H2O2方法的实用性，对实际食品样品中Fe2+ 和H2O2进行了加标回收。本发明实施例在各参数最优条件下，检测了牛奶、纯净水、蜂蜜中的浓度。
除牛奶的所有实样用0.22μm水系针孔滤膜过滤后分析。牛奶处理方法如下，将牛奶与5％的三氯乙酸以1：1的体积比混合，涡旋10min后，在10000rpm下离心10min，取上清液，然后将pH调至7，用0.22μm水系针孔滤膜过滤后分析。
将不同浓度的Fe2+(或H2O2)加入到上述上清液中得到待测液。采用本发明实施例提供的检测Fe2+(或H2O2)的方法，分别扫描荧光光谱和吸收光谱。根据检测出的荧光强度和吸光度，并结合实施例2中的标准曲线拟合出的线性方程，计算出实际食品样品中的Fe2+(或H2O2)浓度，检测结果如表1和表2所示。
表1实际样品中Fe2+的测定
表2实际样品中H2O2的测定
注：1，2代表不同类别
表1和表2列出了加标回收法测定Fe2+和H2O2的结果。与国家标准结果相比，荧光法测定Fe2+ 的回收率在96.54％至104.65％之间，通过比色法测定的回收率在95.53％至104.96％之间(表1)。用荧光法和比色法测定过氧化氢的回收率分别在95.31％至104.37％和95.21％至104.81％之间(表2)。以上结果表明，该方法可用于实际样品中Fe2+和H2O2的测定。
确定最佳反应条件
反应的温度和时间对结果有很大的影响。在不同的温度下，在SQD中添加500μM的Fe2+进行一段时间的反应，发现混合物在25℃时反应最完全，这在图1中25℃时的最高吸光度得到了证实。然后，在25℃下，Fe2+和SQDs的混合体系(SQD/Fe2+)反应不同的时间。当反应时间为4-8分钟时，吸光度达到最高(图2)。在SQD/Fe2+中加入H2O2后，吸光度随反应时间而降低，并在6分钟后几乎保持不变(图3)。因此，将25℃的温度，Fe2+的反应时间为4分钟，H2O2的反应时间为6分钟作为最佳测定条件。
本发明基于SQDs的双信号方法，用于测定水溶液中的铁(II)(Fe2+)和H2O2。由于Fe2+ 与硫上的孤电子对络合，Fe2+可以引起SQD的荧光猝灭，混合溶液的颜色从浅黄色变为深绿色。通过 Fenton反应，H2O2可以恢复SQD的猝灭荧光，混合物的颜色从绿色变为无色。Fe2+的浓度与荧光强度和吸光度分别在2.5-55μM和1.25-500μM范围内具有良好的线性关系。荧光法和比色法的检测限分别为1.41μM和0.54μM。为了测定H2O2，使用这种荧光法和比色法，线性范围分别为1.17mM至 1.97mM和0.867mM至1.50mM，检出限分别为0.03μM和0.06μM。抗干扰测试表明，该双信号方法对测定Fe2+和H2O2具有良好的选择性。此外，还进行了不同矿泉水，纯牛奶和蜂蜜中Fe2+和H2O2的测定。结果表明，该SQDs作为快速，灵敏和选择性测定Fe2+和H2O2的传感器具有巨大的潜力。
Claims (1)
1.一种基于硫量子点SQDs的比色荧光双信号测定食品中的Fe2+和H2O2的方法，其特征在于，按照以下步骤进行：
（1）检测水溶液中的铁Fe2+和H2O2浓度的体系中，最佳反应条件的确定；
A.SQDs/Fe2+体系分别在不同温度条件下反应一定时间，在450nm和612nm处分别测定反应液荧光强度和吸光度，确定荧光、比色检测Fe2+最佳反应温度为25℃；
B.SQDs/Fe2+体系在最佳反应温度条件下，每隔1min在450nm和612nm处分别测定反应液荧光强度和吸光度，确定荧光、比色检测Fe2+最佳反应时间为4分钟；
C.SQDs/Fe2+/H2O2在最佳反应温度条件下，每隔2min在450nm和612nm处分别测定反应液荧光强度和吸光度，确定荧光、比色检测H2O2最佳反应时间为6分钟；
（2）SQDs双信号检测Fe2+和H2O2的选择性
在最佳反应条件下，加入一定浓度的Fe2+、食品中的干扰物，包括Na+，Ag+，K+，NH4 +，Hg2+，Cu2+，Zn2+，Cd2+，Ca2+，Mn2+，Mg2+，NO3 -，HCO3 -，CO3 2-和SO4 2-，然后分别在450nm和612nm处测定反应液荧光强度和吸光度；在25℃条件下，加入一定浓度的Fe2+反应4分钟后，加入一定浓度H2O2、食品中的干扰物，包括赖氨酸Lys，甲硫氨酸Met，丝氨酸Ser，天冬氨酸Asn，甘氨酸Gly，谷氨酰胺Gln，精氨酸Arg，Na+，K+，NH4 +，Zn2+，Ca2+，Mn2+，Mg2+和Cu2+，反应6分钟后，分别在450nm和612nm处测定反应液荧光强度和吸光度；
（3）检测Fe2+并建立Fe2+标准曲线
在最佳反应条件下，SQDs加入不同浓度的Fe2+，在450nm或612nm下测定反应液荧光强度F或紫外吸光度A；以Fe2+浓度为横坐标，荧光强度F或紫外吸光度A为纵坐标，得到Fe2+标准曲线；
（4）检测H2O2并建立H2O2标准曲线
在最佳反应条件下，加入不同浓度的H2O2，在450nm或612nm下测定反应液荧光强度F或紫外吸光度；以H2O2浓度为横坐标，荧光强度F或紫外吸光度A为纵坐标，得到H2O2标准曲线；
上述硫量子点SQDs的合成方法，如下：
1）先将1.4 g升华硫、4.0 g氢氧化钠、3 mL聚乙二醇-200和50ml蒸馏水混合，在70℃下回流4小时得A液；
2）A液与2%的H2O2等体积混合进一步氧化刻蚀得到B液；
3）然后将B液用4 MNaOH调pH至7，在转速为3500 rpm下离心5 min后留下上清液，即得到SQDs。
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