基于DNA酶和氧化石墨烯的高灵敏荧光生物传感体系的研究
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摘要
生物传感器（Biosensor）是由生物学、物理学、数学、化学、计算机科学等多个学科交叉融合而发展起来的一门新兴而活跃的学科。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。然而随着疾病诊断要求的不断提高与科学研究的不断深入，我们迫切需要发展一些灵敏度高、选择性好、操作简单且成本低廉的生物传感器以便更好地满足科学研究和实际应用的需求。目前提高荧光传感器灵敏度的方法主要有两类：一类是通过使用碳纳米材料等超级猝灭剂提高猝灭效率以降低检测荧光背景，另一类则是利用各种酶切催化循环放大手段提高传感器的灵敏度。
基于以上考虑，综合文献报道，本文主要采用可降低背景信号的荧光猝灭剂以及信号放大技术在提高生物传感器的灵敏度方面做了一些工作，主要内容如下：
（1）基于DNA酶生物传感体系的研究。DNA酶是利用体外筛选的组合生物学技术获得的一段具有催化功能的DNA片段。在第2章中，我们基于氧化石墨烯（GO）对不同长度的单链DNA具有不同的吸附力设计了一种荧光增强型的DNA酶生物传感器用于铅离子的检测。由于底物链和酶链的杂交探针中含有一段单链DNA序列，故该探针可以吸附在氧化石墨烯上，并使底物链5'端的荧光基团靠近GO，从而导致荧光团的荧光被猝灭。当加入Pb~(2+)后，DNA酶的催化活性被激活，并将底物链切割为两部分。含有荧光团的短链DNA（5个碱基）从酶链上解链游离下来。释放出的酶链可继续与未反应的底物链杂交，同时诱发下一轮的催化切割，如此循环，传感体系中就存在很多标记有荧光团的短链DNA。加入GO之后，由于短链DNA与氧化石墨烯结合力很弱，故荧光团远离氧化石墨烯而使其荧光不被猝灭。该传感器的灵敏度很高，对铅离子的检测限为300pM。
上一章报道的DNA酶荧光传感器对酶链有一定的要求，即酶链的环状部分需要有一段长的DNA单链便于杂交探针吸附在GO上。如果环状部分的单链DNA太短或以双链形式存在就会影响杂交探针与GO的结合能力，从而导致传感器的背景荧光增加。因此，利用GO做荧光猝灭剂限制了上述传感器的通用性。在第3章中，我们利用苝二酰亚胺阳离子化合物的团聚体作猝灭剂构建了一个通用的DNA酶生物传感平台。苝二酰亚胺阳离子化合物可以吸附于DNA酶探针上并发生团聚，而其团聚体可以通过荧光共振能量转移猝灭荧光团（FAM）的荧光。铅离子或L-组氨酸的加入使DNA酶的催化活性被激活并将底物链切割为两部分，释放出标记有FAM的短链DNA、另外一段底物链以及DNA酶。被释放的DNA酶可继续同未反应的底物链杂交，同时诱发下一轮的催化切割。由于苝二酰亚胺阳离子化合物主要吸附在酶链和切割后的另外一段底物链上，而标记有FAM的短链DNA只有5个碱基，苝二酰亚胺阳离子化合物很难与其结合，因此FAM的荧光未被猝灭。该传感器具有很高的灵敏度和选择性且通用性好，可用于检测各种目标物。
催化信标是当目标物存在时可以催化DNA酶水解底物链从而产生荧光信号增强的荧光检测体系。一般来说，催化信标由标记荧光团的底物链和标记猝灭团的DNA酶链组成，其中底物链与DNA酶链的比例应严格控制在≤1，而这样限制了催化信标在信号放大检测中的应用。另外，这种催化信标荧光猝灭效率较低，因此具有较高的荧光背景。在第4章中，基于具有特殊茎-环结构的分子信标和与之具有相同配对碱基的双链DNA之间熔链温度的差异，我们提出了一种利用分子信标作为底物链，从而避免对DNA酶修饰且能进行多重循环信号放大的新型荧光催化信标。而且该新型催化信标还可作为荧光放大基团用于目标DNA的检测。其动力学响应范围为0.5nM~300nM，检测下限为200pM。
为了进一步拓宽DNA酶的应用范围，在第5章中，我们基于DNA酶拓扑结构的改变可引起其催化活性的改变构建了一个通用的放大传感平台用于检测DNA序列和酶的活性。由于8-17DNAzyme的部分序列被固定于探针的发夹结构中，故酶的催化活性被抑制。当存在目标物（DNA或甲基化酶）时，目标物和探针作用使其发夹结构被打开，8-17DNA酶从发夹结构中释放出来且催化活性被激活。具有催化活性的8-17DNA酶可与发夹型的分子信标底物杂交形成催化分子信标传感体系。加入锌离子后，DNA酶催化切割分子信标底物，使底物链片段从酶链上游离下来，荧光明显增强。最后游离出来的8-17DNA酶的酶链可重新与分子信标底物杂交，引发下一轮的催化切割，如此循环，实现了目标物的放大检测。此外，我们还利用小分子末端保护策略和DNA酶的信号放大功能实现了蛋白质的放大检测。当不存在链霉亲和素（SA）时，DNA探针被核酸外切酶I（ExoI）切割为单碱基核苷酸，故不能催化切割分子信标底物，因此传感器的背景荧光很低。当存在SA时，探针上标记的生物素和SA结合从而阻止了ExoI切割DNA探针。最后包含8-17DNA酶序列的DNA探针可以催化切割分子信标底物并引起传感体系荧光信号的增强。
（2）在第6章中，我们将氧化石墨烯高的荧光猝灭率与核酸外切酶Ⅲ信号放大功能结合构建了一种高灵敏、快速、单标记的DNA荧光传感器。标记有荧光素（FAM）的DNA探针以单链形式存在故不能被核酸外切酶III切割，因此它可以通过π-π堆积作用吸附于GO表面并且荧光被猝灭。当目标DNA和探针链杂交后，核酸外切酶III沿3'→5'方向将探针链切割成碱基碎片，从而目标DNA被释放出来。释放出的目标DNA可继续和其它DNA探针杂交同时诱发第二次酶切循环反应。当酶切反应结束后加入适量的GO，由于DNA探针被切碎，故FAM远离GO而使其荧光不被猝灭。实验结果表明该方法可实现对目标DNA的高灵敏检测，检测下限为20pM。
（3）在第7章中，我们利用苝二酰亚胺阳离子化合物作为荧光信号的报告分子，富G核苷酸序列作为铅离子的识别探针构建了一种简单、快速和无标记的荧光增强型铅离子传感器。带正电荷的苝二酰亚胺阳离子化合物和带负电荷的DNA探针之间强的静电吸附作用导致苝二酰亚胺阳离子化合物吸附在DNA探针上并使其荧光被猝灭。当加入铅离子后，DNA探针从单链状态折叠成稳定的G四股螺旋结构。此时，苝二酰亚胺阳离子化合物与G四股螺旋结构的吸附力远低于它与单链DNA的吸附力，使得很多苝二酰亚胺阳离子化合物脱离DNA而游离于溶液中，从而导致传感体系的荧光增强。该传感器的动力学响应范围为10nM-10μM，检测限为4nM。
Biosensor is an emerging and active field originating from the interplay ofnumerous subjects, such as biology, physics, mathematics, chemistry and computertechnology. Recent years, as the new detection technologies constantly spring up andget perfected both in the research and practical application, the sensitivity and thespecificity of the biosensor have been largely improved. However, with the researchdeveloping, the development of the sensitive, selective and inexpensive biosensorsare of paramount importance for biomedical research and clinical diagnosis. Twodifferent strategies have been widely employed to improve the sensitivity of a sensor:lower the detection background and signal amplified detection.
In view of the considerations above and many relevant documents, we mainlyutilize the super fluorescence quencher and signal amplified strategy to improve thedetection sensitivity of the biosensor. The major contents are as follows:
(1) Study on the biosensors based on DNAzyme. DNAzymes (also calleddeoxyribozymes or DNA enzymes) are DNA sequences that can catalyze chemicalreactions, such as cleaving ribonucleic acid targets. Based on the remarkabledifference in affinity of GO with ssDNA containing different number of bases inlength, we constructed a graphene–DNAzyme based sensing system for amplifiedfluorescence “turn-on” detection of Pb~(2+)in chapter2. The DNAzyme-substratehybrid containing a large ssDNA loop make the fluorophore close to the GO surfaceto the greatest extent to afford a high quenching efficiency and a low backgroundfluorescence. Upon the addition of Pb~(2+), the DNAzyme was activated and cleaved thesubstrate strand, releasing a short FAM-linked oligonuleotide fragment, a relatedlong oligonuleotide fragment and the DNAzyme strand. The DNAzyme strand canhybridize with another substrate strand and then induce the second cycle of cleavageby binding Pb~(2+). The introduction of GO into the sensing solution will result in weakquenching of the fluorescence of FAM due to the weak affinity of the shortFAM-linked oligonuleotide fragment to GO, and the fluorescence intensity shouldgradually increase with increasing Pb~(2+)concentration added. Our proposed biosensorexhibits a high sensitivity towards target with a detection limit of300pM for Pb~(2+).
In chapter3, we employ cationic perylene derivative as a superquencher toconstruct DNAzyme-based fluorescence catalytic biosensors for the detection of Pb~(2+) and L-histidine. The strong affinity of perylene derivative with DNA drives it to forma complex with the DNAzyme-substrate hybrid, and efficiently quench thefluorescence of the FAM moiety. The introduction of Pb~(2+), however, will activateDNAzyme to catalyze the cleavage of the substrate in the RNA site, releasing a shortFAM-linked oligonuleotide fragment which show weak affinity with perylenederivative, and the introduction of perylene derivative into the system will showweak quenching effect on the FAM’s fluorescence. These results togetherdemonstrate that our design provides a convenient and universal platform foramplified detection of a wide range of targets.
The molecular beacon has the properties of low background and high sensitivity,and the huge difference with the melting temperature of other double-strand DNA.By utilizing the molecular beacons as the substrate sequence, a new kind offluorescent catalytic and molecular beacon has been developed in chapter4.Moreover, this design is willing to be applied in the fluorescence-based signalamplification for the detection of target DNA. A dynamic range from0.5nM to300nM for target DNA was achieved and the detection limit was200pM.
In chapter5, taking advantage of a topological effect of DNAzyme, we designeda new and general amplified sensing platform for nucleic acids and Dam MTaseactivity detection. Because the8-17DNAzyme is caged by partially hybridizing toform a hairpin structure, and is inactive to MB substrate, which provides a lowbackground for the sensing system. In the presence of the target, the hairpin structureis opened, and the8-17DNAzyme is liberated from the caged structure with itscatalytic activity being restored. The activated8-17DNAzyme can hybridize with theMB substrate to form the CAMB system and catalyze the cleavage of the MBsubstrate in the presence of cofactor Zn~(2+). After cleavage, the quenched MBfluorophore/quencher pair was separated from each other, resulting in an obviousincrease of fluorescent signal and a free DNAzyme strand. Eventually, eachtarget-induced activated8-17DNAzyme can undergo many cycles to trigger thecleavage of many MB substrates, achieving an amplified detection signal for thetarget. Furthermore, we constructed an amplified sensing platform for proteindetection based on the terminal protection of small-molecule-linked DNA. In theabsence of SA, the DNA probe was hydrolyzed successively into mononucleotides byExo I and cannot catalyze the cleavage of the MB substrate, therefore, providing azero-background for the sensing system. In the presence of SA, the probe is protectedfrom the degradation by Exo I. Consequently, the probe containing the8-17 DNAzyme sequence can catalyze the cleavage of the hairpin-structured MB substrateto induce a significant fluorescence enhancement.
(2) Using the GO as super fluorescence quencher and the Exo III as anamplifying biocatalyst, we develop a facile, sensitive, cost-effective method for DNAdetection in chapter6. In the absence of target DNA, Exo III is unable to catalyze theremoval of bases from the probe DNA. Upon the addition of GO, the fluorescence ofFAM was significantly quenched because of the strong binding between ssDNA andGO. In the presence of target DNA, Exo III can catalyze the stepwise removal ofmononucleotides of probe DNA from the blunt3′termini, which resulting in thereleasing of the target and fluorophore. The released target DNA can hybridize withanother probe DNA and then initiate a next round of cleavage. The introduction ofGO into the sensing solution will induce weak quenching of the fluorophore becauseof the weak affinity of the fluorophore and GO, and the fluorescence intensitygradually increases with increasing target concentration. The proposed biosensorexhibits high sensitivity, and a detection limit of20pM could be achieved for targetDNA.
(3) In chapter7, we report a simple, rapid and label-free approach forfluorescent “turn-on” detection of Pb~(2+)by using a water-soluble cationic perylenederivative (compound1) as the fluorescence reporter and the G-rich DNA probe forthe specific binding of Pb~(2+). Strong electrostatic interactions between the cationiccompound1and the polyanionic nucleic acid induced the aggregation of compound1and resulted in the fluorescence quenching. Upon the addition of Pb~(2+), theconformation of DNA probe changed from a random-coil to a Pb~(2+)-stabilizedG-quadruplex structure. This conformational change weakened the interactionbetween probe DNA and compound1. As a result, compound1is present in both thefree monomeric and the aggregated form in aqueous solution. Owing to the existenceof the free dye monomer, a fluorescence enhancement was observed. A dynamicrange from10nM to10μM for Pb~(2+)was achieved and the detection limit was4nM.
基于以上考虑，综合文献报道，本文主要采用可降低背景信号的荧光猝灭剂以及信号放大技术在提高生物传感器的灵敏度方面做了一些工作，主要内容如下：
（1）基于DNA酶生物传感体系的研究。DNA酶是利用体外筛选的组合生物学技术获得的一段具有催化功能的DNA片段。在第2章中，我们基于氧化石墨烯（GO）对不同长度的单链DNA具有不同的吸附力设计了一种荧光增强型的DNA酶生物传感器用于铅离子的检测。由于底物链和酶链的杂交探针中含有一段单链DNA序列，故该探针可以吸附在氧化石墨烯上，并使底物链5'端的荧光基团靠近GO，从而导致荧光团的荧光被猝灭。当加入Pb~(2+)后，DNA酶的催化活性被激活，并将底物链切割为两部分。含有荧光团的短链DNA（5个碱基）从酶链上解链游离下来。释放出的酶链可继续与未反应的底物链杂交，同时诱发下一轮的催化切割，如此循环，传感体系中就存在很多标记有荧光团的短链DNA。加入GO之后，由于短链DNA与氧化石墨烯结合力很弱，故荧光团远离氧化石墨烯而使其荧光不被猝灭。该传感器的灵敏度很高，对铅离子的检测限为300pM。
上一章报道的DNA酶荧光传感器对酶链有一定的要求，即酶链的环状部分需要有一段长的DNA单链便于杂交探针吸附在GO上。如果环状部分的单链DNA太短或以双链形式存在就会影响杂交探针与GO的结合能力，从而导致传感器的背景荧光增加。因此，利用GO做荧光猝灭剂限制了上述传感器的通用性。在第3章中，我们利用苝二酰亚胺阳离子化合物的团聚体作猝灭剂构建了一个通用的DNA酶生物传感平台。苝二酰亚胺阳离子化合物可以吸附于DNA酶探针上并发生团聚，而其团聚体可以通过荧光共振能量转移猝灭荧光团（FAM）的荧光。铅离子或L-组氨酸的加入使DNA酶的催化活性被激活并将底物链切割为两部分，释放出标记有FAM的短链DNA、另外一段底物链以及DNA酶。被释放的DNA酶可继续同未反应的底物链杂交，同时诱发下一轮的催化切割。由于苝二酰亚胺阳离子化合物主要吸附在酶链和切割后的另外一段底物链上，而标记有FAM的短链DNA只有5个碱基，苝二酰亚胺阳离子化合物很难与其结合，因此FAM的荧光未被猝灭。该传感器具有很高的灵敏度和选择性且通用性好，可用于检测各种目标物。
催化信标是当目标物存在时可以催化DNA酶水解底物链从而产生荧光信号增强的荧光检测体系。一般来说，催化信标由标记荧光团的底物链和标记猝灭团的DNA酶链组成，其中底物链与DNA酶链的比例应严格控制在≤1，而这样限制了催化信标在信号放大检测中的应用。另外，这种催化信标荧光猝灭效率较低，因此具有较高的荧光背景。在第4章中，基于具有特殊茎-环结构的分子信标和与之具有相同配对碱基的双链DNA之间熔链温度的差异，我们提出了一种利用分子信标作为底物链，从而避免对DNA酶修饰且能进行多重循环信号放大的新型荧光催化信标。而且该新型催化信标还可作为荧光放大基团用于目标DNA的检测。其动力学响应范围为0.5nM~300nM，检测下限为200pM。
为了进一步拓宽DNA酶的应用范围，在第5章中，我们基于DNA酶拓扑结构的改变可引起其催化活性的改变构建了一个通用的放大传感平台用于检测DNA序列和酶的活性。由于8-17DNAzyme的部分序列被固定于探针的发夹结构中，故酶的催化活性被抑制。当存在目标物（DNA或甲基化酶）时，目标物和探针作用使其发夹结构被打开，8-17DNA酶从发夹结构中释放出来且催化活性被激活。具有催化活性的8-17DNA酶可与发夹型的分子信标底物杂交形成催化分子信标传感体系。加入锌离子后，DNA酶催化切割分子信标底物，使底物链片段从酶链上游离下来，荧光明显增强。最后游离出来的8-17DNA酶的酶链可重新与分子信标底物杂交，引发下一轮的催化切割，如此循环，实现了目标物的放大检测。此外，我们还利用小分子末端保护策略和DNA酶的信号放大功能实现了蛋白质的放大检测。当不存在链霉亲和素（SA）时，DNA探针被核酸外切酶I（ExoI）切割为单碱基核苷酸，故不能催化切割分子信标底物，因此传感器的背景荧光很低。当存在SA时，探针上标记的生物素和SA结合从而阻止了ExoI切割DNA探针。最后包含8-17DNA酶序列的DNA探针可以催化切割分子信标底物并引起传感体系荧光信号的增强。
（2）在第6章中，我们将氧化石墨烯高的荧光猝灭率与核酸外切酶Ⅲ信号放大功能结合构建了一种高灵敏、快速、单标记的DNA荧光传感器。标记有荧光素（FAM）的DNA探针以单链形式存在故不能被核酸外切酶III切割，因此它可以通过π-π堆积作用吸附于GO表面并且荧光被猝灭。当目标DNA和探针链杂交后，核酸外切酶III沿3'→5'方向将探针链切割成碱基碎片，从而目标DNA被释放出来。释放出的目标DNA可继续和其它DNA探针杂交同时诱发第二次酶切循环反应。当酶切反应结束后加入适量的GO，由于DNA探针被切碎，故FAM远离GO而使其荧光不被猝灭。实验结果表明该方法可实现对目标DNA的高灵敏检测，检测下限为20pM。
（3）在第7章中，我们利用苝二酰亚胺阳离子化合物作为荧光信号的报告分子，富G核苷酸序列作为铅离子的识别探针构建了一种简单、快速和无标记的荧光增强型铅离子传感器。带正电荷的苝二酰亚胺阳离子化合物和带负电荷的DNA探针之间强的静电吸附作用导致苝二酰亚胺阳离子化合物吸附在DNA探针上并使其荧光被猝灭。当加入铅离子后，DNA探针从单链状态折叠成稳定的G四股螺旋结构。此时，苝二酰亚胺阳离子化合物与G四股螺旋结构的吸附力远低于它与单链DNA的吸附力，使得很多苝二酰亚胺阳离子化合物脱离DNA而游离于溶液中，从而导致传感体系的荧光增强。该传感器的动力学响应范围为10nM-10μM，检测限为4nM。
Biosensor is an emerging and active field originating from the interplay ofnumerous subjects, such as biology, physics, mathematics, chemistry and computertechnology. Recent years, as the new detection technologies constantly spring up andget perfected both in the research and practical application, the sensitivity and thespecificity of the biosensor have been largely improved. However, with the researchdeveloping, the development of the sensitive, selective and inexpensive biosensorsare of paramount importance for biomedical research and clinical diagnosis. Twodifferent strategies have been widely employed to improve the sensitivity of a sensor:lower the detection background and signal amplified detection.
In view of the considerations above and many relevant documents, we mainlyutilize the super fluorescence quencher and signal amplified strategy to improve thedetection sensitivity of the biosensor. The major contents are as follows:
(1) Study on the biosensors based on DNAzyme. DNAzymes (also calleddeoxyribozymes or DNA enzymes) are DNA sequences that can catalyze chemicalreactions, such as cleaving ribonucleic acid targets. Based on the remarkabledifference in affinity of GO with ssDNA containing different number of bases inlength, we constructed a graphene–DNAzyme based sensing system for amplifiedfluorescence “turn-on” detection of Pb~(2+)in chapter2. The DNAzyme-substratehybrid containing a large ssDNA loop make the fluorophore close to the GO surfaceto the greatest extent to afford a high quenching efficiency and a low backgroundfluorescence. Upon the addition of Pb~(2+), the DNAzyme was activated and cleaved thesubstrate strand, releasing a short FAM-linked oligonuleotide fragment, a relatedlong oligonuleotide fragment and the DNAzyme strand. The DNAzyme strand canhybridize with another substrate strand and then induce the second cycle of cleavageby binding Pb~(2+). The introduction of GO into the sensing solution will result in weakquenching of the fluorescence of FAM due to the weak affinity of the shortFAM-linked oligonuleotide fragment to GO, and the fluorescence intensity shouldgradually increase with increasing Pb~(2+)concentration added. Our proposed biosensorexhibits a high sensitivity towards target with a detection limit of300pM for Pb~(2+).
In chapter3, we employ cationic perylene derivative as a superquencher toconstruct DNAzyme-based fluorescence catalytic biosensors for the detection of Pb~(2+) and L-histidine. The strong affinity of perylene derivative with DNA drives it to forma complex with the DNAzyme-substrate hybrid, and efficiently quench thefluorescence of the FAM moiety. The introduction of Pb~(2+), however, will activateDNAzyme to catalyze the cleavage of the substrate in the RNA site, releasing a shortFAM-linked oligonuleotide fragment which show weak affinity with perylenederivative, and the introduction of perylene derivative into the system will showweak quenching effect on the FAM’s fluorescence. These results togetherdemonstrate that our design provides a convenient and universal platform foramplified detection of a wide range of targets.
The molecular beacon has the properties of low background and high sensitivity,and the huge difference with the melting temperature of other double-strand DNA.By utilizing the molecular beacons as the substrate sequence, a new kind offluorescent catalytic and molecular beacon has been developed in chapter4.Moreover, this design is willing to be applied in the fluorescence-based signalamplification for the detection of target DNA. A dynamic range from0.5nM to300nM for target DNA was achieved and the detection limit was200pM.
In chapter5, taking advantage of a topological effect of DNAzyme, we designeda new and general amplified sensing platform for nucleic acids and Dam MTaseactivity detection. Because the8-17DNAzyme is caged by partially hybridizing toform a hairpin structure, and is inactive to MB substrate, which provides a lowbackground for the sensing system. In the presence of the target, the hairpin structureis opened, and the8-17DNAzyme is liberated from the caged structure with itscatalytic activity being restored. The activated8-17DNAzyme can hybridize with theMB substrate to form the CAMB system and catalyze the cleavage of the MBsubstrate in the presence of cofactor Zn~(2+). After cleavage, the quenched MBfluorophore/quencher pair was separated from each other, resulting in an obviousincrease of fluorescent signal and a free DNAzyme strand. Eventually, eachtarget-induced activated8-17DNAzyme can undergo many cycles to trigger thecleavage of many MB substrates, achieving an amplified detection signal for thetarget. Furthermore, we constructed an amplified sensing platform for proteindetection based on the terminal protection of small-molecule-linked DNA. In theabsence of SA, the DNA probe was hydrolyzed successively into mononucleotides byExo I and cannot catalyze the cleavage of the MB substrate, therefore, providing azero-background for the sensing system. In the presence of SA, the probe is protectedfrom the degradation by Exo I. Consequently, the probe containing the8-17 DNAzyme sequence can catalyze the cleavage of the hairpin-structured MB substrateto induce a significant fluorescence enhancement.
(2) Using the GO as super fluorescence quencher and the Exo III as anamplifying biocatalyst, we develop a facile, sensitive, cost-effective method for DNAdetection in chapter6. In the absence of target DNA, Exo III is unable to catalyze theremoval of bases from the probe DNA. Upon the addition of GO, the fluorescence ofFAM was significantly quenched because of the strong binding between ssDNA andGO. In the presence of target DNA, Exo III can catalyze the stepwise removal ofmononucleotides of probe DNA from the blunt3′termini, which resulting in thereleasing of the target and fluorophore. The released target DNA can hybridize withanother probe DNA and then initiate a next round of cleavage. The introduction ofGO into the sensing solution will induce weak quenching of the fluorophore becauseof the weak affinity of the fluorophore and GO, and the fluorescence intensitygradually increases with increasing target concentration. The proposed biosensorexhibits high sensitivity, and a detection limit of20pM could be achieved for targetDNA.
(3) In chapter7, we report a simple, rapid and label-free approach forfluorescent “turn-on” detection of Pb~(2+)by using a water-soluble cationic perylenederivative (compound1) as the fluorescence reporter and the G-rich DNA probe forthe specific binding of Pb~(2+). Strong electrostatic interactions between the cationiccompound1and the polyanionic nucleic acid induced the aggregation of compound1and resulted in the fluorescence quenching. Upon the addition of Pb~(2+), theconformation of DNA probe changed from a random-coil to a Pb~(2+)-stabilizedG-quadruplex structure. This conformational change weakened the interactionbetween probe DNA and compound1. As a result, compound1is present in both thefree monomeric and the aggregated form in aqueous solution. Owing to the existenceof the free dye monomer, a fluorescence enhancement was observed. A dynamicrange from10nM to10μM for Pb~(2+)was achieved and the detection limit was4nM.
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