基于贵金属纳米粒子标记技术的均相免疫分析新方法研究
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摘要
免疫分析利用抗体-抗原间的特异性结合反应对蛋白质、药物、激素、毒素、微生物等物质进行高选择性、高亲和性定性定量检测，已被广泛应用于临床医学、化学、药学等领域。传统的免疫分析方法如酶联吸附免疫方法(ELISA)大多采用非均相模式，操作繁琐，分析时间长，需要抗体的包埋，多次洗涤和显色等步骤。难以满足某些快速检测的要求。金纳米粒子(GNPs)易于合成与修饰，具有生物相容性好、光散射特性高等优点，是一种优异的光学标记探针。共振光散射相关光谱(RLSCS)是一种高灵敏度的单颗粒探测方法，该方法基于贵重金属纳米粒子在高聚焦的微区内由于布朗运动会产生散射光光强波动，通过对波动进行自相关分析可获取微区内粒子浓度、扩散系数等信息。
本文将单颗粒检测方法与贵金属纳米粒子(如金和银纳米粒子)标记技术结合，发展了一些灵敏的均相免疫分析新方法。本论文研究工作主要包括：
1)本文将RLSCS检测方法和金纳米粒子标记技术结合，构建了肝癌标志物甲胎蛋白(AFP)的均相夹心免疫分析新方法。该方法将AFP的两种抗体通过共价键的形式连接到GNPs上，形成金标抗体探针。当两种抗体探针与含有抗原的样品溶液混合，夹心免疫反应使GNPs聚集形成二聚或者多聚体，导致溶液中GNPs的特征扩散时间的变化，能够被RLSCS灵敏地检测到。研究结果表明GNPs的特征扩散时间与抗原浓度的对数值呈线性相关。系统地研究各种因素对检测的影响。在优化的条件下，AFP线性范围为1pmol/L-1nmol/L，检测下限为1pmol/L。将该方法成功地用于人血清中AFP含量的测定，其结果与ELISA法一致。与传统方法相比，该方法操作简单、分析快速、灵敏度高、选择性好，耗样量小。
2)竞争免疫模式可同时适用于大分子和小分子的分析。银纳米粒子(SNPs)作为探针载体具有比GNPs更高的光散射强度。将高灵敏的RLSCS方法与银纳米粒子(SNPs)标记技术相结合，建立了均相竞争免疫分析新方法，用于小分子-雌二醇(E2)和大分子-甲胎蛋白(AFP)分析。此方法将E2抗体和AFP的抗体、抗原通过共价的形式包覆在SNPs表面，E2的半抗原(雌二醇与牛血清白蛋白连接物，BSA-E2)以静电吸附作用连接在SNPs上。当含有E2或AFP的样品溶液与过量的包覆了抗体的SNPs溶液混合，通过免疫反应SNPs@Ab上的一部分位点将被占据，再加入AFP抗原和E2半抗原连接的SNPs，使其占据剩下的位点，同时形成二聚或多聚的免疫产物。溶液中SNPs的特征扩散时间将发生变化，而这种变化能够灵敏的被RLSCS检测到。研究结果表明SNPs的特征扩散时间与E2和AFP加入浓度的对数值呈线性相关。系统地研究了各种因素对检测的影响。在优化的条件下，E2的检测线性范围为10pmol/L-1nmol/L，检测下限为10pmol/L；AFP的检测线性范围为100pmol/L-10nmol/L，检测下限为100pmol/L。将该方法用于人尿液中E2含量和人血清中AFP含量的测定，其结果与传统的ELISA方法所得的结果基本吻合。本法具有灵敏度高、操作简单、分析时间短、普适性强等优点，在临床诊断、食品安全检测和环境分析等方面具有广阔应用前景。
3) GNPs溶液受到激光照射可产生强烈的共振散射光子爆发，光子爆发个数与溶液中GNPs粒子个数呈良好的线性关系。基于检测基线噪声满足高斯分布、纳米粒子光子爆发信号满足泊松分布的特征，发展了光子爆发纳米粒子计数方法。对光子爆发的强度与相应强度下光子爆发个数作图，得到光子爆发强度与个数的关系，通过曲线拟合确定光子爆发的噪声分布区间及其数学期望μ和方差σ，设定阈值=μ+8×σ。通过软件得到光子爆发峰的个数。此方法重现性好、灵敏度高。
4)基于光子爆发计数技术，发展了均相免疫分析新方法。分别建立了前列腺特异抗原(PSA)和甲胎蛋白(AFP)的夹心和竞争免疫分析方法。其方法的原理类似于散射相关光谱方法，采用金纳米粒子标记抗体或抗原。在夹心免疫分析中，免疫反应后GNPs会形成一些二聚或者低聚体，导致了溶液中粒子数目减少，进而引起光子爆发数目减少，这一变化可以通过光子爆发计数检测技术灵敏的表征。我们对构建的四种分析体系进行了系统的优化。其中所得PSA夹心免疫分析的最优条件为以680pmol/L的20nm GNPs为探针进行实验，线性范围为1pmol/L-10nmol/L，检测下限为1pmol/L；PSA竞争免疫分析的最优条件为以1.36nmol/L的20nm GNPs为探针进行实验，线性范围为10pmol/L-10nmol/L，检测下限为10pmol/L；AFP夹心免疫分析的最优条件为以340pmol/L的40nmGNPs为探针进行实验，线性范围为1pmol/L-10nmol/L，检测下限为1pmol/L；AFP竞争免疫分析的最优条件为以170pmol/L的20nm GNPs为探针进行实验，线性范围为1pmol/L-1nmol/L，检测下限为1pmol/L。最后以优化后的系统对实际样品(血清)中PSA和AFP含量进行了定量检测。实验结果与传统的ELISA方法结果基本吻合。此方法的灵敏度高、选择性好、样品处理简单，是一种具有发展潜力的分析检测新方法。该方法与RLSCS方法相比，重现性得到了一定的改善。
Currently, immunoassay is one of the most important analytical methods forproteins, drugs, hormones, toxins, microbes and other substances based on theantibody-antigen specific binding reaction. Due to its high selectivity and affinityimmunoassay has been widely used in clinical medicine, chemistry, pharmaceuticaland other fields. The heterogeneous assay mode was widely applied in traditionalimmunoassays such as enzyme linked immunosorbent assay (ELISA). However, theheterogeneous immunoassays involve antibody immobilization, immune reaction andwashing cycles, and thus this assay is labor-intensive and time-consuming. Generally,homogeneous immunoassay is an attractive detection format because it is amenable toautomation, reduced the risk of contamination and shorten analysis time. The key inhomogeneous immunoassay is to develop a new detection method for quantitativelyand sensitively distinguishing properties of antibodies (or antigen) andantigen-antibody complexes in the reaction solution. In this thesis, combined singleparticle detection methods with noble metal nanoparticles (such as gold or silvernanoparticles) labelling techniques, we devepled homogeneous immunoassays withhigh sensitivity, and main work is included the following parts.
As one of the optical probes, gold nanoparticles (GNPs) have many advantages,such as facile synthesis and modification, strong light scattering properties, goodbiocompatibilty and so on. We established a setup of resonance light scatteringcorrelation spectroscopy (RLSCS). The principle of RLSCS is similar to fluorescencecorrelation spectroscopy (FCS). When GNPs pass through the detection volume ofRLSCS system due to Brownian motion, the fluctuation of the scattering lightintensity can be tracked. Certain information such as the concentration or diffusioncoefficient of the detected nanoparticles can be obtained through the correlation analysis of the light fluctuation.
1. We conjugated two different antibodies (Ab) with GNPs respectively. Inimmunoassays, when two different GNPs labeld with antibodies were mixed in asample containing antigen (Ag) targets, the binding of targets will cause GNPs toform dimers (or oligomers), which leads to the significant increase in thecharacteristic diffusion time of GNPs in the detection volume. The RLSCS methodcan sensitively detect the changes in the characteristic diffusion time of GNPs beforeand after immune reactions. We used this technology in homogeneous immunoassaysfor the liver cancer biomarker alpha-fetoprotein (AFP). The linear range of this assayis from1pmol/L to1nmol/L and the detection limit is1pmol/L for AFP. This newmethod was successfully applied for the direct determination of AFP levels in serafrom healthy subjects and cancer patients. Our results were in good agreement withELISA assays.
2. Competitive immunoassay mode can be applied to both large and smallmolecules. Silver nanoparticles (SNPs) as a probe carrier have stronger plasmonicscattering property than GNPs. The antibody, antigen and hapten were attached to thesurface of SNPs. The antibody-labeled SNPs were firstly mixed with a samplecontaining antigens, and the part of antibody-labeled SNPs was bound to antigens ofinterest in the sample. And then, the antigen-labeled SNPs were added into the mixedsolution above, and they were bound to free antibody-labeled SNPs (excess) to formdimers (or oligomers), which led to the significant increase in the characteristicdiffusion time of SNPs in a tinny detection volume. In the competitive model, thecharacteristic diffusion time of SNPs decreased with an increase of antigenconcentration. The RLSCS can sensitively detects the changes in the characteristicdiffusion time of SNPs before and after the immunoreactions. In order to demonstratethe universality of this new method, small biomolecules,17-β estradiol (E2), andbiomacromolecules AFP, were used as assay models. The linear ranges of this methodwere from10pmol/L to10nmol/L for E2and100pmol/L to10nmol/L for AFP,respectively, and the detection limits were10pmol/L for E2and100pmol/L for AFP,respectively. The presented method was successfully used to the determination of E2levels in human urine and AFP levels in human sera. Our results were in goodagreement with ELISA assays.
3. The photon bursting of GNPs will be generated in a highly focused laser beam (less than1fL) due to the plasmon resonance scattering and Brownian motion ofGNPs. The number of photon burst and the number of particles in solution GNPsshowed a good linear relationship. We observed that the noise shows Gaussiandistribution, and photon burst signal of GNPs meets Poisson distribution. Based onthe behaviors of noise and photon burst signal of GNPs, we established the photonburst counting method. On the plot between the intensity and the number of thephoton burst, the relationship between the intensity and the number of the photonburst can be obtained. Through the curve fitting, the noise distribution range ofphoton burst and its mathematical expectation μ and variance σ were determined, wedefined the threshold equals to μ+8×σ. Through software, the number of photonburst can be obtained. This method has good reproducibility and high sensitivity.
4. Based on the photon burst counting method, we developed the homogeneoussandwich and competitive immunoassay. In assay, sandwich or competitive immunoreactions will cause GNPs to form dimers (or oligomers), which will cause a decreasein the overall number of GNPs in solution and the number of photon burst, thischanges can be detected by photon burst counting technique sensitively. We appliedprostate-specific antigen (PSA) and alpha-fetoprotein (AFP) as modes to evaluate thesystem. The linear range of PSA sandwich immunoassay was1pmol/L-10nmol/L,and the detection limit was1pmol/L; The linear range of PSA competitiveimmunoassay was10pmol/L-10nmol/L, and the detection limit was10pmol/L; Thelinear range of AFP sandwich immunoassay was1pmol/L-10nmol/L, and thedetection limit was1pmol/L; The linear range of AFP competitive immunoassay was1pmol/L-1nmol/L, and the detection limit was1pmol/L. Our results were in goodagreement with ELISA assays.
本文将单颗粒检测方法与贵金属纳米粒子(如金和银纳米粒子)标记技术结合，发展了一些灵敏的均相免疫分析新方法。本论文研究工作主要包括：
1)本文将RLSCS检测方法和金纳米粒子标记技术结合，构建了肝癌标志物甲胎蛋白(AFP)的均相夹心免疫分析新方法。该方法将AFP的两种抗体通过共价键的形式连接到GNPs上，形成金标抗体探针。当两种抗体探针与含有抗原的样品溶液混合，夹心免疫反应使GNPs聚集形成二聚或者多聚体，导致溶液中GNPs的特征扩散时间的变化，能够被RLSCS灵敏地检测到。研究结果表明GNPs的特征扩散时间与抗原浓度的对数值呈线性相关。系统地研究各种因素对检测的影响。在优化的条件下，AFP线性范围为1pmol/L-1nmol/L，检测下限为1pmol/L。将该方法成功地用于人血清中AFP含量的测定，其结果与ELISA法一致。与传统方法相比，该方法操作简单、分析快速、灵敏度高、选择性好，耗样量小。
2)竞争免疫模式可同时适用于大分子和小分子的分析。银纳米粒子(SNPs)作为探针载体具有比GNPs更高的光散射强度。将高灵敏的RLSCS方法与银纳米粒子(SNPs)标记技术相结合，建立了均相竞争免疫分析新方法，用于小分子-雌二醇(E2)和大分子-甲胎蛋白(AFP)分析。此方法将E2抗体和AFP的抗体、抗原通过共价的形式包覆在SNPs表面，E2的半抗原(雌二醇与牛血清白蛋白连接物，BSA-E2)以静电吸附作用连接在SNPs上。当含有E2或AFP的样品溶液与过量的包覆了抗体的SNPs溶液混合，通过免疫反应SNPs@Ab上的一部分位点将被占据，再加入AFP抗原和E2半抗原连接的SNPs，使其占据剩下的位点，同时形成二聚或多聚的免疫产物。溶液中SNPs的特征扩散时间将发生变化，而这种变化能够灵敏的被RLSCS检测到。研究结果表明SNPs的特征扩散时间与E2和AFP加入浓度的对数值呈线性相关。系统地研究了各种因素对检测的影响。在优化的条件下，E2的检测线性范围为10pmol/L-1nmol/L，检测下限为10pmol/L；AFP的检测线性范围为100pmol/L-10nmol/L，检测下限为100pmol/L。将该方法用于人尿液中E2含量和人血清中AFP含量的测定，其结果与传统的ELISA方法所得的结果基本吻合。本法具有灵敏度高、操作简单、分析时间短、普适性强等优点，在临床诊断、食品安全检测和环境分析等方面具有广阔应用前景。
3) GNPs溶液受到激光照射可产生强烈的共振散射光子爆发，光子爆发个数与溶液中GNPs粒子个数呈良好的线性关系。基于检测基线噪声满足高斯分布、纳米粒子光子爆发信号满足泊松分布的特征，发展了光子爆发纳米粒子计数方法。对光子爆发的强度与相应强度下光子爆发个数作图，得到光子爆发强度与个数的关系，通过曲线拟合确定光子爆发的噪声分布区间及其数学期望μ和方差σ，设定阈值=μ+8×σ。通过软件得到光子爆发峰的个数。此方法重现性好、灵敏度高。
4)基于光子爆发计数技术，发展了均相免疫分析新方法。分别建立了前列腺特异抗原(PSA)和甲胎蛋白(AFP)的夹心和竞争免疫分析方法。其方法的原理类似于散射相关光谱方法，采用金纳米粒子标记抗体或抗原。在夹心免疫分析中，免疫反应后GNPs会形成一些二聚或者低聚体，导致了溶液中粒子数目减少，进而引起光子爆发数目减少，这一变化可以通过光子爆发计数检测技术灵敏的表征。我们对构建的四种分析体系进行了系统的优化。其中所得PSA夹心免疫分析的最优条件为以680pmol/L的20nm GNPs为探针进行实验，线性范围为1pmol/L-10nmol/L，检测下限为1pmol/L；PSA竞争免疫分析的最优条件为以1.36nmol/L的20nm GNPs为探针进行实验，线性范围为10pmol/L-10nmol/L，检测下限为10pmol/L；AFP夹心免疫分析的最优条件为以340pmol/L的40nmGNPs为探针进行实验，线性范围为1pmol/L-10nmol/L，检测下限为1pmol/L；AFP竞争免疫分析的最优条件为以170pmol/L的20nm GNPs为探针进行实验，线性范围为1pmol/L-1nmol/L，检测下限为1pmol/L。最后以优化后的系统对实际样品(血清)中PSA和AFP含量进行了定量检测。实验结果与传统的ELISA方法结果基本吻合。此方法的灵敏度高、选择性好、样品处理简单，是一种具有发展潜力的分析检测新方法。该方法与RLSCS方法相比，重现性得到了一定的改善。
Currently, immunoassay is one of the most important analytical methods forproteins, drugs, hormones, toxins, microbes and other substances based on theantibody-antigen specific binding reaction. Due to its high selectivity and affinityimmunoassay has been widely used in clinical medicine, chemistry, pharmaceuticaland other fields. The heterogeneous assay mode was widely applied in traditionalimmunoassays such as enzyme linked immunosorbent assay (ELISA). However, theheterogeneous immunoassays involve antibody immobilization, immune reaction andwashing cycles, and thus this assay is labor-intensive and time-consuming. Generally,homogeneous immunoassay is an attractive detection format because it is amenable toautomation, reduced the risk of contamination and shorten analysis time. The key inhomogeneous immunoassay is to develop a new detection method for quantitativelyand sensitively distinguishing properties of antibodies (or antigen) andantigen-antibody complexes in the reaction solution. In this thesis, combined singleparticle detection methods with noble metal nanoparticles (such as gold or silvernanoparticles) labelling techniques, we devepled homogeneous immunoassays withhigh sensitivity, and main work is included the following parts.
As one of the optical probes, gold nanoparticles (GNPs) have many advantages,such as facile synthesis and modification, strong light scattering properties, goodbiocompatibilty and so on. We established a setup of resonance light scatteringcorrelation spectroscopy (RLSCS). The principle of RLSCS is similar to fluorescencecorrelation spectroscopy (FCS). When GNPs pass through the detection volume ofRLSCS system due to Brownian motion, the fluctuation of the scattering lightintensity can be tracked. Certain information such as the concentration or diffusioncoefficient of the detected nanoparticles can be obtained through the correlation analysis of the light fluctuation.
1. We conjugated two different antibodies (Ab) with GNPs respectively. Inimmunoassays, when two different GNPs labeld with antibodies were mixed in asample containing antigen (Ag) targets, the binding of targets will cause GNPs toform dimers (or oligomers), which leads to the significant increase in thecharacteristic diffusion time of GNPs in the detection volume. The RLSCS methodcan sensitively detect the changes in the characteristic diffusion time of GNPs beforeand after immune reactions. We used this technology in homogeneous immunoassaysfor the liver cancer biomarker alpha-fetoprotein (AFP). The linear range of this assayis from1pmol/L to1nmol/L and the detection limit is1pmol/L for AFP. This newmethod was successfully applied for the direct determination of AFP levels in serafrom healthy subjects and cancer patients. Our results were in good agreement withELISA assays.
2. Competitive immunoassay mode can be applied to both large and smallmolecules. Silver nanoparticles (SNPs) as a probe carrier have stronger plasmonicscattering property than GNPs. The antibody, antigen and hapten were attached to thesurface of SNPs. The antibody-labeled SNPs were firstly mixed with a samplecontaining antigens, and the part of antibody-labeled SNPs was bound to antigens ofinterest in the sample. And then, the antigen-labeled SNPs were added into the mixedsolution above, and they were bound to free antibody-labeled SNPs (excess) to formdimers (or oligomers), which led to the significant increase in the characteristicdiffusion time of SNPs in a tinny detection volume. In the competitive model, thecharacteristic diffusion time of SNPs decreased with an increase of antigenconcentration. The RLSCS can sensitively detects the changes in the characteristicdiffusion time of SNPs before and after the immunoreactions. In order to demonstratethe universality of this new method, small biomolecules,17-β estradiol (E2), andbiomacromolecules AFP, were used as assay models. The linear ranges of this methodwere from10pmol/L to10nmol/L for E2and100pmol/L to10nmol/L for AFP,respectively, and the detection limits were10pmol/L for E2and100pmol/L for AFP,respectively. The presented method was successfully used to the determination of E2levels in human urine and AFP levels in human sera. Our results were in goodagreement with ELISA assays.
3. The photon bursting of GNPs will be generated in a highly focused laser beam (less than1fL) due to the plasmon resonance scattering and Brownian motion ofGNPs. The number of photon burst and the number of particles in solution GNPsshowed a good linear relationship. We observed that the noise shows Gaussiandistribution, and photon burst signal of GNPs meets Poisson distribution. Based onthe behaviors of noise and photon burst signal of GNPs, we established the photonburst counting method. On the plot between the intensity and the number of thephoton burst, the relationship between the intensity and the number of the photonburst can be obtained. Through the curve fitting, the noise distribution range ofphoton burst and its mathematical expectation μ and variance σ were determined, wedefined the threshold equals to μ+8×σ. Through software, the number of photonburst can be obtained. This method has good reproducibility and high sensitivity.
4. Based on the photon burst counting method, we developed the homogeneoussandwich and competitive immunoassay. In assay, sandwich or competitive immunoreactions will cause GNPs to form dimers (or oligomers), which will cause a decreasein the overall number of GNPs in solution and the number of photon burst, thischanges can be detected by photon burst counting technique sensitively. We appliedprostate-specific antigen (PSA) and alpha-fetoprotein (AFP) as modes to evaluate thesystem. The linear range of PSA sandwich immunoassay was1pmol/L-10nmol/L,and the detection limit was1pmol/L; The linear range of PSA competitiveimmunoassay was10pmol/L-10nmol/L, and the detection limit was10pmol/L; Thelinear range of AFP sandwich immunoassay was1pmol/L-10nmol/L, and thedetection limit was1pmol/L; The linear range of AFP competitive immunoassay was1pmol/L-1nmol/L, and the detection limit was1pmol/L. Our results were in goodagreement with ELISA assays.
引文
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