用于碳水化合物类农产品重金属ELISA检测的样品前处理研究
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摘要
近年来,传统的检测技术在应对频发的食品安全问题时愈发凸显其繁琐、耗时、不易现场操作等弊端。ELISA试剂盒作为一种快速、可靠的检测方法,其应用解决了传统检测技术的一些缺陷。但ELISA试剂盒只能检测游离的液态重金属,针对这一情况,研究并建立一种高效、快速并易于现场操作的与ELISA检测技术相适的样品前处理方法具有重要的现实意义。
本文选取菜心、胡萝卜和南瓜作为蔬菜类农产品的代表,以其为研究对象,用稀HCl、HNO3及H2SO4对样品进行浸提,探索蔬菜中重金属Cu、Pb、Cd的最适浸提条件。研究表明,选取浓度为2.0M的稀HCl在40℃下反应20min可获得最佳效果,与国家标准方法中的微波消解法相比,其提取率为64~98%;选取浓度为2.0M的稀HNO3在40℃下反应10min可获得最佳效果,与国标法相比,其提取率为61~100%;选取浓度为2.0M的稀H2SO4在25℃下反应20min可获得最佳效果,与国标法相比,其提取率为57~100%。且稀HCl对菜心中的Cu、Cd,南瓜中的Cu都有很好的提取效果,提取率为87~98%;稀HNO3对菜心中的Cu、Pb、Cd,南瓜中的Cu、Cd都有非常好的提取效果,提取率为90~100%;稀H2SO4对菜心、胡萝卜、南瓜中的Cu,菜心和胡萝卜中的Cd亦有不错的提取效果,提取率为81~100%,上述重金属离子的测定均可选择相应的稀酸浸提法代替国标法来测定其含量。
以苹果、葡萄和香蕉作为水果类农产品的代表,实验表明,使用浓度为2.0-4.0M的稀HCl在25℃下反应15min可获得最佳效果,与国标法相比,其提取率为57~96%;选取浓度为4.0M的稀HNO3在70℃下反应20min可获得最佳效果,与国标法相比,其提取率为55~99%;选取浓度为4.0M的稀H2SO4在4℃下反应20min可获得最佳效果,与国标法相比,其提取率为54~96%。并且稀HNO3对苹果中的Cu、Cd,葡萄和香蕉中的Cu都有非常好的提取效果,其提取率为84~100%,稀HCl及稀H2SO4对于上述重金属离子亦都有很好的提取效果,提取率分别为83~96%、81~96%;稀H2SO4对葡萄中的Cd和苹果中的Pb亦有不错的提取效果,其提取率分别为83%、85%,上述重金属离子的测定均可选择相应的稀酸浸提法代替国标法来测定其含量。
以大米、小麦和糙米作为粮食类农产品的代表,研究表明,使用浓度为2.0M的稀HCl在25℃下反应20min可获得最佳效果,其提取率为37~57%;选取浓度为2.0M的稀HNO3在4℃下反应20min可获得最佳效果,其提取率为30~72%;选取浓度为4.0M的稀H2SO4在25℃下反应20min可获得最佳效果,其提取率为17~66%。对于大米、糙米中的Cu、Pb、Cd,小麦中的Cu、Pb,稀HCl的提取率>50%;对于大米、小麦及糙米中的Cu、Cd,糙米中的Pb,稀HNO3提取率都>50%;对于大米、小麦、糙米中的Cu,大米中的Pb,稀H2SO4的提取率>50%。
加标回收实验结果表明,稀HCl对蔬菜类、水果类及粮食类中的Cu、Pb、Cd的加标回收率分别为69~104%、76~105%、78~100%,与国标法相比,略低于国标法;稀HNO3对蔬菜类、水果类及粮食类中的Cu、Pb、Cd的加标回收率分别为87~109%、87~111%,、82~113%,与国标法相比,两者相近;稀H2SO4对蔬菜类、水果类及粮食类中的Cu、Pb、Cd的加标回收率分别为80~114%、87~103%、86~121%,与国标法相比,两者较为接近,上述结果验证了稀酸浸提法的可靠性。
以三种稀酸的最适反应条件浸提蔬菜类、水果类及粮食类农产品中的重金属Cu、Pb、Cd,分别用石墨炉原子分光光度法(GF-AAS)和ELISA试剂盒检测农产品中重金属的含量,统计分析表明,两种方法线性相关性良好(相关系数R2=0.9515)。表明所建立的稀酸提取碳水化合物类农产品中重金属的前处理方法能够满足ELISA试剂盒检测碳水化合物类农产品中重金属的要求。?
With frequent food safety problem in recent years, the traditional testing technologies of food safety become more tedious, more time-consuming and more difficult to do detection in field. Enzyme-Linked Immunosorbent Assay (ELISA) kit as a rapid, reliable detection method has been applied to solve some disadvantages of the traditional testing technology. But ELISA kit can only detect the liquid free iron of heavy metals, in view of this situation, it would be of realistic and far-reaching significance to develop an efficient, fast, easy sample pretreatment method based on ELISA .
The optimum pretreatment conditions for Cu, Pb and Cd in vegetables such as Chinese cabbage (Brassica campestris L. sub. Chinensis var. utilis Tsen et Lee), carrot(Daucus carota var. L. sativa Hoffm.) and pumpkin(Cucurbita moschata (Duch. ex Lam.) Duch. ex Poir.) were studied with the extracting agent of three dilute acid (HCl, HNO3 and H2SO4). The results showed that the optimum pretreatment conditions were performed as follow: 40℃for 20min with 2.0M HCl, and the extraction rate was 64~98% as compared with the National standard method of microwave digestion; 40℃for 10min with 2.0M HNO3, and the extraction rate was 61~100% as compared with the National standard method; 25℃for 20min with 2.0M H2SO4, and the extraction rate was 57~100% as compared with the National standard method. And for Cu and Cd in Chinese cabbage and Cu in pumpkin, HCl has better extraction rate, the extraction rate was 87~98%; for Cu, Pb and Cd in Chinese cabbage and Cu and Cd in pumpkin, HNO3 has the best extraction rate, the extraction rate was 90~100%; for Cu in Chinese cabbage, carrot and pumpkin and Cd in Chinese cabbage and carrot, H2SO4 has better extraction rate, the extraction rate was 81~100%, all of them could substitute for the traditional pretreatment methods.
The optimum pretreatment conditions of Cu, Pb and Cd extraction for fruit such as apple(Malus pumila Mill.), grape(Vitis vinifera L.) and banana(Musa paradisiacal L.) were investigated with the extracting agent of three dilute acid (HCl, HNO3 and H2SO4) ,too. The results showed that the optimum pretreatment conditions were performed as follow: 25℃for 15min with 2.0-4.0M HCl, and the extraction rate was 57~96% as compared with the National standard method; 70℃for 20min with 4.0M HNO3, and the extraction rate was 55~99% as compared with the National standard method; 4℃for 20min with 4.0M H2SO4, and the extraction rate was 54~96% as compared with the National standard method. And for Cu and Cd in apple and Cu in grape and banana, HNO3 has better extraction rate as well as HCl and H2SO4, the extraction rate were 84~100%, 83~96%,81~96%,respectively; for Cd in grape and Pb in apple, H2SO4 has better extraction rate, the extraction rate were 83%, 85%, respectively, all of them could substitute for the traditional pretreatment methods.
The optimum pretreatment conditions of Cu, Pb and Cd extraction for grain such as rice(Oryza sativa L.), wheat(Triticum aestivum L.) and brown rice(Oryza sativa L.) were also investigated with the extracting agent of three dilute acid (HCl, HNO3 and H2SO4) . The results showed that the optimum pretreatment conditions were performed as follow: 25℃for 20min with 2.0M HCl, and the extraction rate compared by the National standard method was 37~57%; 4℃for 20min with 2.0M HNO3, and the extraction rate compared by the National standard method was 30~72%; 25℃for 20min with 4.0M H2SO4, and the extraction rate compared by the National standard method was 17~66%. And for Cu, Pb and Cd in rice and brown rice and Cu and Pb in wheat, HCl was of more than 50% extraction rate; for Cu and Cd of rice, wheat and brown rice and Pb in brown rice, HNO3 was of more than 50% extraction rate; for Cu in rice, wheat and brown rice and Pb in rice, H2SO4 was of more than 50% extraction rate. The standard recovery test shows that, the recovery rates of Cu, Pb and Cd in vegetables, fruits and grains extracted by dilute HCl were 69~104%, 76~105%, 78~100%, which is slightly lower than the National standard method; the recovery rates of Cu, Pb and Cd in vegetables, fruits and grains extracted by dilute HNO3 were 87~109%, 87~111%, 82~113%, which is close to the National standard method; the recovery rates of Cu, Pb and Cd in vegetables, fruits and grains extracted by dilute H2SO4 were 69~104%, 76~105%, 78~100%, which was also close to the National standard method, the above results verify the reliability of acid extraction.
Finally, The both group data of Cu concentrations of carbohydrate produces, extracted with dilute acid pretreatment method, and determined by graphite furnace atomic absorption spectrometry(GF-AAS) method and ELISA assay respectively, were significantly correlated(R2=0.9515), this showed that dilute acid pretreatment method could meet the requirements of detecting heavy metals(Cu, Pb and Cd) of carbohydrate produces with ELISA kit.
本文选取菜心、胡萝卜和南瓜作为蔬菜类农产品的代表,以其为研究对象,用稀HCl、HNO3及H2SO4对样品进行浸提,探索蔬菜中重金属Cu、Pb、Cd的最适浸提条件。研究表明,选取浓度为2.0M的稀HCl在40℃下反应20min可获得最佳效果,与国家标准方法中的微波消解法相比,其提取率为64~98%;选取浓度为2.0M的稀HNO3在40℃下反应10min可获得最佳效果,与国标法相比,其提取率为61~100%;选取浓度为2.0M的稀H2SO4在25℃下反应20min可获得最佳效果,与国标法相比,其提取率为57~100%。且稀HCl对菜心中的Cu、Cd,南瓜中的Cu都有很好的提取效果,提取率为87~98%;稀HNO3对菜心中的Cu、Pb、Cd,南瓜中的Cu、Cd都有非常好的提取效果,提取率为90~100%;稀H2SO4对菜心、胡萝卜、南瓜中的Cu,菜心和胡萝卜中的Cd亦有不错的提取效果,提取率为81~100%,上述重金属离子的测定均可选择相应的稀酸浸提法代替国标法来测定其含量。
以苹果、葡萄和香蕉作为水果类农产品的代表,实验表明,使用浓度为2.0-4.0M的稀HCl在25℃下反应15min可获得最佳效果,与国标法相比,其提取率为57~96%;选取浓度为4.0M的稀HNO3在70℃下反应20min可获得最佳效果,与国标法相比,其提取率为55~99%;选取浓度为4.0M的稀H2SO4在4℃下反应20min可获得最佳效果,与国标法相比,其提取率为54~96%。并且稀HNO3对苹果中的Cu、Cd,葡萄和香蕉中的Cu都有非常好的提取效果,其提取率为84~100%,稀HCl及稀H2SO4对于上述重金属离子亦都有很好的提取效果,提取率分别为83~96%、81~96%;稀H2SO4对葡萄中的Cd和苹果中的Pb亦有不错的提取效果,其提取率分别为83%、85%,上述重金属离子的测定均可选择相应的稀酸浸提法代替国标法来测定其含量。
以大米、小麦和糙米作为粮食类农产品的代表,研究表明,使用浓度为2.0M的稀HCl在25℃下反应20min可获得最佳效果,其提取率为37~57%;选取浓度为2.0M的稀HNO3在4℃下反应20min可获得最佳效果,其提取率为30~72%;选取浓度为4.0M的稀H2SO4在25℃下反应20min可获得最佳效果,其提取率为17~66%。对于大米、糙米中的Cu、Pb、Cd,小麦中的Cu、Pb,稀HCl的提取率>50%;对于大米、小麦及糙米中的Cu、Cd,糙米中的Pb,稀HNO3提取率都>50%;对于大米、小麦、糙米中的Cu,大米中的Pb,稀H2SO4的提取率>50%。
加标回收实验结果表明,稀HCl对蔬菜类、水果类及粮食类中的Cu、Pb、Cd的加标回收率分别为69~104%、76~105%、78~100%,与国标法相比,略低于国标法;稀HNO3对蔬菜类、水果类及粮食类中的Cu、Pb、Cd的加标回收率分别为87~109%、87~111%,、82~113%,与国标法相比,两者相近;稀H2SO4对蔬菜类、水果类及粮食类中的Cu、Pb、Cd的加标回收率分别为80~114%、87~103%、86~121%,与国标法相比,两者较为接近,上述结果验证了稀酸浸提法的可靠性。
以三种稀酸的最适反应条件浸提蔬菜类、水果类及粮食类农产品中的重金属Cu、Pb、Cd,分别用石墨炉原子分光光度法(GF-AAS)和ELISA试剂盒检测农产品中重金属的含量,统计分析表明,两种方法线性相关性良好(相关系数R2=0.9515)。表明所建立的稀酸提取碳水化合物类农产品中重金属的前处理方法能够满足ELISA试剂盒检测碳水化合物类农产品中重金属的要求。?
With frequent food safety problem in recent years, the traditional testing technologies of food safety become more tedious, more time-consuming and more difficult to do detection in field. Enzyme-Linked Immunosorbent Assay (ELISA) kit as a rapid, reliable detection method has been applied to solve some disadvantages of the traditional testing technology. But ELISA kit can only detect the liquid free iron of heavy metals, in view of this situation, it would be of realistic and far-reaching significance to develop an efficient, fast, easy sample pretreatment method based on ELISA .
The optimum pretreatment conditions for Cu, Pb and Cd in vegetables such as Chinese cabbage (Brassica campestris L. sub. Chinensis var. utilis Tsen et Lee), carrot(Daucus carota var. L. sativa Hoffm.) and pumpkin(Cucurbita moschata (Duch. ex Lam.) Duch. ex Poir.) were studied with the extracting agent of three dilute acid (HCl, HNO3 and H2SO4). The results showed that the optimum pretreatment conditions were performed as follow: 40℃for 20min with 2.0M HCl, and the extraction rate was 64~98% as compared with the National standard method of microwave digestion; 40℃for 10min with 2.0M HNO3, and the extraction rate was 61~100% as compared with the National standard method; 25℃for 20min with 2.0M H2SO4, and the extraction rate was 57~100% as compared with the National standard method. And for Cu and Cd in Chinese cabbage and Cu in pumpkin, HCl has better extraction rate, the extraction rate was 87~98%; for Cu, Pb and Cd in Chinese cabbage and Cu and Cd in pumpkin, HNO3 has the best extraction rate, the extraction rate was 90~100%; for Cu in Chinese cabbage, carrot and pumpkin and Cd in Chinese cabbage and carrot, H2SO4 has better extraction rate, the extraction rate was 81~100%, all of them could substitute for the traditional pretreatment methods.
The optimum pretreatment conditions of Cu, Pb and Cd extraction for fruit such as apple(Malus pumila Mill.), grape(Vitis vinifera L.) and banana(Musa paradisiacal L.) were investigated with the extracting agent of three dilute acid (HCl, HNO3 and H2SO4) ,too. The results showed that the optimum pretreatment conditions were performed as follow: 25℃for 15min with 2.0-4.0M HCl, and the extraction rate was 57~96% as compared with the National standard method; 70℃for 20min with 4.0M HNO3, and the extraction rate was 55~99% as compared with the National standard method; 4℃for 20min with 4.0M H2SO4, and the extraction rate was 54~96% as compared with the National standard method. And for Cu and Cd in apple and Cu in grape and banana, HNO3 has better extraction rate as well as HCl and H2SO4, the extraction rate were 84~100%, 83~96%,81~96%,respectively; for Cd in grape and Pb in apple, H2SO4 has better extraction rate, the extraction rate were 83%, 85%, respectively, all of them could substitute for the traditional pretreatment methods.
The optimum pretreatment conditions of Cu, Pb and Cd extraction for grain such as rice(Oryza sativa L.), wheat(Triticum aestivum L.) and brown rice(Oryza sativa L.) were also investigated with the extracting agent of three dilute acid (HCl, HNO3 and H2SO4) . The results showed that the optimum pretreatment conditions were performed as follow: 25℃for 20min with 2.0M HCl, and the extraction rate compared by the National standard method was 37~57%; 4℃for 20min with 2.0M HNO3, and the extraction rate compared by the National standard method was 30~72%; 25℃for 20min with 4.0M H2SO4, and the extraction rate compared by the National standard method was 17~66%. And for Cu, Pb and Cd in rice and brown rice and Cu and Pb in wheat, HCl was of more than 50% extraction rate; for Cu and Cd of rice, wheat and brown rice and Pb in brown rice, HNO3 was of more than 50% extraction rate; for Cu in rice, wheat and brown rice and Pb in rice, H2SO4 was of more than 50% extraction rate. The standard recovery test shows that, the recovery rates of Cu, Pb and Cd in vegetables, fruits and grains extracted by dilute HCl were 69~104%, 76~105%, 78~100%, which is slightly lower than the National standard method; the recovery rates of Cu, Pb and Cd in vegetables, fruits and grains extracted by dilute HNO3 were 87~109%, 87~111%, 82~113%, which is close to the National standard method; the recovery rates of Cu, Pb and Cd in vegetables, fruits and grains extracted by dilute H2SO4 were 69~104%, 76~105%, 78~100%, which was also close to the National standard method, the above results verify the reliability of acid extraction.
Finally, The both group data of Cu concentrations of carbohydrate produces, extracted with dilute acid pretreatment method, and determined by graphite furnace atomic absorption spectrometry(GF-AAS) method and ELISA assay respectively, were significantly correlated(R2=0.9515), this showed that dilute acid pretreatment method could meet the requirements of detecting heavy metals(Cu, Pb and Cd) of carbohydrate produces with ELISA kit.
引文
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