基于纳米材料和核酸适配体的高灵敏度光学生物传感器研究
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摘要
近年来,生物传感器因具有灵敏度高、选择性好、成本低和方便快捷等优点,被广泛地应用到与生命活动相关的核酸分子、蛋白质、金属离子、小分子和酶的检测当中。纳米材料和功能核酸的发展,为构建新颖的、用于各种目标物检测的生物传感技术提供了全新的设计思路和平台。
本论文作为国家自然科学基金(No.21173071)和教育部高等学校博士点专项基金资助课题(No.20114104110002)的一部分,通过把功能核酸和纳米材料相结合,构建了几种光学生物传感器用于对重金属离子、生物小分子和酶活性及其抑制剂的检测。与传统检测方法相比,本论文所建立的传感器对目标物的检测具有灵敏度高、选择性好、成本低和操作简单等优点,同时初步验证了传感器的实用性。其主要内容如下：
在第二章中构建了一种基于水溶性聚噻吩衍生物和非标记功能核酸的光学传感器,可以简单快速、高灵敏地和高选择性地检测水溶液中的铅离子。该传感器检测原理是利用铅离子诱导富含G碱基的核酸DNA的结构从自由卷曲状态到G-四链体状态,同时使用阳离子水溶性聚噻吩(PMNT)作为信号产生单元来产生光学信号。通过使用特定的核酸序列TBAA (5'-GGAAGGTGTGGAAGG-3'),实现了对铅离子的特异性识别,避免了传统功能核酸传感器使用掩蔽剂(CN-, SCN-)用于消除汞离子的干扰所带来的问题。当溶液中不存在铅离子时,PMNT与DNA探针通过静电相互作用,形成类双链的刚性复合物,使得PMNT骨架的共轭程度增大,溶液显现红色。当溶液中存在铅离子时,铅离子会诱导DNA探针折叠成G-四链体结构,PMNT通过静电作用缠绕在G-四链体表面形成复合物,在该复合物中PMNT的共轭程度低,溶液颜色为黄色。基于以上原理,本传感器实现了对铅离子的高灵敏和特异性检测。本传感器可以在5分钟内快速的检测水溶液中微摩尔浓度的铅离子,利用荧光检测方法可以把检测限提高到纳摩尔级别。该传感器成功的被应用到自来水中铅离子的检测。
在第三章中基于氧化石墨烯对单链DNA和G-四链体DNA的吸附能力不同,设计了一种荧光增强型的G-四链体传感器用于铅离子的检测。通过使用在第二章中筛选出的核酸序列TBAA作为探针,实现了对铅离子特异性的识别。首先在探针的5’末端修饰上荧光发色团,不存在铅离子时,核酸探针在溶液中呈现柔软的单链构象。加入氧化石墨烯,通过单链核酸的碱基与氧化石墨烯的芳香性六元环π-π堆积作用,单链核酸探针吸附在氧化石墨烯表面导致荧光发色团的荧光猝灭。当存在铅离子时,铅离子诱导TBAA从单链构象折叠成G-四链体构象。加入氧化石墨烯,因为G-四链体与氧化石墨烯的作用力比较弱,带有荧光发色团的G-四链体远离氧化石墨烯的表面使荧光发色团的荧光不被猝灭。该传感器对铅离子的最低检测限为400pM,同时该传感器被成功的应用到河水和自来水中铅离子的检测。
在第四章中利用氧化石墨烯对单、双链DNA的吸附能力不同,设计了一个基于氧化石墨烯的生物传感器用于HIV-1逆转录酶RNase H的活性检测及抑制剂筛选。首先在DNA序列的3’末端修饰上荧光发色团,然后与该序列互补的RNA杂交形成DNA/RNA杂交体。不存在RNase H时,因为DNA/RNA与氧化石墨烯的相互作用比较弱,加入氧化石墨烯后DNA/RNA不能稳定的吸附在氧化石墨烯的表面,因此荧光发色团的荧光不会被猝灭。当存在RNase H时,RNase H能够催化DNA/RNA中的RNA裂解成核糖核苷片段从而释放出单链DNA。加入氧化石墨烯后,单链DNA能够强力的吸附在氧化石墨烯的表面导致荧光猝灭。当存在酶抑制剂时,RNase H的酶活性被抑制,RNA/DNA依旧保持双链的状态。加入氧化石墨烯后荧光不被猝灭。该传感器对HIV-1RNase H酶的最低检测限为0.6units mL-1,同时可以进行高通量筛选HIV-1RNase H酶抑制剂。本传感器不仅提供了一个检测HIV-1RNase H酶活性和抑制剂筛选的通用平台,也在抗艾滋病药物的研发以及临床治疗方面表现出潜在的应用价值。
在第五章中发展了一种新型的基于核酸切刻酶辅助目标循环放大的分子适配体信标策略用于ATP高灵敏的检测。切刻酶是一种限制性核酸内切酶,可特异性地识别双链DNA中的核苷酸序列,并对其中的一条链进行切割。在本实验中使用的切刻内切酶是Nt.CviPⅡ,能够特异性的在5’-···↓CCA…-3’位点处切刻双链DNA。在该传感策略中,把ATP的核酸适配体分割成两个短链寡聚核苷酸,Aptl和Apt2。其中Aptl的3’端修饰了荧光发色团。Aptl可以通过分子内自组装的方式形成分子信标。Aptl环状部分可以和Apt2—同作为ATP的适配体。不存在ATP时,因为含有错配的碱基对,Apt2不能够与Aptl中的环状部位杂交。因为Aptl和Apt2含有大的单链结构,加入氧化石墨烯后,通过单链DNA的碱基与氧化石墨烯的芳香环结构之间的π-π堆积作用,Aptl和Apt2吸附在氧化石墨烯的表面导致Aptl的荧光猝灭。当存在ATP时,Apt2和ATP一起作用于Aptl的环状部位打开Aptl的发卡结构形成双链DNA。在这个双链DNA结构中含有核酸切刻酶Nt.CviPII的切割位点。加入Nt.CviPⅡ后,可以把双链DNA中的Aptl切割成两部分,从而使APT和Apt2游离出来。游离出来的APT和Apt2又可以对另外—分子的Aptl分子信标进行互补配对,然后再次被酶切割,从而进行循环,累积许多含有荧光基团的短链DNA。因为被酶切割后Aptl是两条短链DNA,其与氧化石墨烯的相互作用能力比较弱,从而远离氧化石墨烯的表面产生荧光信号,并经过多次循环后实现了信号放大效应。该方法实现了对ATP的高灵敏和高特异性检测,响应动态范围为10nM到1000nM,最低检测限为4nM。该传感器的检测限比传统的方法相降低3个数量级。
Recently, because of their high sensitivity, good selectivity, low cost and short time-consuming, biosensors have been widely applied to the detection of heavy metal ions, small biomoleculars and enzymes. The development of nanomaterials and functional nucleic acids provides the new strategies and platforms for the design of biosensing technology.
As a part of the projects supported by the National Natural Science Foundation of China (No.21173071) and the Research Fund for the Doctoral Program of Higher Education of China (No.20114104110002)), in this doctoral thesis, we have developed a series of optical biosensors for the detection of heavy metal ions, small molecular, enzyme activity and drug screening by combining of the advantages of nanomaterials and functional nucleic acids. Compared with the traditional methods, the proposed detection methods are convenient, sensitive and cost-effective. The practicability of these developed methods was also verified. The detailed contents are described as follows:
In the chapter2, we designed a highly selective and sensitive sensor for detection of Pb2+by using conjugated polymers and label-free oligonucleotides. This method is based on Pb2+-induced G-rich DNA conformation switch from a random-coil to G-quadruplex with a water-soluble polythiophene derivative (PMNT) as "a polymeric stain" to transduce optical signal. We selected a specific sequence oligonucleotide, TBAA (5'-GGAAGGTGTGGAAGG-3'), which can form a G-quadruplex structure upon the addition of Pb2+. This strategy provided a promising alternative to Pb2+determination in the presence of Hg2+instead of the universal masking agent of Hg2+(such as CN-,SCN-). In the absence of Pb2+, PMNT and TBAA probe readily formed an electrostatic PMNT-TBAA complex in aqueous solution. In the complex, PMNT took a highly conjugated and planar conformation with a characteristic red color. In the present of Pb2+, the TBAA probe formed a G-quadruplex structure through the specific Pb2+binding G-quartets. PMNT wound on the surface of G-quadruplex through electrostatic interaction, resulting in the twisting of conjugated backbone. The color of PMNT-G-quadruplex is yellow. By this method, we could identify micromolar Pb2+concentrations within5min even with the naked eye. Furthermore, the detection limit was improved to the nanomolar range by the fluorometric method. We also successfully utilized this biosensor for the determination of Pb2+in tap water samples.
In the chapter3, we designed a highly selective and sensitive sensor for detection of Pb2+by using graphene oxide (GO) and G-quadruplex DNA (G4). Based on Pb+-induced G-rich DNA conformation switch from a random-coil to G-quadruplex and the remarkable difference in the absorbing affinity of GO with ssDNA and G-quadruplex, we constructed a GO-G4based fluorescence "turn-on" sensing system for rapid, sensitive and selective detection of Pb2+by using "mix-and-detect" assay format. We used the specific sequence G-rich ssDNA (TBAA) for the detection of Pb2+which was slected in chapter2. In the absence of Pb2+, the TBAA is in a flexible single strand state. The introduction of GO to the carboxy fluorescein (FAM)-labeled TBAA solution would result in strong binding between nucleotide bases and aromatic structure of GO via π-stacking, bringing the fluorophore into close proximity with the GO surface. Consequently, the fluorescence of FAM is quenched via energy transfer from dyes to GO. However, in the presence of Pb2+, the conformation of the TBAA is switched from a random coil to G-quadruplex complex. The introduction of GO into the sensor solution will result in weak quenching of the fluorescence of FAM due to the weak affinity of G-quadruplex complex to GO, and the fluorescence intensity should gradually increase with the increasing concentration of Pb2+. A detection limit of400pM for Pb2+ions was estimated. We also successfully utilized this biosensor for the determination of Pb2+in tap water and river water samples.
In the chapter4, we have developed a simple and highly sensitive fluorescent biosensor for the detection of HIV-1RNase H activity and inhibition by using graphene oxide (GO) as sensing platform. In our approach, a DNA-RNA substrate was prepared which FAM was labeled at the3'termini of single strand DNA. The FAM labeled DNA-RNA substrate preserves most of the fluorescence when mixed with GO. However, in the present of RNase H, RNase H can cleavage the RNA into fragments, resulting in the dissociation of ssDNA and mononucleotides. The fluorescence intensity was greatly quenched after the addition of GO. The as-proposed method provides a low detection limit of0.6units mL-1for HIV-1RNase H activity analysis. Furthermore, this approach shows potential for high-throughput screening of HIV-1RNase H inhibitors. The method not only provides a universal platform for monitoring activity and inhibition of RNase H but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.
In chapter5, we developed a novel molecular aptamer beacon biosensing strategy for high-sensitive detection of ATP based on Nicking Endonuclease (NEase)-assisted target recycling amplification. NEase is a special family of restriction endonucleases, which can recognize a specific sequence kown as a restriction site along a double-strand DNA and only cleave one strand of it, leaving a nick in the DNA. The NEase used here is Nt.CviPII, which recognizes a simple asymmetric sequence,5'-…↓CCA…-3'. In this assay, the aptamer of ATP was split into two subunits, which was named Aptl and Apt2, respectively. Aptl was designed to form a hairpin structure whose3'-terminal was modified a fluorophore. The Apt2was not labeled any dye molecule. The loop sequence of Aptl and Apt2were combined as the aptamer of ATP. In the absence of ATP, because of the mismatch bases, Apt2can not hybridize with the loop structure of Aptl. Aptl and Apt2were adsorbed onto the surface of graphene oxide through π-stacking interaction between the ring structrue in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. In the present of ATP, Apt2combined with ATP hybridized with Aptl to form double-stranded DNA structure. In this double-stranded DNA, it contains the cleavage sites of Nt.CviPII. The Nt.CviPII can recognize the specific nucleotide sequence and cleave the Aptl into two fragments. After nicking, the ATP and Apt2will dissociate from the probe-target complex. Then, ATP and Apt2can hybridize with another intact Aptl to form a new substrate for NEase and the cycle starts anew. Through such strand-scission cycle, only one target can trigger cleavage of a large quantity of Aptl, which can accumulate more short-strand DNA containing fluorophore. Because of the weak affinity between short-strand DNA with graphene oxide, the introduction of GO into the sensing solution will result in weak quenching of the fluorescence of FAM. After several cycles, it leads to significant amplification of the signal. The results revealed that this strategy offered a sensitive and selective method for the detection of ATP over the concentration range from10nM to1000nM with the detection limit of4nM. The sensitivity of assay is3orders magnitude better than previously reported methods.
本论文作为国家自然科学基金(No.21173071)和教育部高等学校博士点专项基金资助课题(No.20114104110002)的一部分,通过把功能核酸和纳米材料相结合,构建了几种光学生物传感器用于对重金属离子、生物小分子和酶活性及其抑制剂的检测。与传统检测方法相比,本论文所建立的传感器对目标物的检测具有灵敏度高、选择性好、成本低和操作简单等优点,同时初步验证了传感器的实用性。其主要内容如下：
在第二章中构建了一种基于水溶性聚噻吩衍生物和非标记功能核酸的光学传感器,可以简单快速、高灵敏地和高选择性地检测水溶液中的铅离子。该传感器检测原理是利用铅离子诱导富含G碱基的核酸DNA的结构从自由卷曲状态到G-四链体状态,同时使用阳离子水溶性聚噻吩(PMNT)作为信号产生单元来产生光学信号。通过使用特定的核酸序列TBAA (5'-GGAAGGTGTGGAAGG-3'),实现了对铅离子的特异性识别,避免了传统功能核酸传感器使用掩蔽剂(CN-, SCN-)用于消除汞离子的干扰所带来的问题。当溶液中不存在铅离子时,PMNT与DNA探针通过静电相互作用,形成类双链的刚性复合物,使得PMNT骨架的共轭程度增大,溶液显现红色。当溶液中存在铅离子时,铅离子会诱导DNA探针折叠成G-四链体结构,PMNT通过静电作用缠绕在G-四链体表面形成复合物,在该复合物中PMNT的共轭程度低,溶液颜色为黄色。基于以上原理,本传感器实现了对铅离子的高灵敏和特异性检测。本传感器可以在5分钟内快速的检测水溶液中微摩尔浓度的铅离子,利用荧光检测方法可以把检测限提高到纳摩尔级别。该传感器成功的被应用到自来水中铅离子的检测。
在第三章中基于氧化石墨烯对单链DNA和G-四链体DNA的吸附能力不同,设计了一种荧光增强型的G-四链体传感器用于铅离子的检测。通过使用在第二章中筛选出的核酸序列TBAA作为探针,实现了对铅离子特异性的识别。首先在探针的5’末端修饰上荧光发色团,不存在铅离子时,核酸探针在溶液中呈现柔软的单链构象。加入氧化石墨烯,通过单链核酸的碱基与氧化石墨烯的芳香性六元环π-π堆积作用,单链核酸探针吸附在氧化石墨烯表面导致荧光发色团的荧光猝灭。当存在铅离子时,铅离子诱导TBAA从单链构象折叠成G-四链体构象。加入氧化石墨烯,因为G-四链体与氧化石墨烯的作用力比较弱,带有荧光发色团的G-四链体远离氧化石墨烯的表面使荧光发色团的荧光不被猝灭。该传感器对铅离子的最低检测限为400pM,同时该传感器被成功的应用到河水和自来水中铅离子的检测。
在第四章中利用氧化石墨烯对单、双链DNA的吸附能力不同,设计了一个基于氧化石墨烯的生物传感器用于HIV-1逆转录酶RNase H的活性检测及抑制剂筛选。首先在DNA序列的3’末端修饰上荧光发色团,然后与该序列互补的RNA杂交形成DNA/RNA杂交体。不存在RNase H时,因为DNA/RNA与氧化石墨烯的相互作用比较弱,加入氧化石墨烯后DNA/RNA不能稳定的吸附在氧化石墨烯的表面,因此荧光发色团的荧光不会被猝灭。当存在RNase H时,RNase H能够催化DNA/RNA中的RNA裂解成核糖核苷片段从而释放出单链DNA。加入氧化石墨烯后,单链DNA能够强力的吸附在氧化石墨烯的表面导致荧光猝灭。当存在酶抑制剂时,RNase H的酶活性被抑制,RNA/DNA依旧保持双链的状态。加入氧化石墨烯后荧光不被猝灭。该传感器对HIV-1RNase H酶的最低检测限为0.6units mL-1,同时可以进行高通量筛选HIV-1RNase H酶抑制剂。本传感器不仅提供了一个检测HIV-1RNase H酶活性和抑制剂筛选的通用平台,也在抗艾滋病药物的研发以及临床治疗方面表现出潜在的应用价值。
在第五章中发展了一种新型的基于核酸切刻酶辅助目标循环放大的分子适配体信标策略用于ATP高灵敏的检测。切刻酶是一种限制性核酸内切酶,可特异性地识别双链DNA中的核苷酸序列,并对其中的一条链进行切割。在本实验中使用的切刻内切酶是Nt.CviPⅡ,能够特异性的在5’-···↓CCA…-3’位点处切刻双链DNA。在该传感策略中,把ATP的核酸适配体分割成两个短链寡聚核苷酸,Aptl和Apt2。其中Aptl的3’端修饰了荧光发色团。Aptl可以通过分子内自组装的方式形成分子信标。Aptl环状部分可以和Apt2—同作为ATP的适配体。不存在ATP时,因为含有错配的碱基对,Apt2不能够与Aptl中的环状部位杂交。因为Aptl和Apt2含有大的单链结构,加入氧化石墨烯后,通过单链DNA的碱基与氧化石墨烯的芳香环结构之间的π-π堆积作用,Aptl和Apt2吸附在氧化石墨烯的表面导致Aptl的荧光猝灭。当存在ATP时,Apt2和ATP一起作用于Aptl的环状部位打开Aptl的发卡结构形成双链DNA。在这个双链DNA结构中含有核酸切刻酶Nt.CviPII的切割位点。加入Nt.CviPⅡ后,可以把双链DNA中的Aptl切割成两部分,从而使APT和Apt2游离出来。游离出来的APT和Apt2又可以对另外—分子的Aptl分子信标进行互补配对,然后再次被酶切割,从而进行循环,累积许多含有荧光基团的短链DNA。因为被酶切割后Aptl是两条短链DNA,其与氧化石墨烯的相互作用能力比较弱,从而远离氧化石墨烯的表面产生荧光信号,并经过多次循环后实现了信号放大效应。该方法实现了对ATP的高灵敏和高特异性检测,响应动态范围为10nM到1000nM,最低检测限为4nM。该传感器的检测限比传统的方法相降低3个数量级。
Recently, because of their high sensitivity, good selectivity, low cost and short time-consuming, biosensors have been widely applied to the detection of heavy metal ions, small biomoleculars and enzymes. The development of nanomaterials and functional nucleic acids provides the new strategies and platforms for the design of biosensing technology.
As a part of the projects supported by the National Natural Science Foundation of China (No.21173071) and the Research Fund for the Doctoral Program of Higher Education of China (No.20114104110002)), in this doctoral thesis, we have developed a series of optical biosensors for the detection of heavy metal ions, small molecular, enzyme activity and drug screening by combining of the advantages of nanomaterials and functional nucleic acids. Compared with the traditional methods, the proposed detection methods are convenient, sensitive and cost-effective. The practicability of these developed methods was also verified. The detailed contents are described as follows:
In the chapter2, we designed a highly selective and sensitive sensor for detection of Pb2+by using conjugated polymers and label-free oligonucleotides. This method is based on Pb2+-induced G-rich DNA conformation switch from a random-coil to G-quadruplex with a water-soluble polythiophene derivative (PMNT) as "a polymeric stain" to transduce optical signal. We selected a specific sequence oligonucleotide, TBAA (5'-GGAAGGTGTGGAAGG-3'), which can form a G-quadruplex structure upon the addition of Pb2+. This strategy provided a promising alternative to Pb2+determination in the presence of Hg2+instead of the universal masking agent of Hg2+(such as CN-,SCN-). In the absence of Pb2+, PMNT and TBAA probe readily formed an electrostatic PMNT-TBAA complex in aqueous solution. In the complex, PMNT took a highly conjugated and planar conformation with a characteristic red color. In the present of Pb2+, the TBAA probe formed a G-quadruplex structure through the specific Pb2+binding G-quartets. PMNT wound on the surface of G-quadruplex through electrostatic interaction, resulting in the twisting of conjugated backbone. The color of PMNT-G-quadruplex is yellow. By this method, we could identify micromolar Pb2+concentrations within5min even with the naked eye. Furthermore, the detection limit was improved to the nanomolar range by the fluorometric method. We also successfully utilized this biosensor for the determination of Pb2+in tap water samples.
In the chapter3, we designed a highly selective and sensitive sensor for detection of Pb2+by using graphene oxide (GO) and G-quadruplex DNA (G4). Based on Pb+-induced G-rich DNA conformation switch from a random-coil to G-quadruplex and the remarkable difference in the absorbing affinity of GO with ssDNA and G-quadruplex, we constructed a GO-G4based fluorescence "turn-on" sensing system for rapid, sensitive and selective detection of Pb2+by using "mix-and-detect" assay format. We used the specific sequence G-rich ssDNA (TBAA) for the detection of Pb2+which was slected in chapter2. In the absence of Pb2+, the TBAA is in a flexible single strand state. The introduction of GO to the carboxy fluorescein (FAM)-labeled TBAA solution would result in strong binding between nucleotide bases and aromatic structure of GO via π-stacking, bringing the fluorophore into close proximity with the GO surface. Consequently, the fluorescence of FAM is quenched via energy transfer from dyes to GO. However, in the presence of Pb2+, the conformation of the TBAA is switched from a random coil to G-quadruplex complex. The introduction of GO into the sensor solution will result in weak quenching of the fluorescence of FAM due to the weak affinity of G-quadruplex complex to GO, and the fluorescence intensity should gradually increase with the increasing concentration of Pb2+. A detection limit of400pM for Pb2+ions was estimated. We also successfully utilized this biosensor for the determination of Pb2+in tap water and river water samples.
In the chapter4, we have developed a simple and highly sensitive fluorescent biosensor for the detection of HIV-1RNase H activity and inhibition by using graphene oxide (GO) as sensing platform. In our approach, a DNA-RNA substrate was prepared which FAM was labeled at the3'termini of single strand DNA. The FAM labeled DNA-RNA substrate preserves most of the fluorescence when mixed with GO. However, in the present of RNase H, RNase H can cleavage the RNA into fragments, resulting in the dissociation of ssDNA and mononucleotides. The fluorescence intensity was greatly quenched after the addition of GO. The as-proposed method provides a low detection limit of0.6units mL-1for HIV-1RNase H activity analysis. Furthermore, this approach shows potential for high-throughput screening of HIV-1RNase H inhibitors. The method not only provides a universal platform for monitoring activity and inhibition of RNase H but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.
In chapter5, we developed a novel molecular aptamer beacon biosensing strategy for high-sensitive detection of ATP based on Nicking Endonuclease (NEase)-assisted target recycling amplification. NEase is a special family of restriction endonucleases, which can recognize a specific sequence kown as a restriction site along a double-strand DNA and only cleave one strand of it, leaving a nick in the DNA. The NEase used here is Nt.CviPII, which recognizes a simple asymmetric sequence,5'-…↓CCA…-3'. In this assay, the aptamer of ATP was split into two subunits, which was named Aptl and Apt2, respectively. Aptl was designed to form a hairpin structure whose3'-terminal was modified a fluorophore. The Apt2was not labeled any dye molecule. The loop sequence of Aptl and Apt2were combined as the aptamer of ATP. In the absence of ATP, because of the mismatch bases, Apt2can not hybridize with the loop structure of Aptl. Aptl and Apt2were adsorbed onto the surface of graphene oxide through π-stacking interaction between the ring structrue in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. In the present of ATP, Apt2combined with ATP hybridized with Aptl to form double-stranded DNA structure. In this double-stranded DNA, it contains the cleavage sites of Nt.CviPII. The Nt.CviPII can recognize the specific nucleotide sequence and cleave the Aptl into two fragments. After nicking, the ATP and Apt2will dissociate from the probe-target complex. Then, ATP and Apt2can hybridize with another intact Aptl to form a new substrate for NEase and the cycle starts anew. Through such strand-scission cycle, only one target can trigger cleavage of a large quantity of Aptl, which can accumulate more short-strand DNA containing fluorophore. Because of the weak affinity between short-strand DNA with graphene oxide, the introduction of GO into the sensing solution will result in weak quenching of the fluorescence of FAM. After several cycles, it leads to significant amplification of the signal. The results revealed that this strategy offered a sensitive and selective method for the detection of ATP over the concentration range from10nM to1000nM with the detection limit of4nM. The sensitivity of assay is3orders magnitude better than previously reported methods.
引文
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