基于金纳米颗粒修饰丝网印刷电极的胰蛋白酶原-2电化学免疫传感器
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摘要
胰蛋白酶原-2是一种与胰腺疾病相关的生物标志物,在急性胰腺炎和胰腺癌的诊断中发挥重要作用。电化学免疫传感器具有高灵敏性和高特异性的特点,利用其实现对胰蛋白酶原-2的快速、灵敏、简便的定量检测对于临床诊断及即时检验具有重大意义。在本研究中,以胰蛋白酶原-2抗体作为生物分子识别元件,以三电极体系作为信号转换元件,构建基于金纳米颗粒修饰丝网印刷电极的电化学免疫传感器,对胰蛋白酶原-2进行定量检测。在构建过程中,采用化学交联的方法,借助戊二醛的交联作用将金纳米颗粒修饰在丝网印刷电极上,通过金纳米颗粒对抗体的吸附作用可以实现响应信号的放大,从而有效提升灵敏度。通过电化学阻抗谱对传感器的构建过程进行表征,采用差分脉冲伏安法对传感器的检测性能进行评估,结果显示本研究所构建的传感器在胰蛋白酶原-2浓度为2.0-50.0 ng/mL范围内具有良好的线性关系,线性相关系数为0.9954,检测限达0.65 ng/mL。此外,研究还对传感器的准确性与可重复性进行了评估,传感器对在样本中的胰蛋白酶原-2回收率为103.3%-110.5%。结果表明本研究所构建的电化学免疫传感器可实现对胰蛋白酶原-2的定量检测,具有良好的临床应用前景。
Trypsinogen-2 is a biomarker associated with pancreatic diseases and plays an important role in the diagnosis of acute pancreatitis and pancreatic cancer. The electrochemical immunosensor has the characteristics of high sensitivity and specificity.The rapid, sensitive, easy-to-operate and quantitative detection of trypsinogen-2 by electrochemical immunosensor is of great significance for clinical diagnosis and point-of-care testing. In this study, trypsinogen-2 antibody was used as a biomolecule recognition element and a three-electrode system was used as a signal conversion element. An electrochemical immunosensor based on gold nanoparticle modified screen-printed electrode was constructed to quantitatively detect trypsinogen-2. In the process of construction, the chemical cross-linking method was used to modify the gold nanoparticles on the screen printing electrode by the cross-linking action of glutaraldehyde, and the adsorption of the antibody by the gold nanoparticles can realize the amplification of the response signal, thereby effectively increase sensitivity. The sensor construction process was characterized by electrochemical impedance spectroscopy. The detection performance of the sensor was evaluated by differential pulse voltammetry. The results show that the sensor constructed in this study has a good linear relationship in the range of 2.0-50.0 ng/mL of trypsinogen-2 concentration. The correlation coefficient was 0.9954 and the detection limit was 0.65 ng/mL. In addition, the accuracy and repeatability of the sensor were evaluated. The recovery of trypsinogen-2 in the sample was 103.3%-110.5%. The results indicate that the electrochemical immunosensor constructed in this study can achieve quantitative detection of trypsinogen-2 and has a promising potential of clinical application.
Trypsinogen-2 is a biomarker associated with pancreatic diseases and plays an important role in the diagnosis of acute pancreatitis and pancreatic cancer. The electrochemical immunosensor has the characteristics of high sensitivity and specificity.The rapid, sensitive, easy-to-operate and quantitative detection of trypsinogen-2 by electrochemical immunosensor is of great significance for clinical diagnosis and point-of-care testing. In this study, trypsinogen-2 antibody was used as a biomolecule recognition element and a three-electrode system was used as a signal conversion element. An electrochemical immunosensor based on gold nanoparticle modified screen-printed electrode was constructed to quantitatively detect trypsinogen-2. In the process of construction, the chemical cross-linking method was used to modify the gold nanoparticles on the screen printing electrode by the cross-linking action of glutaraldehyde, and the adsorption of the antibody by the gold nanoparticles can realize the amplification of the response signal, thereby effectively increase sensitivity. The sensor construction process was characterized by electrochemical impedance spectroscopy. The detection performance of the sensor was evaluated by differential pulse voltammetry. The results show that the sensor constructed in this study has a good linear relationship in the range of 2.0-50.0 ng/mL of trypsinogen-2 concentration. The correlation coefficient was 0.9954 and the detection limit was 0.65 ng/mL. In addition, the accuracy and repeatability of the sensor were evaluated. The recovery of trypsinogen-2 in the sample was 103.3%-110.5%. The results indicate that the electrochemical immunosensor constructed in this study can achieve quantitative detection of trypsinogen-2 and has a promising potential of clinical application.
引文
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[2]王毅,曾祝伦,谯雅嘉,等.胰蛋白酶原-2在急性胰腺炎诊断中的应用[J].重庆医学,2002,31(5):408-410.
[3]Chang K,Lu W,Zhang K,et al.Rapid urinary trypsinogen-2 test in the early diagnosis of acute pancreatitis:a meta-analysis[J].Clinical Biochemistry2012,45(13-14):1051-1056.
[4]Cao J,Xia C,Cui T,et al.Correlations between serum trypsinogen-2 and pancreatic cancer[J].HepatoGastroenterol,2015,62(138):435-440.
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[6]Nehls O,Gregor M,Klump B.Serum and bile markers for cholangiocarcinoma.[J].Seminars in Liver Disease,2004,24(02):139-154.
[7]Fujimura T,Ohta T,Kitagawa H,et al.Trypsinogen expression and early detection for peritoneal dissemination in gastric cancer[J].Journal of Surgica Oncology,1998,69(2):71-75.
[8]Paju A,Vartiainen J,Haglund C,et al.Expression of trypsinogen-1,trypsinogen-2,and tumor-associated trypsin inhibitor in ovarian cancer:prognostic study on tissue and serum[J].Clinical Cancer Research,200410(14):4761-4768.
[9]Zhang Y,Zhang J,Yan Z H,et al.The clinica evaluation of pepsinogen,trypsinogen-2,CEA,CA50and CA242 in gastric cancer diagnosis[J].Modern Preventive Medicine,2010,37(6):1116-1118.
[10]范振符,范飞舟,杨剑,等.肿瘤相关胰蛋白酶原-2免疫放射分析[J].标记免疫分析与临床,2004,11(1)28-31.
[11]Li X,Campbell-Thompson M,Wasserfall C H,et al Serum trypsinogen levels in type 1 diabetes[J].Diabetes Care,2017,40(4):577-582.
[12]Lempinen M,Isoniemi H,M?kisalo H,et al.Enhanced detection of cholangiocarcinoma with serum trypsinogen-2 in patients with severe bile duc strictures[J].Journal of Hepatology,2007,47(5):677-683.
[13]张艺,张珏,马智鸿,等.胃蛋白酶原与胰蛋白酶原-2联合检测筛查胃癌的意义[J].中国临床医学2009,16(4):548-550.
[14]Itkonen O.Human trypsinogens in the pancreas and in cancer[J].Scandinavian Journal of Clinical&Laboratory Investigation,2010,70(2):136-143.
[15]崔婷婷,曹建彪,郭汉斌,等.血清肿瘤相关性胰蛋白酶原2检测在胰腺癌初筛中的意义[J].医学综述2011,17(7):1092-1094.
[16]崔婷婷,曹建彪,张莉,等.血清胰蛋白酶原-2与急性胰腺炎严重程度分析[J].中国热带医学,201313(6):746-748.
[17]赵书云,赵洁,刘杰,等.血清胰蛋白酶原-2对肝细胞癌的诊断价值[J].解放军医药杂志,2015,27(2):4-7.
[18]Joseph W.Electrochemical biosensors:towards point-of-care cancer diagnostics[J].Biosensors and Bioelectronics,2006,21(10):1887-92.
[19]Monosik R,StredanskyM,Sturdik E.Application of electrochemical biosensors in clinical diagnosis[J]Journal of Clinical Laboratory Analysis,2012,26(1):22-34.
[20]Chutichetpong P,Cheeveewattanagul NSrilohasin P,et al.Rapid screening drug susceptibility test in tuberculosis using sandwich electrochemica immunosensor[J].Analytica Chimica Acta,2018,1025108-117.
[21]冯甜,张弦,杨弦弦,等.电化学免疫传感器检测样品中黄曲霉毒素B1的误差分析[J].国际检验医学杂志,2014,35(8):947-51.
[22]Chinnasamy T,Veerappan S,Senthilkumar K,et al Development of electrochemical based sandwich enzyme linked immunosensor for Cryptosporidium parvum detection in drinking water[J].Journal of Environmenta Monitoring,2011,13(10):2782-2787.
[23]Frens G.Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions[J]Nature Physical Science,1973,241(105):20-22.
[2]王毅,曾祝伦,谯雅嘉,等.胰蛋白酶原-2在急性胰腺炎诊断中的应用[J].重庆医学,2002,31(5):408-410.
[3]Chang K,Lu W,Zhang K,et al.Rapid urinary trypsinogen-2 test in the early diagnosis of acute pancreatitis:a meta-analysis[J].Clinical Biochemistry2012,45(13-14):1051-1056.
[4]Cao J,Xia C,Cui T,et al.Correlations between serum trypsinogen-2 and pancreatic cancer[J].HepatoGastroenterol,2015,62(138):435-440.
[5]Ichikawa Y,Koshikawa N,Hasegawa S,et al Marked increase of trypsin(ogen)in serum of linitis plastica(gastric cancer,borrmann 4)patients.[J].Clinica Cancer Research An Official Journal of the American Association for Cancer Research,2000,6(4):1385-1388
[6]Nehls O,Gregor M,Klump B.Serum and bile markers for cholangiocarcinoma.[J].Seminars in Liver Disease,2004,24(02):139-154.
[7]Fujimura T,Ohta T,Kitagawa H,et al.Trypsinogen expression and early detection for peritoneal dissemination in gastric cancer[J].Journal of Surgica Oncology,1998,69(2):71-75.
[8]Paju A,Vartiainen J,Haglund C,et al.Expression of trypsinogen-1,trypsinogen-2,and tumor-associated trypsin inhibitor in ovarian cancer:prognostic study on tissue and serum[J].Clinical Cancer Research,200410(14):4761-4768.
[9]Zhang Y,Zhang J,Yan Z H,et al.The clinica evaluation of pepsinogen,trypsinogen-2,CEA,CA50and CA242 in gastric cancer diagnosis[J].Modern Preventive Medicine,2010,37(6):1116-1118.
[10]范振符,范飞舟,杨剑,等.肿瘤相关胰蛋白酶原-2免疫放射分析[J].标记免疫分析与临床,2004,11(1)28-31.
[11]Li X,Campbell-Thompson M,Wasserfall C H,et al Serum trypsinogen levels in type 1 diabetes[J].Diabetes Care,2017,40(4):577-582.
[12]Lempinen M,Isoniemi H,M?kisalo H,et al.Enhanced detection of cholangiocarcinoma with serum trypsinogen-2 in patients with severe bile duc strictures[J].Journal of Hepatology,2007,47(5):677-683.
[13]张艺,张珏,马智鸿,等.胃蛋白酶原与胰蛋白酶原-2联合检测筛查胃癌的意义[J].中国临床医学2009,16(4):548-550.
[14]Itkonen O.Human trypsinogens in the pancreas and in cancer[J].Scandinavian Journal of Clinical&Laboratory Investigation,2010,70(2):136-143.
[15]崔婷婷,曹建彪,郭汉斌,等.血清肿瘤相关性胰蛋白酶原2检测在胰腺癌初筛中的意义[J].医学综述2011,17(7):1092-1094.
[16]崔婷婷,曹建彪,张莉,等.血清胰蛋白酶原-2与急性胰腺炎严重程度分析[J].中国热带医学,201313(6):746-748.
[17]赵书云,赵洁,刘杰,等.血清胰蛋白酶原-2对肝细胞癌的诊断价值[J].解放军医药杂志,2015,27(2):4-7.
[18]Joseph W.Electrochemical biosensors:towards point-of-care cancer diagnostics[J].Biosensors and Bioelectronics,2006,21(10):1887-92.
[19]Monosik R,StredanskyM,Sturdik E.Application of electrochemical biosensors in clinical diagnosis[J]Journal of Clinical Laboratory Analysis,2012,26(1):22-34.
[20]Chutichetpong P,Cheeveewattanagul NSrilohasin P,et al.Rapid screening drug susceptibility test in tuberculosis using sandwich electrochemica immunosensor[J].Analytica Chimica Acta,2018,1025108-117.
[21]冯甜,张弦,杨弦弦,等.电化学免疫传感器检测样品中黄曲霉毒素B1的误差分析[J].国际检验医学杂志,2014,35(8):947-51.
[22]Chinnasamy T,Veerappan S,Senthilkumar K,et al Development of electrochemical based sandwich enzyme linked immunosensor for Cryptosporidium parvum detection in drinking water[J].Journal of Environmenta Monitoring,2011,13(10):2782-2787.
[23]Frens G.Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions[J]Nature Physical Science,1973,241(105):20-22.