CN112301095A - 一种超灵敏检测前列腺特异性抗原psa的方法 - Google Patents
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Abstract
本发明涉及一种超灵敏检测前列腺特异性抗原PSA的方法，采用动态光散射(DLS)和透射电镜(TEM)研究了该方法的可行性。如图1A所示，加入0.1μg mL‑1PSA前后，直径变化显著。在没有PSA的情况下，AuNPs的直径为419.7nm，加入0.1mg mL‑1PSA后，其直径减小到46.3nm。结果表明，PSA的存在可以引起AuNPs聚集分散的变化。TEM图像进一步证实这一结果，加入0.1μg mL‑ 1PSA前，AuNPs处于聚集状态，加入0.1μg mL‑ 1PSA后，AuNPs处于分散状态(图1B)。这些结果表明，该方法可以通过测量AuNPs的直径变化来定量PSA。该生物传感器能够实现PSA的高灵敏度检测。
Description
技术领域
本发明属于生物传感器的开发方法，涉及一种超灵敏检测前列腺特异性抗原PSA的方法。
背景技术
前列腺特异性抗原(PSA)是一种特异性的前列腺癌标志物，目前已成为临床诊 断前列腺癌的重要生物标志物[1]，因此，对PSA的快速定量的检测具有重要临床价值 [2]。目前，PSA的检测方法有多种，包括荧光标记法[3]、电化学免疫传感器法[4]、表面 等离子体共振法[5]、电化学发光法等[6]。为了提高灵敏度，还结合滚环放大等放大方法 设计检测方案[7]。聚合酶链反应(PCR)作为核酸扩增和检测的金标准，具有成本低、 灵敏度高、选择性好的优点，但是还没有被用于PSA的检测。
考虑到蛋白质在疾病中的重要作用，PCR被应用到对蛋白质的高灵敏度检测，从而发展了免疫PCR(immuno-PCR,IPCR)[8]。适配体是一种单链DNA或RNA，具 有较好的亲和力，可以与多种不同的目标分子特异性结合。与抗体相比，适配体具有 合成简单、成本低、免疫原性低等优点[9]。因此，基于适配体的免疫PCR检测被开发 为IPCR,[10,11]的替代方法。IPCR中，一个适配体作为亲和配体，通过PCR直接扩增 产生信号，而不需要费力地在抗体和DNA序列之间偶联。该方法已应用于凝血酶、 血小板源生长因子等蛋白的检测[12-16]。为了避免分离或洗涤步骤，通过将适配体与目 标物的结合过程转化为新的扩增DNA序列进行检测，发展了均相PCR检测方法[11,17]。 这种基于适配体的PCR检测方法已被广泛应用于检测各种疾病相关蛋白[17-22]，如抗 原、PDGF-BB和凝血酶等。然而，目前将有同时检测蛋白质和均相PCR结合的研究 较少。
金纳米颗粒(AuNPs)作为一种替代信号报告物在生物传感器中得到了广泛的研究，其具有高的光稳定性，易于与DNA等生物分子合成和功能化，具有较强的光吸 收和散射特性[23]。这些理想的性质导致了基于AuNPs的比色法的发展，这种比色法适 用于包括金属离子、小分子和生物分子在内的一系列分析物[24]，这种方法可以方便地 进行视觉检测，但灵敏度较低[25]。最近，动态光散射(dynamic light sacttering,DLS) 作为一种检测技术被引入到基于AuNPs的分析中，直接用于监测分析物引起的AuNPs 的尺寸变化。DLS是一种常用的光学技术，用于表征悬浮液中颗粒(或大分子)的尺 寸分布[26]。AuNPs可以达到很高的光散射能力，使他检测限低至10-16M，而且他们的 光散射强度与颗粒大小成正比[27-32]。同时，AuNPs的光散射信号远高于大多数生物样 品的干扰物质，直径为80nm的AuNPs的光散射信号甚至比有机染料强105倍。
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发明内容
要解决的技术问题
为了避免现有技术的不足之处，本发明提出一种超灵敏检测前列腺特异性抗原PSA的方法，降低现有技术易出现假阳性的风险，提高现有技术的灵敏度。
技术方案
一种超灵敏检测前列腺特异性抗原PSA的方法，其特征在于步骤如下：
步骤1、制备脱氧核糖核酸DNA修饰AuNPs：将巯基修饰的DNA的Oligo 6和 Oligo 7分别溶解于60.0μL的pH为5.0的醋酸盐缓冲溶液，再加入12.0μL的20.0mM 的三(2-羧乙基)膦(TCEP)，以还原修饰在DNA上的巯基为二硫键，然后将上述溶 液全部转移至10.0mLAuNPs溶液中，室温孵育16.0h；然后在接下来的44.0h内， 向金纳米粒子溶液中加入5.0M的氯化钠NaCl溶液，并使最终溶液中NaCl的浓度为 100.0mM；修饰完成，除去未修饰到金纳米粒子表面的DNA；最后将制备的修饰了 DNA的金纳米粒子稀释到所需浓度；
步骤2、分子内杂交反应：2.0μL 0.4nM的DNA的Oligo 1和Oligo 2在90.0℃ 加热变性10.0分钟并迅速冷却到室温，然后向20.0mM Tris-Cl，pH值7.9、50.0mM 醋酸钾KAc、10.0mM醋酸镁Mg(Ac)2和1.0mM二硫苏糖醇DTT的缓冲溶液加入 2.0μL不同浓度的前列腺特异性抗原：从1.0pg mL-1到1.0μg mL-1，以及室温的DNA 的Oligo 1和Oligo 2混合液，在37.0℃温度下加热30.0分钟；然后再加入0.5U聚合 酶和1.0μL 2.5mM脱氧核糖核苷三磷酸dNTPs使溶液总量是10.0μL，并继续在37℃ 反应30.0分钟，然后90.0℃加热5分钟，使克列诺片段聚合酶Klenow Fragment失去 活性，反应后的溶液作为PSA的聚合酶链反应PCR的扩增模板于步骤3中使用；
步骤3、聚合酶链扩增反应：PCR混合物的总量为50.0μL的溶液，包括小于50.0 μL的步骤2的溶液、1U Taq酶、200.0μM dNTPs、2.5mM氯化镁MgCl2、4.0nM步 骤1中DNA修饰的AuNPs、1×缓冲溶液、1.0μM Oligo 3、1.0μM Oligo 4、1.0μM Oligo 5；
当PSA存在时，Oligo 1和Oligo 2为DNA/DNA双链结构，PSA识别并结合Oligo 1形成PSA/Oligo 1复合物，与PSA混合后释放Oligo 2，反应终止；当PSA不存在时， Oligo 1和Oligo 2在克列诺片段聚合酶的作用下以彼此为模板延伸，得到两条更长的 DNA链，这两条长DNA链在PCR循环中，通过正向引物和反向引物Oligo 3和Oligo 4识别新模板进行PCR扩增，并在每个周期的延伸过程中对Oligo 5进行切割，其中， Oligo 5包含两个部分，一部分与新扩增模板DNA互补，另一部分与AuNPs上修饰的 DNA的Oligo 6和Oligo 7互补，因此在PCR扩增前AuNPs处于聚集状态，随着PCR 循环周期的延长，AuNPs的聚集程度会逐渐减弱，PSA浓度变化会影响溶液中AuNPs 的直径变化，通过测定直径变化可以定量PSA
PCR扩增条件是：首先94℃变性3分钟，接着以94℃20s，48℃30s，72℃30 s的程序循环30次，最后为保证延伸充分进行，72℃温度条件下继续延伸3分钟； PCR反应后溶液中AuNPs的直径变化用动态光散射方法检测；
步骤4、动态光散射粒子直径的测量：采用Nano/Zeta电位计动态光散射粒径测量方法检测步骤3的溶液直径变化；操作条件：温度25.0℃，入射角度90.0°，入射激 光波长683.0nm，激光功率100.0mW；所有测量的尺寸均以强度平均值为基础，每个 粒径为五个测量值的平均值；
所述DNA序列为：
所述金纳米粒子AuNPs的制备：将2.0mL38.8 mM柠檬酸钠溶液快速加入煮沸的1.0mM氯金酸HAuCl4溶液中搅拌，待溶液颜色由淡黄色变为酒红色后，再回流搅拌 15.0分钟；然后通过连续搅拌将溶液冷却到室温，最后AuNPs通过0.4μm尼龙滤膜 过滤收集。
所述步骤1的除去未修饰到金纳米粒子表面的DNA是：将溶液放置在13800.0rpm转速的离心机离心三次，每次离心30.0分钟，以除去未修饰到金纳米粒子表面的DNA， 每次用pH 8.0的三羟甲基氨基甲烷-醋酸Tris-Ac缓冲溶液洗涤。
有益效果
本发明提出的一种超灵敏检测前列腺特异性抗原PSA的方法，利用Taq聚合酶的复制和酶切特性，切断连接DNA修饰的AuNPs的链，使本来处于聚集状态的AuNPs 分散。AuNPs的分散导致AuNPs的大小发生变化，DLS对其进行了高度敏感的测量。
采用动态光散射(DLS)和透射电镜(TEM)研究了该方法的可行性。如图1A 所示，加入0.1μg mL-1PSA前后，直径变化显著。在没有PSA的情况下，AuNPs的 直径为419.7nm，加入0.1mg mL-1PSA后，其直径减小到46.3nm。结果表明，PSA 的存在可以引起AuNPs聚集分散的变化。TEM图像进一步证实这一结果，加入0.1μg mL-1PSA前，AuNPs处于聚集状态，加入0.1μgmL-1PSA后，AuNPs处于分散状态 (图1B)。这些结果表明，该方法可以通过测量AuNPs的直径变化来定量PSA。
对分析物的线性响应是分析的关键，因此该方法对不同浓度的PSA存在时AuNPs溶液的直径进行了DLS检测。由图2可知，随着PSA浓度的增加，AuNPs的平均直 径逐渐减小且AuNPs平均直径与PSA浓度的线性关系，结果显示在10.0pg mL-1到1.0μg mL-1范围内，AuNPs平均直径随PSA浓度的对数线性减小。线性回归方程D＝ 318.3-46.66log10C(C:pgmL-1)和检出限7.0pg mL-1(3δ/斜率)。这些结果表明，该生 物传感器能够实现PSA的高灵敏度检测。
附图说明
图1：(A)动态光散射的结果；(B)透射电镜的结果(a)没有PSA的PCR反应产物； (b)0.1mg mL-1PSA存在时的溶液直径。
图2：金纳米粒子的水合直径与PSA浓度对数变化的线性关系，PSA的浓度从10.0pg mL-1到1.0μg mL-1。误差棒表示三次测量值的标准差。
具体实施方式
现结合实施例、附图对本发明作进一步描述：
实例一：通过加标回收率研究该检测方法在复杂生物体质中的检测能力，即在没有被测物质的样品基质中加入定量的标准物质，按样品的处理步骤分析，得到的结果 与理论值的比值。具体过程如下：首先在分子内杂交反应的缓冲液中加入5％人血清 和100.0pgmL-1的标准PSA样品，经过分子内杂交反应和聚合酶链扩增反应，用本发 明方法检测到的PSA浓度是91.70pg mL-1，回收率是91.7％，相对标准偏差(RSD) 是4.3％。
实例二：通过加标回收率研究该检测方法在复杂生物体质中的检测能力，即在没有被测物质的样品基质中加入定量的标准物质，按样品的处理步骤分析，得到的结果 与理论值的比值。具体过程如下：首先在分子内杂交反应的缓冲液中加入5％人血清 和1.0ngmL-1的标准PSA样品，经过分子内杂交反应和聚合酶链扩增反应，用本发明 方法检测到的PSA浓度是0.98ng mL-1，回收率是98.0％，相对标准偏差(RSD)是 1.9％。
实例三：通过加标回收率研究该检测方法在复杂生物体质中的检测能力，即在没有被测物质的样品基质中加入定量的标准物质，按样品的处理步骤分析，得到的结果 与理论值的比值。具体过程如下：首先在分子内杂交反应的缓冲液中加入5％人血清 和10.0ngmL-1的标准PSA样品，经过分子内杂交反应和聚合酶链扩增反应，用本发 明方法检测到的PSA浓度是9.3ng mL-1，回收率是93.0％，相对标准偏差(RSD)是 3.1％。
基于PCR的蛋白质检测需要将蛋白质的信息转化为核酸的检测。因此，我们设想，含有靶蛋白适配体的单链DNA(ssDNA)亲和探针可以作为我们系统中PCR的双链 DNA(dsDNA)模板，从而实现对蛋白质的灵敏度和普遍性检测。依据适配体也能够 结合互补的DNA序列形成一个双链结构，我们设计了一种基于适配体与AuNPs检测 的方法，该方法通过将DNA/DNA双链结构转换为DNA/目标复合物的结果来实现。 图1所示的原理图显示了基于AuNPs的的检测原理，通过双链DNA与复合物的结构 变化到双链DNA的结构来实现。Oligo 1和Oligo 2为DNA/DNA双链结构，PSA识 别并结合Oligo 1形成DNA/target complex，与PSA混合后释放Oligo 2，反应终止。 当PSA不存在时，Oligo 1和Oligo 2在exo-聚合酶的作用下以彼此为模板延伸，得到 两条更长的DNA链，这两条长DNA链在PCR循环中，通过正向引物和反向引物(Oligo 3和Oligo 4)识别新模板进行PCR扩增，并在每个周期的延伸过程中对Oligo 5进行 切割。其中，Oligo 5包含两个部分，一部分与新扩增模板DNA互补，另一部分与AuNPs 上修饰的DNA(Oligo 6和Oligo 7)互补，因此在PCR扩增前AuNPs处于聚集状态，随着PCR循环周期的延长，AuNPs的聚集程度会逐渐减弱。因此，实现了将PSA浓 度转化为AuNPs的直径变化，并利用DLS对直径变化进行测定实现定量检测PSA。
基于PCR的PSA检测需要将PSA的信息转化为核酸的检测。采用动态光散射 (DLS)和透射电镜(TEM)研究了该方法的可行性。如图2所示，加入0.1μg mL-1PSA 前后，水合直径变化显著。在没有PSA的情况下，PCR产物的水合直径为419.7nm， 加入0.1mg mL-1PSA后，其水合直径减小到46.3nm。结果表明，PSA的存在可以引 起AuNPs聚集分散的变化。TEM图像进一步证实这一结果，加入0.1μg mL-1PSA前， AuNPs处于聚集状态，加入0.1μg mL-1PSA后，AuNPs处于分散状态。这些结果表 明，该方法可以通过测量AuNPs的尺寸变化来检测PSA。同时这些结果也表明，DLS 比吸收光谱更敏感，是一种更灵敏的检测PCR产物的技术。
对分析物的线性响应是分析的关键，因此该方法对不同浓度的PSA存在时金纳米粒溶液的直径进行了DLS检测。随着PSA浓度的增加，AuNPs的平均直径逐渐减小。 图2为AuNPs平均直径与PS浓度的线性关系，结果显示在10.0pg mL-1到1.0μg mL-1范围内，AuNPs平均直径随PSA浓度的对数线性减小。线性回归方程D＝318.3- 46.66log10C(C:pg mL-1)和检出限7.0pg mL-1(3δ/斜率)。这些结果表明，该生物传感 器能够实现PSA的高灵敏度检测。
为了研究该检测方法在复杂生物体质中的检测能力，我们进行了加入回收实验。如表1-2所示，在每个含有5％人血清的缓冲液中加入不同浓度的PSA，其回收率为 94.6％-97.7％，相对标准偏差(RSD)为2.8％-5.3％。这些结果表明，DLS可以准确 地检测到这些样品中的PSA，表明其在生物系统中检测PSA蛋白的潜在应用价值。
表1-2人血清的PSA加入回收法
Claims (3)
1.一种超灵敏检测前列腺特异性抗原PSA的方法，其特征在于步骤如下：
步骤1、制备脱氧核糖核酸DNA修饰AuNPs：将巯基修饰的DNA的Oligo 6和Oligo 7分别溶解于60.0μL的pH为5.0的醋酸盐缓冲溶液，再加入12.0μL的20.0mM的三(2-羧乙基)膦(TCEP)，以还原修饰在DNA上的巯基为二硫键，然后将上述溶液全部转移至10.0mL AuNPs溶液中，室温孵育16.0h；然后在接下来的44.0h内，向金纳米粒子溶液中加入5.0M的氯化钠NaCl溶液，并使最终溶液中NaCl的浓度为100.0mM；修饰完成，除去未修饰到金纳米粒子表面的DNA；最后将制备的修饰了DNA的金纳米粒子稀释到所需浓度；
步骤2、分子内杂交反应：2.0μL 0.4nM的DNA的Oligo 1和Oligo 2在90.0℃加热变性10.0分钟并迅速冷却到室温，然后向20.0mM Tris-Cl，pH值7.9、50.0mM醋酸钾KAc、10.0mM醋酸镁Mg(Ac)2和1.0mM二硫苏糖醇DTT的缓冲溶液加入2.0μL不同浓度的前列腺特异性抗原：从1.0pg mL-1到1.0μg mL-1，以及室温的DNA的Oligo 1和Oligo 2混合液，在37.0℃温度下加热30.0分钟；然后再加入0.5U聚合酶和1.0μL 2.5mM脱氧核糖核苷三磷酸dNTPs使溶液总量是10.0μL，并继续在37℃反应30.0分钟，然后90.0℃加热5分钟，使克列诺片段聚合酶Klenow Fragment失去活性，反应后的溶液作为PSA的聚合酶链反应PCR的扩增模板于步骤3中使用；
步骤3、聚合酶链扩增反应：PCR混合物的总量为50.0μL的溶液，包括小于50.0μL的步骤2的溶液、1U Taq酶、200.0μM dNTPs、2.5mM氯化镁MgCl2、4.0nM步骤1中DNA修饰的AuNPs、1×缓冲溶液、1.0μM Oligo 3、1.0μM Oligo 4、1.0μM Oligo 5；
当PSA存在时，Oligo 1和Oligo 2为DNA/DNA双链结构，PSA识别并结合Oligo 1形成PSA/Oligo 1复合物，与PSA混合后释放Oligo 2，反应终止；当PSA不存在时，Oligo 1和Oligo 2在克列诺片段聚合酶的作用下以彼此为模板延伸，得到两条更长的DNA链，这两条长DNA链在PCR循环中，通过正向引物和反向引物Oligo 3和Oligo 4识别新模板进行PCR扩增，并在每个周期的延伸过程中对Oligo 5进行切割，其中，Oligo 5包含两个部分，一部分与新扩增模板DNA互补，另一部分与AuNPs上修饰的DNA的Oligo 6和Oligo 7互补，因此在PCR扩增前AuNPs处于聚集状态，随着PCR循环周期的延长，AuNPs的聚集程度会逐渐减弱，PSA浓度变化会影响溶液中AuNPs的直径变化，通过测定直径变化可以定量PSA
PCR扩增条件是：首先94℃变性3分钟，接着以94℃20s，48℃30s，72℃30s的程序循环30次，最后为保证延伸充分进行，72℃温度条件下继续延伸3分钟；PCR反应后溶液中AuNPs的直径变化用动态光散射方法检测；
步骤4、动态光散射粒子直径的测量：采用Nano/Zeta电位计动态光散射粒径测量方法检测步骤3的溶液直径变化；操作条件：温度25.0℃，入射角度90.0°，入射激光波长683.0nm，激光功率100.0mW；所有测量的尺寸均以强度平均值为基础，每个粒径为五个测量值的平均值；
所述DNA序列为：
2.根据权利要求1所述超灵敏检测前列腺特异性抗原PSA的方法，其特征在于：所述金纳米粒子AuNPs的制备：将2.0mL38.8 mM柠檬酸钠溶液快速加入煮沸的1.0mM氯金酸HAuCl4溶液中搅拌，待溶液颜色由淡黄色变为酒红色后，再回流搅拌15.0分钟；然后通过连续搅拌将溶液冷却到室温，最后AuNPs通过0.4μm尼龙滤膜过滤收集。
3.根据权利要求1所述超灵敏检测前列腺特异性抗原PSA的方法，其特征在于：所述步骤1的除去未修饰到金纳米粒子表面的DNA是：将溶液放置在13800.0rpm转速的离心机离心三次，每次离心30.0分钟，以除去未修饰到金纳米粒子表面的DNA，每次用pH 8.0的三羟甲基氨基甲烷-醋酸Tris-Ac缓冲溶液洗涤。
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