基于DNA适配体芯片上电致化学发光生物传感器的制备及应用研究
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摘要
适配体是经过SELEX技术筛选合成出来的单链DNA或者RNA短链。山于适配体具有制备方便、稳定性好、亲和力强,且能高效、特异性的结合各种生物目标分子,使其越来越受研究工作者的关注。近年来,DNA适配体已经成为临床、药物、食品、卫生及环境检测等领域的研究热点。
量子点具有独特的小尺寸效应和荧光特性以及良好的生物兼容性,已被广泛的应用到各个领域。电致化学发光分析法(ECL)因不需要光源、背景干扰小、较高的灵敏度、较宽的线性范围以及良好的重现性和选择性,使其逐渐成为分析检测中的重要手段。这些技术的有效结合使得高灵敏度、高选择性,甚至在线、实时、活体检测等成为可能。本论文在上述基础上开展了以下研究工作：
设计了一种新型、灵敏、简单、仅用一条适配体构建的ATP三明治夹心型电致化学发光生物传感器。首先在ITO电极上原位还原低表面密度的纳米金,然后再组装上适配体的一段断链。利用适配体两条断链天然构型之间的吸引来完成对目标分子的识别。体系的ECL信号随着ATP的浓度增加而增大。依此建立的方法的线性范围为0.2～100 nmol/L。检出限为0.02 nmol/L。方法具有高选择性和高灵敏度,适用于其它生物小分子。
基于量子点芯片电致化学发光,构建了一种新型的DNA适配体生物传感器用于检测凝血酶。首先刻蚀芯片电极,然后在ITO电极上原位合成纳米金。然后通过巯基连接上凝血酶适配体,再通过温育固定上凝血酶目标物。而没有结合凝血酶的DNA适配体与5′-生物素标记的互补DNA低聚核苷酸(cDNA)杂交形成双链DNA低聚核苷酸结构,再结合亲和素标记的量子点。此传感器的电化学信号与凝血酶浓度成反比,依此建立了凝血酶的分析方法。方法具有选择性好、稳定性高和灵敏度高等特点。
Aptamers are single stranded DNA molecules or RNA that produced by SELEX technology. Aptamers have received widely attention because of its merits, such as simple synthesis, good stability, high affinity and specificity to many biologic targets. Recently, aptamers have been fleetly developed and used in many fields such as clinical pharmacy, food hygiene, environmental supervision, etc.
Quantum dots (QDs) have received tremendous attention for their possible luminescent applications in aqueous solution. With size tunable narrow emission spectra and broad excitation spectra, they have been widely used as multicolored luminescent probes, biological labels and bioimaging. Electrogenerated chemiluminescence (ECL) has many advantages such as needless special light, low background, higher sensitivity, wide calibration ranges and better selectivity, so it has great application potentials in analytical chemistry. The method based above techniques is highly sensitive and selective, can be used in real-time, on-line and in-vivo analysis. The main content is listed as follows: A novel electrogenerated chemiluminescence (ECL) sandwich assay for the determination of small molecule is designed employing a single aptamer sequence as molecular recognition element for ATP as a model analyte and quantum dots served as an ECL label. This sandwich-type sensor is fabricated on an indium tin oxide chip covered with a low surface coverage of gold nanoparticles. A single ATP aptamer, split into two fragments, is used to recognize the target and construct the sandwich-type ECL biosensors. An enhanced ECL signal is generated upon recognition of the target ATP, attributed to a change in the conformation of the ECL probe from random coil-like configuration on the probe-modified film, in close proximity to the sensor interface. The integrated ECL intensity versus the concentration of ATP was linear in the range from 0.2 to 100 nM. The detection limit was 0.02 nM. This work demonstrates that the combination of a highly binding aptamer to analyte with a highly sensitive ECL technique to design The DNA aptamber biosensor is a great promising approach for the determination of small molecule drugs. A novel electrogenerated chemiluminescent biosensor based on DNA aptamer was constructed for the detection of thrombin. A low surface coverage of gold nanoparticles was firstly reduced on an ITO electrode on a chip. Thrombin was inclubated with probes inmobilized on gold nanoparticles. The no-label probes were hybridized with the 5'-biotin modified cDNA oligonucleotides and then coupled to Avidin-QDs. The ECL signal of the biosensor decreased with the increase of target protein. The analytical method was developed based on this. The biosensor has good stability, high sensitivity and specificity.
量子点具有独特的小尺寸效应和荧光特性以及良好的生物兼容性,已被广泛的应用到各个领域。电致化学发光分析法(ECL)因不需要光源、背景干扰小、较高的灵敏度、较宽的线性范围以及良好的重现性和选择性,使其逐渐成为分析检测中的重要手段。这些技术的有效结合使得高灵敏度、高选择性,甚至在线、实时、活体检测等成为可能。本论文在上述基础上开展了以下研究工作：
设计了一种新型、灵敏、简单、仅用一条适配体构建的ATP三明治夹心型电致化学发光生物传感器。首先在ITO电极上原位还原低表面密度的纳米金,然后再组装上适配体的一段断链。利用适配体两条断链天然构型之间的吸引来完成对目标分子的识别。体系的ECL信号随着ATP的浓度增加而增大。依此建立的方法的线性范围为0.2～100 nmol/L。检出限为0.02 nmol/L。方法具有高选择性和高灵敏度,适用于其它生物小分子。
基于量子点芯片电致化学发光,构建了一种新型的DNA适配体生物传感器用于检测凝血酶。首先刻蚀芯片电极,然后在ITO电极上原位合成纳米金。然后通过巯基连接上凝血酶适配体,再通过温育固定上凝血酶目标物。而没有结合凝血酶的DNA适配体与5′-生物素标记的互补DNA低聚核苷酸(cDNA)杂交形成双链DNA低聚核苷酸结构,再结合亲和素标记的量子点。此传感器的电化学信号与凝血酶浓度成反比,依此建立了凝血酶的分析方法。方法具有选择性好、稳定性高和灵敏度高等特点。
Aptamers are single stranded DNA molecules or RNA that produced by SELEX technology. Aptamers have received widely attention because of its merits, such as simple synthesis, good stability, high affinity and specificity to many biologic targets. Recently, aptamers have been fleetly developed and used in many fields such as clinical pharmacy, food hygiene, environmental supervision, etc.
Quantum dots (QDs) have received tremendous attention for their possible luminescent applications in aqueous solution. With size tunable narrow emission spectra and broad excitation spectra, they have been widely used as multicolored luminescent probes, biological labels and bioimaging. Electrogenerated chemiluminescence (ECL) has many advantages such as needless special light, low background, higher sensitivity, wide calibration ranges and better selectivity, so it has great application potentials in analytical chemistry. The method based above techniques is highly sensitive and selective, can be used in real-time, on-line and in-vivo analysis. The main content is listed as follows: A novel electrogenerated chemiluminescence (ECL) sandwich assay for the determination of small molecule is designed employing a single aptamer sequence as molecular recognition element for ATP as a model analyte and quantum dots served as an ECL label. This sandwich-type sensor is fabricated on an indium tin oxide chip covered with a low surface coverage of gold nanoparticles. A single ATP aptamer, split into two fragments, is used to recognize the target and construct the sandwich-type ECL biosensors. An enhanced ECL signal is generated upon recognition of the target ATP, attributed to a change in the conformation of the ECL probe from random coil-like configuration on the probe-modified film, in close proximity to the sensor interface. The integrated ECL intensity versus the concentration of ATP was linear in the range from 0.2 to 100 nM. The detection limit was 0.02 nM. This work demonstrates that the combination of a highly binding aptamer to analyte with a highly sensitive ECL technique to design The DNA aptamber biosensor is a great promising approach for the determination of small molecule drugs. A novel electrogenerated chemiluminescent biosensor based on DNA aptamer was constructed for the detection of thrombin. A low surface coverage of gold nanoparticles was firstly reduced on an ITO electrode on a chip. Thrombin was inclubated with probes inmobilized on gold nanoparticles. The no-label probes were hybridized with the 5'-biotin modified cDNA oligonucleotides and then coupled to Avidin-QDs. The ECL signal of the biosensor decreased with the increase of target protein. The analytical method was developed based on this. The biosensor has good stability, high sensitivity and specificity.
引文
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