基于量子点荧光探针的高灵敏蛋白质检测方法
|
摘要
通过制备量子点荧光检测探针,构建了一种基于量子点探针和免疫磁珠的蛋白质检测方法,实现了对蛋白肿瘤标志物癌胚抗原(carcinoembryonic antigen,CEA)的高灵敏定量检测.在该检测体系中,若存在有靶标蛋白,其与量子点检测探针以及捕获探针之间会发生免疫反应形成三明治结构,利用磁力分离器对免疫复合物进行富集后,通过检测富集在磁珠表面的量子点荧光信号,可实现对靶标蛋白的定量.该方法的检测灵敏度为38 pg/mL,线性范围为0.39~50ng/mL,临床质控样本检验结果表明,该方法准确度高,可重复性好,可应用于临床样本检测.该量子点探针检测体系具有灵敏度高、特异性好、样品消耗量低等优点,在疾病早期诊断方面具有广阔的应用前景.
In this study,based on the unique properties of quantum dots(QDs)and immune magnetic beads,an fluorescent assay was built to detect low concentration carcinoembryonic anti-gen(CEA).The assay was carried out in a sandwich formation in which two antibodies simultaneously recognized two separate binding sites on the same target CEA.After enrichment with magnetic separator,the fluorescent intensities produced by the QDs,which were confined to the surface of the beads,could be read efficiently to quantify the concentration of the CEA.For this assay,the limit of detection was 38 pg/mL with the linear range was from0.39 to 50 ng/mL,and it has been proved with high accuracy and repeatability in tumor marker control.With the high sensitivity,specificity and low sample consumption,this method takes a big step to the early diagnosis of diseases.
In this study,based on the unique properties of quantum dots(QDs)and immune magnetic beads,an fluorescent assay was built to detect low concentration carcinoembryonic anti-gen(CEA).The assay was carried out in a sandwich formation in which two antibodies simultaneously recognized two separate binding sites on the same target CEA.After enrichment with magnetic separator,the fluorescent intensities produced by the QDs,which were confined to the surface of the beads,could be read efficiently to quantify the concentration of the CEA.For this assay,the limit of detection was 38 pg/mL with the linear range was from0.39 to 50 ng/mL,and it has been proved with high accuracy and repeatability in tumor marker control.With the high sensitivity,specificity and low sample consumption,this method takes a big step to the early diagnosis of diseases.
引文
[1]WAGNER M K,LI F,LI J,et al.Use of quantum dots in the development of assays for cancer biomarkers[J].Anal Bioanal Chem,2010,397(8):3213-3224.
[2]LI X,ASMITANANDA T,GAO L,et al.Biomarkers in the lung cancer diagnosis:A clinical perspective[J].Neoplasma,2012,59(5):500-507.
[3]SUNG H J,CHO J Y.Biomarkers for the lung cancer diagnosis and their advances in proteomics[J].BMB Rep,2008,41(9):615-625.
[4]SIEGEL R,MA J,ZOU Z,et al.Cancer statistics,2014[J].CA Cancer J Clin,2014,64(1):9-29.
[5]MITSUHASHI N,TAKAHASHI T,SAKURAI H,et al.Establishment and characterization of a new human lung poorly differentiated adenocarcinoma cell line,GLL-1,producing carcinoembryonic antigen(CEA)and CA19-9[J].Lung Cancer,1995,12(1/2):13-24.
[6]GRUNNET M,SORENSEN J B.Carcinoembryonic antigen(CEA)as tumor marker in lung cancer[J].Lung Cancer,2012,76(2):138-143.
[7]JIA C P,ZHONG X Q,HUA B,et al.Nano-ELISA for highly sensitive protein detection[J].Biosens Bioelectron,2009,24(9):2836-2841.
[8]XIANG D S,ZENG G P,HE Z K.Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes[J].Biosens Bioelectron,2011,26(11):4405-4410.
[9]PENG Z A,PENG X.Formation of high-quality CdTe,CdSe,and CdS nanocrystals using CdO as precursor[J].J Am Chem Soc,2001,123(1):183-184.
[10]PETRYAYEVA E,ALGAR W R,MEDINTZ I L.Quantum dots in bioanalysis:A review of applications across various platforms for fluorescence spectroscopy and imaging[J].Appl Spectrosc,2013,67(3):215-252.
[11]KLIMOV V I.Spectral and dynamical properties of multiexcitons in semiconductor nanocrystals[J].Annu Rev Phys Chem,2007,58:635-673.
[12]SMITH A M,NIE S.Semiconductor nanocrystals:Structure,properties,and band gap engineering[J].Acc Chem Res,2010,43(2):190-200.
[13]YU X,XIA H S,SUN Z D,et al.On-chip dual detection of cancer biomarkers directly in serum based on self-assembled magnetic bead patterns and quantum dots[J].Biosens Bioelectron,2013,41:129-136.
[14]PARK H,HWANG M P,LEE J W,et al.Harnessing immunomagnetic separation and quantum dot-based quantification capacities for the enumeration of absolute levels of biomarker[J].Nanotechnology,2013,24(28):285103.
[15]DUNBAR S A.Applications of Luminex xMAP technology for rapid,high-throughput multiplexed nucleic acid detection[J].Clin Chim Acta,2006,363(1-2):71-82.
[16]GOKARNA A,JIN L H,HWANG J S,et al.Quantum dot-based protein micro-and nanoarrays for detection of prostate cancer biomarkers[J].Proteomics,2008,8(9):1809-1818.
[17]CHENG X,PU X,JUN P,et al.Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips[J].Int J Nanomedicine,2014(9):5619-5626.
[18]ZHU X,DUAN D,PUBLICOVER N G.Magnetic bead based assay for C-reactive protein using quantum-dot fluorescence labeling and immunoaffinity separation[J].Analyst,2010,135(2):381-389.
[19]ZHOU L,OU L J,CHU X,et al.Aptamer-based rolling circle amplification:A platform for electrochemical detection of protein[J].Anal Chem,2007,79(19):7492-7500.
[20]WHITEAKER J R,ZHAO L,ZHANG H Y,et al.Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers[J].Anal Biochem,2007,362(1):44-54.
[21]LIU M,JIA C,HUANG Y,et al.Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes[J].Analyst,2010,135(2):327-331.
[22]SWIERCZEWSKA M,LIU G,LEE S,et al.High-sensitivity nanosensors for biomarker detection[J].Chem Soc Rev,2012,41(7):2641-2655.
[23]ESTEVE-TURRILLAS F A,ABAD-FUENTES A.Applications of quantum dots as probes in immunosensing of small-sized analytes[J].Biosens Bioelectron,2013,41:12-29.
[24]WANG J,MOUNTZIARIS T J.Homogeneous immunoassays based on fluorescence emission intensity variations of zinc selenide quantum dot sensors[J].Biosens Bioelectron,2013,41:143-149.
[2]LI X,ASMITANANDA T,GAO L,et al.Biomarkers in the lung cancer diagnosis:A clinical perspective[J].Neoplasma,2012,59(5):500-507.
[3]SUNG H J,CHO J Y.Biomarkers for the lung cancer diagnosis and their advances in proteomics[J].BMB Rep,2008,41(9):615-625.
[4]SIEGEL R,MA J,ZOU Z,et al.Cancer statistics,2014[J].CA Cancer J Clin,2014,64(1):9-29.
[5]MITSUHASHI N,TAKAHASHI T,SAKURAI H,et al.Establishment and characterization of a new human lung poorly differentiated adenocarcinoma cell line,GLL-1,producing carcinoembryonic antigen(CEA)and CA19-9[J].Lung Cancer,1995,12(1/2):13-24.
[6]GRUNNET M,SORENSEN J B.Carcinoembryonic antigen(CEA)as tumor marker in lung cancer[J].Lung Cancer,2012,76(2):138-143.
[7]JIA C P,ZHONG X Q,HUA B,et al.Nano-ELISA for highly sensitive protein detection[J].Biosens Bioelectron,2009,24(9):2836-2841.
[8]XIANG D S,ZENG G P,HE Z K.Magnetic microparticle-based multiplexed DNA detection with biobarcoded quantum dot probes[J].Biosens Bioelectron,2011,26(11):4405-4410.
[9]PENG Z A,PENG X.Formation of high-quality CdTe,CdSe,and CdS nanocrystals using CdO as precursor[J].J Am Chem Soc,2001,123(1):183-184.
[10]PETRYAYEVA E,ALGAR W R,MEDINTZ I L.Quantum dots in bioanalysis:A review of applications across various platforms for fluorescence spectroscopy and imaging[J].Appl Spectrosc,2013,67(3):215-252.
[11]KLIMOV V I.Spectral and dynamical properties of multiexcitons in semiconductor nanocrystals[J].Annu Rev Phys Chem,2007,58:635-673.
[12]SMITH A M,NIE S.Semiconductor nanocrystals:Structure,properties,and band gap engineering[J].Acc Chem Res,2010,43(2):190-200.
[13]YU X,XIA H S,SUN Z D,et al.On-chip dual detection of cancer biomarkers directly in serum based on self-assembled magnetic bead patterns and quantum dots[J].Biosens Bioelectron,2013,41:129-136.
[14]PARK H,HWANG M P,LEE J W,et al.Harnessing immunomagnetic separation and quantum dot-based quantification capacities for the enumeration of absolute levels of biomarker[J].Nanotechnology,2013,24(28):285103.
[15]DUNBAR S A.Applications of Luminex xMAP technology for rapid,high-throughput multiplexed nucleic acid detection[J].Clin Chim Acta,2006,363(1-2):71-82.
[16]GOKARNA A,JIN L H,HWANG J S,et al.Quantum dot-based protein micro-and nanoarrays for detection of prostate cancer biomarkers[J].Proteomics,2008,8(9):1809-1818.
[17]CHENG X,PU X,JUN P,et al.Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips[J].Int J Nanomedicine,2014(9):5619-5626.
[18]ZHU X,DUAN D,PUBLICOVER N G.Magnetic bead based assay for C-reactive protein using quantum-dot fluorescence labeling and immunoaffinity separation[J].Analyst,2010,135(2):381-389.
[19]ZHOU L,OU L J,CHU X,et al.Aptamer-based rolling circle amplification:A platform for electrochemical detection of protein[J].Anal Chem,2007,79(19):7492-7500.
[20]WHITEAKER J R,ZHAO L,ZHANG H Y,et al.Antibody-based enrichment of peptides on magnetic beads for mass-spectrometry-based quantification of serum biomarkers[J].Anal Biochem,2007,362(1):44-54.
[21]LIU M,JIA C,HUANG Y,et al.Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes[J].Analyst,2010,135(2):327-331.
[22]SWIERCZEWSKA M,LIU G,LEE S,et al.High-sensitivity nanosensors for biomarker detection[J].Chem Soc Rev,2012,41(7):2641-2655.
[23]ESTEVE-TURRILLAS F A,ABAD-FUENTES A.Applications of quantum dots as probes in immunosensing of small-sized analytes[J].Biosens Bioelectron,2013,41:12-29.
[24]WANG J,MOUNTZIARIS T J.Homogeneous immunoassays based on fluorescence emission intensity variations of zinc selenide quantum dot sensors[J].Biosens Bioelectron,2013,41:143-149.