几种典型环境雌激素检测的双层类脂膜电化学免疫传感技术研究
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摘要
生物膜结构和功能复杂，双层类脂膜(BLMs)作为模拟生物膜模型已被广泛接受。修饰了活性物质的BLMs可作为离子转运的通道，抗原-抗体键合的骨架，氧化还原反应的双极性电极或是能量转化的器件，在研究各类生物分子与膜的相互作用，了解生物膜功能，开发新型生物传感器方面发挥了重要作用。环境雌激素是一类存在于环境中，具有类似生物体内雌激素活性的化学物质。越来越多的证据显示,许多被人类不合理释放于环境中的化学污染物具有雌激素的生物效应。建立准确、灵敏、高效的环境雌激素检测筛查方法对把握环境质量、预防环境雌激素对人类造成的危害具有重要意义，现有环境雌激素的检测筛查技术尚需进一步的补充完善。
本研究针对制约传感器发展的固定化关键技术，以17β-雌二醇，己烯雌酚，双酚A等典型环境雌激素为代表，研究活性物质(水溶性抗体和膜受体)在BLMs上的修饰，分别以雌激素抗体和膜受体(mER)作为功能元件，构建基于BLMs的新型电化学免疫传感器，并初步用于环境雌激素的检测和筛查，主要研究内容包括：
1.固体支撑稳定BLMs体系的构建
(1)成膜液组成和配比的优化：卵磷脂和胆固醇作为成膜液主要成分，研究了不同卵磷脂和胆固醇浓度和配比对BLMs稳定性的影响。
(2)不同支撑材料的选择及成膜技术研究：膜片电极作为支撑材料,分别尝试了直接沾取成膜，成膜后再次自组装及脂质体成膜等几种不同的BLMs制备方法，考察了不同方法形成脂膜的性能。
(3) BLMs的稳定性考察：琼脂作为支撑材料，进一步考察了膜片电极和电化学铂电极上琼脂支撑BLMs的制备方法，构建了琼脂支撑稳定的BLMs体系。
2.精氨酸多肽偶联抗体制备技术研究
(1)适合偶联修饰物的筛查及偶联方法研究：实验筛选了氨基壬烷，8个精氨酸组成的细胞穿膜肽（R8）等抗体偶联修饰物。以17β-雌二醇抗体为代表，采用碳化二亚胺法实现了R8与抗体的偶联。对反应条件进行了优化，实验最后选择EDC与R8反应的摩尔比为10:1，活化时间为20min，在优化好的条件下，MALDI-TOF-MS分析结果表明R8与抗17β-雌二醇抗体偶联比约为5:1。
(2)修饰前后抗体的活性鉴定：SPR技术考察了抗体修饰前后亲和常数。17β-雌二醇抗体修饰前后的亲和常数分别为3.8509×10~(-5)和2.3233×10~(-6)，亲和能力略有增加，提示R8修饰不影响17β-雌二醇抗体的结合位点及结合活性，可满足下一步的测定要求。实验同时观察到相同摩尔浓度抗体修饰后与抗原结合引起SPR信号响应增大，进一步证实了R8对抗体的修饰。
(3)修饰位点的初步研究：对修饰位点进行了初步研究。利用MALDI-TOF-MS技术，采用trypin酶解方法对修饰前后的雌二醇抗体进行了酶解，修饰R8后抗体在1496.8528Da，1624.9769Da，2013.2524Da处比较强的肽指纹峰消失，在低分子量(<600Da)范围内的肽指纹峰增加。实验合成了适用于MALDI-TOF-MS的新基质[CHC][NH3+-Si-SBA-15-Si-NH3+][CHC]，去除了基质信号的干扰，考察了低分子量范围内肽指纹峰的变化。
3. R8偶联抗体修饰BLMs电化学免疫传感技术研究
(1)抗体修饰BLMs的研制：优化了卵磷脂和胆固醇的浓度，卵磷脂浓度为20%，与胆固醇的质量比在4:1-4.5:1之间时，形成的BLMs非常稳定。采用两步自组装方法制备了17β-雌二醇修饰的BLMs，原子力扫描电镜测试了空白成膜液和自组装后成膜液的表面形态，空白的成膜液AFM扫描结果非常光滑，在自组装R8修饰雌二醇抗体后，其AFM扫描结果可看到片片突起，修饰雌二醇抗体后相同电压下BLMs的电流响应值降低。实验对抗体的自组装时间进行了优化，选择30分钟的自组装时间。
(2)17β-雌二醇抗体修饰BLMs传感器构建：以修饰了雌二醇抗体的BLMs作为识别元件，采用竞争结合的方法，利用线性扫描法考察了17β-雌二醇对该生物传感体系的响应。对17β-雌二醇竞争结合物E2-BSA的浓度进行了优化，在检测体系中加入150μL1mg/mLE2-BSA时与抗体的结合基本达到饱和。传感器对17β-雌二醇的响应范围为0.01-0.20ng/mL，最低检出浓度0.01ng/mL，并初步应用于自来水样的测定。在优化好的条件下，考察了体系的抗干扰性能，双酚A，己烯雌酚，4-壬基酚等常见环境雌激素对本体系均无响应。
(3)己烯雌酚抗体修饰BLMs传感器构建：在优化好的条件下，利用R8对己烯雌酚抗体进行修饰，制备了己烯雌酚抗体修饰的BLMs，竞争结合法测定了己烯雌酚对该生物传感体系的响应，建立的传感器响应体系对己烯雌酚的响应范围为0.10-0.20ng/mL，最低检出浓度为0.10ng/mL，传感器对雌二醇，双酚A，4-壬基酚等常见环境雌激素显示了良好的抗干扰性能，也进一步验证了修饰方法和检测体系的可行性。
4. mER修饰BLMs电化学免疫传感技术研究
(1) mER修饰BLMs的构建：利用生物膜上mER与卵磷脂和胆固醇组成成膜液良好的生物相容性实现mER与成膜液的融合，原子力扫描电镜扫描观察到不同于空白成膜液的粗糙突起，进一步证实了mER在成膜液中的存在。
(2)方法条件优化：对成膜条件进行了优化，mER溶液与空白成膜液1:1混合时间为5min。修饰mER的BLMs稳定性良好，在0-1.0V范围内的线性扫描结果表明：相同扫描电压下与空白相比修饰了mER的BLMs膜电流略有增加，也进一步证实了修饰物在BLMs上的镶嵌。
(3) mER修饰BLMs对典型雌激素的响应：采用线性扫描伏安方法考察了mER修饰BLMs对典型雌激素雌二醇的响应，该体系对17β-雌二醇方法的检出限为1×10~(-10)mol/L，17β-雌二醇浓度大于1×10~(-8)mol/L时结合基本达到饱和。该体系对浓度为1×10~(-5)mol/L双酚A可产生响应，1×10~(-10)mol/L17β-雌二醇和1×10~(-5)mol/L双酚A同时作用时存在协同效应，传感器的响应增强。
本研究建立了水溶性抗体及膜受体两种不同的BLMs表面活性物质固定化方法，通过对成膜条件和修饰方法的摸索，建立了基于细胞穿膜肽R8修饰的BLMs生物传感技术并应用于水中17β-雌二醇，己烯雌酚的检测。建立了基于mER的BLMs生物传感技术并以典型环境雌激素雌二醇和双酚A进行了验证，考察了协同作用效果。相关的研究进一步拓展和完善了BLMs的应用范围，建立的检测技术作为现有环境雌激素检测和筛查方法的有益补充，具有较好的理论价值和应用前景。
Biomembranes are fundamental to life.The structure and functions of biomembranes arecomplicated. BLMs has served as one of the most useful models for biological membranes.Thelipid bilayer, after suitable modification, acts as a conduit for ion transport, as a framework forantigen–antibody binding, as a bipolar electrode for redox reactions, and as a reactor for energyconversion. Furthermore, a modified lipid bilayer performs as a transducer for signaltransduction (i.e. sensing), and numerous other functions as well. Environmental estrogens(EEs)are a kind of chemical compounds which exist in the environment. It has been proved that EEshave estrogenic activity similar to the in vivo estrogens. More and more evidences show lots ofchemical contaminants had similar function of estrogens and were being released in theenvironment.With the growing concern on EEs, people are more foused on the detection orscreening methods for EEs.It is very important to monitor the concentration of estrogen andpredict the pollution of environmental estrogen.
Foused on immobilized method for developing of novel biosensor，we have constructednovel BLM electrochemical immunosensors with EEs antibody estrogen or membranereceptor(mER) as a functional element. The biosensor was fabricated to directly detect17β-estradiol, diethylstilbestrol and bisphenol A, and the properties of the modified electrodeswere characterized by Linear sweep voltammetry.
1. Study on Solid supported BLMs system
Lecithin and cholesterol were used as the main component of the membrance formingsolution.The preparation method and the stability of the BLMs were studied using Patch clampelectrode or agar as supporting material.The effects of lecithin and cholesterol concentrationsand ratios on the stability of BLMs was studied. The stable BLMs was obtained by agarsupported electrode.
2. Study on preparation of arginine-riched polypeptide modified antibody
Different kinds of hydrophobic groups was screened.The coupling of8-arginine-containingcell penetrating peptide (R8) and anti-17β-estradiol antibody was realized by carbodiimide method.The reaction conditions were optimized. The molar ratio of EDC and R8was10:1andthe activation time for the reaction was about20min.Under optimized conditions, the couplingratio of R8and anti-17β-estradiol antibody was about5:1. Surface plasmon resonancetechnology was used to study the affinity constant of antibody. Affinity constants of Ab-E2was3.8509×10~(-5)and that of R8-Ab-E2was2.3233×10~(-6).The results suggested the modification ofR8to antibody does not affect the binding site of E2with estradiol antibody. The response of ofR8-Ab-E2was high than that of Ab-E2in the same molar concentration.The resluts furtherconfirmed the modification of R8to antibodies. Peptide mass fingerprinting(PMF) of R8-Ab-E2and Ab-E2was Compared.Trypin was used as the enzymatic reagents.The PMF was changedwhen the Ab-E2was modified by R8. The peptide fingerprint peaks of Ab-E2at774.2623Da，1102.4167Da，1496.6604Da，1624.7266Da，2012.9243Da was disappear when it was modifiedby R8.However new peptide fingerprint peaks was appeared in low mass charge ratio area. A newMatrix [CHC][NH3+-Si-SBA-15-Si-NH3+][CHC] was synthesised in order to remove thesignal interference of matrix.
3. Study on novel BLMs electrochemical immunosensor modified by R8coupling antibody
A novel arginine-riched polypeptide modified BLM electrochemical biosensor wasproposed for determination of17β-Estradiol and diethylstilbestrol. Themodification ofantibodies on BLM was performed at the same time with the formation of BLMs.With sameratio of lecithin and cholesterol, higher concentration of lecithin was more easy to formBLMs.TheBLMs was very stable with20%lecithin when the ratio of lecithin and cholesterolwas4:1to4.5:1. A two-step self-assembly method was used to prepared estradiol antibodymodified BLMs.The AFM was used to test the blank BLMs-forming solution and the BLMSforming solution after self-assembly.The results show it was very smooth for blankBLMs-forming soulution.The patches of projections were observed on BLMS forming solutionafter self-assembly.The current was lower when R8-Ab-E2was modified on BLMS. Theself-assembly time for antibody incorporated on BLMs was optimized and30minutes wasselected for the fixed process. The response on17β-estradiol to the biosensor was studied byLinear sweep voltammetry. Competitive assay experiments demonstrated the feasibility of thebiosensor with an response concentration of17β-estradiol ranging from0.01to0.20ng/mL anda detection limit of0.01ng/mL. Diethylstilbestrol antibody was further used to validate thefeasibility of the system. The response concentration of diethylstilbestrol ranging from0.1to0.2 ng/mL and a detection limit of0.10ng/mL.The biosensor showed good has been used fordetection of EEs in running water.
4. Study on novel BLMs electrochemical immunosensor modified by mER
A novel mER modified BLMs electrochemical biosensor was proposed for determinationof environmental estrogens. mER modified BLMs forming solution was achieved withliquid-liquid extraction method.The rough projections was observed when mER modified BLMsforming solution was scan by the atomic force scanning electron microscope.The conditons onliquid-liquid extraction method was optimized. One part mER solution and one part blankmembrance forming solution was mixed about5min.The mER modified BLMs showed goodstability. Compared with blank solution, the membrane currents of mER modified BLM wasincreased slightly, which further confirmed modification of mER on the BLM. Linear sweepvoltammetry method was used to detect the response of mER modified BLM to17β-estradiol.The detection limit of the system to17β-estradiol was1×10~(-10)mol/L.The responsewas saturated when the concentration of17β-estradiol was high than1×10~(-8)mol/L.The systemcan produce response to1×10~(-5)mol/L bisphenol A. The response of the sensor was increasedwhen1×10~(-10)mol/L17β-estradiol and1×10~(-5)mol/L bisphenol A was coexist.
This research established two kinds of new immobilized methods for active materialincorportaed into BLMs. A self-assembled method was developed for incorporatingwater-soluble antibody into BLM. A peptide composed of R8was used as an intramolecularanchor that allows the incorporation of modified antibody into BLM. Competitive assayexperiments demonstrated the feasibility of the biosensor for determination of17β-estradiol anddiethylstilbestrol. The results suggested that arginine-rich peptides can be applied inelectrochemical sensors for the immunoassay and investigation of the interactions of antibodywith other molecules on BLM. mER modified BLM biosensor show good response to17β-estradiol and Bisphenol A. Related research is further expanding and improving the rangeof BLMs applications.It was a useful complement to other technology with better theoreticalvalue and application prospect. Further studies involving look for suitalbe electroactivesubstance to improve the sensibility of the syetem.
本研究针对制约传感器发展的固定化关键技术，以17β-雌二醇，己烯雌酚，双酚A等典型环境雌激素为代表，研究活性物质(水溶性抗体和膜受体)在BLMs上的修饰，分别以雌激素抗体和膜受体(mER)作为功能元件，构建基于BLMs的新型电化学免疫传感器，并初步用于环境雌激素的检测和筛查，主要研究内容包括：
1.固体支撑稳定BLMs体系的构建
(1)成膜液组成和配比的优化：卵磷脂和胆固醇作为成膜液主要成分，研究了不同卵磷脂和胆固醇浓度和配比对BLMs稳定性的影响。
(2)不同支撑材料的选择及成膜技术研究：膜片电极作为支撑材料,分别尝试了直接沾取成膜，成膜后再次自组装及脂质体成膜等几种不同的BLMs制备方法，考察了不同方法形成脂膜的性能。
(3) BLMs的稳定性考察：琼脂作为支撑材料，进一步考察了膜片电极和电化学铂电极上琼脂支撑BLMs的制备方法，构建了琼脂支撑稳定的BLMs体系。
2.精氨酸多肽偶联抗体制备技术研究
(1)适合偶联修饰物的筛查及偶联方法研究：实验筛选了氨基壬烷，8个精氨酸组成的细胞穿膜肽（R8）等抗体偶联修饰物。以17β-雌二醇抗体为代表，采用碳化二亚胺法实现了R8与抗体的偶联。对反应条件进行了优化，实验最后选择EDC与R8反应的摩尔比为10:1，活化时间为20min，在优化好的条件下，MALDI-TOF-MS分析结果表明R8与抗17β-雌二醇抗体偶联比约为5:1。
(2)修饰前后抗体的活性鉴定：SPR技术考察了抗体修饰前后亲和常数。17β-雌二醇抗体修饰前后的亲和常数分别为3.8509×10~(-5)和2.3233×10~(-6)，亲和能力略有增加，提示R8修饰不影响17β-雌二醇抗体的结合位点及结合活性，可满足下一步的测定要求。实验同时观察到相同摩尔浓度抗体修饰后与抗原结合引起SPR信号响应增大，进一步证实了R8对抗体的修饰。
(3)修饰位点的初步研究：对修饰位点进行了初步研究。利用MALDI-TOF-MS技术，采用trypin酶解方法对修饰前后的雌二醇抗体进行了酶解，修饰R8后抗体在1496.8528Da，1624.9769Da，2013.2524Da处比较强的肽指纹峰消失，在低分子量(<600Da)范围内的肽指纹峰增加。实验合成了适用于MALDI-TOF-MS的新基质[CHC][NH3+-Si-SBA-15-Si-NH3+][CHC]，去除了基质信号的干扰，考察了低分子量范围内肽指纹峰的变化。
3. R8偶联抗体修饰BLMs电化学免疫传感技术研究
(1)抗体修饰BLMs的研制：优化了卵磷脂和胆固醇的浓度，卵磷脂浓度为20%，与胆固醇的质量比在4:1-4.5:1之间时，形成的BLMs非常稳定。采用两步自组装方法制备了17β-雌二醇修饰的BLMs，原子力扫描电镜测试了空白成膜液和自组装后成膜液的表面形态，空白的成膜液AFM扫描结果非常光滑，在自组装R8修饰雌二醇抗体后，其AFM扫描结果可看到片片突起，修饰雌二醇抗体后相同电压下BLMs的电流响应值降低。实验对抗体的自组装时间进行了优化，选择30分钟的自组装时间。
(2)17β-雌二醇抗体修饰BLMs传感器构建：以修饰了雌二醇抗体的BLMs作为识别元件，采用竞争结合的方法，利用线性扫描法考察了17β-雌二醇对该生物传感体系的响应。对17β-雌二醇竞争结合物E2-BSA的浓度进行了优化，在检测体系中加入150μL1mg/mLE2-BSA时与抗体的结合基本达到饱和。传感器对17β-雌二醇的响应范围为0.01-0.20ng/mL，最低检出浓度0.01ng/mL，并初步应用于自来水样的测定。在优化好的条件下，考察了体系的抗干扰性能，双酚A，己烯雌酚，4-壬基酚等常见环境雌激素对本体系均无响应。
(3)己烯雌酚抗体修饰BLMs传感器构建：在优化好的条件下，利用R8对己烯雌酚抗体进行修饰，制备了己烯雌酚抗体修饰的BLMs，竞争结合法测定了己烯雌酚对该生物传感体系的响应，建立的传感器响应体系对己烯雌酚的响应范围为0.10-0.20ng/mL，最低检出浓度为0.10ng/mL，传感器对雌二醇，双酚A，4-壬基酚等常见环境雌激素显示了良好的抗干扰性能，也进一步验证了修饰方法和检测体系的可行性。
4. mER修饰BLMs电化学免疫传感技术研究
(1) mER修饰BLMs的构建：利用生物膜上mER与卵磷脂和胆固醇组成成膜液良好的生物相容性实现mER与成膜液的融合，原子力扫描电镜扫描观察到不同于空白成膜液的粗糙突起，进一步证实了mER在成膜液中的存在。
(2)方法条件优化：对成膜条件进行了优化，mER溶液与空白成膜液1:1混合时间为5min。修饰mER的BLMs稳定性良好，在0-1.0V范围内的线性扫描结果表明：相同扫描电压下与空白相比修饰了mER的BLMs膜电流略有增加，也进一步证实了修饰物在BLMs上的镶嵌。
(3) mER修饰BLMs对典型雌激素的响应：采用线性扫描伏安方法考察了mER修饰BLMs对典型雌激素雌二醇的响应，该体系对17β-雌二醇方法的检出限为1×10~(-10)mol/L，17β-雌二醇浓度大于1×10~(-8)mol/L时结合基本达到饱和。该体系对浓度为1×10~(-5)mol/L双酚A可产生响应，1×10~(-10)mol/L17β-雌二醇和1×10~(-5)mol/L双酚A同时作用时存在协同效应，传感器的响应增强。
本研究建立了水溶性抗体及膜受体两种不同的BLMs表面活性物质固定化方法，通过对成膜条件和修饰方法的摸索，建立了基于细胞穿膜肽R8修饰的BLMs生物传感技术并应用于水中17β-雌二醇，己烯雌酚的检测。建立了基于mER的BLMs生物传感技术并以典型环境雌激素雌二醇和双酚A进行了验证，考察了协同作用效果。相关的研究进一步拓展和完善了BLMs的应用范围，建立的检测技术作为现有环境雌激素检测和筛查方法的有益补充，具有较好的理论价值和应用前景。
Biomembranes are fundamental to life.The structure and functions of biomembranes arecomplicated. BLMs has served as one of the most useful models for biological membranes.Thelipid bilayer, after suitable modification, acts as a conduit for ion transport, as a framework forantigen–antibody binding, as a bipolar electrode for redox reactions, and as a reactor for energyconversion. Furthermore, a modified lipid bilayer performs as a transducer for signaltransduction (i.e. sensing), and numerous other functions as well. Environmental estrogens(EEs)are a kind of chemical compounds which exist in the environment. It has been proved that EEshave estrogenic activity similar to the in vivo estrogens. More and more evidences show lots ofchemical contaminants had similar function of estrogens and were being released in theenvironment.With the growing concern on EEs, people are more foused on the detection orscreening methods for EEs.It is very important to monitor the concentration of estrogen andpredict the pollution of environmental estrogen.
Foused on immobilized method for developing of novel biosensor，we have constructednovel BLM electrochemical immunosensors with EEs antibody estrogen or membranereceptor(mER) as a functional element. The biosensor was fabricated to directly detect17β-estradiol, diethylstilbestrol and bisphenol A, and the properties of the modified electrodeswere characterized by Linear sweep voltammetry.
1. Study on Solid supported BLMs system
Lecithin and cholesterol were used as the main component of the membrance formingsolution.The preparation method and the stability of the BLMs were studied using Patch clampelectrode or agar as supporting material.The effects of lecithin and cholesterol concentrationsand ratios on the stability of BLMs was studied. The stable BLMs was obtained by agarsupported electrode.
2. Study on preparation of arginine-riched polypeptide modified antibody
Different kinds of hydrophobic groups was screened.The coupling of8-arginine-containingcell penetrating peptide (R8) and anti-17β-estradiol antibody was realized by carbodiimide method.The reaction conditions were optimized. The molar ratio of EDC and R8was10:1andthe activation time for the reaction was about20min.Under optimized conditions, the couplingratio of R8and anti-17β-estradiol antibody was about5:1. Surface plasmon resonancetechnology was used to study the affinity constant of antibody. Affinity constants of Ab-E2was3.8509×10~(-5)and that of R8-Ab-E2was2.3233×10~(-6).The results suggested the modification ofR8to antibody does not affect the binding site of E2with estradiol antibody. The response of ofR8-Ab-E2was high than that of Ab-E2in the same molar concentration.The resluts furtherconfirmed the modification of R8to antibodies. Peptide mass fingerprinting(PMF) of R8-Ab-E2and Ab-E2was Compared.Trypin was used as the enzymatic reagents.The PMF was changedwhen the Ab-E2was modified by R8. The peptide fingerprint peaks of Ab-E2at774.2623Da，1102.4167Da，1496.6604Da，1624.7266Da，2012.9243Da was disappear when it was modifiedby R8.However new peptide fingerprint peaks was appeared in low mass charge ratio area. A newMatrix [CHC][NH3+-Si-SBA-15-Si-NH3+][CHC] was synthesised in order to remove thesignal interference of matrix.
3. Study on novel BLMs electrochemical immunosensor modified by R8coupling antibody
A novel arginine-riched polypeptide modified BLM electrochemical biosensor wasproposed for determination of17β-Estradiol and diethylstilbestrol. Themodification ofantibodies on BLM was performed at the same time with the formation of BLMs.With sameratio of lecithin and cholesterol, higher concentration of lecithin was more easy to formBLMs.TheBLMs was very stable with20%lecithin when the ratio of lecithin and cholesterolwas4:1to4.5:1. A two-step self-assembly method was used to prepared estradiol antibodymodified BLMs.The AFM was used to test the blank BLMs-forming solution and the BLMSforming solution after self-assembly.The results show it was very smooth for blankBLMs-forming soulution.The patches of projections were observed on BLMS forming solutionafter self-assembly.The current was lower when R8-Ab-E2was modified on BLMS. Theself-assembly time for antibody incorporated on BLMs was optimized and30minutes wasselected for the fixed process. The response on17β-estradiol to the biosensor was studied byLinear sweep voltammetry. Competitive assay experiments demonstrated the feasibility of thebiosensor with an response concentration of17β-estradiol ranging from0.01to0.20ng/mL anda detection limit of0.01ng/mL. Diethylstilbestrol antibody was further used to validate thefeasibility of the system. The response concentration of diethylstilbestrol ranging from0.1to0.2 ng/mL and a detection limit of0.10ng/mL.The biosensor showed good has been used fordetection of EEs in running water.
4. Study on novel BLMs electrochemical immunosensor modified by mER
A novel mER modified BLMs electrochemical biosensor was proposed for determinationof environmental estrogens. mER modified BLMs forming solution was achieved withliquid-liquid extraction method.The rough projections was observed when mER modified BLMsforming solution was scan by the atomic force scanning electron microscope.The conditons onliquid-liquid extraction method was optimized. One part mER solution and one part blankmembrance forming solution was mixed about5min.The mER modified BLMs showed goodstability. Compared with blank solution, the membrane currents of mER modified BLM wasincreased slightly, which further confirmed modification of mER on the BLM. Linear sweepvoltammetry method was used to detect the response of mER modified BLM to17β-estradiol.The detection limit of the system to17β-estradiol was1×10~(-10)mol/L.The responsewas saturated when the concentration of17β-estradiol was high than1×10~(-8)mol/L.The systemcan produce response to1×10~(-5)mol/L bisphenol A. The response of the sensor was increasedwhen1×10~(-10)mol/L17β-estradiol and1×10~(-5)mol/L bisphenol A was coexist.
This research established two kinds of new immobilized methods for active materialincorportaed into BLMs. A self-assembled method was developed for incorporatingwater-soluble antibody into BLM. A peptide composed of R8was used as an intramolecularanchor that allows the incorporation of modified antibody into BLM. Competitive assayexperiments demonstrated the feasibility of the biosensor for determination of17β-estradiol anddiethylstilbestrol. The results suggested that arginine-rich peptides can be applied inelectrochemical sensors for the immunoassay and investigation of the interactions of antibodywith other molecules on BLM. mER modified BLM biosensor show good response to17β-estradiol and Bisphenol A. Related research is further expanding and improving the rangeof BLMs applications.It was a useful complement to other technology with better theoreticalvalue and application prospect. Further studies involving look for suitalbe electroactivesubstance to improve the sensibility of the syetem.
引文
[1] Leventis P.A.,Grinstein S., The Distribution and Function of Phosphatidylserine inCellular Membranes,Annu. Rev. Biophys.,2010,39:407-427.
[2] Muller P., Rudin D. O., Tien H. T., Wescott W. C.,Reconstitution of cell membranestructure invitro and its transformation into anexcitable system, Nature,1962,194:979-980.
[3] Ottova A.，Tien H. T., The40th anniversary of bilayer lipid membrane research,Bioelectrochem.,2002,56:171–173.
[4] Thomas J. M.，Sidney A. S.,Roles of Bilayer Material Properties in Function andDistribution of Membrane Proteins,Annu. Rev. Biophys. Biomol.Struct.,2006,35:177-198.
[5]毕只初，类脂双层（BLM）概念及其实验进展—BLM的40年，生物化学与生物物理进展，2001，28（4）：447-454.
[6] Bolton J.R.,Solar Power,Fuels.New York:Academic Press,1977:167-225
[7] Tien.H.,毕之初,从双层类脂膜到生物分子电子器件的研究，化学传感器，2000，20（2）：1-17.
[8] Yoshikawa K.， Hayashi H.,Shimooka T.,Terada H., Isnii T., Stable phospholipidmembrane supported on porous filter paper， Biochem.Biophys.Res.Conunun.,1987,45:109.
[9] Thompson M.,Lennox R.B.,Mcclelland R.A., Structure and electrochemical properties ofmicrofiltration filter-lipid membrane systems,Anal.Chem.,1982,76:54.
[10]Boyle K.O.,Siddiqi F.A.,Tien H.T., Antigen-Antibody-Complement Reaction Studies onMicro Bilayer Lipid Membranes,Immunol. Invest.,1984,85:103.
[11]Kocherginsky N.M., Osak I.S.， Bromberg L.E.,Karyagin V.A.,MosbovskyY.S., Themodeling of biological membrane properties by means of filters impregnated withlipid-like substances,J.Memb.Sci.,1987,30:3946.
[12]Vázqueza M.I., Romero V., Hierrezuelo J., Rico R., López-Romero J.M., López-RamírezM.R., Benavente J.,Effect of lipid nanoparticles inclusion on transport parametersthrough regenerated cellulose membranes,J. Memb. Sci.,2011,370(1–2):70–75.
[13]Benavente J., Vázquez M.I., Hierrezuelo J., López-Romero J.M., Physicochemical andtransport parameters for a lipid coated regenerated cellulose membrane,Vac.,2011,85(12):1067–1070.
[14]Mittal K.L.,Shah D.O.,Surfactant in solution,Plenum,1991(11)，1:8-9.
[15]Ottova-Leitmannova A.,Matynski T.,Wardak A.,Tien H.T.,Birge R.,MolecularElectronics and bioelectronics,Ads. Chem. Series,1994,240,439-454.
[16]Tien, H.T., Ottova, A.L., Supported planar lipid bilayers (s-BLMs) as electrochemicalbiosensors,Electrochim,Act.,1998,43(23),3587–3610.
[17]Wu Z., Wang B., Cheng Z., Yang X., Dong S., Wang E., A Draft Sequence of the RiceGenome (Oryza sativa L. ssp. indica),Biosensors&Bioelec-tronics,2001,16(1–2),47–52.
[18]Xia W.， Li Y.Y.， Wan Y.J., Chen T., Wei J., Lin Y., Xu S.Q., Electrochemical biosensorfor estrogenic substance using lipid bilayers modified by Au nanoparticles, Biosens.Bioelectron.,2010,25:2253–2258.
[19]Zhou H.Y., Liu N., Gao Z.X.， Wang H.G., Fang Y.J., A novel one-step method toincorporate ss DNA into bilayer lipid membranes supported on an agar electrode,Electrochem. Commun.,2008,10:787-790.
[20]Liu N., Gao Z.X., Zhou H.Y., Yue M.X., Detection of SEB gene by bilayer lipidmembranes nucleic acid biosensor supported by modified patch-clamp pipette electrode,Biosens. Bioelectron.,2007,22:2371-2376;
[21]Stephen A. Sarles, John D. W. Madden and Donald J. Leo，Hair cell inspiredmechanotransduction with a gel-supported, artificial lipid membrane，Soft Matter,2011,7,4644-4653
[22]张彦莉，郭忠先，沈含熙。双层类脂膜的修饰及其在分析化学中的应用，分析化学，1999，27（9）：1096-1102.
[23]田心棣，以双层脂膜为基础的生物传感器及其应用与展望，谢明贵译。生物化学与生物物理进展，1992,19（2）：100-104；
[24]St pniewski M.， Kepczynski M.，D. Jamróz，Nowakowska M.， Rissanen S.，Vattulainen I.， Róg T.,Interaction of Hematoporphyrin with Lipid Membranes, J. Phys.Chem. B,2012,116(16),4889–4897.
[25]Nikolelis D. P.， Petropoulou S. E., Mitrokotsa M. V., A minisensor for the rapidscreening of atenolol in pharmaceutical preparations based on surface-stabilized bilayerlipid membranes with incorporated DNA, Electrochem. Commun.,2002,58:107–112.
[26]Liu N., Gao Z.N., Zhou H.Y., Yue M.X., Detection of SEB gene by bilayer lipidmembranes nucleic acid biosensor supported by modified patch-clamp pipette electrode,Biosens. Bioelectron.,2007,22:2371–2376.
[27]Woller J.G.，B rjesson K.，Svedhem S.，Albinsson B., Reversible Hybridization of DNAAnchored to a Lipid Membrane via Porphyrin, Langmuir,2012,28(4),1944–1953.
[28]Zhou H.Y., Liu N., Gao Z.X., Wang H.Y.,Fang Y.J., A novel one-step method toincorporate ssDNA into bilayer lipid membranes supported on an agar electrode,Electrochem.,2008:10:787–790.
[29]Tong Y.H., Han X.J., Song Y.H., Jiang J.G., Wang E.K., Characterization and property ofDNA incorporated bilayer lipid membranes, Biophys. Chem.,2003,105:1–9.
[30]Trojanowicz M., MiernikA Bilayer lipid membrane glucose biosensor with improvedstability and sensitivity Electrochimica Acta,2011,46(7):1053-1061;
[31]Hianik T., nejdarkova M., ervenanska Z., Miernik A., Krawczyn′T., Krawczyk S.,Trojanowicz M., Bilayer lipid membrane glucose biosensors with improved stability andsensitivity,Chem.Anal.,1997,42:901.
[32]Trojanowicz M.，Miernik A., Bilayer lipid membrane glucose biosensors with improvedstability and sensitivity, Electrochim. Acta,2011,46:1053-1061.
[33]Rhotena M. C.，Burgess J. D.，Hawkridge F. M., The reaction of cytochromecfromdifferent species with cytochrome coxidase immobilized in an electrode supported lipidbilayer membrane. Electroanal. Chem.,2002,534,143-150.
[34]Ueda Y.，Morigaki K.， Tatsu Y.，Yumoto N.，Imaishi H., Immobilization and activityassay of cytochrome P450on patterned lipid membranes, Biochem. Biophys. Res.Commun.,2007,355:926–931.
[35]Torchilin V.P.,Weissig V.，邓意辉，徐晖译.脂质体，化学工业出版社，2007，9:53–57
[36]Wang L.G.,Li Y.H.,Tien H.T.,Bioelectrochem.Bioenerg.,1995:36(2):145-147;
[37]谭学才，李荫，梁汝萍等.双层类脂膜非标记乙型肝炎表面抗原免疫传感器，分析测试学报，2005，24（5）：1-5.
[38]Vockenroth I. K., Atanasova P. P., Long J. R., Functional incorporation of the poreforming segment of AChR M2into tethered bilayer lipid membranes, Biochim. Biophys.Acta,2007,1768:1114-1120.
[39]Xia W.，Li Y.Y.，Wan Y.J., Chen T., Wei J., Lin Y., Xu S.Q., Electrochemical biosensorfor estrogenic substance using lipid bilayers modified by Au nanoparticles,Biosens,Bioelectron.,2010,25:2253–2258.
[40]Dutseva E. A.， Antonenko Y. N.， Kotova E.A.， Pfeifer J. R.， Koert U., Sensitizedphotoinactivation of minigramicidin channels in bilayer lipid membranes,Biochim.Biophys. Acta,2007,1768:1230–1237.
[41]Vockenroth I. K.， Atanasova P. P.，Long J. R.，Toby A., Jenkins A.， Knoll W.， K perI., Functional incorporation of the pore forming segment of AChR M2into tetheredbilayer lipid membranes, Biochim. Biophys. Acta,2007,1768:1114–1120.
[42]Phung T., Zhang Yanli, Dunlop J.， Dalziel J., Bilayer lipid membranes supported onTeflon filters:A functional environment for ion channels, Biosens.Bioelectron.,2011,26:3127–3135.
[43]Sima W.J., Lee J.W., Shi S.K., Song K.B., Oh J.E., Assessment of fates of estrogens inwastewater and sludge from various types of wastewater treatment plants, Chemosphere,2011,82(10):1448–1453.
[44]宋文婷，陆光华，李湘鸣，张海珍，秦健.长江（南京段）环境雌激素的污染特征，生态环境学报，2009,18(5):1615-1619.
[45]万毅，胡建英，邵兵.固相萃取-GC-MS法检测环境中痕量农药类内分泌干扰物，环境科学，2002，23（增刊）：79-82.
[46]梁增辉，何世华，孙成均，等.引起青蛙畸形的环境内分泌干扰物的初步研究,环境与健康杂志,2002,19(6):419-421.
[47]许雷,冉勇,龚剑,等.珠江广州河段及其邻近河流表层沉积物中烷基酚的污染状况，生态环境,2007,16（6）：1615-1619.
[48]吴世闽,贾瑷,彭辉,巫晓琴,董兆敏,胡建英,辽东湾海水中甾体雌激素的检测及生态风险评价，中国环境科学,2011,31(11)：1904~1909.
[49]Jiang W.W., Yan Y., Ma M., Wang D.H., Luo Q., Wang Z.J., Satyanarayanan S.K.,Assessment of source water contamination by estrogenic disrupting compounds inChina,J. Env.Sci.,2012,24(2):320–328.
[50]Chighizola C.， L.Meroni P. The role of environmental estrogens and autoimmunity,Autoimmun,Rev.2012,11:A493–A501；
[51]McLachlan J.A., Functional toxicology: a new approach to detect biologically activexenobiotics, Environ. Health Perspect.,1993,101(5):386-387.
[52]Colborn T., Saal F. S., Soto A.M., Developmental effects of endocrine-disruptionchemicals in wildlife and humans.Environ, Health Perspect.,1993,101(5):378-384.
[53]Meyera M. R., Prossnitz E. R., The G protein-coupled estrogen receptor GPER/GPR30as a regulator of cardiovascular function, Vasc. Pharm.,2011,55:17–25.
[54]Soto A.M., Sonnenschein C., Environmental causes of cancer: endocrine disruptors ascarcinogens, nat. rev.,2010,6(7):363-70.
[55]Miao S., Gao Z.X.,Kou Z.Q., Xu G.F.,Su C.J., Liu N.,Influence of Bisphenol A ondeveloping rat estrogen receptors and some cytokines: A two-generational study, Jour.Tox.Env. Heal.,2008,71(15):1000-1008.
[56]Ingerslev F.， Halling-S rensen B.， Halling-S rensen B.,Evaluation of AnalyticalChemical Methods for Detection of Estrogens in the Environment，Working Report No.44,2003，Arbejdsrapport fra Milj styrelsen.
[57]Baronti C., Curini R., D'Ascenzo G., Corcia A.D., Gentili A., Samperi R., MonitoringNatural and Synthetic Estrogens at Activated Sludge Sewage Treatment Plants and in aReceiving River Water, Environ. Sci. Technol.,2000,34:5059-5066.
[58]Shore L.S., Gurevitz M., Shemesh M., Estrogen as an Environmental Pollutant, Bull.Environ. Contam. Toxicol.,1993,51:361-366；
[59]Kuch H.M. and Ballschmiter K., Determination of endocrine-disrupting phenoliccompounds and estrogens in surface and drinking water by HRGC-(NCI)-MS in thepicogram per liter range,Environ. Sci. Technol.,2001,35:3201–3206.
[60] Zuo Y. G., Zhang K., Lin Y. J., Microwave-accelerated derivatization for thesimultaneous gas chromatographic–mass spectrometric analysis of natural and syntheticestrogenic steroids,Chromatogr. A,2007,1148:211–218.
[61]Anastasia A., Dimitra V., Determination of phenolic and steroid endocrine disruptingcompounds in environmental matrices,Env Sci Pollut Res,2008,15(3):228-236.
[62]Hu R.K., Zhang L.F., Yang Z. G. Picogram determination of estrogens in water usinglarge volume injection gas chromatography–mass spectrometry., Anal. Bioanal. Chem.,2008,390:349–359.
[63]Koh Y. K. K., Chiu T. Y., Boobis A., Cartmell E., Lester J.N., Scrimshaw,M.D. Determination of steroid estrogens in wastewater by high performance liquidchromatography–tandem mass spectrometry,Chromatogr. A.,2007,1173:81–87.
[64]Sun L., Yong W., Chu X.G., Chub X.g., Lin J.M., Simultaneous determination of15steroidal oral contraceptives in water using solid-phase disk extraction followed by highperformance liquid chromatography–tandem mass spectrometry, Chromatogr. A.,2009,1216:5416–5423.
[65]Watabe Y., Kondo T., Nishikawa T., Fujita T., Kaya K., Hosoya K., Fully automatedliquid chromatography–mass spectrometry determination of17β-estradiol in river water,J.Chromatogr.,2006,A1120:252–59.
[66]David M., Vlastimil K., Enhancing sensitivity of liquid chromatographic/ion-traptandem mass spectrometric determination of estrogens by on-line pre-columnderivatization, Chromatogr. A,2008,1192:248–253.
[67]李剑,马梅,,饶凯锋,王子健.酵母双杂交技术构建重组人雌激素受体基因酵母,生态毒理学报,2008，3（1）：21-26.
[68]Wozei E., Hermanowicz S.W., Holman H-Y. N., Developing a biosensor for estrogens inwater samples: Study of the real-time response of live cells of the estrogen-sensitive yeaststrain RMY/ER-ERE using fluorescence microscopy, Biosens. Bioelectron.,2006,21(8):1654–1658.
[69]Ana L.S. Passos, Patr′cia I.S. Pinto, Deborah M. Power, Adelino V.M. Canario，A yeastassay based on the gilthead sea bream (teleost fish) estrogen receptor bfor monitoringestrogen mimics，Ecotoxicology and Environmental Safety,2009,72:1529–1537.
[70]Volpe G., Fares G., Quadri F., Draisci R., Ferrettib G., Marchiafavab C., MosconeaD., Palleschi G.,A disposable immunosensor for detection of17β-estradiol innon-extracted bovine serum,Analytica. Chimica. Acta.,2006,572:11–16.
[71]Zacco E., Pividori M. I., Alegret S., Galve R., Electrochemical magnetoimmunosensingstrategy for the detection of pesticides residues,MarcoAnal. Chem.,2006,78(6):1780–1788.
[72]Gao Z.X.,Liu N., Cao Q.L., Zhang L., Wang S.Q.,Yao W., Chao F.H.Immunochip for thedetection of five kinds of chemicals: Atrazine, nonylphenol,17-beta estradiol, paraverineand chloramphenicol, Biosens. Bioelectron.,2009,24:1445-1450.
[73]Liu N., Su P., Gao Z.X., Zhu M.X., Fang Y.J., Chao F.H., Simultaneous detection forthree kinds of veterinary drugs: Chloramphenicol, clenbuterol and17-beta-estradiol byhigh-throughput suspension array technology, Anal. Chim. Acta.,2009,632:128-134.
[74]Pemberton R. M., Mottram T. T., Hart J. P., Development of a screen-printed carbonelectrochemical immunosensor for picomolar concentrations of estradiol in human serumextracts, J. Biochem. Biophys. Methods.,2005,63:201–212.
[75]Butler D., Guilbault G. G., Disposable amperometric immunosensor for the detection of17-β estradiol using screen-printed electrodes, Sens. Actuators, B.,2006,113:692–699.
[76]刘楠，SEB基因的膜片电极支撑双层类脂膜核酸传感器研究，硕士毕业论文，2006；
[77]Coronado, R. Latorre. Phoseholipid bilayers made from monolayers on patch0clamp pipteites.Biophysical Journal,1983,43(2):231-236;
[78]刘振伟主编，实用膜片钳技术，军事医学科学出版社，2006，第一版：135-136；
[79]朱彤，王韫秀，顾兆伟，聂宏光，郝丽英。雌激素膜受体非基因组效应的研究进展，实用药物与临床，2007，10（4）：233-235;
[80]Jianqing Liu, Baoan Ning, Ming Liu, Yanan Sun, Zhiyong Sun, Yihong Zhang, XianjunFan, Zhixian Gao. Construction of ribosome display library based on lipocalin scaffoldand screening anticalins with specificity for estradiol, analyst,2012,137:2470-2479.
[1] Labadie P., Budzinski H., Determination of steroidal hormone profiles along the Jalled’Eysines River (near Bordeaux, France),Environ. Sci. Technol.,2005.39:5113–5120.
[2] Hanselman T.A., Graetz D.A., Wilkie A.C., Szabo N.J., Diaz C.S., Determination ofsteroidal estrogens in flushed dairy manure wastewater by gas chromatography–massspectrometry,J. Environ. Qual.,2006,35:695–700.
[3] Stanford B.D., Weinberg H.S., Isotope dilution for quantitation of steroid estrogens andnonylphenols by gas chromatography with tandem mass spec-trometry in septic, soil, andgroundwater matrices, J. Chromatogr., A2007,1176(1-2):26–36.
[4] Peng X., Yu Y., Tang.C, Tan J., Huang Q., Wang Z., Occurrence of steroid estrogens,endocrine-disrupting phenols and acid pharmaceutical residues in urban riverine water ofthe Pearl River Delta, South China,Sci. Total Environ.,2008.397:158–166.
[5] Zhang Z.L., Rhind S. M., Kerr C., Osprey M., Kyle C. E., Selective pressurized liquidextraction of estrogenic compounds in soil and analysis by gas chromatography-massspectrometry,Chem J., Anal. Acta,2011,685:29–35.
[6] Trinh T., Harden N. B., Coleman H. M., Khan S. J., A novel reusable ionic liquidchemically bonded fused-silica fiber for headspace solid-phase microextraction/gaschromatography-flame ionization detection of methyl tert-butyl ether in a gasolinesample, Chromatogr. A,2011,1218:1668–1676.
[7] Guo F., Liu Q., Qu G.B., Song S.J., Sun J.T., Shi J.B., Jiang G.B., Simultaneousdetermination of five estrogens and four androgens in water samples by onlinesolid-phase extraction coupled with high-performance liquid chromatography-tandemmass spectrometry.Chromatogr. A,2013,1281:9–18.
[8] Beck I.C., Bruhn R., Gandrass J., Analysis of estrogenic activity in coastal surface watersof the Baltic Sea using the yeast estrogen screen,Chemosphere.,2006.63:1870–78.
[9] Gentili A., Perret D., Marchese S., Mastropasqua R., Curini R., DiCorcia A., Analysis offree estrogens and their conjugates in sewage and river waters by solid-phase extractionthen liquid chromatography electrospray-tandem mass spectrometry,Chromatographia,2002,26:25–32.
[10]Reddy S., Iden C.R., Brownawell B.J., Analysis of steroid conjugates in sewage influentand effluent by liquid chromatography-tandem mass spec-trometry, Anal.Chem.,2005,77:7032–38.
[11]Yamamoto A., Kakutani N., Yamamoto K., Kamiura T., Myiakoda H., Steroid hormoneprofiles of urban and tidal rivers using LC/MS/MS equipped with electrospray ionizationand atmospheric pressure photoionization sources, Environ.Sci.Technol.,2006,40:4132–37.
[12] Koh Y.K., Chiu T.Y., Boobis A., Cartmell E., Lester J.N., Scrimshaw M.D.,Determination of steroid estrogens in wastewater by high performance liquidchromatography–tandem mass spectrometry,J. Chromatogr. A2007,1173:81–87.
[13]Palmer P.M.,Wilson L.R., O’Keefe P., Sheridan R., King T., Chen C.Y., Sources ofpharmaceutical pollution in the New York City Watershed, Sci. TotalEnviron.,2008,394:90–102.
[14]Ciofi L., Fibbi D., Chiuminatto U., Coppinic E., Checchini L., Bubba M. D.,Fully-automated on-line solid phase extraction coupled to high-performance liquidchromatography-tandem mass spectrometric analysis at sub-ng/L levels of selectedestrogens in surface water and wastewater, J. Chromatogr. A,2013,1283:53–61.
[15]Hamid H., Eskicioglu C.,Fate of estrogenic hormones in wastewater and sludgetreatment: A review of properties and analytical detection techniques in sludge matrix,Water Res.,2012,46:5813-5833.
[16]He Y.J., Chen W., Zheng X.Y., Wang X.N., Huang X., Fate and removal of typicalpharmaceuticals and personal care products by three different treatment processes,Sci.Total Environ.,2013,447:248–254.
[17]Tom íková H., Aufartová J., Solich P., Nováková L.,High-sensitivity analysis offemale-steroid hormones in environmental samples, TrAC.,2012,34:35-38.
[18]Liu X.Q., Wong D.K., Picogram-detection of estradiol at an electrochemicalimmunosensor with a gold nanoparticle Protein G-(LC-SPDP)-scaffold, Talanta.,2009,77:1437-1443.
[19]Pemberton R.M., Mottram T.T., Hart J.P., Development of a screen-printed carbonelectrochemical immunosensor for picomolar concentrations of estradiol in human serumextracts,J. Biochem. Biophys. Methods.,2005,63:201-212.
[20]Piao M.H., Noh H.B., Rahman M. A., Won M.S.,.Shim Y.B. Label-free detection ofbisphenol A using a potentiometric immunosensor,Electroanalysis,2008.20(1):30-37.
[21]Centi S., Laschi S., Fránek M., Mascini M., A disposable immunomagneticelectrochemical sensor based on functionalised magnetic beads and carbon-basedscreen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls(PCBs),Anal. Chim. Acta.,2005,538(1-2):205-212.
[22]Eugenia S., Marco M., Sonia C., Anthony P.F., Detection of polychlorinatedbiphenyls(PCBs) in milk using a disposable immunomagnetic electrochemicalsensor,Anal. Lett,2007,40:1371–1385.
[23]Evtugyn G.A., Eremin S.A., Shaljamova R.P., Ismagilova A.R.,Budnikov H.C.,Amperometric immunosensor for nonylphenol determination based on peroxidaseindicating reaction, Biosens. Bioelectron.,2006,22:56–62.
[24]Esseghaier C., Helali S., Fredj H. B., Tlili A., Abdelghani A.,Polypyrrole-neutravidinlayer for impedimetric biosensor, Sens. Actuators.,2008.131(2):584-589.
[25]Valera E., Mu iz D., Rodríguez A., Fabrication of flexible interdigitated μ-electrodes(FIDμEs) for the development of a conductimetric immunosensor for atrazine detectionbased on antibodies labelled with gold nanoparticles. Microelectron.Eng.,2010，87:167-173；
[26] Helali S., Fredj B.H., Cherif K., Abdelghani A.,Martelet C., Jaffrezic-Renault N.Surface plasmon resonance and impedance spectroscopy on gold electrode for biosensorapplication,Mater. Sci. Eng. C.,2008,28(5-6):588-593.
[27] Yao W.,Gao Z.X.,Cheng Y.Y., Quartz crystal microbalance for the detection of carbarylusing molecularly imprintedpolymers as recognition element, J. Sep. Sci.2009,32:3334-3339.
[28]李剑,马梅,,饶凯锋,王子健.酵母双杂交技术构建重组人雌激素受体基因酵母,生态毒理学报,2008，3（1）：21-26.
[29]Wozei E., Hermanowicz S.W., Holman H.Y., Developing a biosensor for estrogens inwater samples: Study of the real-time response of live cells of the estrogen-sensitive yeaststrain RMY/ER-ERE using fluorescence microscopy, Biosens. Bioelectron.,2006,21(8):1654-1658.
[30]Passos A.L., Pinto P.I., Power D.M., Canario A.V., A yeast assay based on the giltheadsea bream (teleost fish) estrogen receptor bfor monitoring estrogen mimics,Ecotoxicol.Environ.Saf.,2009,72:1529-1537.
[31]宋文婷，陆光华，李湘鸣，张海珍，秦健.长江（南京段）环境雌激素的污染特征，生态环境学报，2009,18(5):1615-1619.
[32]Jiang W.W., Yan Y., Ma M, Wang D.H., Luo Q., Wang Z.J., Satyanarayanan S.K.,Assessment of source water contamination by estrogenic disrupting compounds inChina,J. Environ. Sci.2012.24(2):320–328.
[33]Gao Z., Liu N., Cao Q., Zhang L., Wang S., Yao W., Chao F., Immunochip for thedetection of five kinds of chemicals: atrazine, nonylphenol,17-beta estradiol, paraverineand chloramphenicol. Biosens. Bioelectron.,2009,24:1445-1450.
[34]Liu N., Gao Z., Ma H., Su P., Ma X., Li X., Ou G., Simultaneous and rapid detection ofmultiple pesticide and veterinary drug residues by suspension array technology, Biosens.Bioelectron.,2013，41：710-716.
[35]Liu N., Su P., Gao Z., Zhu M., Fang Y., Chao F., Simultaneous detection for three kindsof veterinary drugs: Chloramphenicol, clenbuterol and17-beta-estradiol byhigh-throughput suspension array technology, An. Chimica. Acta,2009,632:128-134.
[2] Muller P., Rudin D. O., Tien H. T., Wescott W. C.,Reconstitution of cell membranestructure invitro and its transformation into anexcitable system, Nature,1962,194:979-980.
[3] Ottova A.，Tien H. T., The40th anniversary of bilayer lipid membrane research,Bioelectrochem.,2002,56:171–173.
[4] Thomas J. M.，Sidney A. S.,Roles of Bilayer Material Properties in Function andDistribution of Membrane Proteins,Annu. Rev. Biophys. Biomol.Struct.,2006,35:177-198.
[5]毕只初，类脂双层（BLM）概念及其实验进展—BLM的40年，生物化学与生物物理进展，2001，28（4）：447-454.
[6] Bolton J.R.,Solar Power,Fuels.New York:Academic Press,1977:167-225
[7] Tien.H.,毕之初,从双层类脂膜到生物分子电子器件的研究，化学传感器，2000，20（2）：1-17.
[8] Yoshikawa K.， Hayashi H.,Shimooka T.,Terada H., Isnii T., Stable phospholipidmembrane supported on porous filter paper， Biochem.Biophys.Res.Conunun.,1987,45:109.
[9] Thompson M.,Lennox R.B.,Mcclelland R.A., Structure and electrochemical properties ofmicrofiltration filter-lipid membrane systems,Anal.Chem.,1982,76:54.
[10]Boyle K.O.,Siddiqi F.A.,Tien H.T., Antigen-Antibody-Complement Reaction Studies onMicro Bilayer Lipid Membranes,Immunol. Invest.,1984,85:103.
[11]Kocherginsky N.M., Osak I.S.， Bromberg L.E.,Karyagin V.A.,MosbovskyY.S., Themodeling of biological membrane properties by means of filters impregnated withlipid-like substances,J.Memb.Sci.,1987,30:3946.
[12]Vázqueza M.I., Romero V., Hierrezuelo J., Rico R., López-Romero J.M., López-RamírezM.R., Benavente J.,Effect of lipid nanoparticles inclusion on transport parametersthrough regenerated cellulose membranes,J. Memb. Sci.,2011,370(1–2):70–75.
[13]Benavente J., Vázquez M.I., Hierrezuelo J., López-Romero J.M., Physicochemical andtransport parameters for a lipid coated regenerated cellulose membrane,Vac.,2011,85(12):1067–1070.
[14]Mittal K.L.,Shah D.O.,Surfactant in solution,Plenum,1991(11)，1:8-9.
[15]Ottova-Leitmannova A.,Matynski T.,Wardak A.,Tien H.T.,Birge R.,MolecularElectronics and bioelectronics,Ads. Chem. Series,1994,240,439-454.
[16]Tien, H.T., Ottova, A.L., Supported planar lipid bilayers (s-BLMs) as electrochemicalbiosensors,Electrochim,Act.,1998,43(23),3587–3610.
[17]Wu Z., Wang B., Cheng Z., Yang X., Dong S., Wang E., A Draft Sequence of the RiceGenome (Oryza sativa L. ssp. indica),Biosensors&Bioelec-tronics,2001,16(1–2),47–52.
[18]Xia W.， Li Y.Y.， Wan Y.J., Chen T., Wei J., Lin Y., Xu S.Q., Electrochemical biosensorfor estrogenic substance using lipid bilayers modified by Au nanoparticles, Biosens.Bioelectron.,2010,25:2253–2258.
[19]Zhou H.Y., Liu N., Gao Z.X.， Wang H.G., Fang Y.J., A novel one-step method toincorporate ss DNA into bilayer lipid membranes supported on an agar electrode,Electrochem. Commun.,2008,10:787-790.
[20]Liu N., Gao Z.X., Zhou H.Y., Yue M.X., Detection of SEB gene by bilayer lipidmembranes nucleic acid biosensor supported by modified patch-clamp pipette electrode,Biosens. Bioelectron.,2007,22:2371-2376;
[21]Stephen A. Sarles, John D. W. Madden and Donald J. Leo，Hair cell inspiredmechanotransduction with a gel-supported, artificial lipid membrane，Soft Matter,2011,7,4644-4653
[22]张彦莉，郭忠先，沈含熙。双层类脂膜的修饰及其在分析化学中的应用，分析化学，1999，27（9）：1096-1102.
[23]田心棣，以双层脂膜为基础的生物传感器及其应用与展望，谢明贵译。生物化学与生物物理进展，1992,19（2）：100-104；
[24]St pniewski M.， Kepczynski M.，D. Jamróz，Nowakowska M.， Rissanen S.，Vattulainen I.， Róg T.,Interaction of Hematoporphyrin with Lipid Membranes, J. Phys.Chem. B,2012,116(16),4889–4897.
[25]Nikolelis D. P.， Petropoulou S. E., Mitrokotsa M. V., A minisensor for the rapidscreening of atenolol in pharmaceutical preparations based on surface-stabilized bilayerlipid membranes with incorporated DNA, Electrochem. Commun.,2002,58:107–112.
[26]Liu N., Gao Z.N., Zhou H.Y., Yue M.X., Detection of SEB gene by bilayer lipidmembranes nucleic acid biosensor supported by modified patch-clamp pipette electrode,Biosens. Bioelectron.,2007,22:2371–2376.
[27]Woller J.G.，B rjesson K.，Svedhem S.，Albinsson B., Reversible Hybridization of DNAAnchored to a Lipid Membrane via Porphyrin, Langmuir,2012,28(4),1944–1953.
[28]Zhou H.Y., Liu N., Gao Z.X., Wang H.Y.,Fang Y.J., A novel one-step method toincorporate ssDNA into bilayer lipid membranes supported on an agar electrode,Electrochem.,2008:10:787–790.
[29]Tong Y.H., Han X.J., Song Y.H., Jiang J.G., Wang E.K., Characterization and property ofDNA incorporated bilayer lipid membranes, Biophys. Chem.,2003,105:1–9.
[30]Trojanowicz M., MiernikA Bilayer lipid membrane glucose biosensor with improvedstability and sensitivity Electrochimica Acta,2011,46(7):1053-1061;
[31]Hianik T., nejdarkova M., ervenanska Z., Miernik A., Krawczyn′T., Krawczyk S.,Trojanowicz M., Bilayer lipid membrane glucose biosensors with improved stability andsensitivity,Chem.Anal.,1997,42:901.
[32]Trojanowicz M.，Miernik A., Bilayer lipid membrane glucose biosensors with improvedstability and sensitivity, Electrochim. Acta,2011,46:1053-1061.
[33]Rhotena M. C.，Burgess J. D.，Hawkridge F. M., The reaction of cytochromecfromdifferent species with cytochrome coxidase immobilized in an electrode supported lipidbilayer membrane. Electroanal. Chem.,2002,534,143-150.
[34]Ueda Y.，Morigaki K.， Tatsu Y.，Yumoto N.，Imaishi H., Immobilization and activityassay of cytochrome P450on patterned lipid membranes, Biochem. Biophys. Res.Commun.,2007,355:926–931.
[35]Torchilin V.P.,Weissig V.，邓意辉，徐晖译.脂质体，化学工业出版社，2007，9:53–57
[36]Wang L.G.,Li Y.H.,Tien H.T.,Bioelectrochem.Bioenerg.,1995:36(2):145-147;
[37]谭学才，李荫，梁汝萍等.双层类脂膜非标记乙型肝炎表面抗原免疫传感器，分析测试学报，2005，24（5）：1-5.
[38]Vockenroth I. K., Atanasova P. P., Long J. R., Functional incorporation of the poreforming segment of AChR M2into tethered bilayer lipid membranes, Biochim. Biophys.Acta,2007,1768:1114-1120.
[39]Xia W.，Li Y.Y.，Wan Y.J., Chen T., Wei J., Lin Y., Xu S.Q., Electrochemical biosensorfor estrogenic substance using lipid bilayers modified by Au nanoparticles,Biosens,Bioelectron.,2010,25:2253–2258.
[40]Dutseva E. A.， Antonenko Y. N.， Kotova E.A.， Pfeifer J. R.， Koert U., Sensitizedphotoinactivation of minigramicidin channels in bilayer lipid membranes,Biochim.Biophys. Acta,2007,1768:1230–1237.
[41]Vockenroth I. K.， Atanasova P. P.，Long J. R.，Toby A., Jenkins A.， Knoll W.， K perI., Functional incorporation of the pore forming segment of AChR M2into tetheredbilayer lipid membranes, Biochim. Biophys. Acta,2007,1768:1114–1120.
[42]Phung T., Zhang Yanli, Dunlop J.， Dalziel J., Bilayer lipid membranes supported onTeflon filters:A functional environment for ion channels, Biosens.Bioelectron.,2011,26:3127–3135.
[43]Sima W.J., Lee J.W., Shi S.K., Song K.B., Oh J.E., Assessment of fates of estrogens inwastewater and sludge from various types of wastewater treatment plants, Chemosphere,2011,82(10):1448–1453.
[44]宋文婷，陆光华，李湘鸣，张海珍，秦健.长江（南京段）环境雌激素的污染特征，生态环境学报，2009,18(5):1615-1619.
[45]万毅，胡建英，邵兵.固相萃取-GC-MS法检测环境中痕量农药类内分泌干扰物，环境科学，2002，23（增刊）：79-82.
[46]梁增辉，何世华，孙成均，等.引起青蛙畸形的环境内分泌干扰物的初步研究,环境与健康杂志,2002,19(6):419-421.
[47]许雷,冉勇,龚剑,等.珠江广州河段及其邻近河流表层沉积物中烷基酚的污染状况，生态环境,2007,16（6）：1615-1619.
[48]吴世闽,贾瑷,彭辉,巫晓琴,董兆敏,胡建英,辽东湾海水中甾体雌激素的检测及生态风险评价，中国环境科学,2011,31(11)：1904~1909.
[49]Jiang W.W., Yan Y., Ma M., Wang D.H., Luo Q., Wang Z.J., Satyanarayanan S.K.,Assessment of source water contamination by estrogenic disrupting compounds inChina,J. Env.Sci.,2012,24(2):320–328.
[50]Chighizola C.， L.Meroni P. The role of environmental estrogens and autoimmunity,Autoimmun,Rev.2012,11:A493–A501；
[51]McLachlan J.A., Functional toxicology: a new approach to detect biologically activexenobiotics, Environ. Health Perspect.,1993,101(5):386-387.
[52]Colborn T., Saal F. S., Soto A.M., Developmental effects of endocrine-disruptionchemicals in wildlife and humans.Environ, Health Perspect.,1993,101(5):378-384.
[53]Meyera M. R., Prossnitz E. R., The G protein-coupled estrogen receptor GPER/GPR30as a regulator of cardiovascular function, Vasc. Pharm.,2011,55:17–25.
[54]Soto A.M., Sonnenschein C., Environmental causes of cancer: endocrine disruptors ascarcinogens, nat. rev.,2010,6(7):363-70.
[55]Miao S., Gao Z.X.,Kou Z.Q., Xu G.F.,Su C.J., Liu N.,Influence of Bisphenol A ondeveloping rat estrogen receptors and some cytokines: A two-generational study, Jour.Tox.Env. Heal.,2008,71(15):1000-1008.
[56]Ingerslev F.， Halling-S rensen B.， Halling-S rensen B.,Evaluation of AnalyticalChemical Methods for Detection of Estrogens in the Environment，Working Report No.44,2003，Arbejdsrapport fra Milj styrelsen.
[57]Baronti C., Curini R., D'Ascenzo G., Corcia A.D., Gentili A., Samperi R., MonitoringNatural and Synthetic Estrogens at Activated Sludge Sewage Treatment Plants and in aReceiving River Water, Environ. Sci. Technol.,2000,34:5059-5066.
[58]Shore L.S., Gurevitz M., Shemesh M., Estrogen as an Environmental Pollutant, Bull.Environ. Contam. Toxicol.,1993,51:361-366；
[59]Kuch H.M. and Ballschmiter K., Determination of endocrine-disrupting phenoliccompounds and estrogens in surface and drinking water by HRGC-(NCI)-MS in thepicogram per liter range,Environ. Sci. Technol.,2001,35:3201–3206.
[60] Zuo Y. G., Zhang K., Lin Y. J., Microwave-accelerated derivatization for thesimultaneous gas chromatographic–mass spectrometric analysis of natural and syntheticestrogenic steroids,Chromatogr. A,2007,1148:211–218.
[61]Anastasia A., Dimitra V., Determination of phenolic and steroid endocrine disruptingcompounds in environmental matrices,Env Sci Pollut Res,2008,15(3):228-236.
[62]Hu R.K., Zhang L.F., Yang Z. G. Picogram determination of estrogens in water usinglarge volume injection gas chromatography–mass spectrometry., Anal. Bioanal. Chem.,2008,390:349–359.
[63]Koh Y. K. K., Chiu T. Y., Boobis A., Cartmell E., Lester J.N., Scrimshaw,M.D. Determination of steroid estrogens in wastewater by high performance liquidchromatography–tandem mass spectrometry,Chromatogr. A.,2007,1173:81–87.
[64]Sun L., Yong W., Chu X.G., Chub X.g., Lin J.M., Simultaneous determination of15steroidal oral contraceptives in water using solid-phase disk extraction followed by highperformance liquid chromatography–tandem mass spectrometry, Chromatogr. A.,2009,1216:5416–5423.
[65]Watabe Y., Kondo T., Nishikawa T., Fujita T., Kaya K., Hosoya K., Fully automatedliquid chromatography–mass spectrometry determination of17β-estradiol in river water,J.Chromatogr.,2006,A1120:252–59.
[66]David M., Vlastimil K., Enhancing sensitivity of liquid chromatographic/ion-traptandem mass spectrometric determination of estrogens by on-line pre-columnderivatization, Chromatogr. A,2008,1192:248–253.
[67]李剑,马梅,,饶凯锋,王子健.酵母双杂交技术构建重组人雌激素受体基因酵母,生态毒理学报,2008，3（1）：21-26.
[68]Wozei E., Hermanowicz S.W., Holman H-Y. N., Developing a biosensor for estrogens inwater samples: Study of the real-time response of live cells of the estrogen-sensitive yeaststrain RMY/ER-ERE using fluorescence microscopy, Biosens. Bioelectron.,2006,21(8):1654–1658.
[69]Ana L.S. Passos, Patr′cia I.S. Pinto, Deborah M. Power, Adelino V.M. Canario，A yeastassay based on the gilthead sea bream (teleost fish) estrogen receptor bfor monitoringestrogen mimics，Ecotoxicology and Environmental Safety,2009,72:1529–1537.
[70]Volpe G., Fares G., Quadri F., Draisci R., Ferrettib G., Marchiafavab C., MosconeaD., Palleschi G.,A disposable immunosensor for detection of17β-estradiol innon-extracted bovine serum,Analytica. Chimica. Acta.,2006,572:11–16.
[71]Zacco E., Pividori M. I., Alegret S., Galve R., Electrochemical magnetoimmunosensingstrategy for the detection of pesticides residues,MarcoAnal. Chem.,2006,78(6):1780–1788.
[72]Gao Z.X.,Liu N., Cao Q.L., Zhang L., Wang S.Q.,Yao W., Chao F.H.Immunochip for thedetection of five kinds of chemicals: Atrazine, nonylphenol,17-beta estradiol, paraverineand chloramphenicol, Biosens. Bioelectron.,2009,24:1445-1450.
[73]Liu N., Su P., Gao Z.X., Zhu M.X., Fang Y.J., Chao F.H., Simultaneous detection forthree kinds of veterinary drugs: Chloramphenicol, clenbuterol and17-beta-estradiol byhigh-throughput suspension array technology, Anal. Chim. Acta.,2009,632:128-134.
[74]Pemberton R. M., Mottram T. T., Hart J. P., Development of a screen-printed carbonelectrochemical immunosensor for picomolar concentrations of estradiol in human serumextracts, J. Biochem. Biophys. Methods.,2005,63:201–212.
[75]Butler D., Guilbault G. G., Disposable amperometric immunosensor for the detection of17-β estradiol using screen-printed electrodes, Sens. Actuators, B.,2006,113:692–699.
[76]刘楠，SEB基因的膜片电极支撑双层类脂膜核酸传感器研究，硕士毕业论文，2006；
[77]Coronado, R. Latorre. Phoseholipid bilayers made from monolayers on patch0clamp pipteites.Biophysical Journal,1983,43(2):231-236;
[78]刘振伟主编，实用膜片钳技术，军事医学科学出版社，2006，第一版：135-136；
[79]朱彤，王韫秀，顾兆伟，聂宏光，郝丽英。雌激素膜受体非基因组效应的研究进展，实用药物与临床，2007，10（4）：233-235;
[80]Jianqing Liu, Baoan Ning, Ming Liu, Yanan Sun, Zhiyong Sun, Yihong Zhang, XianjunFan, Zhixian Gao. Construction of ribosome display library based on lipocalin scaffoldand screening anticalins with specificity for estradiol, analyst,2012,137:2470-2479.
[1] Labadie P., Budzinski H., Determination of steroidal hormone profiles along the Jalled’Eysines River (near Bordeaux, France),Environ. Sci. Technol.,2005.39:5113–5120.
[2] Hanselman T.A., Graetz D.A., Wilkie A.C., Szabo N.J., Diaz C.S., Determination ofsteroidal estrogens in flushed dairy manure wastewater by gas chromatography–massspectrometry,J. Environ. Qual.,2006,35:695–700.
[3] Stanford B.D., Weinberg H.S., Isotope dilution for quantitation of steroid estrogens andnonylphenols by gas chromatography with tandem mass spec-trometry in septic, soil, andgroundwater matrices, J. Chromatogr., A2007,1176(1-2):26–36.
[4] Peng X., Yu Y., Tang.C, Tan J., Huang Q., Wang Z., Occurrence of steroid estrogens,endocrine-disrupting phenols and acid pharmaceutical residues in urban riverine water ofthe Pearl River Delta, South China,Sci. Total Environ.,2008.397:158–166.
[5] Zhang Z.L., Rhind S. M., Kerr C., Osprey M., Kyle C. E., Selective pressurized liquidextraction of estrogenic compounds in soil and analysis by gas chromatography-massspectrometry,Chem J., Anal. Acta,2011,685:29–35.
[6] Trinh T., Harden N. B., Coleman H. M., Khan S. J., A novel reusable ionic liquidchemically bonded fused-silica fiber for headspace solid-phase microextraction/gaschromatography-flame ionization detection of methyl tert-butyl ether in a gasolinesample, Chromatogr. A,2011,1218:1668–1676.
[7] Guo F., Liu Q., Qu G.B., Song S.J., Sun J.T., Shi J.B., Jiang G.B., Simultaneousdetermination of five estrogens and four androgens in water samples by onlinesolid-phase extraction coupled with high-performance liquid chromatography-tandemmass spectrometry.Chromatogr. A,2013,1281:9–18.
[8] Beck I.C., Bruhn R., Gandrass J., Analysis of estrogenic activity in coastal surface watersof the Baltic Sea using the yeast estrogen screen,Chemosphere.,2006.63:1870–78.
[9] Gentili A., Perret D., Marchese S., Mastropasqua R., Curini R., DiCorcia A., Analysis offree estrogens and their conjugates in sewage and river waters by solid-phase extractionthen liquid chromatography electrospray-tandem mass spectrometry,Chromatographia,2002,26:25–32.
[10]Reddy S., Iden C.R., Brownawell B.J., Analysis of steroid conjugates in sewage influentand effluent by liquid chromatography-tandem mass spec-trometry, Anal.Chem.,2005,77:7032–38.
[11]Yamamoto A., Kakutani N., Yamamoto K., Kamiura T., Myiakoda H., Steroid hormoneprofiles of urban and tidal rivers using LC/MS/MS equipped with electrospray ionizationand atmospheric pressure photoionization sources, Environ.Sci.Technol.,2006,40:4132–37.
[12] Koh Y.K., Chiu T.Y., Boobis A., Cartmell E., Lester J.N., Scrimshaw M.D.,Determination of steroid estrogens in wastewater by high performance liquidchromatography–tandem mass spectrometry,J. Chromatogr. A2007,1173:81–87.
[13]Palmer P.M.,Wilson L.R., O’Keefe P., Sheridan R., King T., Chen C.Y., Sources ofpharmaceutical pollution in the New York City Watershed, Sci. TotalEnviron.,2008,394:90–102.
[14]Ciofi L., Fibbi D., Chiuminatto U., Coppinic E., Checchini L., Bubba M. D.,Fully-automated on-line solid phase extraction coupled to high-performance liquidchromatography-tandem mass spectrometric analysis at sub-ng/L levels of selectedestrogens in surface water and wastewater, J. Chromatogr. A,2013,1283:53–61.
[15]Hamid H., Eskicioglu C.,Fate of estrogenic hormones in wastewater and sludgetreatment: A review of properties and analytical detection techniques in sludge matrix,Water Res.,2012,46:5813-5833.
[16]He Y.J., Chen W., Zheng X.Y., Wang X.N., Huang X., Fate and removal of typicalpharmaceuticals and personal care products by three different treatment processes,Sci.Total Environ.,2013,447:248–254.
[17]Tom íková H., Aufartová J., Solich P., Nováková L.,High-sensitivity analysis offemale-steroid hormones in environmental samples, TrAC.,2012,34:35-38.
[18]Liu X.Q., Wong D.K., Picogram-detection of estradiol at an electrochemicalimmunosensor with a gold nanoparticle Protein G-(LC-SPDP)-scaffold, Talanta.,2009,77:1437-1443.
[19]Pemberton R.M., Mottram T.T., Hart J.P., Development of a screen-printed carbonelectrochemical immunosensor for picomolar concentrations of estradiol in human serumextracts,J. Biochem. Biophys. Methods.,2005,63:201-212.
[20]Piao M.H., Noh H.B., Rahman M. A., Won M.S.,.Shim Y.B. Label-free detection ofbisphenol A using a potentiometric immunosensor,Electroanalysis,2008.20(1):30-37.
[21]Centi S., Laschi S., Fránek M., Mascini M., A disposable immunomagneticelectrochemical sensor based on functionalised magnetic beads and carbon-basedscreen-printed electrodes (SPCEs) for the detection of polychlorinated biphenyls(PCBs),Anal. Chim. Acta.,2005,538(1-2):205-212.
[22]Eugenia S., Marco M., Sonia C., Anthony P.F., Detection of polychlorinatedbiphenyls(PCBs) in milk using a disposable immunomagnetic electrochemicalsensor,Anal. Lett,2007,40:1371–1385.
[23]Evtugyn G.A., Eremin S.A., Shaljamova R.P., Ismagilova A.R.,Budnikov H.C.,Amperometric immunosensor for nonylphenol determination based on peroxidaseindicating reaction, Biosens. Bioelectron.,2006,22:56–62.
[24]Esseghaier C., Helali S., Fredj H. B., Tlili A., Abdelghani A.,Polypyrrole-neutravidinlayer for impedimetric biosensor, Sens. Actuators.,2008.131(2):584-589.
[25]Valera E., Mu iz D., Rodríguez A., Fabrication of flexible interdigitated μ-electrodes(FIDμEs) for the development of a conductimetric immunosensor for atrazine detectionbased on antibodies labelled with gold nanoparticles. Microelectron.Eng.,2010，87:167-173；
[26] Helali S., Fredj B.H., Cherif K., Abdelghani A.,Martelet C., Jaffrezic-Renault N.Surface plasmon resonance and impedance spectroscopy on gold electrode for biosensorapplication,Mater. Sci. Eng. C.,2008,28(5-6):588-593.
[27] Yao W.,Gao Z.X.,Cheng Y.Y., Quartz crystal microbalance for the detection of carbarylusing molecularly imprintedpolymers as recognition element, J. Sep. Sci.2009,32:3334-3339.
[28]李剑,马梅,,饶凯锋,王子健.酵母双杂交技术构建重组人雌激素受体基因酵母,生态毒理学报,2008，3（1）：21-26.
[29]Wozei E., Hermanowicz S.W., Holman H.Y., Developing a biosensor for estrogens inwater samples: Study of the real-time response of live cells of the estrogen-sensitive yeaststrain RMY/ER-ERE using fluorescence microscopy, Biosens. Bioelectron.,2006,21(8):1654-1658.
[30]Passos A.L., Pinto P.I., Power D.M., Canario A.V., A yeast assay based on the giltheadsea bream (teleost fish) estrogen receptor bfor monitoring estrogen mimics,Ecotoxicol.Environ.Saf.,2009,72:1529-1537.
[31]宋文婷，陆光华，李湘鸣，张海珍，秦健.长江（南京段）环境雌激素的污染特征，生态环境学报，2009,18(5):1615-1619.
[32]Jiang W.W., Yan Y., Ma M, Wang D.H., Luo Q., Wang Z.J., Satyanarayanan S.K.,Assessment of source water contamination by estrogenic disrupting compounds inChina,J. Environ. Sci.2012.24(2):320–328.
[33]Gao Z., Liu N., Cao Q., Zhang L., Wang S., Yao W., Chao F., Immunochip for thedetection of five kinds of chemicals: atrazine, nonylphenol,17-beta estradiol, paraverineand chloramphenicol. Biosens. Bioelectron.,2009,24:1445-1450.
[34]Liu N., Gao Z., Ma H., Su P., Ma X., Li X., Ou G., Simultaneous and rapid detection ofmultiple pesticide and veterinary drug residues by suspension array technology, Biosens.Bioelectron.,2013，41：710-716.
[35]Liu N., Su P., Gao Z., Zhu M., Fang Y., Chao F., Simultaneous detection for three kindsof veterinary drugs: Chloramphenicol, clenbuterol and17-beta-estradiol byhigh-throughput suspension array technology, An. Chimica. Acta,2009,632:128-134.