基于氧化石墨烯的荧光生物传感新方法研究
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摘要
生物分子的快速灵敏检测一直以来都是生物医学和分析化学领域十分关注的课题。现代社会的快速发展对生物传感技术提出了更高的要求，发展新的分析方法和技术以应对越来越多的分析难题和社会热点问题为分析化学工作者提出了严峻的挑战。荧光生物传感技术具有灵敏度高和响应速度快等优点，被广泛应用于各种生物分子的检测，包括蛋白质、生物小分子、核酸和酶活性等。传统的荧光生物传感技术往往需要多重荧光标记和一系列的放大方法来提高灵敏度，从而增加了检测的成本和实验的复杂性。另外，传统的荧光传感技术还存在较高背景荧光的干扰。因此进一步改进和发展新的荧光传感技术，以提高生物分析的性能和降低检测的成本有十分重要的意义。
氧化石墨烯(GO)由于其独特的DNA吸附性能和通用型荧光淬灭剂的性质，被广泛应用于生物分子的检测。基于GO的荧光传感器与传统荧光传感器相比有诸多优势，例如改进了检测灵敏度、降低了检测成本和增大了信背比等，这表明基于GO的荧光传感器在生物分子检测方面有很大的应用前景。这类传感器大多依赖GO能强烈吸附荧光染料标记的单链DNA同时淬灭其荧光，目标物与荧光单链DNA结合使其构象转变，进而使荧光染料与GO距离拉大，荧光恢复，从而实现对目标物的检测。但是这类传感器的检测限一般有限且需要荧光标记，有待进一步改进。将DNA嵌入染料与GO相结合进行生物分子检测已有报道，其检测性能显著改善，我们推断此类非标荧光传感器有望在生物分子检测中得到广泛应用。
本论文针对以上问题，在综合文献报道的基础之上，基于GO独特的DNA吸附和荧光染料淬灭性能发展了一系列简单、快速、灵敏的荧光生物传感新方法用于DNA酶活性、核酸和三磷酸腺苷(ATP)等生物分子的检测，具体内容如下：
第2章中，基于发夹引物和聚合延伸，结合GO独特的DNA吸附和通用型荧光淬灭性能发展了一种新型非标荧光检测方法用于DNA3'磷酸酶的检测及其抑制剂的定量表征。本实验设计了一条3'磷酸化的DNA发夹引物，只有经DNA3'磷酸酶的去磷酸作用后，发夹引物才能发生聚合延伸生成双链DNA产物。由于GO与双链DNA的结合力较弱不能有效淬灭染色双链产物的荧光，因此得到较强的荧光信号。无DNA3'磷酸酶时，发夹引物不能去磷酸化引发聚合延伸，进而被GO吸附淬灭，得到较弱的背景信号，从而实现对目标DNA3'磷酸酶的灵敏检测。该方法可实现对两种DNA3'磷酸酶(T4多聚核苷酸激酶和小虾碱性磷酸酶)活性的同时检测，检测限分别为0.07U/mL和0.003U/mL。另外，该方法不仅可以对DNA3'磷酸酶的抑制剂进行定量表征，还适用于复杂生物环境中目标物的测定。本方法设计简单、操作方便、灵敏度高且选择性好，有望成为DNA3'磷酸酶检测的一种很好的选择并能应用于DNA损伤修复机理的研究。
第3章中，基于等温循环链置换聚合反应(ICSDPR)和DNA嵌入染料的光学特性，结合GO独特的吸附和淬灭性能发展了一种非标放大荧光DNA检测方法。实验设计了一条3'磷酸化的发夹探针，只有加入目标DNA与发夹杂交引发其构象转变，引物才能与发夹打开的茎端杂交，然后在聚合酶作用下发生ICSDPR。经过多步的循环放大，产生大量的双链DNA产物，进而得到放大的荧光信号。无目标DNA时，发夹探针不能发生构象转变和ICSDPR，经SYBR Green I(SG)染色和GO吸附后，生成较低的背景信号，从而实现对目标DNA的放大荧光检测。该方法首次提出在GO平台上结合SG和ICSDPR放大检测DNA，有较宽的线性范围和高选择性，检测限为4pM，跟基于GO的均相DNA检测相比有明显优势。该方法设计简单、方便经济，有望成为DNA灵敏检测的一种常用方法。
第4章中，基于ATP依赖的DNA连接反应和GO独特的吸附和淬灭性能，发展了一种高灵敏的ATP检测新方法。实验设计的三种DNA探针退火杂交形成带有单链缺口的荧光双链DNA，在辅助因子ATP和T4DNA连接酶同时存在下，缺口被连接形成完整的双链DNA。连接产物有较高的熔链温度能在实验加热条件下保持双链结构，不被GO有效淬灭，得到较强的荧光信号。无ATP时，DNA连接反应不能发生，三链退火的复合物在实验加热条件下解链进而被GO吸附，荧光淬灭，从而实现对目标ATP的灵敏检测。该方法简单快速、灵敏度高选择性好，检测限达0.3nM，跟大多数无放大的均相ATP适配体传感方法相比有明显优势，跟电化学适配体方法的检测限有可比性。该方法具有高选择性，不仅能使ATP从其它三磷酸核苷中分离开来，还可使ATP与其同系物区分开来。
第5章中，基于核酸外切酶Ⅲ(ExoⅢ)对酶切底物的选择性和GO对单双链DNA结合力的差别发展了一种非标荧光ATP检测方法。实验设计的5'磷酸化的发夹探针能跟互补DNA退火杂交形成带单链缺口的双链DNA产物。在辅助因子ATP和连接酶同时存在时，缺口被连接形成完整的长茎端发夹产物，且靠近3'端的碱基硫代处理。这种发夹产物不能被ExoⅢ酶切，经SG染色和GO混合后，得到显著的荧光信号。无ATP时，缺口不能被连接进而被ExoⅢ酶切，互补DNA被降解同时释放发夹探针，染色后的发夹被GO吸附淬灭，生成较低的背景信号，从而实现对目标ATP的灵敏检测。该方法设计巧妙新颖，有较高的灵敏度和选择性，检测限达0.2nM。
第6章中，基于限制性核酸内切酶酶切的特异性和GO对不同的DNA结构结合力大小的差别发展了一种新型荧光方法用于检测核酸内切酶的活性及其抑制剂。我们设计了一条单标记荧光发夹探针，发夹的茎端序列包含有内切酶的识别位点。当目标内切酶存在时，发夹探针被酶切成三部分，其中带荧光染料的DNA短片段与GO结合力较小而游离到溶液中，生成较强的荧光信号。无目标内切酶时，发夹荧光探针被GO吸附，发生荧光共振能量转移荧光淬灭，得到较弱的背景信号，从而实现对目标内切酶的灵敏检测。该方法设计简单、操作方便、经济快捷，为进一步研究蛋白质与DNA的相互作用机理提供一个新的选择，对内切酶抑制剂的定量表征又使其在新药研发方面有一定的应用潜力。
Rapid and sensitive detection of biomolecules has always been the researchinterest in biomedical and analytical chemistry. The rapid development of modernsociety imposes higher requirements on biosensing technology. Developing newanalytical techniques for solving more and more analytical problems and social crisishas proposed a great challenge for analysts. Fluorescent biosensing technology hasbeen widely applied in the detection of various biomolecules, including protein, smallmolecules, nucleic acids and enzyme activity, attributing to its analytical advantages ofhigh sensitivity and rapid response time. In order to improve the sensitivity, traditionalfluorescent biosensing technology usually requires multiple-fluorophore labeling anddifferent amplification techniques, which might add cost and complexity for the assay.
In addition, traditional fluorescent biosensing technology suffers from a relativelyhigh background signal. Therefore, it is of great importance to develop new fluorescentbiosensing techniques so as to enhance the bioanalytical performance and reduce thecost.
Due to its unique DNA absorbing ability and universal quenching properties forfluorophores, graphene oxide (GO) has been widely applied in biomolecular detection.GO-based fluorescent biosensors show several advantages compared with thetraditional ones, including improved assay sensitivity, low cost and enhanced signal tobackground ratio and so on. These advantages reveal that GO-based fluorescentbiosensors hold great potential in biomolecular detection. Most of these biosensorsrely on the fact that GO can adsorb the dye-labeled single-stranded DNA (ssDNA) andeffectively quench its fluorescence, the binding of analyte with dye-labeled ssDNAresults in the departure of the dye and GO, and the recovery of fluorescence which canbe used for target detection. However, these biosensors require fluorophore-labeledprobes and show relatively poor detection limits, which need further improvements.There has been report for biomolecular detection based on the combination of DNAinteracting dyes and GO, which shows an improved detection limit and it is expectedthat this kind of biosensor may find wide applications in biomolecular detection.
Focusing on the above questions and making use of the achievements of previousresearchers, this thesis developed several novel fluorescent biosensing methods for thedetection of DNA enzyme activity, DNA and adenosine triphosphate (ATP) based on the unique DNA adsorption and fluorescence quenching property of GO. The detailsare described in following chapters.
In Chapter2, we developed a novel label-free fluorescent strategy for thedetection of DNA3' phosphatase and its inhibitors based on hairpin primer andpolymerase elongation. Only in the presence of DNA3' phosphatase, DNA hairpinprimer with3'-phosphorylated end can be dephosphated, then polymerase elongationwas initiated to produce the double-stranded DNA (dsDNA). Because GO shows weakbinding affinity toward the SYBR Green I (SG) stained dsDNA that it cannoteffectively quench the fluorescence, thus one can obtain a strong fluorescence signal.Whereas, the hairpin primer would not be dephosphated in the absence of DNA3'phosphatase and its polymerase elongation would not occur. Adsorption of hairpinprimer by GO generates a rather weak background signal. This method can realize thesimultaneous detection of two kinds of DNA3' phosphatase (T4PNKP and SAP) withachieved detection limits of0.07U/mL and0.003U/mL, respectively. In addition, thismethod is able to quantitatively evaluate the inhibition effect of the inhibitorcompounds and also applicable for complex biological sample.
In Chapter3, a label-free sensitive fluorescent DNA biosensor was presentedbased on isothermal circular strand-displacement polymerization reaction (ICSDPR)combined with GO binding. This biosensor relies on the hybridization of target DNAwith hairpin probe as the prerequisite for ICSDPR and the preferential binding of GOto stained single stranded DNA (ssDNA) over double stranded one to improve thesignal to background ratio. By combining the properties of GO and SG with theICSDPR amplification, this fluorescent DNA biosensor displays a wide dynamic rangefrom0.01nM to10nM and a low detection limit of4pM. The proposed strategy issimple, cost-effective and sensitive, which might provide a promising method ofchoice for convenient DNA detection.
In Chapter4, a simple, amplification-free and sensitive fluorescent biosensor forATP detection was developed based on the ATP-dependent enzymatic reaction(ATP-DER) and GO binding. In this assay, two half DNA probes anneal with adye-labeled template to form a DNA duplex substrate with a single-stranded nick. Theformed nick could be sealed by T4DNA ligase in the presence of ATP, and theresulting unnicked DNA duplex was resistant to thermal denaturation. Whereas, theligation reaction would not happen in the absence of ATP, and the nicked DNA duplexwas subject to thermal denaturation, which leads to the separation of two half DNAprobes and template. After addition of GO, the fluorescence of the nicked DNA duplex was greatly quenched, whereas efficient fluorescence quenching did not occur to theunnicked DNA duplex and high fluorescence intensity was obtained. The proposedstrategy was simple, amplification-free and showed high selectivity to ATP that coulddistinguish ATP from its analogues. The results revealed that the method allowedsensitive quantitative assay of ATP with a wide linear response range from0.5nM to100nM and a low detection limit of0.3nM.
In Chapter5, we present a novel, label-free exonuclease Ⅲ-aided fluorescentassay strategy for ATP based on the ATP-dependent enzymatic reaction and GO. In thisassay, hairpin probe with5' phosphorylated end anneals with complementary DNA toform a dsDNA complex with a single-stranded nick, which can be ligated by T4DNAligase in the presence of its cofactor ATP, resulting in a hairpin product with a stem oflong dsDNA. Because the five phosphorothioate nucleosides near the3' end of hairpinprobe can hamper the Exo Ⅲ cleavage, thus the hairpin product after ligation couldkeep intact upon treatment with Exo Ⅲ. After addition of GO, a strong fluorescencesignal was observed. Whereas, the single-stranded nick cannot be ligated in theabsence of ATP, Exo Ⅲ cleaves the annealed dsDNA complex from the nick site anddigests the C-DNA, releasing the hairpin probe. GO selectively adsorb the hairpinprobe leading to efficient quenching of the fluorescence. This sensor displays animproved sensitivity and a wide linear range within the ATP concentration from1nMto200nM with a low detection limit of0.2nM. The proposed approach is simple,cost-effective and convenient, which might create a new methodology for developingsensitive ATP biosensor.
In Chapter6, we developed a simple and novel fluorescent strategy for thedetection of endonuclease activity and its inhibitors based on hairpin probe and GO. Inthis assay, we designed a single dye-labeled hairpin probe that contains the recognitionsite for the endonuclease in the stem. Upon treatment with the target endonuclease,hairpin probe was digested into three pieces. One of the digested products, thedye-labeled short DNA would not be absored by GO, leads to a strong fluorescencesignal. In the absence of endonuclease, hairpin probe kept intact and was immediatelyabsorbed by GO, resulting in an effective fluorescence quenching. In addition, thismethod was applicable for inhibitor quantification. Given the simplicity, convenienceof this approach, the proposed method may provide an alternative approach for thestudy of the interaction between protein and DNA.
氧化石墨烯(GO)由于其独特的DNA吸附性能和通用型荧光淬灭剂的性质，被广泛应用于生物分子的检测。基于GO的荧光传感器与传统荧光传感器相比有诸多优势，例如改进了检测灵敏度、降低了检测成本和增大了信背比等，这表明基于GO的荧光传感器在生物分子检测方面有很大的应用前景。这类传感器大多依赖GO能强烈吸附荧光染料标记的单链DNA同时淬灭其荧光，目标物与荧光单链DNA结合使其构象转变，进而使荧光染料与GO距离拉大，荧光恢复，从而实现对目标物的检测。但是这类传感器的检测限一般有限且需要荧光标记，有待进一步改进。将DNA嵌入染料与GO相结合进行生物分子检测已有报道，其检测性能显著改善，我们推断此类非标荧光传感器有望在生物分子检测中得到广泛应用。
本论文针对以上问题，在综合文献报道的基础之上，基于GO独特的DNA吸附和荧光染料淬灭性能发展了一系列简单、快速、灵敏的荧光生物传感新方法用于DNA酶活性、核酸和三磷酸腺苷(ATP)等生物分子的检测，具体内容如下：
第2章中，基于发夹引物和聚合延伸，结合GO独特的DNA吸附和通用型荧光淬灭性能发展了一种新型非标荧光检测方法用于DNA3'磷酸酶的检测及其抑制剂的定量表征。本实验设计了一条3'磷酸化的DNA发夹引物，只有经DNA3'磷酸酶的去磷酸作用后，发夹引物才能发生聚合延伸生成双链DNA产物。由于GO与双链DNA的结合力较弱不能有效淬灭染色双链产物的荧光，因此得到较强的荧光信号。无DNA3'磷酸酶时，发夹引物不能去磷酸化引发聚合延伸，进而被GO吸附淬灭，得到较弱的背景信号，从而实现对目标DNA3'磷酸酶的灵敏检测。该方法可实现对两种DNA3'磷酸酶(T4多聚核苷酸激酶和小虾碱性磷酸酶)活性的同时检测，检测限分别为0.07U/mL和0.003U/mL。另外，该方法不仅可以对DNA3'磷酸酶的抑制剂进行定量表征，还适用于复杂生物环境中目标物的测定。本方法设计简单、操作方便、灵敏度高且选择性好，有望成为DNA3'磷酸酶检测的一种很好的选择并能应用于DNA损伤修复机理的研究。
第3章中，基于等温循环链置换聚合反应(ICSDPR)和DNA嵌入染料的光学特性，结合GO独特的吸附和淬灭性能发展了一种非标放大荧光DNA检测方法。实验设计了一条3'磷酸化的发夹探针，只有加入目标DNA与发夹杂交引发其构象转变，引物才能与发夹打开的茎端杂交，然后在聚合酶作用下发生ICSDPR。经过多步的循环放大，产生大量的双链DNA产物，进而得到放大的荧光信号。无目标DNA时，发夹探针不能发生构象转变和ICSDPR，经SYBR Green I(SG)染色和GO吸附后，生成较低的背景信号，从而实现对目标DNA的放大荧光检测。该方法首次提出在GO平台上结合SG和ICSDPR放大检测DNA，有较宽的线性范围和高选择性，检测限为4pM，跟基于GO的均相DNA检测相比有明显优势。该方法设计简单、方便经济，有望成为DNA灵敏检测的一种常用方法。
第4章中，基于ATP依赖的DNA连接反应和GO独特的吸附和淬灭性能，发展了一种高灵敏的ATP检测新方法。实验设计的三种DNA探针退火杂交形成带有单链缺口的荧光双链DNA，在辅助因子ATP和T4DNA连接酶同时存在下，缺口被连接形成完整的双链DNA。连接产物有较高的熔链温度能在实验加热条件下保持双链结构，不被GO有效淬灭，得到较强的荧光信号。无ATP时，DNA连接反应不能发生，三链退火的复合物在实验加热条件下解链进而被GO吸附，荧光淬灭，从而实现对目标ATP的灵敏检测。该方法简单快速、灵敏度高选择性好，检测限达0.3nM，跟大多数无放大的均相ATP适配体传感方法相比有明显优势，跟电化学适配体方法的检测限有可比性。该方法具有高选择性，不仅能使ATP从其它三磷酸核苷中分离开来，还可使ATP与其同系物区分开来。
第5章中，基于核酸外切酶Ⅲ(ExoⅢ)对酶切底物的选择性和GO对单双链DNA结合力的差别发展了一种非标荧光ATP检测方法。实验设计的5'磷酸化的发夹探针能跟互补DNA退火杂交形成带单链缺口的双链DNA产物。在辅助因子ATP和连接酶同时存在时，缺口被连接形成完整的长茎端发夹产物，且靠近3'端的碱基硫代处理。这种发夹产物不能被ExoⅢ酶切，经SG染色和GO混合后，得到显著的荧光信号。无ATP时，缺口不能被连接进而被ExoⅢ酶切，互补DNA被降解同时释放发夹探针，染色后的发夹被GO吸附淬灭，生成较低的背景信号，从而实现对目标ATP的灵敏检测。该方法设计巧妙新颖，有较高的灵敏度和选择性，检测限达0.2nM。
第6章中，基于限制性核酸内切酶酶切的特异性和GO对不同的DNA结构结合力大小的差别发展了一种新型荧光方法用于检测核酸内切酶的活性及其抑制剂。我们设计了一条单标记荧光发夹探针，发夹的茎端序列包含有内切酶的识别位点。当目标内切酶存在时，发夹探针被酶切成三部分，其中带荧光染料的DNA短片段与GO结合力较小而游离到溶液中，生成较强的荧光信号。无目标内切酶时，发夹荧光探针被GO吸附，发生荧光共振能量转移荧光淬灭，得到较弱的背景信号，从而实现对目标内切酶的灵敏检测。该方法设计简单、操作方便、经济快捷，为进一步研究蛋白质与DNA的相互作用机理提供一个新的选择，对内切酶抑制剂的定量表征又使其在新药研发方面有一定的应用潜力。
Rapid and sensitive detection of biomolecules has always been the researchinterest in biomedical and analytical chemistry. The rapid development of modernsociety imposes higher requirements on biosensing technology. Developing newanalytical techniques for solving more and more analytical problems and social crisishas proposed a great challenge for analysts. Fluorescent biosensing technology hasbeen widely applied in the detection of various biomolecules, including protein, smallmolecules, nucleic acids and enzyme activity, attributing to its analytical advantages ofhigh sensitivity and rapid response time. In order to improve the sensitivity, traditionalfluorescent biosensing technology usually requires multiple-fluorophore labeling anddifferent amplification techniques, which might add cost and complexity for the assay.
In addition, traditional fluorescent biosensing technology suffers from a relativelyhigh background signal. Therefore, it is of great importance to develop new fluorescentbiosensing techniques so as to enhance the bioanalytical performance and reduce thecost.
Due to its unique DNA absorbing ability and universal quenching properties forfluorophores, graphene oxide (GO) has been widely applied in biomolecular detection.GO-based fluorescent biosensors show several advantages compared with thetraditional ones, including improved assay sensitivity, low cost and enhanced signal tobackground ratio and so on. These advantages reveal that GO-based fluorescentbiosensors hold great potential in biomolecular detection. Most of these biosensorsrely on the fact that GO can adsorb the dye-labeled single-stranded DNA (ssDNA) andeffectively quench its fluorescence, the binding of analyte with dye-labeled ssDNAresults in the departure of the dye and GO, and the recovery of fluorescence which canbe used for target detection. However, these biosensors require fluorophore-labeledprobes and show relatively poor detection limits, which need further improvements.There has been report for biomolecular detection based on the combination of DNAinteracting dyes and GO, which shows an improved detection limit and it is expectedthat this kind of biosensor may find wide applications in biomolecular detection.
Focusing on the above questions and making use of the achievements of previousresearchers, this thesis developed several novel fluorescent biosensing methods for thedetection of DNA enzyme activity, DNA and adenosine triphosphate (ATP) based on the unique DNA adsorption and fluorescence quenching property of GO. The detailsare described in following chapters.
In Chapter2, we developed a novel label-free fluorescent strategy for thedetection of DNA3' phosphatase and its inhibitors based on hairpin primer andpolymerase elongation. Only in the presence of DNA3' phosphatase, DNA hairpinprimer with3'-phosphorylated end can be dephosphated, then polymerase elongationwas initiated to produce the double-stranded DNA (dsDNA). Because GO shows weakbinding affinity toward the SYBR Green I (SG) stained dsDNA that it cannoteffectively quench the fluorescence, thus one can obtain a strong fluorescence signal.Whereas, the hairpin primer would not be dephosphated in the absence of DNA3'phosphatase and its polymerase elongation would not occur. Adsorption of hairpinprimer by GO generates a rather weak background signal. This method can realize thesimultaneous detection of two kinds of DNA3' phosphatase (T4PNKP and SAP) withachieved detection limits of0.07U/mL and0.003U/mL, respectively. In addition, thismethod is able to quantitatively evaluate the inhibition effect of the inhibitorcompounds and also applicable for complex biological sample.
In Chapter3, a label-free sensitive fluorescent DNA biosensor was presentedbased on isothermal circular strand-displacement polymerization reaction (ICSDPR)combined with GO binding. This biosensor relies on the hybridization of target DNAwith hairpin probe as the prerequisite for ICSDPR and the preferential binding of GOto stained single stranded DNA (ssDNA) over double stranded one to improve thesignal to background ratio. By combining the properties of GO and SG with theICSDPR amplification, this fluorescent DNA biosensor displays a wide dynamic rangefrom0.01nM to10nM and a low detection limit of4pM. The proposed strategy issimple, cost-effective and sensitive, which might provide a promising method ofchoice for convenient DNA detection.
In Chapter4, a simple, amplification-free and sensitive fluorescent biosensor forATP detection was developed based on the ATP-dependent enzymatic reaction(ATP-DER) and GO binding. In this assay, two half DNA probes anneal with adye-labeled template to form a DNA duplex substrate with a single-stranded nick. Theformed nick could be sealed by T4DNA ligase in the presence of ATP, and theresulting unnicked DNA duplex was resistant to thermal denaturation. Whereas, theligation reaction would not happen in the absence of ATP, and the nicked DNA duplexwas subject to thermal denaturation, which leads to the separation of two half DNAprobes and template. After addition of GO, the fluorescence of the nicked DNA duplex was greatly quenched, whereas efficient fluorescence quenching did not occur to theunnicked DNA duplex and high fluorescence intensity was obtained. The proposedstrategy was simple, amplification-free and showed high selectivity to ATP that coulddistinguish ATP from its analogues. The results revealed that the method allowedsensitive quantitative assay of ATP with a wide linear response range from0.5nM to100nM and a low detection limit of0.3nM.
In Chapter5, we present a novel, label-free exonuclease Ⅲ-aided fluorescentassay strategy for ATP based on the ATP-dependent enzymatic reaction and GO. In thisassay, hairpin probe with5' phosphorylated end anneals with complementary DNA toform a dsDNA complex with a single-stranded nick, which can be ligated by T4DNAligase in the presence of its cofactor ATP, resulting in a hairpin product with a stem oflong dsDNA. Because the five phosphorothioate nucleosides near the3' end of hairpinprobe can hamper the Exo Ⅲ cleavage, thus the hairpin product after ligation couldkeep intact upon treatment with Exo Ⅲ. After addition of GO, a strong fluorescencesignal was observed. Whereas, the single-stranded nick cannot be ligated in theabsence of ATP, Exo Ⅲ cleaves the annealed dsDNA complex from the nick site anddigests the C-DNA, releasing the hairpin probe. GO selectively adsorb the hairpinprobe leading to efficient quenching of the fluorescence. This sensor displays animproved sensitivity and a wide linear range within the ATP concentration from1nMto200nM with a low detection limit of0.2nM. The proposed approach is simple,cost-effective and convenient, which might create a new methodology for developingsensitive ATP biosensor.
In Chapter6, we developed a simple and novel fluorescent strategy for thedetection of endonuclease activity and its inhibitors based on hairpin probe and GO. Inthis assay, we designed a single dye-labeled hairpin probe that contains the recognitionsite for the endonuclease in the stem. Upon treatment with the target endonuclease,hairpin probe was digested into three pieces. One of the digested products, thedye-labeled short DNA would not be absored by GO, leads to a strong fluorescencesignal. In the absence of endonuclease, hairpin probe kept intact and was immediatelyabsorbed by GO, resulting in an effective fluorescence quenching. In addition, thismethod was applicable for inhibitor quantification. Given the simplicity, convenienceof this approach, the proposed method may provide an alternative approach for thestudy of the interaction between protein and DNA.
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