CN117517426A - 一种基于纳米酶构建检测1,5-脱水葡萄糖醇的双模式电化学传感器 - Google Patents
一种基于纳米酶构建检测1,5-脱水葡萄糖醇的双模式电化学传感器 Download PDFInfo
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Abstract
一种基于纳米酶构建检测1,5‑AG的双模式电化学传感器,以具有大比表面积,高电导率和良好过氧化氢催化活性的MXene‑RGO/Pt@PdNPs纳米酶为基础,利用PROD酶催化氧化1,5‑AG生成果糖和H2O2,使MXene‑RGO/Pt@Pd NPs纳米酶催化H2O2生成H2O和O2,产生的O2进而氧化缓冲液中的亚铁氰化钾,产生电子转移,利用DPV法和i‑t法进行扫描,通过两个信号的相互对比和校正,减少检测误差。最后通过标准曲线确定血清中1,5‑AG的浓度。该方法成本低、选择性好,灵敏度高,DPV最低检测限为0.488μg/mL,i‑t最低检测限为0.814μg/mL。
Description
技术领域
本发明属于生物检测领域,具体涉及一种基于纳米酶构建检测1,5-AG的双模式电化学传感器。
背景技术
1,5-脱水葡萄糖醇(1,5-AG)是葡萄糖代谢的检测标志物。目前用于1,5-AG的检测方法主要有气相-液相薄层色谱、反相色谱柱和吡喃糖氧化酶(PROD)固定柱全酶法、液相色谱负离子电喷雾串联质谱(LC-MS/MS)和电化学阻抗谱改良等。公开号CN 112630216A的发明专利,涉及一种1,5-AG测定试剂盒及采用紫外等分析仪检测1,5-AG浓度的方法;公开号CN 107703071B的发明专利,涉及一种检测1,5-AG的试剂盒,能够有效消除1~20mM内源性葡萄糖的干扰。但由于这些方法存在着样品前处理繁杂、成本高、需要专业人员使用,且在实际样品中易受到其他物质交叉影响等缺点;因此,建立一种快速、灵敏、操作简便的1,5-AG检测方法。
发明内容
本发明所要解决的技术问题是提供一种基于二维碳化钛-还原氧化石墨烯-铂@钯(MXene-RGO/Pt@PdNPs)纳米酶构建双模式电化学传感器,实现1,5-AG的检测,差分脉冲伏安法(DPV法)检测1,5-AG的最低检测限为0.488μg/mL,安培法(i-t法)检测1,5-AG的最低检测限为0.814μg/mL。
本发明检测原理为:利用导电性高、稳定性强的二维碳化钛(MXene),比表面积大、吸附性能良好及电子转移能力强的还原氧化石墨烯(RGO),催化活性强的金属Pt和Pd,制备了MXene-RGO/Pt@PdNPs纳米酶,再通过i-t技术将Au NPs电沉积在电极上以增强导电性,最后加入PROD酶,构建电化学生物传感界面,当1,5-AG导入到生物传感界面后,1,5-AG会在PROD酶的催化作用下发生水解,生成果糖(1,5-AF)和H2O2,MXene-RGO/Pt@PdNPs纳米酶就会催化H2O2生成H2O和O2,同时将传感器放入铁氰化钾缓冲液中进行DPV和i-t扫描后,H2O2在复合材料的催化作用下产生O2,再氧化缓冲液中的亚铁氰化钾,从而发生电子转移,产生电流信号,采用DPV和i-t双模式记录氧化还原峰值电流变化,实现1,5-AG的检测。与现有的方法对比,该方法操作相对较简单,特异性高,且通过两信号的相互校正,可以有效地将环境变化带来的影响归一化,减少检测误差。
本发明按照以下步骤进行:
步骤1:MXene-RGO/Pt@Pd纳米酶的制备
(1)二维碳化钛(MXene)的制备:MXene固体粉末加入纯水,搅拌均匀,再将其超声破碎,得MXene溶液。
(2)还原性氧化石墨烯(RGO)的制备:称取单层氧化石墨烯(GO)固体置于纯水中,搅拌均匀,再将溶液进行超声破碎,最后加入抗坏血酸(AA)进行还原,搅拌后即可得到RGO溶液。
(3)二维碳化钛-还原氧化石墨烯(MXene-RGO)的制备:将RGO溶液和MXene溶液搅拌均匀,得到Mxene-RGO溶液。
(4)二维碳化钛-还原氧化石墨烯-铂@钯(MXene-RGO/Pt@PdNPs)纳米酶的制备:将六氯铂酸钠(Na2PtCl6)和六氯钯酸钠(Na2PdCl6)加入MXene-RGO溶液中,再加入水合肼,搅拌均匀,得到MXene-RGO/Pt@PdNPs纳米酶。
步骤2:验证MXene-RGO/Pt@Pd NPs纳米酶的催化活性
利用3,3′,5,5′-四甲基联苯胺/邻苯二胺(TMB/OPD)体系验证MXene-RGO/Pt@PdNPs纳米酶的过氧化氢催化活性。观察溶液是否变深蓝色和深黄色,使用紫外分光光度计检测TMB和OPD溶液的氧化产物在652nm和450nm处的吸光度。
步骤3:电极的修饰与电化学传感器的构建
(1)AuNPs/SPE的制备:将丝网印刷电极(SPE)置于稀硫酸(H2SO4)溶液中,采用循环伏安法(CV)进行活化,将活化后得SPE浸入氯金酸(HAuCl4)溶液中,利用i-t进行恒电位沉积,得AuNPs/SPE电极;
(2)PROD/MXene-RGO/Pt@Pd/Au NPs/SPE传感界面的制备:滴加MXene-RGO/Pt@Pd溶液在Au NPs/SPE上,孵育,再加入PROD酶,得到PROD/MXene-RGO/Pt@Pd/AuNPs/SPE工作电极,最后用牛血清蛋白(BSA)溶液封闭,成功构建出所需要的工作电极。
步骤4:检测1,5-AG的条件优化和1,5-AG的工作曲线绘制
(1)将标准1,5-AG溶液滴加到步骤3得到的电化学传感器中,孵育、清洗、吹干,得到1,5-AG/PROD/MXene-RGO/Pt@Pd/AuNPs/SPE工作电极;
(2)将工作电极放入铁氰化钾/亚铁氰化钾缓冲溶液中,采用电化学工作站的DPV法和i-t法扫描,记录其电流。
(3)分别对孵育温度、复合材料浓度、缓冲液pH值、PROD酶浓度和孵育时间等实验条件进行优化,获得检测1,5-AG的最优实验条件。
(3)分别对不同浓度的1,5-AG进行检测,记录峰值电流;根据传感器的电流响应值与1,5-AG浓度的关系,绘制工作曲线,计算出该方法的最低检测限。
步骤5:实际血清样本中1,5-AG的检测
(1)在步骤3得到的电化学传感器上,滴加待测实际血清样本,进行孵育、得到工作电极。
(2)将工作电极放入缓冲液中,采用电化学工作站的DPV法和i-t法扫描,记录其电流值。
(3)根据步骤4所述的工作曲线,得到所述待测实际样本中1,5-AG的浓度。
进一步,所述步骤1中,Na2PtCl6和Na2PdCl6为20mmol/L。
进一步,所述步骤2中,TMB和OPD为50mmol/L。
进一步,所述步骤3中,H2SO4溶液为0.5mol/L,CV扫描电压为-1.2V~1.2V,扫描20段。
进一步,所述步骤3中,使用的HAuCl4溶液的质量分数为0.01%,沉积时间为180s。
进一步,所述步骤3中,MXene-RGO/Pt@Pd溶液和PROD溶液的浓度为1.00mg/mL。
优选,所述步骤4中,1,5-AG检测的孵育温度为35℃,材料浓度为0.65mg/mL,缓冲液pH为7.40,PROD酶浓度为1.00mg/mL,孵育时间为40min。
其中,步骤1提供了一种具有大比表面积、高电导率、高过氧化氢催化活性的MXene-RGO/Pt@Pd纳米酶;步骤2通过验证纳米酶活性,对步骤4,5的成功检测是必不可少的;通过加入PROD酶,为步骤3,4,5提供检测传感界面;步骤3构成特异性识别1,5-AG的生物传感界面,是步骤4和步骤5中1,5-AG的电化学检测中必不可少的关键步骤;步骤4中1,5-AG工作曲线为步骤5的实际血清样本检测提供依据。可见步骤1-5相互支撑,共同作用,才能利用以MXene-RGO/Pt@Pd、PROD等构建传感器实现1,5-AG的检测。
本发明与现有技术相比具有如下优点:
本发明充分利用MXene的高导电性和吸附性,以及RGO的强负载能力、催化性质,并结合具有高催化活性的Pt和Pd,成功制备了具备优异的过氧化氢催化活性的MXene-RGO/Pt@Pd纳米酶;再利用PROD酶能够特异性识别1,5-AG,为血清样本中1,5-AG的检测提供了新的方法。该传感器特异性强、稳定性高、重现性好,DPV最低检测限为0.488μg/mL,i-t最低检测限为0.814μg/mL。采用加标法对血清样本中1,5-AG进行特异性检测,DPV法检测1,5-AG的回收率为90.30%~102.44%,i-t法检测1,5-AG的回收率为95.60%~106.20%,回收率良好。证明本发明检测血清样本中1,5-AG的操作简便,灵敏度高。
附图说明
图1基于MXene-RGO/Pt@Pd NPs纳米酶的双模式电化学传感器用于1,5-AG检测的实验原理图;
图2纳米复合材料的透射电镜(TEM)图,(A)MXene,(B)MXene-RGO,(C)MXene-RGO/Pt@Pd;
图3验证酶活性的现象图和紫外光谱图;(A)TMB实验现象图,(B)TMB实验紫外光谱图,(C)OPD实验现象图,(D)OPD实验紫外光谱图;
图4基于MXene-RGO/Pt@Pd NPs纳米酶的双模式电化学传感器检测不同1,5-AG浓度的DPV曲线(A)与DPV标准曲线(B);
图5基于MXene-RGO/Pt@Pd NPs纳米酶的双模式电化学传感器检测不同1,5-AG浓度的i-t曲线(A)与i-t标准曲线(B)。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
一种基于MXene-RGO/Pt@PdNPs纳米酶的双模式电化学传感器用于1,5-AG检测的实验原理图见图1。首先采用电沉积技术将Au NPs修饰在活化的丝网印刷电极表面;将制备好的MXene-RGO/Pt@PdNPs纳米酶修饰在Au NPs/SPE,其中,MXene具有高导电性,稳定性强优点;RGO具有较好的电子转移能力,较大的比表面积以及良好的吸附性能;而金属Pt和Pd的结合可以增强催化活性,再结合MXene和RGO原有的催化性质,使得MXene-RGO/Pt@PdNPs纳米酶具有很强的催化活性。接着加入PROD酶,构建电化学生物传感界面。当1,5-AG导入到生物传感界面后,1,5-AG会在PROD酶的催化作用下发生水解,生成1,5-anhydrofuctose(1,5-AF)和H2O2,MXene-RGO/Pt@PdNPs纳米酶就会催化H2O2生成H2O和O2,同时将传感器放入铁氰化钾缓冲液中进行DPV和i-t扫描,H2O2在复合材料催化作用下产生O2,进而氧化缓冲液中亚铁氰化钾,发生电子转移,从而产生电流信号,以此可采用DPV和i-t双模式记录氧化还原峰值电流变化,基于此,构建1,5-AG传感器,通过两个信号的相互对比和校正,可以有效降低环境变化带来的影响,减少检测误差。实施步骤如下:
步骤1:MXene-RGO/Pt@Pd纳米酶的制备
(1)二维碳化钛(MXene)的制备:取20mg MXene固体粉末倒入烧杯中,加入20mL纯水,搅拌均匀,用超声波细胞粉碎机破碎2h,得MXene溶液。
(2)还原性氧化石墨烯(RGO)的制备:称取20mg单层氧化石墨烯(GO)固体放入烧杯中,加入20mL纯水,混合均匀,在超声波粉碎机破碎1.5h,加入100mg抗坏血酸(AA)进行还原,搅拌12h,即可得到RGO溶液。
(3)二维碳化钛-还原氧化石墨烯(MXene-RGO)的制备:取10mLRGO溶液和10mLMXene溶液搅拌均匀,得到Mxene-RGO溶液。
(4)二维碳化钛-还原氧化石墨烯-铂@钯(MXene-RGO/Pt@PdNPs)纳米酶的制备:在10mL MXene-RGO溶液中,加入2mL 20mmol/L的六氯铂酸钠(Na2PtCl6)和六氯钯酸钠(Na2PdCl6)溶液,再加入10μL水合肼,搅拌12h,得MXene-RGO/Pt@PdNPs纳米酶。
采用JEM-1200EX型透射电子显微镜(TEM)对纳米复合材料进行形貌表征,如图2所示。图2A为MXene的TEM图,呈现不规则的颗粒状;图2B为MXene-RGO的TEM图,可观察到褶皱絮状结构的RGO上面吸附有MXene颗粒;图2C为MXene-RGO/Pt@Pd的TEM图,褶皱絮状结构上有许多黑点,这是还原形成的Pt、Pd纳米粒子,表明MXene-RGO/Pt@Pd纳米复合材料制备成功。
步骤2:验证MXene-RGO/Pt@Pd NPs纳米酶的催化活性
验证酶活性的现象和紫外光谱图,如图3所示。取10μL 1mg/mL的MXene-RGO/Pt@PdNPs纳米酶复合材料,接着加入150μL 125mmol/L的磷酸盐缓冲液(pH=5.0),再依次加入加入20μL 50mmol/L的TMB溶液和20μL 100mmol/L的H2O2溶液,振荡均匀,室温静置10min,观察到溶液变成蓝色,如图3(A),最后使用UV-6100紫外分光光度计测量在652nm处TMB溶液的氧化产物oxTMB的吸光度,如图3(B)。同理,将浓度为50mmol/L的TMB换成50mmol/L的OPD溶液,其他条件不变,观察到溶液变成深黄色,如图3(C)所示,使用紫外分光光度计测量在450nm处OPD溶液的氧化产物的吸光度,如图3(D)所示。由此说明MXene-RGO/Pt@Pd NPs纳米酶在TMB-H2O2和OPD-H2O2显色体系中的具有优异的类过氧化物酶活性。
步骤3:电极的修饰与电化学传感器的构建
(1)将SPE电极浸入0.5mol/LH2SO4溶液中进行循环伏安法扫描,在-1.2V~1.2V的电压范围内扫描20段;扫描完成后用纯水洗净,晾干,得到活化的SPE电极。
(2)将活化后的SPE电极浸入HAuCl4溶液中,搅拌,在0.4V恒电位下沉积180s,沉积金完成后用纯水清洗3次,用洗耳球吹干,得AuNPs/SPE。
(3)在SPE电极表面滴加2.5μLMxene-RGO/Pt@PdNPs溶液,在孵育箱恒温孵育30min,滴加2.5μL吡喃糖氧化酶(PROD)至SPE上,孵育30min,即可得到PROD/MXene-RGO/Pt@Pd/Au NPs/SPE。至此便成功构建出了所需要的电化学传感器。
步骤4:检测1,5-AG的条件优化和1,5-AG的工作曲线绘制
(1)2.5μL标准1,5-AG溶液滴加到PROD/MXene-RGO/Pt@Pd/Au NPs/SPE电化学传感界面中,孵育30min,得到1,5-AG/PROD/MXene-RGO/Pt@Pd/Au NPs/SPE工作电极;
(2)将1,5-AG/PROD/MXene-RGO/Pt@Pd/Au NPs/SPE工作电极放入铁氰化钾/亚铁氰化钾缓冲溶液中,采用DH7000C电化学工作站的差分脉冲伏安法(DPV)和安培法(i-t)扫描,对孵育温度、复合材料浓度、缓冲液pH值、PROD酶浓度和孵育时间等实验条件进行优化,获得检测1,5-AG的最优条件,分别为最佳孵育温度为35℃,最佳材料浓度为0.65mg/mL,最佳缓冲液pH为7.40,最佳PROD酶浓度为1.00mg/mL,最佳孵育时间为40min。
(2)将上述所得到的工作电极置于pH值为7.4铁氰化钾/亚铁氰化钾缓冲溶液中,采用DH7000C电化学工作站中DPV法和i-t法进行扫描,记录其电流值。不同1,5-AG浓度的DPV曲线和i-t曲线如图4和图5所示,随着1,5-AG浓度的增加,DPV和i-t电流值随之增大。1,5-AG浓度在0.1μg/mL-100.0μg/mL范围内,传感器电流响应值(Y)与1,5-AG浓度(X)之间呈线性关系,DPV工作曲线为Y=0.1045X+17.2315,相关系数为0.9925,i-t标准曲线线性方程为Y=0.0352X+4.7989,R2=0.9818。由公式CLOD=3Sd/b(Sd表示三次空白对照组的标准偏差,b表示工作曲线的斜率)计算得到DPV法最低检测限为0.488μg/mL,i-t法最低检测限为0.814μg/mL。
步骤5:实际血清样本中1,5-AG的检测
(1)收集已知1,5-AG浓度的两种类型血清样本2份(正常人血清:样本1;糖尿病患者血清:样本2)。将这两种血清与三种不同浓度的1,5-AG溶液以1:1的比例混合在一起,混合后加入浓度分别为20.00μg/mL、50.00μg/mL、80.00μg/mL的1,5-AG,振荡均匀,制成6种不同浓度的混合液。再将其制成1,5-AG/PROD/MXene-RGO/Pt@Pd/Au NPs/SPE工作电极,在相同条件下分别采用DH7000C电化学工作站中的DPV法和i-t法进行扫描。本实验所用血清样本均来自中国人民解放军第924医院(中国桂林)广西代谢病研究重点实验室,并遵循中国人民解放军第924医院广西代谢病研究重点实验室伦理委员会要求。
(2)通过步骤4所得到的1,5-AG工作曲线,计算各血清样本中1,5-AG的浓度,每个血清样本进行三次实验,检测结果见表1和表2。由表1和表2可知,所构建电化学传感器测得的1,5-AG浓度的DPV回收率在90.30%~102.44%之间,相对标准偏差在1.23%~5.03%之间;i-t回收率在95.60%~106.20%之间,相对标准偏差在1.67%~5.57%之间,表明该双模式电化学传感器有望用于实际血清样本检测1,5-AG。
表1 DPV法检测实际血清样本中1,5-AG的检测结果
表2 i-t法检测实际血清样本中1,5-AG的检测结果
Claims (6)
1.一种基于纳米酶构建检测1,5-AG的双模式电化学传感器,按以下步骤进行:
步骤1:二维碳化钛-还原氧化石墨烯-铂@钯纳米酶MXene-RGO/Pt@PdNPs的制备
(1)二维碳化钛MXene的制备:称取MXene固体粉末置于纯水中,超声破碎,得到MXene溶液;
(2)还原性氧化石墨烯RGO的制备:氧化石墨烯GO溶于纯水,超声破碎,加入抗坏血酸AA,搅拌,得到RGO溶液;
(3)二维碳化钛-还原性氧化石墨烯Mxene-RGO的制备:取制备好的RGO溶液和MXene溶液混合搅拌,得到Mxene-RGO溶液;
(4)二维碳化钛-还原氧化石墨烯-铂@钯MXene-RGO/Pt@PdNPs纳米酶的制备:取MXene-RGO溶液,加入六氯铂酸钠Na2PtCl6和六氯钯酸钠Na2PdCl6,再加入水合肼,搅拌,得到MXene-RGO/Pt@PdNPs纳米酶;
步骤2:验证MXene-RGO/Pt@PdNPs纳米酶的催化活性
采用3,3′,5,5′-四甲基联苯胺/邻苯二胺TMB/OPD体系验证MXene-RGO/Pt@PdNPs纳米酶的过氧化氢催化活性;观察溶液是否变深蓝色/深黄色,使用紫外分光光度计检测TMB和OPD溶液的氧化产物在652nm和450nm处的吸光度;
步骤3:电极的修饰与生物传感界面的构建
(1)AuNPs/SPE的制备:裸电极SPE置于稀H2SO4中活化,活化后的SPE在质量分数为0.01%的氯金酸HAuCl4溶液中进行电沉积,沉积时间为180s,得到Au NPs/SPE;
(2)PROD/MXene-RGO/Pt@Pd/Au NPs/SPE传感界面的制备:向Au NPs/SPE滴加MXene-RGO/Pt@Pd溶液,孵育,再滴加吡喃糖氧化酶PROD,得PROD/MXene-RGO/Pt@Pd/Au NPs/SPE传感界面,构建出所需要的电化学传感器;
步骤4:检测1,5-AG的条件优化和1,5-AG的工作曲线绘制
(1)将标准1,5-AG溶液滴加到步骤3得到的电化学传感器中,孵育,得到1,5-AG/PROD/MXene-RGO/Pt@Pd/AuNPs/SPE工作电极;
(2)将工作电极放入铁氰化钾/亚铁氰化钾缓冲溶液中,采用电化学工作站的差分脉冲伏安法DPV和安培法i-t扫描,记录其电流;
(3)对孵育温度、复合材料浓度、缓冲液pH值、PROD酶浓度和孵育时间等实验条件进行优化,获得检测1,5-AG的最优条件;
(4)分别对不同浓度的1,5-AG进行检测,记录峰值电流;根据传感器的电流响应值与1,5-AG浓度的关系,绘制工作曲线,计算出最低检测限;
步骤5:实际血清样本中1,5-AG的检测
(1)在步骤3得到的电化学传感器上,滴加待测实际血清样本,孵育得到工作电极;
(2)将工作电极放入缓冲液中,采用电化学工作站的DPV法和i-t法扫描,记录其电流值;
(3)根据步骤4所述的工作曲线,得到所述待测实际血清样本中1,5-AG的浓度。
2.按照权利要求1所述的传感器,其特征在于:步骤1中,Na2PtCl6和Na2PdCl6浓度为20mmol/L。
3.按照权利要求1所述的传感器,其特征在于:步骤2中,TMB和OPD浓度为50mmol/L。
4.按照权利要求1所述的传感器,其特征在于:步骤3中所述H2SO4溶液浓度为0.5mol/L,CV扫描电压为-1.2V~1.2V,扫描20段。
5.按照权利要求1所述的传感器,其特征在于:步骤3中所述MXene-RGO/Pt@Pd溶液和PROD溶液的浓度为1.0mg/mL。
6.按照权利要求1所述的传感器,其特征在于:步骤4中,1,5-AG检测的孵育温度为35℃,材料浓度为0.65mg/mL,缓冲液pH值为7.40,PROD酶浓度为1.00mg/mL,孵育时间为40min。
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