心肌细胞电位传感器在海洋生物毒素检测中的研究
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摘要
目前海洋毒素对人类健康的威胁越加严重,尤其是麻痹性毒素。提出了一种基于细胞电位传感器检测贝类的生物毒性的新方法,采用心肌细胞与微电子加工技术的微阵列芯片(MEA)相结合,构建了新型的心肌细胞电位传感器。通过心肌细胞与MEA芯片的紧密耦合,实现了实时监测心肌细胞电位的变化,输出稳定和一致性好的胞外场电位信号。通过检测海洋中危害较大的石房蛤麻痹性毒素(STX),分析细胞的信号特征参数,实验结果表明,STX毒素对心肌细胞的电生理活动有明显的抑制作用,对脉冲幅值和脉冲频率有明显的浓度依赖性抑制作用。常规标准的生物毒性检测方法小鼠生物法的检出限为40 g/100 g,而心肌细胞电位传感器可检测的下限为1.004 ng/m L,检测范围为25 nmol/L~1 600 nmol/L,通过对比表明该方法具有较好的检出限且检测方法相对简单,易于规范化和标准化。通过进一步的完善,该方法在海洋毒素的快速检测应用方面具有良好的发展前景。
With the rapid development of modern industry,the threat from marine toxins to human health has be-come increasingly serious,especially the paralytic biological poison. This paper presents a new method of cellbased potential sensor to detect shellfish toxicity. The cardiomyocyte combined with microelectrode array(MEA)bymicroelectronic processing technique which construct a novel cardiomyocyte potential sensor. The sensor can not on-ly real-time monitoring,but also could output good extracellular field potential signal with stability and consistency,which cardiomyocytes seeded on the MEA high-closely. By testing saxitoxin(STX)and analyzing the characteristicsof signal parameters,the experimental results illustrate the STX have obvious inhibitory effect for electrical activityof cardiomyocyte. And the peak potential amplitude and frequency have obvious inhibition of concentration depen-dence,and presented the different concentration sensitivity. The LOD(limit of detection)of conventional standardlaboratory toxicity detection on mouse bioassay method is 40 μg/100 g,while the LOD and detection range of cardio-myocyte potential sensor is 1.004 ng/m L and 25 nmol/L~1 600 nmol/L,respectively. By comparative experiments,this method has great LOD and the detection is relatively simple and easy to fit standardization. With further im-provement,the method has good prospects for development on detection rapidly toxicity of marine toxins.
With the rapid development of modern industry,the threat from marine toxins to human health has be-come increasingly serious,especially the paralytic biological poison. This paper presents a new method of cellbased potential sensor to detect shellfish toxicity. The cardiomyocyte combined with microelectrode array(MEA)bymicroelectronic processing technique which construct a novel cardiomyocyte potential sensor. The sensor can not on-ly real-time monitoring,but also could output good extracellular field potential signal with stability and consistency,which cardiomyocytes seeded on the MEA high-closely. By testing saxitoxin(STX)and analyzing the characteristicsof signal parameters,the experimental results illustrate the STX have obvious inhibitory effect for electrical activityof cardiomyocyte. And the peak potential amplitude and frequency have obvious inhibition of concentration depen-dence,and presented the different concentration sensitivity. The LOD(limit of detection)of conventional standardlaboratory toxicity detection on mouse bioassay method is 40 μg/100 g,while the LOD and detection range of cardio-myocyte potential sensor is 1.004 ng/m L and 25 nmol/L~1 600 nmol/L,respectively. By comparative experiments,this method has great LOD and the detection is relatively simple and easy to fit standardization. With further im-provement,the method has good prospects for development on detection rapidly toxicity of marine toxins.
引文
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[25]于兵,曹际娟,尤永莉,等.ELISA与小白鼠生物法检测贝类中麻痹性贝毒的比较[J].检验检疫科学,2005,15(1):32-35.
[2]Hinder S L,Hays G C,Brooks C J,et al.Toxic Marine Microalgae and Shellfish Poisoning in the Britishisles:History,Review of Epidemiology,and Future Implications[J].Environmental Health,2011,10(1):54.
[3]Fleming L E,Jerez E,Stephan W B B,et al.Evaluation of Harmful Algal Bloom Outreach Activities[J].Marine Drugs,2007,5(4):208-219.
[4]Yan T,Zhou M J.Environmental and Health Effects Associated with Harmful Algal Bloom and Marine Algal Toxins in China[J].Biomedical and Environmental Sciences,2004,17(2):165-176.
[5]Jiang T,Xu Y,Li Y,et al.Seasonal dynamics of Alexandrium Tamarense and Occurrence of Paralytic Shellfish Poisoning Toxins in Bivalves in Nanji Islands,East China Sea[J].Marine and Freshwater Research,2014,65(4):350-358.
[6]Lin C,Liu Z S,Tan C Y,et al.Contamination of Commercially Available Seafood by Key Diarrhetic Shellfish Poisons along the Coast of China[J].Environmental Science and Pollution Research,2015,22(2):1545-1553.
[7]Jahnke H G,Steel D,Fleischer S,et al.A Novel 3D Label-Free Monitoring System of h ES-Derived Cardiomyocyte Clusters:A Step forward to in vitro Cardiotoxicity Testing[J].Plo S One,2013,8(7):e68971.
[8]John E H,Flynn K J.Modelling Changes in Paralytic Shellfish Toxin Content of Dinoflagellates in Response to Nitrogen and Phosphorus Supply[J].Marine Ecology Progress Series,2002,225:147-160.
[9]Ledoux M,Hall S.Proficiency Testing of Eight French Laboratories in Using the AOAC Mouse Bioassay for Paralytic Shellfish Poisoning:Interlaboratory Collaborative Study[J].Journal of AOAC International,2000,83(2):305-310.
[10]Mons M,Speijers G.Paralytic Shellfish Poisoning:A Review[J].Journal of the American Veterinary Medical Association,1978,171(11):1178-1180.
[11]Campas M,Prieto IM N B,MARTY J-L.Biosensors to Detect Marine Toxins:Assessing Seafood Safety[J].Talanta,2007,72(3):884-895.
[12]Oshima Y.Postcolumn Derivatization Liquid Chromatographic Method for Pralytic Shellfish Toxins[J].Journal of Aoac International,1995,78(2):528-532.
[13]van de Riet J M,Gibbs R S,Chou F W,et al.Liquid Chromatographic Post Column Oxidation Method for Analysis of Paralytic Shellfish Toxins in Mussels,Clams,Scallops,and Oysters:SingleLaboratory Validation[J].Journal of AOAC International,2009,92(6):1690-1704.
[14]Aravanis A M,Debusschere B D,Chruscinski A J,et al.A Genetically Engineered Cell-Based Biosensor for Functional Classification of Agents[J].Biosensors and Bioelectronics,2001,16(7):571-577.
[15]Kim S B,Bae H,Cha J M,et al.A Cell-Based Biosensor for RealTime Detection of Cardiotoxicity Using Lensfree Imaging[J].Lab on A Chip,2011,11(10):1801-1807.
[16]王天星,黎洪波,苏凯麒,等.基于细胞电阻抗传感器的细胞多生理参数分析系统设计[J].传感技术学报,2014,27(12):1589-1595.
[17]Wang B,Barahona M,Buck M.A Modular Cell-Based Biosensor Using Engineered Genetic Logic Circuits to Detect and Integrate Multiple Environmental Signals[J].Biosensors and Bioelectronics,2013,40(1):368-376.
[18]Yeung C-K,Sommerhage F,Wrobel G,et al.To Establish a Pharmacological Experimental Platform for the Study of Cardiac Hypoxia Using the Microelectrode Array[J].Journal of Pharmacological and Toxicological Methods,2009,59(3):146-152.
[19]Xu B,Jacquir S,Laurent G,et al.Phase Space Reconstruction of an Experimental Model of Cardiac Field Potential in Normal and Arrhythmic Conditions;Proceedings of the Engineering in Medicine and Biology Society(EMBC),2013 35th Annual International Conference of the IEEE,F,2013[C].IEEE.
[20]徐莹,余辉,张威,等.基于MEMS技术的微电极阵列细胞传感器[J].自然科学进展,2007,17(9):1265-1272.
[21]Kaneko T,Nomura F,Hattori A,et al.Improvement of Electrical Stimulation Protocol for Simultaneous Measurement of Extracellular Potential With on-Chip Multi-Electrode Array System[J].Japanese Journal of Applied Physics,2012,51(6S):06FK02.
[22]Xiao L,Hu Z,Zhang W,et al.Evaluation of Doxorubicin Toxicity on Cardiomyocytes Using a Dual Functional Extracellular Biochip[J].Biosensors and Bioelectronics,2010,26(4):1493-1499.
[23]Meyer T,Boven K-H,G Nther E,等.Micro-Electrode Arrays in Cardiac Safety Pharmacology[J].Drug Safety,2004,27(11):763-772.
[24]胡靓,秦臻,王琴,等.海马神经细胞网络传感器及其记录5-HT对网络活动的作用[J].传感技术学报,2015,(7):947-952.
[25]于兵,曹际娟,尤永莉,等.ELISA与小白鼠生物法检测贝类中麻痹性贝毒的比较[J].检验检疫科学,2005,15(1):32-35.