薄膜Lamb波传感器用于胃蛋白酶原I检测
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摘要
为满足大规模胃癌早期筛查对胃蛋白酶原I(PGI)检测高灵敏度、高效率、操作简单、样品量少的需求,本文构建了一种PGI抗体功能化薄膜型Lamb波生物传感器。对传感器检测腔薄膜进行PGI抗体自组装修饰,传感器检测腔表面修饰的PGI抗体将样品中PGI抗原特异性的捕获并固定在检测腔薄膜表面,Lamb波传感器薄膜表面质量增加导致其A0模式中心频率发生移动,且频率移动量与检测腔表面吸附物质质量增加量正相关,实现对样本中PGI抗原浓度的检测。实验结果表明:PGI抗体功能化薄膜Lamb波生物传感器对PGI抗原实测灵敏度约为102.114 Hz/ng/mL,理论最低检测限(LOD)为0.176ng/mL,单个样本检测时间为40min,与现有基于光学检测法PGI检测技术相比,具有检测系统简单、操作简单、不需要专业人员操作等显著优势,且比多数光学检测法LOD更低,比电化学法PGI检测技术LOD低两个数量级。结果表明,本文提出的PGI抗体免疫功能化薄膜型Lamb波生物传感器对PGI检测且具有检测下限低、灵敏度高、检测效率高、操作简单、无需样品预处理等特点,满足大规模早期胃癌筛查的基本需求。
To achieve the requirements of high sensitivity,high efficiency,simple operation,and low sample size for the detection of pepsinogen I(PGI)in large-scale early screening of gastric cancer,athin-film Lamb wave device with PGI antibody functionality was constructed in this study.The thin film in the device detection cavity was modified with self-assembly of the PGI antibody,which specifically captured and immobilized the PGI antigen in the samples on the surface of the thin film.The resultant increase of mass on the surface led to change of the A0 mode center frequency,and the variation was positively correlated with the increment of mass absorbed on the chamber surface.Thus,the PGI antigen concentration in the samples was detected.The results showed that,when measuring the level of the PGI antigen,the thin-film resonance Lamb wave biosensor with PGI antibody functionality had a sensitivity of about 102.114 Hz/lg(ng/mL),theoretical limit of detection(LOD)of 0.176 ng/mL,and detection time for one sample of 40 min.Compared with the existing PGI detection technology based on optical detection,the Lamb wave sensor has the advantages of being a simple detection system with simple operation and no need for professional operators,and lower LOD than most optical detection methods,two orders lower than that of the electrochemical method of PGI detection.In conclusion,the thin-film resonance Lamb wave biosensor with PGI antibody functionality proposed in this paper has the advantages of low LOD,high sensitivity,high detection efficiency,and simple operation,which can satisfy the basic requirements of large-scale early gastric cancer screening.
To achieve the requirements of high sensitivity,high efficiency,simple operation,and low sample size for the detection of pepsinogen I(PGI)in large-scale early screening of gastric cancer,athin-film Lamb wave device with PGI antibody functionality was constructed in this study.The thin film in the device detection cavity was modified with self-assembly of the PGI antibody,which specifically captured and immobilized the PGI antigen in the samples on the surface of the thin film.The resultant increase of mass on the surface led to change of the A0 mode center frequency,and the variation was positively correlated with the increment of mass absorbed on the chamber surface.Thus,the PGI antigen concentration in the samples was detected.The results showed that,when measuring the level of the PGI antigen,the thin-film resonance Lamb wave biosensor with PGI antibody functionality had a sensitivity of about 102.114 Hz/lg(ng/mL),theoretical limit of detection(LOD)of 0.176 ng/mL,and detection time for one sample of 40 min.Compared with the existing PGI detection technology based on optical detection,the Lamb wave sensor has the advantages of being a simple detection system with simple operation and no need for professional operators,and lower LOD than most optical detection methods,two orders lower than that of the electrochemical method of PGI detection.In conclusion,the thin-film resonance Lamb wave biosensor with PGI antibody functionality proposed in this paper has the advantages of low LOD,high sensitivity,high detection efficiency,and simple operation,which can satisfy the basic requirements of large-scale early gastric cancer screening.
引文
[1]CHEN W,ZHENG R,BAADE PD,et al..Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[2]JEMAL A,BRAY F,CENTER MM,et al..Global cancer statistics[J].CA Cancer J Clin,2011,61(2):69-90.
[3]TAKAHASHI T,SAIKAWA Y,KITAGAWA Y.Gastric cancer:current status of diagnosis and treatment[J].Cancers(Basel),2013,5(1):48-63.
[4]CORREA P.Human gastric carcinogenesis:a multistep and multifactorial process—first American cancer society award lecture on cancer epidemiology and prevention[J].Cancer Res,1992,52(24):6735-6740.
[5]CHISATO H,DAISUKE S,HIDEO Y,et al..The Japanese guidelines for gastric cancer screening[J].Jpn J Clin Oncol,2008,38(4):259-267.
[6]ZHANG XM,LI JX,ZHANG GY,et al..The value of serum pepsinogen levels for the diagnosis of gastric diseases in Chinese Han people in midsouth China[J].BMC Gastroenterol,2014,14(1):3-8.
[7]YUAN Y.Population-based gastric cancer screening in Zhuanghe,Liaoning,from 1997to 2011[J].Zhonghua Zhong Liu Za Zhi,2012,34(7):538-542.
[8]LORENTE S.Helicobacter pylori stimulates pepsinogen secretion from isolated human peptic cells[J].Gut,2002,50(1):13-18.
[9]FOSTER C,AKTAR A,KOPF D,et al..Pepsinogen C:a type 2cell-specific protease[J].AM J Physiol-lung C,2004,286(2):382-387.
[10]KORSTANJE A,DEN HARTOG G,BIEMOND I,et al..The serological gastric biopsy:a non-endoscopical diagnostic approach in management of the dyspeptic patient:significance for primary care based on a survey of the literature[J].Scand J Gastroenterol Suppl,2002,37(236):22-26.
[11]SAMLOFF IM.Cellular localization of group I pepsinogens in human gastric mucosa by immunofluorescence[J].Gastroenterology,1971,61(2):185-188.
[12]HUANG SC,MIKI K,SANO J,et al..Pepsinogens I andⅡin gastric cancer:an immunohistochemical study using monoclonal antibodies[J].Jpn J Cancer Res,1988,79(10):1139-1146.
[13]刘言厚,吕芳,杨树林,等.血清PGⅡ水平与胃疾病及幽门螺杆菌感染关系研究[J].胃肠病学和肝病学杂志,2015,24(2):166-169.LIU Y H,LV F,YANG SH L,et al..Association between serum pepsinogenⅡand gastric disease and Helicobacter pylori infection[J].Chin J Gastroenter Hepatol,2015,24(2):166-169.(in Chinese)
[14]SIPPONEN P,SAMLOFF IM,SAUKKONEN M,et al..Serum pepsinogensⅠandⅡand gastric mucosal histology after partial gastrectomy[J].Gut,1985,26(11):1179-1182.
[15]TANAKA Y,MINE K,NAKAI Y,et al..Serum pepsinogen I concentrations in peptic ulcer patients in relation to ulcer location and stage[J].Gut,1991,32(8):849-852.
[16]KODAMA M,KOYAMA K,TSUBURAYA Y,et al..Group I pepsinogen for early detection of gastric cancer recurrence after total gastrectomy[J].World J Surg,1990,14(1):94-99.
[17]徐倩.胃黏膜“血清学活检”方法及质量控制[J].胃肠病学和肝病学杂志,2015,24(2):130-132.XU Q.Methods and quality control of serological gastric biopsy[J].Chin J Gastroenter Hepatol,2015,24(2):130-132.(in Chinese)
[18]FAN J,XIAO H,ZHANG J,et al..A magnetic nanoparticle-labeled immunoassay with europium and samarium for simultaneous quantification of serum pepsinogenⅠandⅡ[J].Br J Biomed Sci,2017,71(3):1-6.
[19]CHO EJ,KIM HK,JEONG TD,et al..Method evaluation of pepsinogenⅠ/Ⅱassay based on chemiluminescent immunoassays and comparison with other test methods[J].Clin Chim Acta,2016,452:149-154.
[20]YASUKAWA T,HIRANO Y,MOTOCHI N,et al..Enzyme immunosensing of pepsinogens 1and2by scanning electrochemical microscopy[J].Biosens Bioelectron,2007,22(12):3099-3104.
[21]WU F,MAO M,CEN Y,et al..Copolymerization of Eu(TTA)3Phen doped styrene and methyl methacrylate nanoparticles and use in quantitative detection of pepsinogen[J].Rsc.Advances,2017,7(20):12217-12223.
[22]XIE Y,ZHI X,SU H,et al..A novel electrochemical microfluidic chip combined with multiple biomarkers for early diagnosis of gastric cancer[J].Nanoscale Res Lett,2015,10(1):477-485.
[23]朱国民,邱峰.三种血清胃蛋白酶原检测方法在胃癌筛查中的比较分析[J].检验医学,2012,27(11):961-962.ZHU G M,QIU F.Comparative analysis on the screening of gastric cancer in the three kinds of serum pepsinogen detection method[J].Laboratory Medicine,2012,27(11):961-962.(in Chinese)
[24]WEI JC,LIANG Y,QIANG K,et al..The serum pepsinogen test as a predictor of Kazakh gastric cancer[J].Sci Rep,2017,7:43536-43543.
[25]DELLA V B,SAKAN,FUNARI R,et al..Flexible immunosensor for the detection of salivaryα-amylase in body fluids[J].Talanta,2017,174(1):52-58.
[26]LEE DS,LEE JH,LUO J,et al..A surface acoustic wave-based immunosensing device using a nanocrystalline ZnO film on Si[J].J Nanosci Nanotechnol,2009,9(12):7181-7185.
[27]LEE S,KIM KB,KIM YI.Love wave SAW biosensors for detection of antigen-antibody binding and comparison with SPR biosensor[J].Food Sci Biotechnol,2011,20(5):1413-1418.
[28]孔慧,李传宇,周连群,等.薄膜谐振Lamb波传感器测量液体流速矢量的方法[J].光学精密工程,2017,25(1):155-162.KONG H,LI CH Y,ZHOU L Q,et al..A method for fluid velocity vector measurement using thin film Lamb wave resonator[J].Opt.Precision Eng.,2017,25(1):155-162.(in Chinese)
[29]程兆明,张宇川,陈定騊,等.血清胃蛋白酶原亚群的放免测定对胃癌及其它胃部疾病的诊断意义[J].江苏大学学报:医学版,2001,11(2):150-152.CHENG ZH M,ZHANG Y CH,CHEN DT.The diagnostic significance of serum pepsinogens PGⅠandⅡs radioimmunoassay applied to detect gastric cancer and other stomach diseases[J].J Zhenjiang Medical College,2001,11(2):150-152.(in Chinese)
[30]LARSEN JB,HVAS AM.Predictive value of whole blood and plasma coagulation tests for intra-and postoperative bleeding risk:a systematic review[J].Semin Thromb Hemost,2017,43(7):772-805.
[31]DUHAMEL R,ROBERT L,JIA H,et al..Sensitivity of a Lamb wave sensor with 2microm AlN membrane[J].Ultrasonics,2006,44(Suppl 1):e893-e897.
[32]HE H,ZHOU L,WANG Y,et al..Detection of trace microcystin-LR on a 20 MHz QCM sensor coated with in situ self-assembled MIPs[J].Talanta,2015,131(131):8-13.
[33]ARVAND M,FARAHPOUR M,ARDAKI MS.Electrochemical characterization of in situ functionalized gold organosulfur self-assembled monolayer with conducting polymer and carbon nanotubes for determination of rutin[J].Talanta,2018,176:92-101.
[34]DRAKE AW,KLAKAMP SL.A strategic and systematic approach for the determination of biosensor regeneration conditions[J].J Immunol Methods,2011,71(1-2):165-169.
[35]PIRICH CL,DE FREITAS RA,TORRESI RM,et al..Piezoelectric immunochip coated with thin films of bacterial cellulose nanocrystals for dengue detection[J].Biosens Bioelectron,2017,92:47-53.
[36]何皓,张涛,姚佳,等.多孔分子印迹膜修饰的表面等离子体共振微囊藻毒素LR检测传感器[J].光学精密工程,2015,23(3):723-728.HE H,ZHANG T,YAO J,et al..Surface plasmon resonance sensor coated with poriferous MIPs for microcystin-LR detection[J].Opt.Precision Eng.,2015,23(3):723-728.(in Chinese)
[2]JEMAL A,BRAY F,CENTER MM,et al..Global cancer statistics[J].CA Cancer J Clin,2011,61(2):69-90.
[3]TAKAHASHI T,SAIKAWA Y,KITAGAWA Y.Gastric cancer:current status of diagnosis and treatment[J].Cancers(Basel),2013,5(1):48-63.
[4]CORREA P.Human gastric carcinogenesis:a multistep and multifactorial process—first American cancer society award lecture on cancer epidemiology and prevention[J].Cancer Res,1992,52(24):6735-6740.
[5]CHISATO H,DAISUKE S,HIDEO Y,et al..The Japanese guidelines for gastric cancer screening[J].Jpn J Clin Oncol,2008,38(4):259-267.
[6]ZHANG XM,LI JX,ZHANG GY,et al..The value of serum pepsinogen levels for the diagnosis of gastric diseases in Chinese Han people in midsouth China[J].BMC Gastroenterol,2014,14(1):3-8.
[7]YUAN Y.Population-based gastric cancer screening in Zhuanghe,Liaoning,from 1997to 2011[J].Zhonghua Zhong Liu Za Zhi,2012,34(7):538-542.
[8]LORENTE S.Helicobacter pylori stimulates pepsinogen secretion from isolated human peptic cells[J].Gut,2002,50(1):13-18.
[9]FOSTER C,AKTAR A,KOPF D,et al..Pepsinogen C:a type 2cell-specific protease[J].AM J Physiol-lung C,2004,286(2):382-387.
[10]KORSTANJE A,DEN HARTOG G,BIEMOND I,et al..The serological gastric biopsy:a non-endoscopical diagnostic approach in management of the dyspeptic patient:significance for primary care based on a survey of the literature[J].Scand J Gastroenterol Suppl,2002,37(236):22-26.
[11]SAMLOFF IM.Cellular localization of group I pepsinogens in human gastric mucosa by immunofluorescence[J].Gastroenterology,1971,61(2):185-188.
[12]HUANG SC,MIKI K,SANO J,et al..Pepsinogens I andⅡin gastric cancer:an immunohistochemical study using monoclonal antibodies[J].Jpn J Cancer Res,1988,79(10):1139-1146.
[13]刘言厚,吕芳,杨树林,等.血清PGⅡ水平与胃疾病及幽门螺杆菌感染关系研究[J].胃肠病学和肝病学杂志,2015,24(2):166-169.LIU Y H,LV F,YANG SH L,et al..Association between serum pepsinogenⅡand gastric disease and Helicobacter pylori infection[J].Chin J Gastroenter Hepatol,2015,24(2):166-169.(in Chinese)
[14]SIPPONEN P,SAMLOFF IM,SAUKKONEN M,et al..Serum pepsinogensⅠandⅡand gastric mucosal histology after partial gastrectomy[J].Gut,1985,26(11):1179-1182.
[15]TANAKA Y,MINE K,NAKAI Y,et al..Serum pepsinogen I concentrations in peptic ulcer patients in relation to ulcer location and stage[J].Gut,1991,32(8):849-852.
[16]KODAMA M,KOYAMA K,TSUBURAYA Y,et al..Group I pepsinogen for early detection of gastric cancer recurrence after total gastrectomy[J].World J Surg,1990,14(1):94-99.
[17]徐倩.胃黏膜“血清学活检”方法及质量控制[J].胃肠病学和肝病学杂志,2015,24(2):130-132.XU Q.Methods and quality control of serological gastric biopsy[J].Chin J Gastroenter Hepatol,2015,24(2):130-132.(in Chinese)
[18]FAN J,XIAO H,ZHANG J,et al..A magnetic nanoparticle-labeled immunoassay with europium and samarium for simultaneous quantification of serum pepsinogenⅠandⅡ[J].Br J Biomed Sci,2017,71(3):1-6.
[19]CHO EJ,KIM HK,JEONG TD,et al..Method evaluation of pepsinogenⅠ/Ⅱassay based on chemiluminescent immunoassays and comparison with other test methods[J].Clin Chim Acta,2016,452:149-154.
[20]YASUKAWA T,HIRANO Y,MOTOCHI N,et al..Enzyme immunosensing of pepsinogens 1and2by scanning electrochemical microscopy[J].Biosens Bioelectron,2007,22(12):3099-3104.
[21]WU F,MAO M,CEN Y,et al..Copolymerization of Eu(TTA)3Phen doped styrene and methyl methacrylate nanoparticles and use in quantitative detection of pepsinogen[J].Rsc.Advances,2017,7(20):12217-12223.
[22]XIE Y,ZHI X,SU H,et al..A novel electrochemical microfluidic chip combined with multiple biomarkers for early diagnosis of gastric cancer[J].Nanoscale Res Lett,2015,10(1):477-485.
[23]朱国民,邱峰.三种血清胃蛋白酶原检测方法在胃癌筛查中的比较分析[J].检验医学,2012,27(11):961-962.ZHU G M,QIU F.Comparative analysis on the screening of gastric cancer in the three kinds of serum pepsinogen detection method[J].Laboratory Medicine,2012,27(11):961-962.(in Chinese)
[24]WEI JC,LIANG Y,QIANG K,et al..The serum pepsinogen test as a predictor of Kazakh gastric cancer[J].Sci Rep,2017,7:43536-43543.
[25]DELLA V B,SAKAN,FUNARI R,et al..Flexible immunosensor for the detection of salivaryα-amylase in body fluids[J].Talanta,2017,174(1):52-58.
[26]LEE DS,LEE JH,LUO J,et al..A surface acoustic wave-based immunosensing device using a nanocrystalline ZnO film on Si[J].J Nanosci Nanotechnol,2009,9(12):7181-7185.
[27]LEE S,KIM KB,KIM YI.Love wave SAW biosensors for detection of antigen-antibody binding and comparison with SPR biosensor[J].Food Sci Biotechnol,2011,20(5):1413-1418.
[28]孔慧,李传宇,周连群,等.薄膜谐振Lamb波传感器测量液体流速矢量的方法[J].光学精密工程,2017,25(1):155-162.KONG H,LI CH Y,ZHOU L Q,et al..A method for fluid velocity vector measurement using thin film Lamb wave resonator[J].Opt.Precision Eng.,2017,25(1):155-162.(in Chinese)
[29]程兆明,张宇川,陈定騊,等.血清胃蛋白酶原亚群的放免测定对胃癌及其它胃部疾病的诊断意义[J].江苏大学学报:医学版,2001,11(2):150-152.CHENG ZH M,ZHANG Y CH,CHEN DT.The diagnostic significance of serum pepsinogens PGⅠandⅡs radioimmunoassay applied to detect gastric cancer and other stomach diseases[J].J Zhenjiang Medical College,2001,11(2):150-152.(in Chinese)
[30]LARSEN JB,HVAS AM.Predictive value of whole blood and plasma coagulation tests for intra-and postoperative bleeding risk:a systematic review[J].Semin Thromb Hemost,2017,43(7):772-805.
[31]DUHAMEL R,ROBERT L,JIA H,et al..Sensitivity of a Lamb wave sensor with 2microm AlN membrane[J].Ultrasonics,2006,44(Suppl 1):e893-e897.
[32]HE H,ZHOU L,WANG Y,et al..Detection of trace microcystin-LR on a 20 MHz QCM sensor coated with in situ self-assembled MIPs[J].Talanta,2015,131(131):8-13.
[33]ARVAND M,FARAHPOUR M,ARDAKI MS.Electrochemical characterization of in situ functionalized gold organosulfur self-assembled monolayer with conducting polymer and carbon nanotubes for determination of rutin[J].Talanta,2018,176:92-101.
[34]DRAKE AW,KLAKAMP SL.A strategic and systematic approach for the determination of biosensor regeneration conditions[J].J Immunol Methods,2011,71(1-2):165-169.
[35]PIRICH CL,DE FREITAS RA,TORRESI RM,et al..Piezoelectric immunochip coated with thin films of bacterial cellulose nanocrystals for dengue detection[J].Biosens Bioelectron,2017,92:47-53.
[36]何皓,张涛,姚佳,等.多孔分子印迹膜修饰的表面等离子体共振微囊藻毒素LR检测传感器[J].光学精密工程,2015,23(3):723-728.HE H,ZHANG T,YAO J,et al..Surface plasmon resonance sensor coated with poriferous MIPs for microcystin-LR detection[J].Opt.Precision Eng.,2015,23(3):723-728.(in Chinese)