CN108680742A - The rapid detection method of the semicarbazides determination of residual amount in seawater and marine sediment - Google Patents
The rapid detection method of the semicarbazides determination of residual amount in seawater and marine sediment Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 45
- 239000013535 sea water Substances 0.000 title claims abstract description 40
- 239000013049 sediment Substances 0.000 title claims abstract description 35
- 150000003349 semicarbazides Chemical class 0.000 title claims description 17
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000010931 gold Substances 0.000 claims abstract description 14
- 229910052737 gold Inorganic materials 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 25
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 17
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000001212 derivatisation Methods 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- 238000004458 analytical method Methods 0.000 claims description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000010586 diagram Methods 0.000 claims description 3
- 239000000084 colloidal system Substances 0.000 claims 3
- 235000011167 hydrochloric acid Nutrition 0.000 claims 3
- 235000011121 sodium hydroxide Nutrition 0.000 claims 3
- 238000005259 measurement Methods 0.000 claims 1
- 230000010355 oscillation Effects 0.000 claims 1
- 239000006228 supernatant Substances 0.000 claims 1
- DUIOPKIIICUYRZ-UHFFFAOYSA-N semicarbazide group Chemical group NNC(=O)N DUIOPKIIICUYRZ-UHFFFAOYSA-N 0.000 abstract description 20
- 239000003344 environmental pollutant Substances 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 231100000719 pollutant Toxicity 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 19
- 238000012360 testing method Methods 0.000 description 13
- 238000003908 quality control method Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- IAIWVQXQOWNYOU-FPYGCLRLSA-N nitrofural Chemical compound NC(=O)N\N=C\C1=CC=C([N+]([O-])=O)O1 IAIWVQXQOWNYOU-FPYGCLRLSA-N 0.000 description 1
- 229960001907 nitrofurazone Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
本发明公开了海水及海洋沉积物中氨基脲残留量测定的快速检测方法，涉及海洋污染物检测方法领域，公开了一种海水及海洋沉积物中氨基脲残留量测定的快速检测方法，在于使用免疫胶体金法进行测定。实现了首次建立海水和海洋沉积物中氨基脲残留量测定的免疫胶体金快速检测方法，为海水和海洋沉积物中氨基脲污染状况监测提供技术支持的技术效果。
The invention discloses a rapid detection method for the determination of semicarbazide residues in seawater and marine sediments, relates to the field of detection methods for marine pollutants, and discloses a rapid detection method for the determination of semicarbazide residues in seawater and marine sediments. Immunocolloidal gold method for determination. Achieved the first establishment of the immunocolloidal gold rapid detection method for the determination of semicarbazide residues in seawater and marine sediments, and provided technical support for the monitoring of semicarbazide pollution in seawater and marine sediments.
Description
技术领域technical field
本发明涉及海洋污染物检测方法领域，尤其涉及海水及海洋沉积物中氨基脲残留量测定的快速检测方法。The invention relates to the field of marine pollutant detection methods, in particular to a rapid detection method for the determination of semicarbazide residues in seawater and marine sediments.
背景技术Background technique
氨基脲是制取硝基呋喃类等药物的工业原料，也可作为测定酮、醛的试剂、色谱分析及分离激素精油的溶剂。从毒理学角度考虑， 氨基脲具有潜在的弱毒性及致癌性。目前，国内对海洋环境中氨基脲污染状况的研究发现，通过各种途径产生的氨基脲最终汇入大海，成为一种新型的环境污染物。因此建立海洋环境中氨基脲定性、定量检测方法，是评价氨基脲是否会影响海洋环境所必需的。Semicarbazide is an industrial raw material for preparing nitrofuran and other drugs, and it can also be used as a reagent for the determination of ketones and aldehydes, a solvent for chromatographic analysis and separation of hormone essential oils. From a toxicological point of view, semicarbazides are potentially weakly toxic and carcinogenic. At present, domestic research on the pollution of semicarbazides in the marine environment has found that semicarbazides produced through various channels eventually flow into the sea and become a new type of environmental pollutants. Therefore, the establishment of qualitative and quantitative detection methods for semicarbazides in the marine environment is necessary to evaluate whether semicarbazides will affect the marine environment.
现有氨基脲检测方法多为液相色谱串联质法，对实验人员和实验条件要求较高，检测时间较长，成本较高，检测所用仪器价格昂贵，不利于海水和海洋沉积物中氨基脲残留量的简单、快速的测定。免疫胶体金快速检测方法是将胶体金免疫技术和层析技术相结合的分析方法，具有快速、简便、低成本和安全等优点。目前，使用免疫胶体金快速检测方法对氨基脲进行测定的检测对象均为水产品，尚无利用免疫胶体金快检技术开展海水和海洋沉积物中氨基脲残留量测定方法的研究。Most of the existing semicarbazide detection methods are liquid chromatography tandem mass method, which has high requirements for experimenters and experimental conditions, long detection time, high cost, and expensive instruments for detection, which is not conducive to semicarbazide in seawater and marine sediments. Simple and fast determination of residues. The immunocolloidal gold rapid detection method is an analysis method combining colloidal gold immunological technology and chromatography technology, which has the advantages of rapidity, simplicity, low cost and safety. At present, the detection objects of semicarbazides are all aquatic products using the rapid detection method of immunocolloidal gold, and there is no research on the determination of semicarbazide residues in seawater and marine sediments using the rapid detection method of immunocolloidal gold.
发明内容Contents of the invention
本发明的目的是提供一种海水及海洋沉积物中氨基脲残留量测定的快速检测方法，首次建立海水和海洋沉积物中氨基脲残留量测定的免疫胶体金快速检测方法，为海水和海洋沉积物中氨基脲污染状况监测提供技术支持。The purpose of the present invention is to provide a rapid detection method for the determination of semicarbazide residues in seawater and marine sediments, and establish an immunocolloidal gold rapid detection method for the determination of semicarbazide residues in seawater and marine sediments for the first time. Provide technical support for the monitoring of semicarbazide pollution in food.
为解决上述问题，本发明公开了一种海水及海洋沉积物中氨基脲残留量测定的快速检测方法，其特征在于使用免疫胶体金法进行测定。In order to solve the above problems, the present invention discloses a rapid detection method for the determination of semicarbazide residues in seawater and marine sediments, which is characterized in that the determination is carried out by using an immunocolloidal gold method.
本发明还公开了海水及海洋沉积物中氨基脲残留量测定的快速检测方法，包括如下步骤：The invention also discloses a rapid detection method for determining the residual amount of semicarbazide in seawater and marine sediments, which includes the following steps:
（1）海水样品用0.45μm纤维滤膜过滤，去除海水中悬浮物，分析时准确量取经过滤的海水20ml于50mL离心管中；海洋沉积物样品沥去海水，不需干燥，分析时准确称取2.0g沉积物样品于50mL离心管中；(1) Seawater samples are filtered with a 0.45μm fiber membrane to remove suspended solids in seawater. During analysis, accurately measure 20ml of filtered seawater into a 50mL centrifuge tube; drain seawater from marine sediment samples without drying, and accurately weigh them during analysis. Take 2.0g sediment sample in a 50mL centrifuge tube;
（2）向样品中加入5mL盐酸溶液和200μL衍生化试剂溶液，振荡2min，置于60℃水浴锅中，孵育30min；(2) Add 5 mL of hydrochloric acid solution and 200 μL of derivatization reagent solution to the sample, shake for 2 min, place in a 60°C water bath, and incubate for 30 min;
（3）加入5mL磷酸氢二钾溶液和800μL氢氧化钠溶液，振荡1min，再加入10mL乙酸乙酯，振荡2min，然后置于离心机中4000r/min，离心5min；(3) Add 5 mL of dipotassium hydrogen phosphate solution and 800 μL of sodium hydroxide solution, shake for 1 minute, then add 10 mL of ethyl acetate, shake for 2 minutes, then place in a centrifuge at 4000 r/min, and centrifuge for 5 minutes;
（4）准确移取上层液体5mL于15mL离心管中，60℃下吹干；(4) Accurately pipette 5mL of the upper liquid into a 15mL centrifuge tube, and blow dry at 60°C;
（5）向吹干的离心管中加入2mL正己烷，振荡1min，再准确加入500μL复溶试剂，振荡1min，然后置于离心机中4000r/min，离心1min，取下层溶液待测；(5) Add 2 mL of n-hexane to the dried centrifuge tube, shake for 1 min, then accurately add 500 μL of reconstitution reagent, shake for 1 min, then place in a centrifuge at 4000 r/min, centrifuge for 1 min, and take the lower layer solution for testing;
（6）用移液枪移取100μL待测液于免疫胶体金快速检测卡加样孔中，室温反应5min，根据结果判读示意图判读结果。(6) Use a pipette gun to pipette 100 μL of the test solution into the sample well of the immunocolloidal gold rapid detection card, react at room temperature for 5 minutes, and interpret the results according to the schematic diagram.
进一步的，其中所述盐酸溶液浓度为0.20 mol/L：量取密度为ρ=1.19 g/mL浓盐酸0.60 mL，用水稀释至100 mL。Further, wherein the concentration of the hydrochloric acid solution is 0.20 mol/L: measure 0.60 mL of concentrated hydrochloric acid with a density of ρ=1.19 g/mL, and dilute to 100 mL with water.
进一步的，其中所述磷酸氢二钾溶液浓度为0.1mol/L：准确称取1.74 g磷酸氢二钾，溶于100 mL水中制成。Further, the concentration of the dipotassium hydrogen phosphate solution is 0.1mol/L: accurately weigh 1.74 g of dipotassium hydrogen phosphate, and dissolve it in 100 mL of water.
进一步的，其中所述氢氧化钠溶液浓度为1.0 mol/L：准确称取4.00 g氢氧化钠，溶于100 mL水中制成。Further, the concentration of the sodium hydroxide solution is 1.0 mol/L: accurately weigh 4.00 g of sodium hydroxide and dissolve it in 100 mL of water.
进一步的，其中所述氨基脲免疫胶体金快速检测盒，其中含有检测卡、衍生化试剂和复溶试剂。Further, the semicarbazide immune colloidal gold rapid detection kit contains a detection card, a derivatization reagent and a reconstitution reagent.
进一步的，其中所述电子天平的精度为0.01g。Further, the precision of the electronic balance is 0.01g.
进一步的，其中所述室温为20~25℃。Further, wherein the room temperature is 20-25°C.
本发明的有益效果在于：通过海水和海洋环境中的氨基脲在酸性条件下，与衍生化试剂发生衍生化反应，衍生物经乙酸乙酯液-液萃取，正己烷净化后，用免疫胶体金快速检测卡进行测定。The beneficial effects of the present invention are: the semicarbazide in seawater and marine environment undergoes a derivatization reaction with a derivatization reagent under acidic conditions; Rapid test card for determination.
建立海水和海洋沉积物中氨基脲残留量测定的免疫胶体金快速检测方法，与现有海水和海洋沉积物中氨基脲检测方法相比。免疫胶体金快速检测方法对实验人员和实验条件要求较低，不需要液相色谱串联质谱仪等昂贵仪器，将衍生化时间由16h缩短为30min，极大缩短检测时间，可以对海水和海洋沉积物进行现场检测。通过此方法，海水中定性检测限为0.1μg/L，海洋沉积物中定性检测限为1.0μg/kg。Establish an immunocolloidal gold rapid detection method for the determination of semicarbazide residues in seawater and marine sediments, and compare it with the existing detection methods for semicarbazides in seawater and marine sediments. The rapid detection method of immunocolloidal gold has lower requirements on experimenters and experimental conditions, and does not require expensive instruments such as liquid chromatography tandem mass spectrometers, shortens the derivatization time from 16h to 30min, greatly shortens the detection time, and can detect seawater and marine sediments. on-site testing. By this method, the qualitative detection limit in seawater is 0.1 μg/L, and the qualitative detection limit in marine sediment is 1.0 μg/kg.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.
图1是本发明的结果判读依据图；Fig. 1 is the result interpretation basis figure of the present invention;
图2是本发明的实验结果例图。Fig. 2 is an illustration of the experimental results of the present invention.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention.
准备如下试剂及仪器Prepare the following reagents and instruments
盐酸溶液浓度为0.20 mol/L：量取密度为ρ=1.19 g/mL浓盐酸0.60 mL，用水稀释至100mL。The concentration of hydrochloric acid solution is 0.20 mol/L: Measure 0.60 mL of concentrated hydrochloric acid with a density of ρ=1.19 g/mL and dilute it to 100 mL with water.
磷酸氢二钾溶液浓度为0.1mol/L：准确称取1.74 g磷酸氢二钾，溶于100 mL水中制成。The concentration of dipotassium hydrogen phosphate solution is 0.1mol/L: Accurately weigh 1.74 g of dipotassium hydrogen phosphate and dissolve it in 100 mL of water.
氢氧化钠溶液浓度为1.0 mol/L：准确称取4.00 g氢氧化钠，溶于100 mL水中制成。The concentration of sodium hydroxide solution is 1.0 mol/L: Accurately weigh 4.00 g of sodium hydroxide and dissolve it in 100 mL of water.
氨基脲免疫胶体金快速检测盒，其中含有检测卡、衍生化试剂和复溶试剂。Semicarbazide immunocolloidal gold rapid detection kit, which contains detection card, derivatization reagent and reconstitution reagent.
以及正己烷、乙酸乙酯、醋酸纤维滤膜0.45μm、电子天平精度为0.01g，水浴锅、离心机、氮吹仪或样品浓缩仪、移液枪。And n-hexane, ethyl acetate, cellulose acetate filter membrane 0.45μm, electronic balance precision 0.01g, water bath, centrifuge, nitrogen blowing device or sample concentrator, pipette gun.
具体的测试步骤包括如下：The specific test steps include the following:
（1）海水样品用0.45μm纤维滤膜过滤，去除海水中悬浮物，分析时准确量取经过滤的海水20ml于50mL离心管中；海洋沉积物样品沥去海水，不需干燥，分析时准确称取2.0g沉积物样品于50mL离心管中；(1) Seawater samples are filtered with a 0.45μm fiber membrane to remove suspended solids in seawater. During analysis, accurately measure 20ml of filtered seawater into a 50mL centrifuge tube; drain seawater from marine sediment samples without drying, and accurately weigh them during analysis. Take 2.0g sediment sample in a 50mL centrifuge tube;
（2）向样品中加入5mL盐酸溶液和200μL衍生化试剂溶液，振荡2min，置于60℃水浴锅中，孵育30min；(2) Add 5 mL of hydrochloric acid solution and 200 μL of derivatization reagent solution to the sample, shake for 2 min, place in a 60°C water bath, and incubate for 30 min;
（3）加入5mL磷酸氢二钾溶液和800μL氢氧化钠溶液，振荡1min，再加入10mL乙酸乙酯，振荡2min，然后置于离心机中4000r/min，离心5min；(3) Add 5 mL of dipotassium hydrogen phosphate solution and 800 μL of sodium hydroxide solution, shake for 1 minute, then add 10 mL of ethyl acetate, shake for 2 minutes, then place in a centrifuge at 4000 r/min, and centrifuge for 5 minutes;
（4）准确移取上层液体5mL于15mL离心管中，60℃下吹干；(4) Accurately pipette 5mL of the upper liquid into a 15mL centrifuge tube, and blow dry at 60°C;
（5）向吹干的离心管中加入2mL正己烷，振荡1min，再准确加入500μL复溶试剂，振荡1min，然后置于离心机中4000r/min，离心1min，取下层溶液待测；(5) Add 2 mL of n-hexane to the dried centrifuge tube, shake for 1 min, then accurately add 500 μL of reconstitution reagent, shake for 1 min, then place in a centrifuge at 4000 r/min, centrifuge for 1 min, and take the lower layer solution for testing;
（6）用移液枪移取100μL待测液于免疫胶体金快速检测卡加样孔中，室温反应5min，根据结果判读示意图判读结果。(6) Use a pipette gun to pipette 100 μL of the test solution into the sample well of the immunocolloidal gold rapid detection card, react at room temperature for 5 minutes, and interpret the results according to the schematic diagram.
如图1所示的结果判读依据图例所示，测试能够出现三种结果：The result interpretation shown in Figure 1 is based on the legend, and the test can produce three results:
1、阴性：质控线C显色，检测线T比质控线C显色深或一样深，表示样品中不含有待检物或其浓度低于检测限；1. Negative: The quality control line C develops color, and the detection line T is darker or the same darker than the quality control line C, indicating that the sample does not contain the substance to be tested or its concentration is lower than the detection limit;
2、阳性：质控线C显色，检测线T比质控线C显色浅或T线不显色，则表示样品中待检物浓度高于检测限；2. Positive: if the quality control line C develops color, and the detection line T is lighter than the quality control line C or the T line does not develop color, it means that the concentration of the substance to be tested in the sample is higher than the detection limit;
3、无效：质控线C未显色，表明操作步骤不正确或检测卡失效。3. Invalid: The quality control line C has no color, indicating that the operation steps are incorrect or the test card is invalid.
实施例一：Embodiment one:
使用阴性海水样品按照上述步骤进行测试。Perform the test as described above using a negative seawater sample.
实施例二：Embodiment two:
使用阴性海洋沉积物样品按照上述步骤进行测试。The test was performed as described above using a negative marine sediment sample.
实施例三：Embodiment three:
使用空白海洋沉积物样品按照上述步骤进行测试。Use blank marine sediment samples for testing as described above.
实施例四：Embodiment four:
使用阳性海水样品按照上述步骤进行测试。Use positive seawater samples for testing as described above.
实施例五：Embodiment five:
使用阳性海洋沉积物样品按照上述步骤进行测试。Use a positive marine sediment sample to perform the test as described above.
实施例六：Embodiment six:
使用空白海洋沉积物加标样品按照上述步骤进行测试。Use blank marine sediment spiked samples for testing as described above.
由附图2可见，该方法能够准确的对海水及海洋沉积物中氨基脲残留量进行快速测定，为海水和海洋沉积物氨基脲污染状况监测提供技术支持。It can be seen from Figure 2 that the method can accurately and quickly measure the semicarbazide residues in seawater and marine sediments, and provide technical support for the monitoring of semicarbazide pollution status in seawater and marine sediments.
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