纳米及生物放大技术构建信号增强型电化学适体传感器的研究
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摘要
电化学适体传感器通过测定适体与目标物作用前后电化学信号的变化来实现对目标分析物的定量检测,具有操作简单、响应快速、灵敏度高、选择性好等优点。将材料、生物及化学等多种放大技术结合,是实现高灵敏检测的有效方法。本文主要从功能化纳米复合物的制备,电极敏感界面的构建,新型信号放大手段的运用以及检测通量的提高等方面来构建电化学适体传感器,并对其原理及性能等进行了探索和研究。研究工作分为以下几个部分：
1.双酶功能化的空心铂钴纳米链为信号探针构建凝血酶电化学适体传感器
采用模板法原位还原合成空心铂钴纳米链,用于修饰电活性物质二茂铁标记的凝血酶适体,再与葡萄糖氧化酶和辣根过氧化物模拟酶结合,制得适体和双酶功能化的空心铂钴纳米链复合物。本研究基于该纳米复合材料作为信号探针成功制得了一种夹心型的凝血酶电化学适体传感器。利用该空心纳米链大的比表面积,可有效提高电活性物质和双酶的固载量,增强电化学响应信号。当检测底液中存在葡萄糖时,葡萄糖氧化酶首先催化葡萄糖产生H202,生成的H202再通过辣根过氧化物模拟酶和纳米铂的催化,从而进一步增强二茂铁的电化学响应信号,提高检测的灵敏度。实验结果表明,利用空心铂钴纳米链作为固载基质可有效放大分析信号,提高适体传感器的灵敏度。
2.葡萄糖氧化酶功能化复合物的直接电化学及电催化放大构建凝血酶适体传感器
在玻碳电极表面修饰一层树枝状聚氨基胺-碳纳米管复合物膜作为传感器的敏感界面,利用聚氨基胺表面大量的官能团以及碳纳米管空心管状结构具有大的比表面积,在增加修饰电极有效面积的同时提高凝血酶适体的捕获量。此外,本研究制备了纳米铂功能化的还原石墨烯复合物(PtNPs@rGO),利用该复合物良好的生物相容性及大的活性表面在提高葡萄糖氧化酶(GOD)和适体固载量的同时保持其良好的生物活性,并以此构建了夹心型凝血酶适体传感器。利用葡萄糖氧化酶的直接电子传递及催化作用,并结合纳米铂的协调催化来实现检测信号的放大,提高检测的灵敏度。本实验对制备的复合纳米材料进行了表征,并对传感器的响应性能进行了研究。结果表明该方法切实可行,具有检测限低、选择性好等特点。
3.酶的直接电化学和生物放大技术构建凝血酶适体传感器
本研究利用辣根过氧化物酶的直接电化学,并结合两种新型生物放大技术,即目标物循环和杂交链式反应,实现了对于凝血酶的高灵敏检测。首先将巯基标记的捕获探针与凝血酶适体链互补形成双链并通过Au-S键固载到纳米金修饰的玻碳电极表面；当目标物凝血酶存在时,适体-凝血酶复合物的形成使得双链被破坏,同时在外切酶作用下将与凝血酶结合的适体进行剪切,使得目标物凝血酶重新被释放和循环。随后,将电极表面得到的单链捕获探针作为引发剂,与另外两条两端标记生物素的发夹型DNA进行杂交链式反应,使得电极表面含有大量生物素标记的双链聚合物结构,从而可以引入大量亲和素标记的辣根过氧化物酶,利用辣根过氧化物酶的直接电子传递以及其自身的催化提高检测的电化学信号,进一步提高传感器的灵敏度。
4.石墨烯复合纳米材料与生物放大技术构建信号增强型电化学适体传感器用于内毒素的超灵敏检测
目前,利用内毒素适体作为分子识别元件构建适体传感器来检测内毒素的方法少有报告,且灵敏度并不理想。因此,本研究利用三条DNA通过部分杂交形成的Y型连接体标记和剪切酶诱导的目标物循环放大,并结合纳米材料的信号放大构建了一种新型的信号增强型电化学适体传感器用于内毒素的超灵敏检测。石墨烯与电活性物质甲苯胺蓝通过π-π堆积作用形成的复合物能有效提高甲苯胺蓝的固载量,放大电化学信号,从而提高传感器的灵敏度。同时,Y型连接体标记的应用使得剪切酶的识别位点不局限于特点序列的目标DNA,只要将其中两条DNA(捕获探针和辅助探针)的碱基序列进行相应的改变,该方法对于其他目标生物分子的检测具有通用性。5.碳纳米管和石墨烯双重放大的电化学适体传感器用于多组分的同时检测
为了提高适体传感器的检测通量,本研究以功能化的石墨烯纳米复合物作为信号标记,构建了一种新型的夹心型电化学适体传感器,实现了在同一敏感界面对于血小板源性生长因子和凝血酶的同时检测。采用还原石墨烯作为载体通过化学键合作用修饰不同的电活性物质,随后在其表面原位还原产生纳米铂,用于固载对应的不同适体及葡萄糖氧化酶和辣根过氧化物酶,形成了多重标记的石墨烯纳米复合物。同时,采用纳米金包覆的单壁碳纳米管增加电极的有效表面积来增加适体的捕获量,进一步增加石墨烯纳米复合物的固载量。实验结果表明该复合物具有良好的电化学氧化还原活性,结合纳米铂与双酶良好的协同电催化活性,进一步放大电化学信号,从而实现了对于两种目标蛋白同时检测的高特异性和高灵敏度。
Electrochemical aptasensors are valuable analytical tools for monitoring target analytes through the changes of electrochemical signals before and after the binding between aptamers and target analytes, which hold the advantages of high sensitivity, good selectivity, rapid response and simple operation. Using a variety of amplification technology such as chemical, material and biological combination, could effectively enhance the detection sensitivity. Therefore, this research focuses on the preparation of functionalized nanocomposite, the construction of sensitive interface, the application of novel signal amplification strategies and the enhancement of detection efficiency to construct the electrochemical aptasensors. The research contents are mainly as follows:
1. Bi-enzyme functionalized hollow PtCo nanochains as labels for an electrochemical aptasensor
Hollow PtCo nanochains (HPtCoNCs) were synthesized by employing Co nanoparticles produced in situ as templates, which were used for the immobilization of ferrocene-labeled secondary thrombin aptamer (Fc-TBA2) and further functionalized by bi-enzyme—glucose oxidase (GOD) and horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme). This work successfully constructed a sandwich-type electrochemical aptasensor for thrombin detection using the as-prepared multi-functionalized nanomaterials as tracer labels. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, glucose is rapidly oxidized into gluconic acid accompanying with the generation of H2O2, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors.
2. Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin
Poly(amino-amine) dendrimers-encapsulated carbon nanotubes (PAMAM-CNTs) with high surface area was modified on the surface of glassy carbon electrode as the sensor platform to enhance effective area of the modified electrode and increase the immobilization of primary thrombin aptamers (TBA1). In addition, reduced graphene oxide (rGO) was employed to support platinum nanoparticles (PtNPs), forming PtNPs@rGO nanocomposite with good biocompatibility for the decoration of glucose oxidase (GOD) and secondary thrombin aptamers (TBA2) with high loading amount and good biological activity. With the excellent direct electron transfer of GOD and the synergistic electrocatalysis of GOD and PtNPs, a new sandwich-type electrochemical aptasensor was constructed for ultrasensitive detection of thrombin. The preparation and characterization of nanocomposites, as well as the construction and performance of the aptasensor, were also studied. The results showed that this method is feasible with low detection limit and good selectivity.
3. Amperometric aptasensor for thrombin detection using enzyme-mediated direct electrochemistry and DNA-based signal amplification strategy
This work developed an electrochemical aptasensor for highly sensitive detection of thrombin based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using two typical biotechnology amplification—exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) for signal amplification. To construct the aptasensor, double-stranded DNA (dsDNA) of the thiolated capture probe and thrombin binding aptamer was immobilized on gold nanoparticles (AuNPs) modified electrode through Au-S bond. In the presence of thrombin, the formation of aptamer-thrombin complex could result in the dissociation of aptamer from the dsDNA. Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The resulted single stranded capture probe was used as the initiator to trigger the HCR of two biotin-labeled hairpin helper DNAs and lead to the formation of extended dsDNA polymers on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical detection signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of exonuclease-catalyzed analyte recycling and HCR, as well as the inherent electroactivity and electrocatalytic activity of HRP, which exhibited high sensitivity for thrombin determination.
4. A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplification
To date, using lipopolysaccharide (LPS) binding aptamer as molecular recognition element of aptasensors for detection LPS have been rarely reported, and the sensitivity of these aptasensors are dissatisfactory. Thus, this work described a new signal-on electrochemical aptasensor for ultrasensitive detection of LPS by combining the three-way DNA junction acided enzymatic target recycling and nanotechnology for amplification. Toluidine blue (Tb), a kind of aromatic molecules with electrochemical activity, can decorate graphene (Gra) to obtain the Tb-Gra nanocomposite through π-π stacking, which not only improved the solubility and self-assembly properties of Gra, but also increased the immobilization of Tb and enhanced the electrochemical signal. Moreover, the application of three-way DNA junction makes the recognition site of restrictive endonuclease unconstrained on the specified sequence of target DNA. Thus, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of capture probe and assistant probe.
5. Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of carbon nanotubes and multi-labeled graphene sheets
To improve the detection efficiency of aptasensor, this work fabricated a sandwich-type electrochemical aptasensor for one-spot simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin using graphene-nanocomposites as tracer labels. Reduced graphene oxide sheets (rGO) were used as matrices to immobilize the different redox probes, which were subsequently coated with the platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGO nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bi-enzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGO nanocomposites as tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized carbon nanotubes (AuNPs@CNTs) as the sensor platform could enhance the surface area of electrode to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results showed that multi-labeled PtNPs-redox probes-rGO nanocomposites display satisfactory electrochemical redox activity and highly electrocatalytic activity of PtNPs and bi-enzyme, which exhibited high sensitivity and specificity for detection of proteins.
1.双酶功能化的空心铂钴纳米链为信号探针构建凝血酶电化学适体传感器
采用模板法原位还原合成空心铂钴纳米链,用于修饰电活性物质二茂铁标记的凝血酶适体,再与葡萄糖氧化酶和辣根过氧化物模拟酶结合,制得适体和双酶功能化的空心铂钴纳米链复合物。本研究基于该纳米复合材料作为信号探针成功制得了一种夹心型的凝血酶电化学适体传感器。利用该空心纳米链大的比表面积,可有效提高电活性物质和双酶的固载量,增强电化学响应信号。当检测底液中存在葡萄糖时,葡萄糖氧化酶首先催化葡萄糖产生H202,生成的H202再通过辣根过氧化物模拟酶和纳米铂的催化,从而进一步增强二茂铁的电化学响应信号,提高检测的灵敏度。实验结果表明,利用空心铂钴纳米链作为固载基质可有效放大分析信号,提高适体传感器的灵敏度。
2.葡萄糖氧化酶功能化复合物的直接电化学及电催化放大构建凝血酶适体传感器
在玻碳电极表面修饰一层树枝状聚氨基胺-碳纳米管复合物膜作为传感器的敏感界面,利用聚氨基胺表面大量的官能团以及碳纳米管空心管状结构具有大的比表面积,在增加修饰电极有效面积的同时提高凝血酶适体的捕获量。此外,本研究制备了纳米铂功能化的还原石墨烯复合物(PtNPs@rGO),利用该复合物良好的生物相容性及大的活性表面在提高葡萄糖氧化酶(GOD)和适体固载量的同时保持其良好的生物活性,并以此构建了夹心型凝血酶适体传感器。利用葡萄糖氧化酶的直接电子传递及催化作用,并结合纳米铂的协调催化来实现检测信号的放大,提高检测的灵敏度。本实验对制备的复合纳米材料进行了表征,并对传感器的响应性能进行了研究。结果表明该方法切实可行,具有检测限低、选择性好等特点。
3.酶的直接电化学和生物放大技术构建凝血酶适体传感器
本研究利用辣根过氧化物酶的直接电化学,并结合两种新型生物放大技术,即目标物循环和杂交链式反应,实现了对于凝血酶的高灵敏检测。首先将巯基标记的捕获探针与凝血酶适体链互补形成双链并通过Au-S键固载到纳米金修饰的玻碳电极表面；当目标物凝血酶存在时,适体-凝血酶复合物的形成使得双链被破坏,同时在外切酶作用下将与凝血酶结合的适体进行剪切,使得目标物凝血酶重新被释放和循环。随后,将电极表面得到的单链捕获探针作为引发剂,与另外两条两端标记生物素的发夹型DNA进行杂交链式反应,使得电极表面含有大量生物素标记的双链聚合物结构,从而可以引入大量亲和素标记的辣根过氧化物酶,利用辣根过氧化物酶的直接电子传递以及其自身的催化提高检测的电化学信号,进一步提高传感器的灵敏度。
4.石墨烯复合纳米材料与生物放大技术构建信号增强型电化学适体传感器用于内毒素的超灵敏检测
目前,利用内毒素适体作为分子识别元件构建适体传感器来检测内毒素的方法少有报告,且灵敏度并不理想。因此,本研究利用三条DNA通过部分杂交形成的Y型连接体标记和剪切酶诱导的目标物循环放大,并结合纳米材料的信号放大构建了一种新型的信号增强型电化学适体传感器用于内毒素的超灵敏检测。石墨烯与电活性物质甲苯胺蓝通过π-π堆积作用形成的复合物能有效提高甲苯胺蓝的固载量,放大电化学信号,从而提高传感器的灵敏度。同时,Y型连接体标记的应用使得剪切酶的识别位点不局限于特点序列的目标DNA,只要将其中两条DNA(捕获探针和辅助探针)的碱基序列进行相应的改变,该方法对于其他目标生物分子的检测具有通用性。5.碳纳米管和石墨烯双重放大的电化学适体传感器用于多组分的同时检测
为了提高适体传感器的检测通量,本研究以功能化的石墨烯纳米复合物作为信号标记,构建了一种新型的夹心型电化学适体传感器,实现了在同一敏感界面对于血小板源性生长因子和凝血酶的同时检测。采用还原石墨烯作为载体通过化学键合作用修饰不同的电活性物质,随后在其表面原位还原产生纳米铂,用于固载对应的不同适体及葡萄糖氧化酶和辣根过氧化物酶,形成了多重标记的石墨烯纳米复合物。同时,采用纳米金包覆的单壁碳纳米管增加电极的有效表面积来增加适体的捕获量,进一步增加石墨烯纳米复合物的固载量。实验结果表明该复合物具有良好的电化学氧化还原活性,结合纳米铂与双酶良好的协同电催化活性,进一步放大电化学信号,从而实现了对于两种目标蛋白同时检测的高特异性和高灵敏度。
Electrochemical aptasensors are valuable analytical tools for monitoring target analytes through the changes of electrochemical signals before and after the binding between aptamers and target analytes, which hold the advantages of high sensitivity, good selectivity, rapid response and simple operation. Using a variety of amplification technology such as chemical, material and biological combination, could effectively enhance the detection sensitivity. Therefore, this research focuses on the preparation of functionalized nanocomposite, the construction of sensitive interface, the application of novel signal amplification strategies and the enhancement of detection efficiency to construct the electrochemical aptasensors. The research contents are mainly as follows:
1. Bi-enzyme functionalized hollow PtCo nanochains as labels for an electrochemical aptasensor
Hollow PtCo nanochains (HPtCoNCs) were synthesized by employing Co nanoparticles produced in situ as templates, which were used for the immobilization of ferrocene-labeled secondary thrombin aptamer (Fc-TBA2) and further functionalized by bi-enzyme—glucose oxidase (GOD) and horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme). This work successfully constructed a sandwich-type electrochemical aptasensor for thrombin detection using the as-prepared multi-functionalized nanomaterials as tracer labels. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, glucose is rapidly oxidized into gluconic acid accompanying with the generation of H2O2, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors.
2. Direct electrochemistry and electrocatalysis of a glucose oxidase-functionalized bioconjugate as a trace label for ultrasensitive detection of thrombin
Poly(amino-amine) dendrimers-encapsulated carbon nanotubes (PAMAM-CNTs) with high surface area was modified on the surface of glassy carbon electrode as the sensor platform to enhance effective area of the modified electrode and increase the immobilization of primary thrombin aptamers (TBA1). In addition, reduced graphene oxide (rGO) was employed to support platinum nanoparticles (PtNPs), forming PtNPs@rGO nanocomposite with good biocompatibility for the decoration of glucose oxidase (GOD) and secondary thrombin aptamers (TBA2) with high loading amount and good biological activity. With the excellent direct electron transfer of GOD and the synergistic electrocatalysis of GOD and PtNPs, a new sandwich-type electrochemical aptasensor was constructed for ultrasensitive detection of thrombin. The preparation and characterization of nanocomposites, as well as the construction and performance of the aptasensor, were also studied. The results showed that this method is feasible with low detection limit and good selectivity.
3. Amperometric aptasensor for thrombin detection using enzyme-mediated direct electrochemistry and DNA-based signal amplification strategy
This work developed an electrochemical aptasensor for highly sensitive detection of thrombin based on direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) using two typical biotechnology amplification—exonuclease-catalyzed target recycling and hybridization chain reaction (HCR) for signal amplification. To construct the aptasensor, double-stranded DNA (dsDNA) of the thiolated capture probe and thrombin binding aptamer was immobilized on gold nanoparticles (AuNPs) modified electrode through Au-S bond. In the presence of thrombin, the formation of aptamer-thrombin complex could result in the dissociation of aptamer from the dsDNA. Subsequently, with the employment of exonuclease, aptamer was selectively digested and thrombin could be released for analyte recycling. The resulted single stranded capture probe was used as the initiator to trigger the HCR of two biotin-labeled hairpin helper DNAs and lead to the formation of extended dsDNA polymers on the electrode surface. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled HRP, resulting in significantly amplified electrochemical detection signal through the direct electrochemistry and electrocatalysis of HRP. The proposed strategy combined the amplification of exonuclease-catalyzed analyte recycling and HCR, as well as the inherent electroactivity and electrocatalytic activity of HRP, which exhibited high sensitivity for thrombin determination.
4. A signal-on electrochemical aptasensor for ultrasensitive detection of endotoxin using three-way DNA junction-aided enzymatic recycling and graphene nanohybrid for amplification
To date, using lipopolysaccharide (LPS) binding aptamer as molecular recognition element of aptasensors for detection LPS have been rarely reported, and the sensitivity of these aptasensors are dissatisfactory. Thus, this work described a new signal-on electrochemical aptasensor for ultrasensitive detection of LPS by combining the three-way DNA junction acided enzymatic target recycling and nanotechnology for amplification. Toluidine blue (Tb), a kind of aromatic molecules with electrochemical activity, can decorate graphene (Gra) to obtain the Tb-Gra nanocomposite through π-π stacking, which not only improved the solubility and self-assembly properties of Gra, but also increased the immobilization of Tb and enhanced the electrochemical signal. Moreover, the application of three-way DNA junction makes the recognition site of restrictive endonuclease unconstrained on the specified sequence of target DNA. Thus, the high sensitivity and specificity make this method versatile for the detection of other biomolecules by changing the corresponding sequences of capture probe and assistant probe.
5. Simultaneous electrochemical detection of multiple analytes based on dual signal amplification of carbon nanotubes and multi-labeled graphene sheets
To improve the detection efficiency of aptasensor, this work fabricated a sandwich-type electrochemical aptasensor for one-spot simultaneous sensitive detection of platelet-derived growth factor (PDGF) and thrombin using graphene-nanocomposites as tracer labels. Reduced graphene oxide sheets (rGO) were used as matrices to immobilize the different redox probes, which were subsequently coated with the platinum nanoparticles (PtNPs) to form the PtNPs-redox probes-rGO nanocomposites. With the employment of the as prepared nanocomposites, a signal amplification strategy was described based on bi-enzyme (glucose oxidase and horseradish peroxidase) modified PtNPs-redox probes-rGO nanocomposites as tracer labels for secondary aptamers (Apt II) through sandwiched assay. Gold nanoparticles functionalized carbon nanotubes (AuNPs@CNTs) as the sensor platform could enhance the surface area of electrode to capture a large amount of primary aptamers (Apt I), thus amplifying the detection response. The experiment results showed that multi-labeled PtNPs-redox probes-rGO nanocomposites display satisfactory electrochemical redox activity and highly electrocatalytic activity of PtNPs and bi-enzyme, which exhibited high sensitivity and specificity for detection of proteins.
引文
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