基于滚环DNA扩增和纳米金的生物传感技术研究
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摘要
近年来新的分子生物学技术不断涌现，并在科学研究和实际应用中得到不断的发展和完善，使生物分子检测的灵敏度和特异性都得到很大的提高。但随着疾病诊断水平的不断提高和科学研究的不断深入，对生物分子检测方法的要求也越来越高。因此，我们迫切需要开发一些高灵敏性、高特异性、高通量性和高准确性的定性或定量方法，以便更好地满足研究和实际应用的需要。针对上述问题，本论文在蛋白质和核酸的分析和检测方面发展了一系列新的高灵敏性的生物传感技术，并通过分析实际样品以及对照经典的检测方法，初步验证了这些技术的可行性、可靠性及准确性。
（1）核酸适体，是通过体外筛选技术从寡核苷酸库中随机筛选出的一段短的与特定的目标分子具有高亲和力和高特异性的寡核苷酸探针。在第2章，我们利用核酸适体可与抗体相媲美的特点，基于滚环DNA扩增技术（RCA），构建了一种新型的高灵敏性的检测蛋白质的电化学适体传感器，并实现了对模型蛋白—血小板源生长因子B链（PDGF-BB）的高灵敏性和特异性检测。该方法首先采用巯基乙胺自组装技术和戊二醛交联技术将PDGF-BB抗体固定在金电极表面，然后通过抗体-抗原-核酸适体免疫夹心反应，将带有RCA引物的PDGF-BB核酸适体连接在金电极上。该适体-引物探针在连接反应组分和滚环扩增反应组分中对环形DNA模板进行滚环放大反应，接着利用生物素标记的探针捕获扩增产物，再通过生物素-亲和素特异性结合将碱性磷酸酶标记的链霉亲和素捕捉在金电极上，结合酶的生物催化银沉积反应放大分析信号，实现对人血清中PDGF-BB的检测，动态响应范围宽达4个数量级，从10 fM到100 pM，检测下限可达10 fM，比文献所报道的高3个数量级。
（2）上章构建的核酸适体-RCA电化学免疫传感器实现了对模型蛋白PDGF-BB的高灵敏性检测，但是到目前为止，已经筛选出的核酸适体种类有限，不能满足大量蛋白质检测的需要，且核酸适体在蛋白质表面的非特异性吸附会产生一定的背景干扰。第3章中，我们在此实验的基础上，利用脂质体包埋DNA技术和滚环扩增技术，构建了一种超灵敏性的免疫分析方法用于前列腺特异性抗原（PSA）的检测。首先将PSA抗体吸附在微孔板上，免疫夹心反应后，将表面包被了PSA抗体，里面包埋了RCA引物的脂质体固定在微孔板上，然后溶解脂质体，释放其内包埋的RCA引物。接着加入滚环扩增反应组分进行RCA反应，随后加入生物素标记的探针和荧光素标记的探针与扩增产物杂交，再用亲和素标记的微珠捕获反应产物，最后检测荧光信号。由于该方法结合了脂质体包埋引物探针和RCA两步放大反应，分析的灵敏度得到了显著的提高，响应动态范围从0.1 fg/mL到0.1ng/mL，检测下限为0.08 fg/mL。
（3）第4章中，鉴于RCA技术的高灵敏度，结合核酸外切酶I（Exo I）末端保护技术，发展了一种高灵敏性的检测模型分析物—叶酸结合蛋白的光学生物传感技术。该方法利用叶酸与叶酸结合蛋白的特异性结合，保护叶酸标记的单链DNA不被Exo I水解，作为后续RCA反应的成环模板。当DNA链完整存在时，引发滚环DNA扩增反应，随后扩增产物与Taqman探针杂交，在核酸外切酶III（Exo III）作用下引发酶切循环放大反应，产生荧光信号。结果表明，该方法可实现对叶酸结合蛋白的高灵敏性检测，响应动态范围从1 pM到1 nM，检测下限为1 pM。
（4）第5章中，我们在上章末端保护技术的基础上，基于核酸外切酶III末端保护技术和纳米金凝聚变色效应，提出了一种新型、快速、简便、灵敏的比色传感策略用于序列特异性DNA结合蛋白的检测。首先是包含有目标结合蛋白的结合序列的互补金标探针杂交形成纳米金团聚体，溶液呈紫色。目标结合蛋白存在时，与其特定结合序列的特异性结合，保护纳米金团聚体不被Exo III水解，溶液保持紫色不变。当目标结合蛋白不存在时，Exo III水解DNA双链，释放纳米金团聚体，纳米金呈单颗粒分散状态，溶液变红色。结果表明，序列特异性DNA结合蛋白在0~120 nM范围内呈良好的线性关系，检测下限为10 nM。
（5）第6章中，建立了一种基于纳米金凝聚变色效应的检测T4多聚核苷酸激酶（T4 PNK）活性的新方法。鉴于寡核苷酸链标记的纳米金能够稳定存在于一定浓度的盐离子缓冲溶液中，利用5′羟基修饰的分子信标作为金标探针，有T4 PNK存在时，金标探针5′羟基磷酸化，λ核酸外切酶被激活，酶切分子信标茎部双链DNA片段，接着在RecJ核酸外切酶作用下降解纳米金上修饰的残余的单链DNA，使得纳米金在一定盐离子浓度下团聚，溶液变成蓝紫色。结果表明，T4 PNK激酶的检
测的线性响应范围为0~4 U/mL，检测下限为0.24 U/mL。
（6）第7章中，提出了一种基于胞嘧啶-银离子-胞嘧啶特定结构（C-Ag~+-C）的非标记纳米金比色传感技术用于检测银离子（Ag~+）。无Ag~+存在时，富C链吸附在纳米金表面，增加了纳米金表面的负电荷，从而增加了它们的排斥力，在一定盐离子浓度下保护纳米金不团聚，溶液呈红色。当Ag~+存在时，富C链通过C-Ag~+-C配对形成准双链结构，不能吸附在纳米金表面，没有DNA链保护的纳米金颗粒在一定盐离子浓度下发生团聚，颜色由红色变成蓝色。结果表明，Ag~+浓度在0.5~6μM浓度范围内呈良好的线性关系，检测下限为0.1μM。同时，该方法还具有高的选择性和特异性，且简单、快速、成本低、便于普及。
In recent years, a seris of new biology techniques have been developed as a powerful platform for sensitively detection of proteins and DNA. However, with the development of the scientific research, greater sensitivity and specificity techniques are required. Therefore, the development of the high sensitivity, selectivity and accuracy strategies, as well as proved a high performance platform for proteins and DNA assays are of paramount importance for biomedical research and clinical diagnosis. In this thesis, a series of novel biosensing strategies were developed for ultrasensitive detection of proteins and DNA. The proposed methods were implemented in the analysis of realistic biological samples and were in good agreement with the classical methods. These results primarily proved that the proposed technology was feasible, reliable and accurate. The detailed content was described as follows:
(1) Aptamers are nucleic acids (DNA or RNA) that selectively bind to low-molecular-weight organic or inorganic substrates or to macromolecules such as proteins. In chapter 2, based on rolling circle amplification, a novel versatile electrochemical aptasensor was developed for the ultrasensitive detection of model analyte PDGF-BB. First, PDGF-BB antibody was immobilized on the electrode surface via the SAM of cysteamine and the bifunctional linker of glutaraldehyde and then used to capture the protein target. The surface-captured protein was then sandwiched by an aptamer-primer complex. The aptamer-primer sequence mediated an in situ RCA reaction that generated hundreds of copies of a circular DNA template. Detection of the amplified copies via enzymatic silver deposition then allowed enormous sensitivity enhancement in the assay of target protein. In conclusion, the presented method exhibited a linear correlation to target concentration through a 4-decade range of 10 fM~100 pM, and a detection limit as low as 10 fM. The wide dynamic range of the presented sensor was about 3 orders of magnitude larger than those shown previously.
(2) We have reported an aptamer-based immuno-RCA assay for protein detection in chapter 2. However, aptamers for most important protein biomarkers are currently unavailable, and like the DNA-antibody conjugates, aptamers are also exposed to the risk of electrostatic adsorption on proteins as well as chemical or enzymatic degradation. In chapter 3, we reported for the first time a DNA encapsulating liposome based RCA immunoassay, liposome-RCA immunoassay, as an alternative strategy. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg/mL to 0.1 ng/mL with a limit of detection as low as 0.08 fg/mL and a high dose-response sensitivity.
(3) In chapter 4, based on rolling circle amplification and terminal protection assay, a novel photochemical biosensing strategy was developed for the ultrasensitive detection of model analyte folate receptor (FR). This assay was based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule could be protected from the degradation by exonuclease I (Exo I) when the small molecule moiety was bound to its protein target. The small-molecule-linked protected DNA was served as a template of the ligation probe to trigger the rolling circle amplification. And then generated a long tandem repeated sequences that could be selectively and sensitively detected by an exonuclease III-aided oligonucleotide recycling assay. This strategy was demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor. And quasilinear correlation was obtained in the concentration range from 1 pM to 1 nM with a readily achieved detection limit of 1 pM.
(4) In chapter 5, a novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNAbinding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. In the absence of the DNA-binding protein, Exo III will stepwisely and nonprocessively digested the double-stranded DNA. This caused the dissociation of the AuNP aggregates into dispersed AuNPs with a concomitant purple-to-red color change for the solution. In the presence of the DNA-binding protein, tight binding of the proteins to the consensus sequences induced steric hindrance to Exo III in approaching the 3′-termini, which protected the antisense strands from Exo III-mediated digestion. This retained stable AuNP aggregates in the solution with no color change obtained. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM.
(5) In chapter 6, we described a novel colorimetric assay method for detecting of the activity and kinetics of T4 polynucleotide kinase (PNK) by using of molecular beacons-modified gold nanoparticles (AuNPs) coupled withλexonuclease (λexo) and recj exonuclease (recj exo) cleavage. The assay was performed at salt concentrations so that DNA-modified AuNPs were barely stabilized by the electrostatic and steric stabilization. The 5′-hydroxyl group of the molecular beacons modified AuNPs was first phosphorylated in the presence of T4 PNK, then initiatedλexo and recj exo cleavage. Enzymatic cleavage of DNA chains on the AuNP surface destabilized the AuNPs, resulting in a rapid aggregation and a red-to-purple color change. In conclusion, the presented method exhibited a linear correlation to target concentration with a linear range of 0~4 U/mL and a detection limit of 0.24 U/mL.
(6) In chapter 6, a rapid, simple colorimetric sensor with unmodified AuNPs for highly sensitive and selective detection of silver ion was developed. It was based on the specific recognition property of Ag~+ with a cytosine-cytosine mismatched base pair. In the absence of Ag~+, single strand oligonucleotide adsorbed on the surface of AuNPs and protected them from aggregation with the addition of high concentration salt. In contrast, the presence of Ag~+ drived the formation of C-Ag~+-C duplex structure and enabled the oligonucleotide to be desorbed from the surface of the AuNPs, resulting in the aggregation of AuNPs. The results revealed that the method allowed a specific, simple, and rapid assay of Ag~+ with a linear response range from 0.5μM to 6μM and a detection limit of 0.1μM.
（1）核酸适体，是通过体外筛选技术从寡核苷酸库中随机筛选出的一段短的与特定的目标分子具有高亲和力和高特异性的寡核苷酸探针。在第2章，我们利用核酸适体可与抗体相媲美的特点，基于滚环DNA扩增技术（RCA），构建了一种新型的高灵敏性的检测蛋白质的电化学适体传感器，并实现了对模型蛋白—血小板源生长因子B链（PDGF-BB）的高灵敏性和特异性检测。该方法首先采用巯基乙胺自组装技术和戊二醛交联技术将PDGF-BB抗体固定在金电极表面，然后通过抗体-抗原-核酸适体免疫夹心反应，将带有RCA引物的PDGF-BB核酸适体连接在金电极上。该适体-引物探针在连接反应组分和滚环扩增反应组分中对环形DNA模板进行滚环放大反应，接着利用生物素标记的探针捕获扩增产物，再通过生物素-亲和素特异性结合将碱性磷酸酶标记的链霉亲和素捕捉在金电极上，结合酶的生物催化银沉积反应放大分析信号，实现对人血清中PDGF-BB的检测，动态响应范围宽达4个数量级，从10 fM到100 pM，检测下限可达10 fM，比文献所报道的高3个数量级。
（2）上章构建的核酸适体-RCA电化学免疫传感器实现了对模型蛋白PDGF-BB的高灵敏性检测，但是到目前为止，已经筛选出的核酸适体种类有限，不能满足大量蛋白质检测的需要，且核酸适体在蛋白质表面的非特异性吸附会产生一定的背景干扰。第3章中，我们在此实验的基础上，利用脂质体包埋DNA技术和滚环扩增技术，构建了一种超灵敏性的免疫分析方法用于前列腺特异性抗原（PSA）的检测。首先将PSA抗体吸附在微孔板上，免疫夹心反应后，将表面包被了PSA抗体，里面包埋了RCA引物的脂质体固定在微孔板上，然后溶解脂质体，释放其内包埋的RCA引物。接着加入滚环扩增反应组分进行RCA反应，随后加入生物素标记的探针和荧光素标记的探针与扩增产物杂交，再用亲和素标记的微珠捕获反应产物，最后检测荧光信号。由于该方法结合了脂质体包埋引物探针和RCA两步放大反应，分析的灵敏度得到了显著的提高，响应动态范围从0.1 fg/mL到0.1ng/mL，检测下限为0.08 fg/mL。
（3）第4章中，鉴于RCA技术的高灵敏度，结合核酸外切酶I（Exo I）末端保护技术，发展了一种高灵敏性的检测模型分析物—叶酸结合蛋白的光学生物传感技术。该方法利用叶酸与叶酸结合蛋白的特异性结合，保护叶酸标记的单链DNA不被Exo I水解，作为后续RCA反应的成环模板。当DNA链完整存在时，引发滚环DNA扩增反应，随后扩增产物与Taqman探针杂交，在核酸外切酶III（Exo III）作用下引发酶切循环放大反应，产生荧光信号。结果表明，该方法可实现对叶酸结合蛋白的高灵敏性检测，响应动态范围从1 pM到1 nM，检测下限为1 pM。
（4）第5章中，我们在上章末端保护技术的基础上，基于核酸外切酶III末端保护技术和纳米金凝聚变色效应，提出了一种新型、快速、简便、灵敏的比色传感策略用于序列特异性DNA结合蛋白的检测。首先是包含有目标结合蛋白的结合序列的互补金标探针杂交形成纳米金团聚体，溶液呈紫色。目标结合蛋白存在时，与其特定结合序列的特异性结合，保护纳米金团聚体不被Exo III水解，溶液保持紫色不变。当目标结合蛋白不存在时，Exo III水解DNA双链，释放纳米金团聚体，纳米金呈单颗粒分散状态，溶液变红色。结果表明，序列特异性DNA结合蛋白在0~120 nM范围内呈良好的线性关系，检测下限为10 nM。
（5）第6章中，建立了一种基于纳米金凝聚变色效应的检测T4多聚核苷酸激酶（T4 PNK）活性的新方法。鉴于寡核苷酸链标记的纳米金能够稳定存在于一定浓度的盐离子缓冲溶液中，利用5′羟基修饰的分子信标作为金标探针，有T4 PNK存在时，金标探针5′羟基磷酸化，λ核酸外切酶被激活，酶切分子信标茎部双链DNA片段，接着在RecJ核酸外切酶作用下降解纳米金上修饰的残余的单链DNA，使得纳米金在一定盐离子浓度下团聚，溶液变成蓝紫色。结果表明，T4 PNK激酶的检
测的线性响应范围为0~4 U/mL，检测下限为0.24 U/mL。
（6）第7章中，提出了一种基于胞嘧啶-银离子-胞嘧啶特定结构（C-Ag~+-C）的非标记纳米金比色传感技术用于检测银离子（Ag~+）。无Ag~+存在时，富C链吸附在纳米金表面，增加了纳米金表面的负电荷，从而增加了它们的排斥力，在一定盐离子浓度下保护纳米金不团聚，溶液呈红色。当Ag~+存在时，富C链通过C-Ag~+-C配对形成准双链结构，不能吸附在纳米金表面，没有DNA链保护的纳米金颗粒在一定盐离子浓度下发生团聚，颜色由红色变成蓝色。结果表明，Ag~+浓度在0.5~6μM浓度范围内呈良好的线性关系，检测下限为0.1μM。同时，该方法还具有高的选择性和特异性，且简单、快速、成本低、便于普及。
In recent years, a seris of new biology techniques have been developed as a powerful platform for sensitively detection of proteins and DNA. However, with the development of the scientific research, greater sensitivity and specificity techniques are required. Therefore, the development of the high sensitivity, selectivity and accuracy strategies, as well as proved a high performance platform for proteins and DNA assays are of paramount importance for biomedical research and clinical diagnosis. In this thesis, a series of novel biosensing strategies were developed for ultrasensitive detection of proteins and DNA. The proposed methods were implemented in the analysis of realistic biological samples and were in good agreement with the classical methods. These results primarily proved that the proposed technology was feasible, reliable and accurate. The detailed content was described as follows:
(1) Aptamers are nucleic acids (DNA or RNA) that selectively bind to low-molecular-weight organic or inorganic substrates or to macromolecules such as proteins. In chapter 2, based on rolling circle amplification, a novel versatile electrochemical aptasensor was developed for the ultrasensitive detection of model analyte PDGF-BB. First, PDGF-BB antibody was immobilized on the electrode surface via the SAM of cysteamine and the bifunctional linker of glutaraldehyde and then used to capture the protein target. The surface-captured protein was then sandwiched by an aptamer-primer complex. The aptamer-primer sequence mediated an in situ RCA reaction that generated hundreds of copies of a circular DNA template. Detection of the amplified copies via enzymatic silver deposition then allowed enormous sensitivity enhancement in the assay of target protein. In conclusion, the presented method exhibited a linear correlation to target concentration through a 4-decade range of 10 fM~100 pM, and a detection limit as low as 10 fM. The wide dynamic range of the presented sensor was about 3 orders of magnitude larger than those shown previously.
(2) We have reported an aptamer-based immuno-RCA assay for protein detection in chapter 2. However, aptamers for most important protein biomarkers are currently unavailable, and like the DNA-antibody conjugates, aptamers are also exposed to the risk of electrostatic adsorption on proteins as well as chemical or enzymatic degradation. In chapter 3, we reported for the first time a DNA encapsulating liposome based RCA immunoassay, liposome-RCA immunoassay, as an alternative strategy. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg/mL to 0.1 ng/mL with a limit of detection as low as 0.08 fg/mL and a high dose-response sensitivity.
(3) In chapter 4, based on rolling circle amplification and terminal protection assay, a novel photochemical biosensing strategy was developed for the ultrasensitive detection of model analyte folate receptor (FR). This assay was based on our new finding that single-stranded DNA (ssDNA) terminally tethered to a small molecule could be protected from the degradation by exonuclease I (Exo I) when the small molecule moiety was bound to its protein target. The small-molecule-linked protected DNA was served as a template of the ligation probe to trigger the rolling circle amplification. And then generated a long tandem repeated sequences that could be selectively and sensitively detected by an exonuclease III-aided oligonucleotide recycling assay. This strategy was demonstrated for quantitative analysis of the interaction of folate with a tumor biomarker of folate receptor. And quasilinear correlation was obtained in the concentration range from 1 pM to 1 nM with a readily achieved detection limit of 1 pM.
(4) In chapter 5, a novel exonuclease III (Exo III) protection-based colorimetric biosensing strategy was developed for rapid, sensitive, and visual detection of sequence-specific DNAbinding proteins. This strategy relied on the protection of DNA-cross-linked gold nanoparticle (AuNP) aggregates from Exo III-mediated digestion by specific interactions of target proteins with their binding sequences. In the absence of the DNA-binding protein, Exo III will stepwisely and nonprocessively digested the double-stranded DNA. This caused the dissociation of the AuNP aggregates into dispersed AuNPs with a concomitant purple-to-red color change for the solution. In the presence of the DNA-binding protein, tight binding of the proteins to the consensus sequences induced steric hindrance to Exo III in approaching the 3′-termini, which protected the antisense strands from Exo III-mediated digestion. This retained stable AuNP aggregates in the solution with no color change obtained. The results revealed that the method allowed a specific, simple, and quantitative assay of the target protein with a linear response range from 0 to 120 nM and a detection limit of 10 nM.
(5) In chapter 6, we described a novel colorimetric assay method for detecting of the activity and kinetics of T4 polynucleotide kinase (PNK) by using of molecular beacons-modified gold nanoparticles (AuNPs) coupled withλexonuclease (λexo) and recj exonuclease (recj exo) cleavage. The assay was performed at salt concentrations so that DNA-modified AuNPs were barely stabilized by the electrostatic and steric stabilization. The 5′-hydroxyl group of the molecular beacons modified AuNPs was first phosphorylated in the presence of T4 PNK, then initiatedλexo and recj exo cleavage. Enzymatic cleavage of DNA chains on the AuNP surface destabilized the AuNPs, resulting in a rapid aggregation and a red-to-purple color change. In conclusion, the presented method exhibited a linear correlation to target concentration with a linear range of 0~4 U/mL and a detection limit of 0.24 U/mL.
(6) In chapter 6, a rapid, simple colorimetric sensor with unmodified AuNPs for highly sensitive and selective detection of silver ion was developed. It was based on the specific recognition property of Ag~+ with a cytosine-cytosine mismatched base pair. In the absence of Ag~+, single strand oligonucleotide adsorbed on the surface of AuNPs and protected them from aggregation with the addition of high concentration salt. In contrast, the presence of Ag~+ drived the formation of C-Ag~+-C duplex structure and enabled the oligonucleotide to be desorbed from the surface of the AuNPs, resulting in the aggregation of AuNPs. The results revealed that the method allowed a specific, simple, and rapid assay of Ag~+ with a linear response range from 0.5μM to 6μM and a detection limit of 0.1μM.
引文
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