CN111189813B - 一种以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测gsh的方法 - Google Patents
一种以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测gsh的方法 Download PDFInfo
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Abstract
本发明属于荧光检测技术领域,具体公开了一种以戊二醛‑壳聚糖非共轭荧光聚合物为荧光探针检测GSH的方法,所述方法以戊二醛‑壳聚糖非共轭荧光聚合物(GCPF)为荧光探针,以MnO2纳米片为荧光淬灭剂来检测谷胱甘肽(GSH)。本发明可用于定量测定GSH,线性范围为0.5~50μM,最低检测限为(LOD)为84nM,其他离子和生物分子对GSH的检测无干扰,选择性高。将本方法用于检测血清样品中的GSH,其回收率为101.48~103.23%,在生物分析中的具有较好的准确性。
Description
技术领域
本发明涉及荧光检测技术领域,特别是涉及一种以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测GSH的方法。
背景技术
谷胱甘肽(GSH)是哺乳动物细胞中含量最丰富的非蛋白质硫醇,是一种关键的水溶性硫醇化三肽。GSH是至关重要的内源性抗氧化剂,在各种生物过程(例如代谢排毒和调节氧化还原稳态)中起着核心作用。更重要的是,细胞里GSH的水平异常已被证明与多种疾病直接相关,例如癌症、心脏病、肝病和阿尔茨海默氏病。由于其具有生物学意义,因此建立GSH的检测方法用于健康评估和疾病诊断已成为一个重要的研究课题。
目前测定GSH的技术有很多,例如高效液相色谱(HPLC)、表面增强拉曼散射、电化学、电致发光(ECL)、比色法分析、磁共振波谱、荧光光谱和光声成像。在这些方法中,荧光法具有响应时间快、操作简单、成本低、灵敏度高和分析特异性高等优点。然而,已报道的大多数荧光探针存在选择性低、敏感性低、荧光稳定性差、受其他离子或生物小分子的干扰性较大等问题,也有些荧光探针合成方法复杂,还有些荧光探针具有高毒性,容易造成环境二次污染,因此现有的荧光探针大多并不能用于实际应用。
因此,探索一种具有简便的合成方法且良好的水溶性和高荧光性质的荧光探针,将其应用在谷胱甘肽的检测中是十分重要的。
然而,已报道的小分子GSH荧光探针存在选择性不专一、难以响应GSH动态变化、难以检测低浓度GSH、灵敏度易受生物体系复杂环境影响等缺点。这是由于这些反应型荧光探针与GSH之间发生的是不可逆反应,并且反应速度依赖于探针和GSH的高浓度和单一合适的反应条件。同时,生物体内还富含其他含巯基的生物分子,如半胱氨酸(Cys)和高半胱氨酸(Hcy),它们的结构与GSH相似,含有的巯基同样能够与探针分子发生相似的反应,使得探针表现为对GSH识别的不专一。因此,开发能够在复杂环境的生物体系中真正专一识别GSH,并且具有宽的GSH动态响应窗口的荧光探针成为当前的迫切需要。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供一种以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测GSH的方法,用于解决现有技术中用于检测GSH的荧光探针选择性低、敏感性低、荧光稳定性差、受其他离子或生物小分子的干扰性较大等问题。
为实现上述目的及其他相关目的,本发明第一方面提供一种以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测GSH的方法,所述方法以戊二醛-壳聚糖非共轭荧光聚合物(GCPF)为荧光探针,以MnO2纳米片为荧光淬灭剂来检测谷胱甘肽(GSH)。
进一步,所述戊二醛-壳聚糖非共轭荧光聚合物(GCPF)的制备方法为:在室温下,将壳聚糖溶于水中制得壳聚糖水溶液,然后向壳聚糖水溶液逐滴加入冰醋酸(HAc)溶液,搅拌10~20min,再加入戊二醛溶液,搅拌反应5~8h后,获得黄色透明的荧光GCPF溶液。
可选地,所述壳聚糖水溶液的浓度为0.1~0.2g/L,所述戊二醛溶液的浓度为4.0~5.0M,戊二醛溶液的加入量为0.10~0.20mL。
可选地,所述冰醋酸(HAc)溶液的浓度为1.5~2.0M,所述冰醋酸(HAc)溶液的加入量为1~2mL。
进一步,所述MnO2纳米片的制备方法为:将MnCl2·4H2O水溶液添加到四甲基氢氧化铵(TMA·OH)和H2O2的混合溶液中,在室温下搅拌反应6~12h,通过离心获得MnO2纳米片。
可选地,所述MnCl2·4H2O水溶液的浓度为0.2~0.4M,MnCl2·4H2O水溶液的用量为7.5~15mL;所述混合溶液中四甲基氢氧化铵(TMA·OH)的浓度为0.5~0.8M,所述混合溶液中H2O2的浓度为3wt%,所述混合溶液的用量为15~30mL。
进一步,所述方法对GSH的检测范围为0.5~50μM,最低检测限(LOD)为84nM。
进一步,检测GSH时,GCPF浓度为5~50M,MnO2纳米片浓度为0.005~0.5mg/mL,GCPF与MnO2纳米片的体积用量比为1:1;优选地,GCPF浓度为50M,MnO2纳米片浓度为0.05mg/mL。
进一步,检测GSH时,检测体系的pH为4.0~7.0,优选为6.0;温度为10~30℃,优选为25℃。
本发明第二方面提供一种用于检测GSH的荧光探针的制备方法,所述荧光探针为戊二醛-壳聚糖非共轭荧光聚合物(GCPF),其制备方法为:在室温下,将壳聚糖溶于水中制得壳聚糖水溶液,然后向壳聚糖水溶液逐滴加入冰醋酸(HAc)溶液,搅拌10~20min,再加入戊二醛溶液,搅拌反应5~8h后,获得黄色透明的荧光GCPF溶液。
可选地,所述壳聚糖水溶液的浓度为0.1~0.2g/L,所述戊二醛溶液的浓度为4.0~5.0M,戊二醛溶液的加入量为0.10~0.20mL。
可选地,所述冰醋酸(HAc)溶液的浓度为1.5~2.0M,所述冰醋酸(HAc)溶液的加入量为1~2mL。
进一步,所述戊二醛-壳聚糖非共轭荧光聚合物(GCPF)与MnO2纳米片构成GCPF-MnO2,用于检测GSH。
进一步,检测GSH时,GCPF浓度为5~50M,MnO2纳米片浓度为0.005~0.5mg/mL,GCPF与MnO2纳米片的体积用量比为1:1;优选地,GCPF浓度为50M,MnO2纳米片浓度为0.05mg/mL。
进一步,检测GSH时,检测体系的pH为4.0~7.0,优选为6.0;温度为10-30℃,优选为25℃。
本发明第三方面提供一种采用上述制备方法制得的用于检测GSH的荧光探针。
进一步,所述荧光探针在MnO2纳米片存在下选择性地检测GSH。
本发明第四方面提供上述荧光探针在检测GSH上的应用。
进一步,所述荧光探针对GSH的检测范围为0.5~50μM,最低检测限(LOD)为84nM。
如上所述,本发明的以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测GSH的方法,具有以下有益效果:
本发明基于荧光共振能量转移(FRET)的原理,开发了一种以GCPF为探针,基于MnO2纳米片与GCPF之间的FRET构建的荧光增强法传感平台,可以用于定量测定GSH,线性范围为0.5~50μM,最低检测限为(LOD)为84nM,其他离子和生物分子(例如L-赖氨酸、L-苏氨酸、L-缬氨酸、L-谷氨酸和DL-天冬氨酸)对GSH的检测无干扰,表明本方法对GSH的检测具有较高的选择性。将本方法用于检测血清样品中的GSH,其回收率为101.48~103.23%,表明本方法在生物分析中的具有比较好的准确性。
在这个FRET模式中,GCPF作为能量给体,MnO2纳米片作为能量受体。FRET发生后,MnO2纳米片有效地猝灭了GCPF的荧光。当GSH将MnO2纳米片还原成Mn2+离子后,抑制了FRET的发生,使得被猝灭的荧光强度再次增强。本方法中纳米颗粒制备简单、GCPF荧光性能稳定,对GSH的检测具有快速的荧光响应,在未来生物分析中有较好的应用前景。
附图说明
图1显示为本发明检测谷胱甘肽(GSH)的演示原理图。
图2显示为本发明实施例2中GCPF的SEM图(A)和MnO2纳米片的TEM图(B)。
图3显示为本发明实施例2的荧光光谱表征图。图4显示为本发明实施例3中不同含量的MnO2纳米片对GSH检测的影响结果图。
图5显示为本发明实施例3中不同pH对荧光增强法检测GSH的影响结果图。
图6显示为本发明实施例3中不同温度对荧光增强法检测GSH的影响结果图。
图7显示为本发明实施例3中以GCPF为探针荧光增强法检测GSH的动力学行为结果图。
图8显示为本发明实施例4中不同浓度的GSH的条件下体系的荧光光谱图(A)和线性工作曲线图(B)。
图9显示为本发明实施例4中荧光增强法检测GSH的选择性柱状图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
作为一类新型的荧光团,非共轭荧光聚合物及非共轭荧光纳米材料具有优异荧光和光电特性,目前已广泛应用在金属检测、爆炸物质检测、蛋白质传感或细胞成像等领域。本发明采用氨基与羰基化合物的席夫碱反应,将壳聚糖与甲醛和戊二醛(GA)发生交联来合成一种席夫碱类非共轭荧光聚合物,即戊二醛-壳聚糖非共轭荧光聚合物(GCPF),GCPF在分析中具有稳定的荧光,良好的水溶性,高的选择性和灵敏度。
MnO2纳米片是一种超薄半导体材料,具有许多独特的性能。首先,MnO2纳米片具有从250nm到600nm的宽吸收光谱,吸收主峰在374nm,这使得它是一个很好的荧光共振能量转移中的猝灭剂。其次,由于其具有一定的氧化能力,MnO2纳米片可以被还原剂GSH分解为Mn2+离子,从而导致MnO2纳米片的分解。
因此,本发明将壳聚糖和GA在室温下搅拌6h即合成了水溶性GCPF。由于MnO2纳米片的吸收光谱与荧光GCPF的荧光光谱重叠,MnO2纳米片可通过FRET淬灭GCPF的荧光强度,从而构建了GCPF-MnO2的新型的FRET模式。之后,通过GSH将MnO2纳米片转化为Mn2+离子,可以使荧光信号恢复。因此,在GSH存在下,荧光强度再次得以增强。
基于上述原理,本发明中设计了一种以戊二醛-壳聚糖非共轭荧光聚合物(GCPF)和MnO2纳米片构成的荧光传感平台,通过荧光共振能量转移(FRET),能对GSH进行选择性检测(原理如图1所示)。
以下实施例中运用的材料和试剂来源如下:
壳聚糖(脱乙酰度≥95%,结晶度指数>0.57,100–200mPa·s)和戊二醛(GA)水溶液(50%)从阿拉丁试剂公司购买。氯化锰,五水合四甲基氢氧化铵和谷胱甘肽(还原型)购自Sigma-Aldrich(中国北京)。L-缬氨酸,L-苏氨酸,L-谷氨酸,DL-天冬氨酸和L-赖氨酸购自国药控股化学试剂公司(中国上海)。冰醋酸购自成都科龙化学试剂有限公司(中国成都)。0.2M HAc-NaAc缓冲溶液控制溶液的pH。所有实验中所有用水均是超纯水。
以下实施例中运用的仪器如下:
荧光光谱在Perkin Elmer LS55发光光谱仪(Perkin Elmer Instruments,U.K.)上记录。紫外-可见(UV-vis)吸收光谱采用UV2550紫外-可见分光光度计(日本岛津市)记录。透射电子显微镜(TEM)图像是在JEM-2100F透射电子显微镜(JEOL,日本)上获得的。使用来自巩义市雨花仪器有限公司(中国巩义)的ZF-20D紫外分析设备在365nm紫外线下拍摄荧光样品照片。S-25数字式pH计(上海精科工业公司,中国上海)用于检测溶液的酸度。
具体实施过程如下:
实施例1
荧光GCPF、MnO2纳米片的制备及GSH的荧光检测
1、荧光GCPF的制备
首先,在室温下,在剧烈搅拌下,将1.00g壳聚糖溶解于100.0mL水;其次,逐滴加入1mL的1.74M冰醋酸(HAc)溶液,并持续搅拌10分钟;然后快速加入0.10mL的4.73M戊二醛;持续搅拌6h后,获得黄色透明的荧光GCPF溶液。
2、MnO2纳米片的制备
首先,在15秒内将10mL的0.3M MnCl2·4H2O水溶液添加到20mL的0.6M四甲基氢氧化铵(TMA·OH)和3wt%H2O2的混合溶液中;然后在室温下持续恒定搅拌过夜后,通过离心获得MnO2纳米片;将所得MnO2纳米片MnO2纳米片用超纯水和甲醇洗涤三次,以10000rpm离心10分钟;最后,将MnO2纳米片在50℃的真空烘箱中干燥。
使用前,将MnO2纳米片溶解在水中以获得0.5mg/mL的MnO2纳米片水溶液用于后续GSH的检测。
3、GSH的荧光增强检测步骤
在1.5mL的塑料瓶中,依次加入100L的0.2M HAc-NaAc缓冲液(pH 6.0),50L的0.5mg/mL MnO2纳米片,0.5M至50M的GSH,50L的GCPF和适当体积的水,以控制溶液的总体积是1mL,充分旋涡混合后,在室温下反应10分钟,即可表征其荧光光谱。
4、血清样本中GSH的荧光增强测定步骤
牛血清样本是购自Sigma-Aldrich(中国北京),不含GSH。使用加标回收率法来,分别将三种不同浓度的GSH(5M,10M和30M)添加到稀释10倍的血清样品中,其它检测步骤与上述GSH检测步骤相同,计算出各自的加标回收率。
实施例2
GCPF和MnO2纳米片的结构表征及其用于荧光增强法检测GSH的可行性
水溶性的GCPF黄色透明溶液发蓝色荧光,最大激发波长为360nm,最大发射波长为440nm。通过其荧光强度的测定可知,该GCPF溶液保存在4℃的冰箱中可以稳定两个月以上。
从图2所示的电镜图中可以看出:GCPF显示为纳米棒状或线状,垂直直径接近80nm,长度为几微米(图1A)。MnO2纳米片显示为层状结构,具有二维特征形态(图1B)。
为了确认GCPF和MnO2纳米片用于荧光增强法测定GSH的可行性,我们首先用50mMGSH做了初步实验。测试条件为:c(GSH,μM):50;c(MnO2纳米片,mg/mL):0.05;0.02M HAc-NaAc缓冲液:pH 6.0。λex:360nm;λem:440nm;反应时间:10分钟;温度(℃):25(室温)。结果如图3所示,其中内嵌图为在365nm紫外光(上图)和可见光(下图)照射下的样品图片。
如图3所示,GCPF的荧光被MnO2纳米片淬灭了43.5%,这个归因于MnO2纳米片与GCPF之间的FRET。加入GSH后,体系的荧光增强,说明GSH与MnO2纳米片发生氧化还原反应生成Mn2+离子,阻止了FRET的发生。此外,这个荧光猝灭和增强的变化可以在365nm紫外线的照射下,用眼睛直观地观察到,加入GSH后,样品荧光强弱的变化(图3中内嵌图)。由此可见,本发明中开发构建的以GCPF为荧光探针的FRET平台,可以用于荧光增强法测定GSH。
实施例3
荧光增强法检测GSH的条件优化
本实施例优化了不同含量的MnO2纳米片对GSH检测的影响。结果如图4所示,发现随着MnO2纳米片的浓度从0.005增加到0.5mg/mL,GCPF的荧光强度越来越弱;加入GSH后,GCPF的荧光逐渐增强。为了获得最大的信噪比,选择0.05mg/mL的MnO2纳米片作为荧光增强法检测GSH的最佳条件。
同时,本实施例优化了不同pH和温度对荧光增强法检测GSH的影响。结果如图5和图6所示。
综合考虑到荧光的增强和猝灭的差异,选择pH 6.0、室温(25℃)作为荧光增强法测定GSH的最佳条件,研究了以GCPF为探针的荧光增强法测定GSH的动力学行为。测试条件为:反应时间(分钟):瞬间、1、2、4、6、8、10、15、20、25和30。c(GSH,μM):50。c(MnO2,mg/mL):0.05;0.02M HAc-NaAc缓冲溶液:pH 6.0。λex:360nm;λem:440nm;温度(℃):25(室温)。所有误差棒均是由三次测量值结果得出来的。
结果如图7所示,GCPF的荧光强度在30分钟内保持稳定;加入MnO2纳米片后,GCPF的荧光立即淬灭,荧光减弱在30分钟内保持稳定。这表明从荧光GCPF到MnO2纳米片的FRET是一个快速响应的过程,可以在1分钟内看到荧光猝灭的现象。加入GSH后,从加入的瞬间到10分钟,体系的荧光强度逐渐增强,并达到一个稳定的比较高的荧光强度值。这表明GSH和MnO2纳米片的氧化还原反应在10分钟时达到最佳。综合考虑荧光猝灭和增强的效果,10分钟作为检测GSH的最佳条件。
实施例4
荧光增强法检测GSH灵敏度、选择性和加标回收率
在最佳实验条件下,以GCPF为探针用荧光增强法定量测定GSH。测试条件为:c(GSH,μM):0、0.5、1、2、5、10、30和50。c(GSH,μM):50。c(MnO2纳米片,mg/mL):0.05;0.02MHAc-NaAc缓冲溶液:pH 6.0。λex:360nm;λem:440nm;反应时间:10分钟。温度(℃):25(室温)。所有误差棒均是由三次测量值结果得出来的。
结果如图8A所示,随着GSH浓度从0.5μM增加到50μM,荧光强度逐渐增大。荧光强度值与GSH(μM)的浓度之间存在线性关系,线性回归方程为I=131.295+1.154c(μM)(图8B),相关系数(r)为0.9972,检出限(LOD)为84nM。
与其他GSH检测方法相比(表1),本方法的检测限低于大多数报道。可见,本发明的基于GCPF和MnO2纳米片的荧光增强法对GSH的检测有很高的灵敏度。
表1.本方法与其他方法的比较结果
表1中的其他方法来自于以下文献:
1.Peng,C.;Xing,H.;Fan,X.;Xue,Y.;Li,J.;Wang,E.,Glutathione RegulatedInner Filter Effect of MnO2Nanosheets on Boron Nitride Quantum Dots forSensitive Assay.Anal.Chem.2019,91.
2.Zhang,R.;Zhong,X.;Chen,A.-Y.;Liu,J.-L.;Li,S.-K.;Chai,Y.-Q.;Zhuo,Y.;Yuan,R.,Novel Ru(bpy)2(cpaphen)2+/TPrA/TiO2Ternary ECL System:An EfficientPlatform for the Detection of Glutathione with Mn2+as SubstituteTarget.Anal.Chem.2019,91(5),3681-3686.
3.Gao,W.;Liu,Z.;Qi,L.;Lai,J.;Kitte,S.A.;Xu,G.,Ultrasensitiveglutathione detection based on lucigenin cathodic electrochemiluminescence inthe presence of MnO2Nanosheets.Anal.Chem.2016,88(15),7654-7659.
4.Song,B.;Shi,W.;Shi,W.;Qin,X.;Ma,H.;Tan,M.;Zhang,W.;Guo,L.;Yuan,J.,Adual-modal nanoprobe based on Eu(iii)complex–MnO2nanosheet nanocomposites fortime-gated luminescence–magnetic resonance imaging of glutathione in vitroand in vivo.Nanoscale 2019,11(14),6784-6793.
5.Chen,X.X.;Niu,L.Y.;Shao,N.;Yang,Q.Z.,BODIPY-based fluorescent probefor dual-channel detection of nitric oxide and glutathione:Visualization ofcross-talk in living cells.Anal.Chem.2019,91(7),4301-4306.
6.Sun,J.;Liu,F.;Yu,W.;Jiang,Q.;Hu,J.;Liu,Y.;Wang,F.;Liu,X.,Highlysensitive glutathione assay and intracellular imaging with functionalizedsemiconductor quantum dots.Nanoscale 2019,11(11),5014-5020.
7.Wang,F.;Zhou,L.;Zhao,C.;Wang,R.;Fei,Q.;Luo,S.;Guo,Z.;Tian,H.;Zhu,W.-H.,A dual-response BODIPY-based fluorescent probe for the discriminationof glutathione from cystein and homocystein.Chem.Sci.2015,6(4),2584-2589.
8.Zhang,X.;Wu,F.-G.;Liu,P.;Gu,N.;Chen,Z.,Enhanced fluorescence ofgold nanoclusters composed of HAuCl4and histidine by glutathione:Glutathionedetection and selective cancer cell imaging.Small 2014,10(24),5170-5177.
9.Chen,X.;Wang,Y.;Chai,R.;Xu,Y.;Li,H.;Liu,B.,Luminescent lanthanide-based organic/inorganic hybrid materials for discrimination of glutathione insolution and within hydrogels.ACS Appl.Mater.Interfaces 2017,9(15),13554-13563.
10.Dong,Z.-Z.;Lu,L.;Ko,C.-N.;Yang,C.;Li,S.;Lee,M.-Y.;Leung,C.-H.;Ma,D.-L.,A MnO2nanosheet-assisted GSH detection platform using an iridium(iii)complex as a switch-on luminescent probe.Nanoscale 2017,9(14),4677-4682.
为了对比分析本荧光增强法对GSH的检测选择性,本实施例将本方法还同时用于检测其他常见离子和生物小分子,例如金属离子、氨基酸和葡萄糖等。结果如图9所示,其中,GSH、其他不同离子和生物分子的所有浓度均为50μM。c(MnO2纳米片,mg/mL):0.05。0.02M HAc-NaAc缓冲液溶液:pH 6.0。λex:360nm;λem:440nm;反应时间:10分钟;温度(℃):25(室温)。所有误差棒均是由三次测量值结果得出来的。
从图9结果来看,常见离子和生物小分子没有显示出与GSH相似的荧光增强效果,可见,本荧光增强法在检测GSH中比较高的选择性,这将有利于本方法在实际样品中的检测。
最后,我们采用加标回收率法考察本方法应用于实际血清样品时的准确度。在稀释的血清溶液中,分别加入5μM,10μM和30μM GSH,平均回收率分别为102.07%、103.23%和101.48%。所有相对标准差(RSD)均低于5%。可见,本方法在血清样品的检测中是具有一定的准确度和可靠性的。
综上所述,本发明以GCPF为探针,基于MnO2纳米片与GCPF之间的FRET构建的荧光增强法传感平台,可以用于定量测定GSH,线性范围为0.5~50μM。在这个FRET模式中,GCPF作为能量给体,MnO2纳米片作为能量受体。FRET发生后,MnO2纳米片有效地猝灭了GCPF的荧光。当GSH将MnO2纳米片还原成Mn2+离子后,抑制了FRET的发生,使得被猝灭的荧光强度再次增强。本方法中纳米颗粒制备简单、GCPF荧光性能稳定,对GSH的检测具有快速的荧光响应,在未来生物分析中有较好的应用前景。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (9)
1.一种以戊二醛-壳聚糖非共轭荧光聚合物为荧光探针检测GSH的方法,其特征在于,所述方法以戊二醛-壳聚糖非共轭荧光聚合物GCPF为荧光探针,以MnO2纳米片为荧光淬灭剂来检测谷胱甘肽GSH。
2.根据权利要求1所述的方法,其特征在于:所述戊二醛-壳聚糖非共轭荧光聚合物GCPF的制备方法为:在室温下,将壳聚糖溶于水中制得壳聚糖水溶液,然后向壳聚糖水溶液逐滴加入冰醋酸溶液,搅拌10~20min,再加入戊二醛溶液,搅拌反应5~8h后,获得黄色透明的荧光GCPF溶液;
和/或,所述MnO2纳米片的制备方法为:将MnCl2·4H2O水溶液添加到四甲基氢氧化铵TMA·OH和H2O2的混合溶液中,在室温下搅拌反应6~12h,通过离心获得MnO2纳米片。
3.根据权利要求2所述的方法,其特征在于:所述壳聚糖水溶液的浓度为0.1~0.2g/L,所述戊二醛溶液的浓度为4.0~5.0M,戊二醛溶液的加入量为0.10~0.20mL;
和/或,所述冰醋酸溶液的浓度为1.5~2.0M,所述冰醋酸溶液的加入量为1~2mL;
和/或,所述MnCl2·4H2O水溶液的浓度为0.2~0.4M,MnCl2·4H2O水溶液的用量为7.5~15mL;所述混合溶液中四甲基氢氧化铵TMA·OH的浓度为0.5~0.8M,所述混合溶液中H2O2的浓度为3wt%,所述混合溶液的用量为15~30mL。
4.根据权利要求1所述的方法,其特征在于:所述方法对GSH的检测范围为0.5~50μM,最低检测限为84nM。
5.根据权利要求1所述的方法,其特征在于:检测GSH时,GCPF浓度为5~50M,MnO2纳米片浓度为0.005~0.5mg/mL,GCPF与MnO2纳米片的体积用量比为1:1;
和/或,检测GSH时,检测体系的pH为4.0~7.0,温度为10-30℃。
6.根据权利要求5所述的方法,其特征在于:检测GSH时,GCPF浓度为50M,MnO2纳米片浓度为0.05mg/mL。
7.根据权利要求5所述的方法,其特征在于:检测GSH时,检测体系的pH为6.0,温度为25℃。
8.戊二醛-壳聚糖非共轭荧光聚合物GCPF作为荧光探针在检测GSH上的应用,其特征在于,所述戊二醛-壳聚糖非共轭荧光聚合物GCPF的制备方法为:在室温下,将壳聚糖溶于水中制得壳聚糖水溶液,然后向壳聚糖水溶液逐滴加入冰醋酸溶液,搅拌10~20min,再加入戊二醛溶液,搅拌反应5~8h后,获得黄色透明的荧光GCPF溶液;所述荧光探针在MnO2纳米片存在下选择性地检测GSH。
9.根据权利要求8所述的应用,其特征在于:所述壳聚糖水溶液的浓度为0.1~0.2g/L,所述戊二醛溶液的浓度为4.0~5.0M,戊二醛溶液的加入量为0.10~0.20mL;
和/或,所述冰醋酸溶液的浓度为1.5~2.0M,所述冰醋酸溶液的加入量为1~2mL。
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