基于磁球信号放大技术的超灵敏DNA检测方法及其在测定基因表达中的应用
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摘要
DNA是生命现象中不可缺少的生物大分子,是生命体内的基因物质,也是遗传信息的携带者。检测与疾病有关的变异基因对基因筛选、遗传疾病的早期诊断和治疗具有十分深远的意义。因此,随着分子生物学研究的深入DNA的检测手段变得越来越重要。本论文以DNA这一生物大分子为研究对象,将磁球放大技术、核酸杂交技术与光电化学分析技术相结合,研制新型的高灵敏、高选择性的DNA检测方法,并成功应用于检测细胞中的基因表达。主要内容如下：
第一章主要对DNA的检测方法及检测中放大技术进行了简要的综述。DNA检测方法可分为标记型与无标记型,标记型方法主要有荧光分析法、电化学方法、化学发光法、电化学发光法等,无标记的方法涉及到表面等离子共振技术和石英微晶天平法等。本章还介绍了检测DNA中的放大技术,包括核酸体外扩增和检测物信号放大技术两方面,其中基于酶反应、纳米粒子和微磁球的信号放大技术进行了着重介绍。
第二章我们研究了两种以亚微米磁球(MB)为载体标记Ru(bpy)32+-NHS的超灵敏检测人乳腺癌细胞中beta-2-microglobulin (p2M) mRNA的电化学发光(ECL)法。第一种方法是用ECL光谱法。这种方法的方案是：修饰有生物素化的捕捉DNA(B-DNAc)的基底通过杂交反应依次连接上目标DAN(t-DNA)、生物素化的探针DNA (B-DNAp)和链霉亲和素化的磁球(SA-MBs)。然后通过去杂交反应将SA-MBs从基底上释放下来,并标记上Ru(bpy)32+-NHS。标记了Ru(bpy)32+-NHS的MBs固定在金电极上后,利用多通道光学分析仪采集Ru(bpy)32+-NHS的电化学发光光谱图。通过该方法我们测得人乳腺癌细胞中β2M mRNA的检测限达到1.2×10-15mol/L。第二种方法是以碳纳米管(CNTs)作为辅助电极材料包裹标记了Ru(bpy)32+-NHS的MBs,再用ECL方法检测细胞中基因的表达水平,∞2M mRNA的检测限可达3×10-16mol/L。
第三章我们研究了一种新的荧光检测痕量DNA的信号放大方法—磁球表面DNA杂交-去杂交信号放大方法。该方法首先将修饰了B-DNAp的SA-MBs (DNAp-MBs)以1：1的比例连接到基底上的t-DNA上。通过DNA之间去杂交方式将DNAp-MBs释放下来,然后与标记了Cy5的检测DNA (Cy5-DNAd)进行杂交。通过DNAp-MBs和Cy5-DNAd之间去杂交反应,收集释放得到的Cy5-DNAd。而分离得到的DNAA-MBs可以再次与其他的Cy5-DNAd进行杂交-去杂交反应。经多次杂交-去杂交循环过程后可以收集大量Cy5-DNAd。最终,将所有收集到的Cy5-DNAd进行荧光检测。经11次循环放大后t-DNA的检测限为8.5×10-15mol/L。我们将该超灵敏方法用于检测人乳腺癌细胞中RPLP2mRNA的检测。
第四章研究了一种新颖的基于多色编码微珠单分子检测多种DNA的技术,通过同时检测三种DNA及细胞中的多基因表达证明了该技术的可行性。该方法通过精确控制纳米磁球上标记了三种染料(Cy5,FAM和AMCA) DNA的比例构建多色编码纳米球。将不同的颜色编码纳米球标记到不同的t-DNA上后,以普通荧光显微镜摄取多种t-DNA的单分子图象。通过统计t-DNA的数量以同时定量多种t-DNA.我们将该单分子检测技术应用于检测～100细胞中三种基因的表达。
DNA is the genetic material and one of the indispensable biological macromolecules in the phenomenon of life. Detecting mutant genes associated with disease has significant meaning for gene screening, early diagnosis and treatment. Therefore, DNA detection methods become more and more important. This thesis developed several novel methods of DNA detection with high sensitivity and selectivity using magnetic beads as the carrier to amplify signal. This technique was successfully applied to gene expression in the cell. The content as follows:
In chapter one, we reviewed DNA detection methods with and without labels, and signal amplification techniques including amplification of taget DNA and detected substances briefly.
In chapter two, we reported ultrasensitive ECL methods for determination of mRNA in cells using streptavidin-coated magnetic nanobeads (SA-MBs) as the carrier of Ru(bpy)32+-NHS to amplify signal. In this chapter, we used ECL spectrometry to measure gene expression level in cells by using an optical multi-channel analyzer. Using this method, not only the limit of detection for DNA determination was as low as1.2×10-15mol/L, but also the ECL spectrum of Ru(bpy)32+-NHS on the surface of the SA-MBs was obtained. Additionally, we used carbon nanotubes (CNTs) to wrap the Ru(bpy)32+-loaded MBs on the electrode. In this case, the ECL of Ru(bpy)32+was greatly increased. Using this method, DNA of3x10-6mol/L could be detected and mRNA in cells could be quantified.
In chapter three, a new signal amplification strategy based on DNA hybridization-dehybridization reaction on the surface of MBs for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MBs modified with probe DNA (DNAP-MBs) were bound to t-DNA (with a ratio of1:1) captured to a substrate. The DNAp-MBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MBs were then hybridized with other Cy5-DNAd to initiate the next hybridization-dehybridization round. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was8.5×10-15mol/L for11cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2(RPLP2) mRNA in human breast cancer cells.
In chapter four, a novel multiplexed optical coding technique for single-molecule detection (SMD) of DNAs was developed using300-nm-diameter MBs. The encoded MBs were fabricated by hybridizing three kinds of coding DNAs labeled with different dyes (Cy5, FAM and AMCA), respectively, at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated MBs. Different colors from the encoded MBs could be obtained by overlapping three single-primary-color fluorescence images of the MBs corresponding to emission of Cy5(red), FAM (green) and AMCA (blue). Intraccllular multi-gene expression analysis demonstrated the DNA coding technique for SMD.
第一章主要对DNA的检测方法及检测中放大技术进行了简要的综述。DNA检测方法可分为标记型与无标记型,标记型方法主要有荧光分析法、电化学方法、化学发光法、电化学发光法等,无标记的方法涉及到表面等离子共振技术和石英微晶天平法等。本章还介绍了检测DNA中的放大技术,包括核酸体外扩增和检测物信号放大技术两方面,其中基于酶反应、纳米粒子和微磁球的信号放大技术进行了着重介绍。
第二章我们研究了两种以亚微米磁球(MB)为载体标记Ru(bpy)32+-NHS的超灵敏检测人乳腺癌细胞中beta-2-microglobulin (p2M) mRNA的电化学发光(ECL)法。第一种方法是用ECL光谱法。这种方法的方案是：修饰有生物素化的捕捉DNA(B-DNAc)的基底通过杂交反应依次连接上目标DAN(t-DNA)、生物素化的探针DNA (B-DNAp)和链霉亲和素化的磁球(SA-MBs)。然后通过去杂交反应将SA-MBs从基底上释放下来,并标记上Ru(bpy)32+-NHS。标记了Ru(bpy)32+-NHS的MBs固定在金电极上后,利用多通道光学分析仪采集Ru(bpy)32+-NHS的电化学发光光谱图。通过该方法我们测得人乳腺癌细胞中β2M mRNA的检测限达到1.2×10-15mol/L。第二种方法是以碳纳米管(CNTs)作为辅助电极材料包裹标记了Ru(bpy)32+-NHS的MBs,再用ECL方法检测细胞中基因的表达水平,∞2M mRNA的检测限可达3×10-16mol/L。
第三章我们研究了一种新的荧光检测痕量DNA的信号放大方法—磁球表面DNA杂交-去杂交信号放大方法。该方法首先将修饰了B-DNAp的SA-MBs (DNAp-MBs)以1：1的比例连接到基底上的t-DNA上。通过DNA之间去杂交方式将DNAp-MBs释放下来,然后与标记了Cy5的检测DNA (Cy5-DNAd)进行杂交。通过DNAp-MBs和Cy5-DNAd之间去杂交反应,收集释放得到的Cy5-DNAd。而分离得到的DNAA-MBs可以再次与其他的Cy5-DNAd进行杂交-去杂交反应。经多次杂交-去杂交循环过程后可以收集大量Cy5-DNAd。最终,将所有收集到的Cy5-DNAd进行荧光检测。经11次循环放大后t-DNA的检测限为8.5×10-15mol/L。我们将该超灵敏方法用于检测人乳腺癌细胞中RPLP2mRNA的检测。
第四章研究了一种新颖的基于多色编码微珠单分子检测多种DNA的技术,通过同时检测三种DNA及细胞中的多基因表达证明了该技术的可行性。该方法通过精确控制纳米磁球上标记了三种染料(Cy5,FAM和AMCA) DNA的比例构建多色编码纳米球。将不同的颜色编码纳米球标记到不同的t-DNA上后,以普通荧光显微镜摄取多种t-DNA的单分子图象。通过统计t-DNA的数量以同时定量多种t-DNA.我们将该单分子检测技术应用于检测～100细胞中三种基因的表达。
DNA is the genetic material and one of the indispensable biological macromolecules in the phenomenon of life. Detecting mutant genes associated with disease has significant meaning for gene screening, early diagnosis and treatment. Therefore, DNA detection methods become more and more important. This thesis developed several novel methods of DNA detection with high sensitivity and selectivity using magnetic beads as the carrier to amplify signal. This technique was successfully applied to gene expression in the cell. The content as follows:
In chapter one, we reviewed DNA detection methods with and without labels, and signal amplification techniques including amplification of taget DNA and detected substances briefly.
In chapter two, we reported ultrasensitive ECL methods for determination of mRNA in cells using streptavidin-coated magnetic nanobeads (SA-MBs) as the carrier of Ru(bpy)32+-NHS to amplify signal. In this chapter, we used ECL spectrometry to measure gene expression level in cells by using an optical multi-channel analyzer. Using this method, not only the limit of detection for DNA determination was as low as1.2×10-15mol/L, but also the ECL spectrum of Ru(bpy)32+-NHS on the surface of the SA-MBs was obtained. Additionally, we used carbon nanotubes (CNTs) to wrap the Ru(bpy)32+-loaded MBs on the electrode. In this case, the ECL of Ru(bpy)32+was greatly increased. Using this method, DNA of3x10-6mol/L could be detected and mRNA in cells could be quantified.
In chapter three, a new signal amplification strategy based on DNA hybridization-dehybridization reaction on the surface of MBs for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MBs modified with probe DNA (DNAP-MBs) were bound to t-DNA (with a ratio of1:1) captured to a substrate. The DNAp-MBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MBs were then hybridized with other Cy5-DNAd to initiate the next hybridization-dehybridization round. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was8.5×10-15mol/L for11cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2(RPLP2) mRNA in human breast cancer cells.
In chapter four, a novel multiplexed optical coding technique for single-molecule detection (SMD) of DNAs was developed using300-nm-diameter MBs. The encoded MBs were fabricated by hybridizing three kinds of coding DNAs labeled with different dyes (Cy5, FAM and AMCA), respectively, at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated MBs. Different colors from the encoded MBs could be obtained by overlapping three single-primary-color fluorescence images of the MBs corresponding to emission of Cy5(red), FAM (green) and AMCA (blue). Intraccllular multi-gene expression analysis demonstrated the DNA coding technique for SMD.
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