基于荧光染色法的菲律宾帘蛤中微塑料的高效分离研究  

Research on the efficient separation of microplastics in Manila clam based on fluorescent staining

作　　者：姚梦竹 刘吉强 陈翊[3] 沈浩 张宜明[1] YAO Mengzhu;LIU Jiqiang;CHEN Yi;SHEN Hao;ZHANG Yiming(College of Food and Health,Zhejiang A&F University,Hangzhou 311300,China;Key Laboratory of Submarine Geosciences,Second Institute of Oceanography,Ministry of Natural Resources,Hangzhou 310012,China;Yuhang Center for Disease Control and Prevention,Hangzhou 311100,China;Bureau Veritas Consumer Product Shen’ou(Wenzhou)Co.,Ltd,Wenzhou 325027,China)

机构地区：[1]浙江农林大学食品与健康学院,浙江杭州311300 [2]自然资源部第二海洋研究所,自然资源部海底科学重点实验室,浙江杭州310012 [3]杭州市余杭区疾病预防控制中心,浙江杭州311100 [4]必维申瓯质量技术服务温州有限公司,浙江温州325027

出　　处：《中国水产科学》2024年第6期718-730,共13页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(32072298);杭州市农业与社会发展科研引导项目(20220919Y201)。

摘　　要：为建立基于荧光染色技术的贝类中微塑料的高效分离前处理方案,以菲律宾帘蛤(Ruditapes philippinarum)作为研究对象,在10%氢氧化钾(KOH)和蛋白酶K两种消解体系下,考察消解效率、堵膜率和微塑料回收率及红外光谱变化情况,确定最佳消解条件并做优化。优化方案结合尼罗红荧光染色,并用共聚焦显微拉曼光谱仪对疑似荧光颗粒进行验证,确定方案的适用性。实验表明:优化后的KOH消解方案,消解效率提高到(99.80±0.06)%,堵膜率降至(95.78±6.51)%,3~5 mm粒径塑料标准品的回收率为(97.92±3.61)%,400~600μm粒径塑料标准品的回收率为(89.58±4.14)%。经优化的蛋白酶K消解方案,消解效率提升至(99.97±0.02)%,堵膜率降为(94.37±9.85)%,两种粒径的塑料标准品回收率分别为(97.92±3.61)%和(87.08±2.69)%。使用尼罗红溶液染色有效地提高了目视显微观察筛选效率。实验发现蛋白酶K优化组出现众多假阳性荧光亮点,影响显微拉曼测定,而KOH优化组未出现类似的情况。样品共检出0.99个/g(湿重)微塑料,包括聚对苯二甲酸乙二醇酯[polyethylene terephthalate,PET,纤维状,长度为(971.23±22.01)μm]和丙烯腈–丁二烯–苯乙烯共聚物[acrylonitrile butadiene styrene,ABS,颗粒,粒径为(26.88±1.69)μm]。最终采用以10%KOH为消解溶液,超声10 min后,在50℃,150 r/min的水浴中消解4 h,经5%稀盐酸溶液调节pH为优化前处理方案,结合尼罗红荧光染色观察和共聚焦显微拉曼定性,本方案简单、经济且高效,可适用于双壳贝类中微塑料的检测分析。Microplastics are widely present in the atmosphere,soil,and water and can be transferred,enriched,and bioaccumulated through the food chain.The choice of digestion solution depends on test sample characteristics and the digestion time varies from a few hours to several days,which may have detrimental effects on the original plastic by causing dissolution,fracture,and degradation,thereby potentially interfering with experimental data.Each existing detection technology has its advantages and limitations.To establish an efficient pre-separation treatment scheme for shellfish microplastics based on fluorescence staining technology,this study focused on the Manila clam(Ruditapes philippinarum)because they are one of the main indicators of microplastic pollution in shellfish in China.We investigated digestion efficiency,membrane obstruction rate,microplastic recovery rate,as well as changes in the infrared spectrum under two digestion systems:10% potassium hydroxide(KOH)and protease K.Digestion conditions were determined and optimized accordingly.Furthermore,we combined the optimization scheme with Nile red fluorescence staining to validate the unknown fluorescent particles using a confocal micro-Raman spectrometer to confirm the applicability of the proposed scheme.The results showed that under a 10%KOH digestion system,there was an extremely significant difference in digestion efficiency among the three groups(P˂0.01).The highest digestion efficiency was observed in the 4-h group at(99.46±0.49)%with a blocking rate of(126.17±53.30)%.For the protease K digestion system,a significant difference was observed among the three groups(P˂0.05),with the average digestion efficiency being highest in the 16-h group at(99.66±0.08)%.No statistically significant differences were observed in recovery rates of standard microplastic particles among the experimental groups(P>0.05).The major characteristic peaks observed between the experimental and quality control groups were consistent;however,differences existed in peak intensi

关 键 词：微塑料 菲律宾帘蛤 消解 荧光染色 KOH 蛋白酶K 

分 类 号：X55[环境科学与工程—环境工程]