CN116519681A - 一种双原子纳米酶基快速检测头孢氨苄和铅方法 - Google Patents
一种双原子纳米酶基快速检测头孢氨苄和铅方法 Download PDFInfo
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Abstract
本发明公开了一种双原子纳米酶基快速检测头孢氨苄和铅方法,该方法合成Fe、Cu配位的中‑四(4‑羧基苯基)卟吩,高温热解为双原子纳米酶(Cu,Fe‑N‑C),含铁和铜活性位点的共存Cu,Fe‑N‑C由于其协同效应,有较高的过氧化物模拟酶活性,头孢氨苄对Cu,Fe‑N‑C+TMB+H2O2体系有显著的抑制作用,由于头孢氨苄与金属Cu的配合作用,使Cu,Fe‑N‑C与底物的亲和力及活性位点降低,导致体系吸光度降低;而Pb2+的加入使过氧化酶纳米酶的活性得到恢复,由于Pb2+竞争地与头孢氨苄形成配合物,释放出Cu,Fe‑N‑C纳米酶中铁和铜活性位点,导致Cu,Fe‑N‑C与底物的亲和力增加,体系吸光度随之增加;由此建立高灵敏、选择性强头孢氨苄和铅快速检测新方法,定量限分别为0.010和0.012 mg/kg,能够满足国家食品安全的相关要求;本方法具有操作简单、灵敏度高、快速等特点。
Description
技术领域
本发明涉及化学分析检测技术领域,具体为一种双原子纳米酶基快速检测头孢氨苄和铅方法。
背景技术
头孢氨苄是一种 β-内酰胺类抗生素,具有抗菌谱广、无交叉耐药性、低残留、毒副作用小等特点,广泛用于临床医学、兽医界。因此,对头孢氨苄的测定研究具有重要的意义;目前,已报道的头孢氨苄的测定方法有:微生物检测法、高效液相色谱法、光化学荧光分析法、旋光法等。铅离子(Pb2+)是一种不可降解的有毒金属离子。世界卫生组织(WHO)推荐饮用水中Pb2+最大限量为 10μg/L,食品中Pb2+最大限量为0.02-0.5 mg/kg,测定Pb2+的含量是食品安全检测中非常重要的项目。目前,铅的测定方法主要包括原子吸收法(AAS)、原子荧光法(AFS)、电感耦合等离子体质谱(ICP-MS)、电化学法及比色探针法。其中比色探针法由于操作简单、快速、不需要复杂仪器设备而备受关注。为了实现Pb2+的超灵敏度测定,研究通常需要信号放大才能获得良好的灵敏度和低的检测限;
孤立金属原子位于载体上的单原子纳米酶(SAzymes)作为一种具有原子分散活性位点的新型纳米酶,因其具有较高的催化活性、金属原子的最大利用效率以及活性位点的简单模型,在纳米酶的发展中具有重要意义。目前,单原子纳米酶的研究集中在单一金属原子与相关配体的研究,双金属原子位点嵌入氮掺杂碳(FeCu/NC)的纳米酶研究较少。研究小组研究表明,FeCu/NC具有较强的模拟酶活性,增强的性能可归因于Cu和Fe单原子之间独特的协同效应,其中Cu-N4作为电子给体增加了Fe-N4活性中心的电子密度,从而促进了O2活化;
本发明合成Fe、Cu配位的中-四(4-羧基苯基)卟吩,高温热解为双原子纳米酶(Cu,Fe-N-C),含铁和铜活性位点的共存Cu,Fe-N-C由于其协同效应,有较高的过氧化物模拟酶活性,头孢氨苄对Cu,Fe-N-C+TMB+H2O2体系有显著的抑制作用,由于头孢氨苄与金属Cu的配合作用,使Cu,Fe-N-C与底物的亲和力及活性位点降低,导致体系吸光度降低;而Pb2+的加入使过氧化酶纳米酶的活性得到恢复,由于Pb2+竞争地与头孢氨苄形成配合物,释放出Cu,Fe-N-C纳米酶中铁和铜活性位点,导致Cu,Fe-N-C与底物的亲和力增加,体系吸光度随之增加;由此建立高灵敏、选择性强头孢氨苄和铅快速检测新方法,定量限分别为0.010 和0.012mg/kg;能够满足国家食品安全的相关要求,本方法具有操作简单、灵敏度高、快速等特点。
发明内容
针对现有技术的不足,本发明提供了一种双原子纳米酶基快速检测头孢氨苄和铅方法,利用头孢氨苄对Cu,Fe-N-C纳米酶活性的抑制作用及Pb2+对纳米酶抑制体系活性的恢复作用,而建立了头孢氨苄和铅的“关-开”比色快速检测方法;
本发明双原子纳米酶基快速检测头孢氨苄和铅方法的方法如下:
(1)以中-四(4-羧基苯基)卟吩为配体(TCPP),合成Cu,Fe-TCPP配合物,以Cu,Fe-TCPP为前驱进行高温热解并经酸处理,得到双原子Cu,Fe-N-C纳米酶;
(2)在头孢氨苄(CEX)标准溶液中加入Cu,Fe-N-C纳米酶溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液及H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,生成蓝色氧化oxTMB,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,建立吸光度与头孢氨苄浓度的定量关系,绘制标准曲线,得到回归方程;
(3)在头孢氨苄溶液中加入Cu,Fe-N-C纳米酶溶液、Pb2+标准溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液及H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,生成蓝色氧化oxTMB,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,建立吸光度与Pb2+浓度的定量关系,绘制标准曲线,得到回归方程;
(4)在含头孢氨苄的待测样品液中加入Cu,Fe-N-C纳米酶溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液及H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,代入回归方程,计算待测样品液中头孢氨苄浓度;
在含Pb2+的待测样品液中加入Cu,Fe-N-C纳米酶溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液、H2O2及头孢氨苄溶液,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,代入回归方程,计算待测样品液中Pb2+浓度;
所述的Cu,Fe-N-C纳米酶制备如下:
(1)称取2.5-3.0g CuCl2溶于10-15mL甲醇中,超声混合均匀,作为溶液A;4.5-5.5g 中-四(4-羧基苯基)卟吩(TCPP)和2.0-2.5g FeCl3溶于20-25mL甲醇中,超声混合均匀,作为溶液B;将A和B两种溶液均匀混合并80-90℃烘箱干燥老化2h,得到的墨绿色干燥粉末Cu,Fe- TCPP;
(2)称取 4.5-5.5g Cu,Fe-TCPP干燥粉末于石英舟中,管式炉5°C/min升温速率至750℃ 在N2保护下煅烧2-2.5 h得到黑色粉末。粉末于硫酸溶液(H2SO4:H2O=1:9,体积比)中12 h浸泡后用水反复冲洗,干燥后,得到Cu,Fe-N-C纳米酶;
所述的头孢氨苄标准溶液的浓度为0.010~52.76 mg/L,Pb2+标准溶液的浓度为0.012~45.38 mg/L;Cu,Fe-N-C纳米酶浓度为0.2 mg/mL,添加量为100 μL;TMB溶液浓度为5mmol/L,添加量为20-200 μL;H2O2的浓度为50 mmol/L,用量为20-200 μL;
所述离心是在8000-10000 r/min下处理5-10min;
本发明的优点和技术效果:
1、本发明将双金属原子(Fe, Cu)引入以中-四(4-羧基苯基)卟吩为配体的双位点纳米酶中,制备双原子嵌入氮掺杂碳纳米酶(Cu,Fe-N-C),由于Cu和Fe单原子之间独特的协同效应,其中Cu - N4作为电子给体增加了Fe - N4活性中心的电子密度,使Cu,Fe-N-C具有超高的模拟过氧化物酶活性;在H2O2存在下氧化TMB为蓝色oxTMB,而头孢氨苄的加入抑制了Cu,Fe-N-C的过氧化酶活性,由于Cu,Fe-N-C活性位点的降低,当Pb2+存在时,能与头孢氨苄发生配合作用,从而释放Cu,Fe-N-C活性位点,恢复Cu,Fe-N-C活性,导致吸光度提高,从而建立了头孢氨苄和铅比色检测新方法;
2、本发明建立的头孢氨苄和铅比色检测方法,具有高的检测灵敏度,头孢氨苄和铅定量限分别达0.010 和0.012 mg/L,而共存的其他金属离子、其他β-内酰胺类抗生素及共存物不干扰测定,方法具有好的选择性;
3、本发明方法用于各类食品中头孢氨苄和铅残留的检测,有高的回收率,标准物的检测准确,方法具有灵敏度高、特异性强、操作简单、快速等特点;
附图说明
图1为本发明实施例1制备的Cu,Fe-N-C的TEM图;
图2为本发明实施例1中(Cu,Fe-N-C+TMB +H2O2)、(Cu,Fe-N-C+TMB +H2O2+ CEX)及(Cu,Fe-N-C+TMB +H2O2+ CEX + Pb2+)的紫外吸收光谱图;
图3为本发明实施例1Cu,Fe-N-C纳米酶活性位点Cu-N4及 Fe-N4验证结果图;
图4 为实施例1中Cu,Fe-N-C氧化H2O2+TMB的米氏动力学曲线;
图5 为实施例1中Cu,Fe-N-C+CEX氧化H2O2+TMB的米氏动力学曲线;
图 6为实施例1 Cu,Fe-N-C+CEX+ Pb2+氧化H2O2+TMB的米氏动力学曲线;
图7为实施例1中Cu,Fe-N-C+TMB+H2O2体系检测CEX线性紫外-可见吸收光谱(左图)及回归方程(右图);
图8为实施例1中Cu,Fe-N-C+TMB+H2O2+ CEX体系检测Pb2+线性紫外-可见吸收光谱(左图)及回归方程(右图);
图9为实施例1中为共存物质对CEX的影响结果;
图10为实施例1中为共存金属离子对Pb2+的影响结果。
实施方式
下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明,但本发明的保护范围并不仅限于此;
实施例1:牛奶样品中CEX及食用菌中Pb2+的测定
1、Cu,Fe-N-C纳米酶制备
(1)称取2.5g CuCl2溶于10 mL甲醇中,超声混合均匀,作为溶液A;4.5 g 中-四(4-羧基苯基)卟吩(TCPP)和2.0 g FeCl3溶于20 mL甲醇中,超声混合均匀,作为溶液B;将A和B两种溶液均匀混合并80℃烘箱干燥老化2h,得到的墨绿色干燥粉末Cu,Fe- TCPP;
(2)称取 4.5 g Cu,Fe-TCPP干燥粉末于石英舟中,管式炉5°C/min升温速率至750℃ 在N2保护下煅烧2 h得到黑色粉末。粉末于硫酸溶液(H2SO4:H2O=1:9,体积比)中12 h浸泡后用水反复冲洗,干燥后,得到Cu,Fe-N-C纳米酶。图1为制备Cu,Fe-TCPP的TEM图,正如预期的那样,TEM表征证明了纳米酶的纳米片结构,并且在后处理后没有观察到聚集的Cu及Fe物种;
2、Cu,Fe-N-C纳米酶过氧化物酶活性评价:取100 μL浓度为5 mmol/L的TMB,加入0.2 mg/mL Cu,Fe-N-C 100 μL及50 mmol/L的H2O2 100 μL,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲溶液至3 mL,摇匀,静置10 min,10000 r/min离心5 min,取上清液置于654 nm波长处测定吸光度;同时取100 μL 0.2 mg/mL的Cu,Fe-N-C和100 μL 10 mg/L CEX水溶液,加入100 μL浓度为5 mmol/L的TMB 及100 μL浓度为50 mmol/L的H2O2,加入0.1 mmol/L pH4.0 HAc-NaAc缓冲液至3 mL,摇匀,静置5-10 min,10000 r/min离心5 min,取上清液置于654 nm波长处测定吸光度;取100 μL 0.2 mg/mL的Cu,Fe-N-C和100 μL 10 mg/L CEX水溶液,加入100 μL 10 mg/L Pb2+,再加入100 μL浓度为5 mmol/L的TMB 及100 μL浓度为50mmol/L的H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置5-10 min,10000r/min离心5min,取上清液置于654 nm波长处测定吸光度。结果如图2, Cu,Fe-N-C氧化TMB在酸性条件下表现强的过氧化物酶活性,当加入了CEX后,体系的吸光度得到明显抑制,而加入Pb2+后,体系的吸光度得到恢复,吸光度提高。同时参照文献(Anal. Chem. 2020, 92,3373−3379)使用KSCN封闭Cu、Fe位点以确认活性位点,结果如图3,当加入KSCN后,Cu,Fe-N-C+TMB+H2O2体系吸光度明显降低,由此证明,Cu-N4及 Fe-N4作为活性位点;
实验还进行了米氏催化动力学参数测定(图4、图5、图6及表1),加入CEX前、后及加入Pb2+后的米氏常数K m 分别为0.237 mM、1.150 mM和0.534 mM,反应速率常数分别为3.25×10-8 M∙s-1、1.57×10-8 M∙s-1和2.80×10-8 M∙s-1,表明CEX加入降低了Cu,Fe-N-C纳米酶与底物的亲和力及反应速度,而Pb2+加入提高了Cu,Fe-N-C纳米酶与底物的亲和力及反应速度;
表1米氏催化动力学参数
Enzymes | 底物 | K m (mM) | V max (10-8M∙s-1) |
Cu,Fe-N-C | H2O2 | 0.237 | 3.25 |
Cu,Fe-N-C +CEX | H2O2 | 1.150 | 1.57 |
Cu,Fe-N-C+CEX+ Pb2+ | H2O2 | 0.534 | 2.80 |
3、CEX工作曲线制作:在5 mL具塞比色管中加入50 μL 0.5 mg/mL的Cu,Fe-N-C和浓度在0.010~52.76 mg/L CEX标准溶液,加入100 μL浓度为5 mmol/L的TMB 及100 μL浓度为50 mmol/L的H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,静置5-10min,10000 r/min离心5 min,取上清液置于654 nm波长处测定吸光度,CEX浓度为横坐标,吸光度A为纵坐标,绘制标准曲线,得到回归方程,见图7;回归方程、相关系数、相对标准偏差、线性范围等见表2;
4、Pb2+工作曲线制作:在5 mL具塞比色管中加入50 μL 0.5 mg/mL的Cu,Fe-N-C和40 mg/L CEX标准溶液100 μL,加入浓度在0.012~45.38 mg/L Pb2+标准溶液,为加入100 μL浓度为5 mmol/L的TMB 及100 μL浓度为50 mmol/L的H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,静置5-10 min,10000 r/min离心5min,取上清液置于654 nm波长处测定吸光度,CEX浓度为横坐标,吸光度A为纵坐标,绘制标准曲线,得到回归方程,见图8;回归方程、相关系数、相对标准偏差、线性范围等见表2;
表2线性方程、相关系数、相对标准偏差、线性范围
目标物 | 工作曲线 | 相关系数(R2) | 线性范围mg/L | RSD%(n=3) | LOQmg/kg |
CEX | y= 0.029x + 1.551 | 0.995 | 0.010~52.76 | 2.85 | 0.010 |
Pb2+ | y= 0.024x + 0.082 | 0.996 | 0.012~45.38 | 3.10 | 0.012 |
5、方法特异性考察:将CEX和其他共存物质混合,检测共存物质在上述检测体系中对CEX的影响,CEX浓度为5 mg/kg,以上干扰物质浓度为50 mg/kg,图9是共存物质(葡萄糖、谷氨酸、酪氨酸、丝氨酸、精氨酸、亮氨酸、脯氨酸、氨苄西林、克林霉素、氨茶碱、头孢克肟、头孢呋辛、DL -色氨酸、茶碱)对CEX的影响结果,从图中可以看出,Cu,Fe-N-C检测体系有较好的选择特异性,CEX有明显的抑制氧化反应,其它物质几乎没有,方法测定CEX具有好的选择特异性;同时考察了测定Pb2+的选择性,将Pb2+和其他可能共存物质混合,检测金属离子对检测体系中的影响,Pb2+浓度为5 mg/kg,其余干扰物质浓度为50 mg/kg,图10是共存金属离子(Na+、K+、Ca2+、Mg2+、Cu2+、Zn2+、Fe2+、Ni2+、Hg2+等)对Pb2+的影响结果,从图中可以看到,仅有Pb2+对纳米酶催化活性有明显的增强作用,其它物质几乎没有作用,方法具有好的选择特异性;
6、牛奶样品中CEX的测定
(1)样品处理:称取4 mL牛奶于10 mL离心管中,5000 rpm离心10 min。去除上层脂质层;用移液枪小心吸取500 mL中间层至干净试管中,加入2 mL 0.1 mmol/L pH 4.0 HAc-NaAc缓冲液,涡旋混匀。处理后样品稀释5倍;
(2)牛奶样品中CEX的测定:在5 mL具塞比色管中加入50 μL 0.5 mg/mL的Cu,Fe-N-C和上述待测液1mL,加入100 μL浓度为5 mmol/L的TMB 及100 μL浓度为50 mmol/L的H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,静置5-10 min,10000 r/min离心5 min,取上清液置于654 nm波长处测定吸光度,代入回归方程, CEX未检出;
(3)回收率与精密度实验:在牛奶样品中分别添加3个不同浓度的CEX标准溶液;每个浓度平行测定3次,计算加标回收率,并计算出相对标准偏差RSD,结果见表3;测得CEX的加标回收率在96.1%~102.7%,RSD在2.31%~3.86%,本方法有好的的准确性和精密度;
表3 样品加标回收率及RSD(n = 3)
7、大米质控样品Pb2+的测定
(1)样品前处理:选用大米质控样品RMS-A025 作为待测样品(中原标准物质中心);称取干燥后的大米粉样品0.3000 g,置于玻璃消化管中,加入5mL硝酸,旋紧盖塞,浸泡过夜,将样品管转移至反应罐内,将罐体完全封闭,加入氮气,将压力升至 4 000 kPa,外腔温度设置为低于40 ℃,消解升温程序见表4;消解结束完全泄压后,将样品取出,依次转移定容至10mL容量瓶中,混合均匀,待测,并进行空白试验;
表4消解升温程序
步骤 | 温度(℃) | 保持时间/min | 功率/W | 升温时间/min |
1 | 120 | 10 | 1600 | 7 |
2 | 150 | 5 | 1600 | 5 |
3 | 180 | 15 | 1600 | 10 |
(2)样品测定:在5 mL具塞比色管中加入50 μL 0.5 mg/mL的Cu,Fe-N-C和上述待测液1mL和40 mg/L CEX标准溶液100 μL,加入100 μL浓度为5 mmol/L的TMB 及100 μL浓度为50 mmol/L的H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,静置5-10min,10000 r/min离心5 min,取上清液置于654 nm波长处测定吸光度,代入回归方程,计算Pb浓度为1.15 mg/kg,标准值为1.17 ± 0.50mg/kg;
实施例2:猪肉样品中CEX及茶叶中Pb2+的测定
1、Cu,Fe-N-C纳米酶制备
(1)称取2.7 g CuCl2溶于12mL甲醇中,超声混合均匀,作为溶液A;4.7 g 中-四(4-羧基苯基)卟吩(TCPP)和2.3 g FeCl3溶于23mL甲醇中,超声混合均匀,作为溶液B;将A和B两种溶液均匀混合并85℃烘箱干燥老化2h,得到的墨绿色干燥粉末Cu,Fe- TCPP;
(2)称取 5.0 g Cu,Fe-TCPP干燥粉末于石英舟中,管式炉5°C/min升温速率至750℃ 在N2保护下煅烧2.5 h得到黑色粉末。粉末于硫酸溶液(H2SO4:H2O=1:9,体积比)中12 h浸泡后用水反复冲洗,干燥后,得到Cu,Fe-N-C纳米酶;
2、Cu,Fe-N-C纳米酶过氧化物酶活性评价:同实施例1;
3、CEX工作曲线制作:同实施例1;
4、Pb2+工作曲线制作:同实施例1;
5、猪肉样品中CEX含量的测定
(1)样品前处理方法:称取4.00 g已匀浆的样品于50 mL离心管中,加入12 mL乙腈水溶液(15︰2,体积比),均质30 s,于4℃ 4 000 r/min离心5 min,将清液转移至50 mL离心管中;另取一离心管,加入 8 mL乙腈水溶液,洗涤均质器刀头;用玻璃棒捣碎离 心管中的沉淀,加入上述洗涤均质器刀头的溶液,在涡旋混合器上振荡1 min,4 000 r/min离心5min,上清液合并至50 mL离心管中,重复用8 mL乙腈水溶液洗涤刀头并提取一次,合并至50mL离心管中,定容至 50 mL,取20 mL入100 mL容量瓶;
(2)样品中CEX测定:同实施例1,猪肉样品中CEX未检出;
6、茶叶中Pb2+的测定
(1)样品前处理:准确称取茶叶粉末样品 0.5000 g于聚四氟乙烯溶样杯中,分别加 入 5 mL HNO3、2 mLH2O2,混匀,放置过夜;装入外罐,置于微波消解仪中,按 4个工步(0.5 MPa 2 min 400 W、1.0 MPa 2 min 600 W、1.5 MPa 2 min 800 W、2.0 Mpa 2 min1000 W)进行消解;消化后,冷却开罐,将消化液洗入容量瓶中,用高纯水定容至 25 mL,存放备用;同时做试剂空白;
(2)样品测定:同实施例1,茶叶中Pb2+的含量为0.5 mg/kg;
实施例3:鸡肉样品中CEX及蔬菜中Pb2+的测定
1、Cu,Fe-N-C纳米酶制备
(1)称取3.0g CuCl2溶于15 mL甲醇中,超声混合均匀,作为溶液A;5.5 g 中-四(4-羧基苯基)卟吩(TCPP)和2.5g FeCl3溶于25mL甲醇中,超声混合均匀,作为溶液B;将A和B两种溶液均匀混合并90℃烘箱干燥老化2h,得到的墨绿色干燥粉末Cu,Fe- TCPP;
(2)称取5.5g Cu,Fe-TCPP干燥粉末于石英舟中,管式炉5°C/min升温速率至750℃在N2保护下煅烧2.5 h得到黑色粉末。粉末于硫酸溶液(H2SO4:H2O=1:9,体积比)中12 h浸泡后用水反复冲洗,干燥后,得到Cu,Fe-N-C纳米酶;
2、Cu,Fe-N-C纳米酶过氧化物酶活性评价:同实施例1;
3、CEX工作曲线制作:同实施例1;
4、Pb2+工作曲线制作:同实施例1;
5、鸡肉样品中CEX含量的测定
(1)样品前处理方法:同实施例1;
(2)样品中CEX测定:同实施例1,鸡肉样品中CEX未检出;
6、茶叶中Pb2+的测定
(1)样品前处理:选用芹菜标准物质 GBW10048 作为待测样品(地球物理地球化学勘查研究所);准确称取 1.000 g 蔬菜样品,置于玻璃消化管中,依次加入5 mL硝酸、 1 mL双氧水,盖上盖子,将样品管转移至反应罐内,将罐体完全封闭,加入氮气,将压力升至 4000 kPa,外腔温度设置为低于40 ℃,消解升温程序见表5。消解结束完全泄压后,将样品取出,依次转移定容至25 mL容量瓶中,混合均匀,待测,并进行空白试验;
(2)样品测定:同实施例1,芹菜中Pb2+浓度为61.0 µg/kg,标准值为62.3±1.9µg/kg,测定结果在误差范围内;
表5消解升温程序
步骤 | 温度(℃) | 保持时间/min | 功率/W | 升温时间/min |
1 | 110 | 15 | 1500 | 5 |
2 | 150 | 10 | 1500 | 5 |
3 | 190 | 10 | 1500 | 5 |
本发明建立的CEX及Pb2+测定法具有处理步骤少,所用时间短,处理成本低,操作简便,不需要大型仪器设备,在实际检测中具有较强优势。
Claims (4)
1.一种双原子纳米酶基快速检测头孢氨苄和铅方法,其特征在于,包括以下步骤:
(1)以中-四(4-羧基苯基)卟吩为配体(TCPP),合成Cu,Fe-TCPP配合物,以Cu,Fe-TCPP为前驱进行高温热解并经酸处理,得到双原子Cu,Fe-N-C纳米酶;
(2)在头孢氨苄标准溶液中加入Cu,Fe-N-C纳米酶溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液及H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,生成蓝色氧化oxTMB,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,建立吸光度与头孢氨苄浓度的定量关系,绘制标准曲线,得到回归方程;
(3)在头孢氨苄溶液中加入Cu,Fe-N-C纳米酶溶液、Pb2+标准溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液及H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,生成蓝色氧化oxTMB,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,建立吸光度与Pb2+浓度的定量关系,绘制标准曲线,得到回归方程;
(4)在含头孢氨苄的待测样品液中加入Cu,Fe-N-C纳米酶溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液及H2O2,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3 mL,摇匀,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,代入回归方程,计算待测样品液中头孢氨苄浓度;
在含Pb2+的待测样品液中加入Cu,Fe-N-C纳米酶溶液,然后加入3,3',5,5'-四甲基联苯胺(TMB)溶液、H2O2及头孢氨苄溶液,加入0.1 mmol/L pH 4.0 HAc-NaAc缓冲液至3mL,摇匀,静置5-10 min,离心,取上清液置于654 nm波长处测定吸光度,代入回归方程,计算待测样品液中Pb2+浓度。
2.根据权利要求1所述的方法,其特征在于,Cu,Fe-N-C纳米酶制备如下:
(1)称取2.5-3.0 g CuCl2溶于10-15 mL甲醇中,超声混合均匀,作为溶液A;4.5-5.5 g中-四(4-羧基苯基)卟吩(TCPP)和2.0-2.5 g FeCl3溶于20-25 mL甲醇中,超声混合均匀,作为溶液B;将A和B两种溶液均匀混合并80-90℃烘箱干燥老化2h,得到的墨绿色干燥粉末Cu,Fe-TCPP;
(2)称取 4.5-5.5 g Cu,Fe-TCPP干燥粉末于石英舟中,管式炉5°C/min升温速率至750℃ 在N2保护下煅烧2-2.5 h得到黑色粉末。粉末于硫酸溶液(H2SO4:H2O=1:9,体积比)中12h浸泡后用水反复冲洗,干燥后,得到Cu,Fe-N-C纳米酶。
3. 根据权利要求1所述的方法,其特征在于:头孢氨苄标准溶液的浓度为0.010~52.76mg/L,Pb2+标准溶液的浓度为0.012~45.38 mg/L;Cu,Fe-N-C纳米酶浓度为0.2 mg/mL,添加量为100 μL;TMB溶液浓度为5 mmol/L,添加量为20-200 μL;H2O2的浓度为50 mmol/L,用量为20-200 μL。
4.根据权利要求1所述的方法,其特征在于:离心是在8000-10000 r/min下处理5-10min。
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