新兴适配体在高效净化/诊断环境与生物体内污染物的特点及其机理
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摘要
适配体是一类新型识别分子，本质是一段单链的DNA或者RNA，从随机核苷酸文库中筛选而来。它能够通过自身三维构象的变化来特异性地捕捉靶分子，从而达到分离富集靶分子(污染物)的目的。适配体与其它分离富集材料相比，具有自己独特的优势，例如特异亲和性高、分子量小、体外合成、容易化学修饰、低毒性、生物兼容性好等。基于适配体的特性，其已经在化学、医学和生物学上引起了广泛的关注，但是在环境科学领域的相关研究比较匮乏。这个课题试图研究适配体在环境污染净化与健康上的应用及其机理。具体研究方法和结果如下：
(1)适配体固定到琼脂糖颗粒上，然后装配到小柱内，用于饮用水中小分子污染物的净化。适配体不但可以高效净化污水，而且容易再生，达到重复利用的目的。在这部分内容中，我们首次探讨了适配体与小分子污染物的作用机理。适配体吸附小分子污染物符合假二级动力学，且为自发放热反应。适配体与小分子污染物的作用过程可以分为三个步骤，即活化步骤、吸附步骤和杂交步骤；又可以将整个反应过程分为三个阶段，即前期的化学动力学控速阶段，后期的热动力学控速阶段和中间的过渡阶段。这部分研究内容提供了一种净化废水中小分子污染物的新技术，并首次阐述了适配体作用小分子污染物的机理。
(2)适配体通过氨基和羧基作用固定到氧化石墨烯上，增强了氧化石墨烯的水溶性和特异性亲和污染物的能力，并提高了适配体自身抵抗核酸酶降解的能力。我们进一步探讨了pH、温度、离子强度和天然有机质对适配体活性的影响。极端的pH和高浓度的天然有机质会导致适配体失活，从而降低适配体吸附污染物的能力。适配体在低温下具有较强的亲和污染物能力。随着温度升高，适配体吸附污染物的能力出现了轻微的降低。一定范围的离子强度，通过缓解静电排斥和连接架桥作用，增强适配体吸附污染物的能力。这部分研究内容首次详细地分析了环境因素对适配体净化污染物的影响，建立了一种稳定净化污染物的新方法。
(3)适配体在固定到氧化石墨烯上之后，在可见光下促进了氧化石墨烯光催化降解生物型污染物-噬菌体的能力。课题深入地研究了适配体提高石墨烯催化降解生物型污染物的机制。首先噬菌体表面蛋白衣壳被破坏，然后包裹的核酸释放出来。随后，蛋白发生羰基化，核酸发生氧化修饰，氧化石墨烯被还原。在催化过程中，产生的可能活性物种包括羟基自由基、氧自由基和水解离前体物质等。催化过程包含了能量的转化和电子的转移。这部分研究内容提出了一种在可见光下催化降解生物型污染物的新方法，并探索了可能的反应机理。
(4)适配体固定到纳米颗粒上，装载重金属的螯合剂，用来有效地缓解重金属在生物体内的毒性。实验以汞为例，研究了适配体对生物体内汞毒性的影响。适配体纳米颗粒减少了汞在动物肾和脑中的积累，增强了尿液中汞的排泄。行为试验表明了适配体可以缓解受汞污染动物的神经毒性，增强动物的学习和记忆功能。通过血液学研究，实验进一步证实了适配体纳米颗粒具有很好的生物兼容性。适配体纳米颗粒缓解汞毒性的机制包括氧化损伤的降低和关键酶活性的升高。这部分研究内容提出了一种缓解重金属在生物体内毒性的新方法，并探讨了可能的毒性机制。
(5)实验通过溶胶-凝胶的方法将适配体固定到固相微萃取表面，用来快速准确地分析水溶液和和血浆中的生物标志物腺苷。实验首先优化了适配体–固相微萃取的解吸时间和解吸溶剂，然后探讨了温度和离子强度对适配体吸附靶分子的影响，进一步研究了适配体–固相微萃取的存贮和再生能力，最后用适配体固相微萃取成功地分析了血浆中低浓度的腺苷。考虑到固相微萃取具有in vivo取样的能力和在代谢物分析上的优势，生物兼容性的适配体–固相微萃取有望成为一种灵敏诊断环境污染与健康的新装置。
上述的研究结果表明了适配体可以作为一种新型的生物材料用于污染物的分离、富集、净化以及缓解体内污染物毒性，甚至用于环境污染与健康的早期诊断。尽管适配体在环境污染净化与健康上的应用存在一些问题，例如适配体的不稳定性和轻微的毒副作用，但是它的应用潜力值得进一步深入研究。
Aptamers are a new class of single-stranded DNA/RNA molecules selected fromsynthetic nucleic acid libraries for molecular recognition. These single-strandDNA/RNA folded into their specific secondary or tertiary structures are mainlyresponsible for binding target molecules, leading to the separation and accumulationof target molecules (pollutants). Compared to other function materials, aptamer hasunique merits including high affinity, low molecule weight, easy to modification, lowtoxicity and excellent biocompatibility. Given the advantages, aptamer is attractive inchemical, medical, and biological fields, but the related research is rare inenvironmental science. In the present work, the applications and mechanisms ofaptamer were explored in environmental purification and health. The methods andresults are presented as following:
(1) Aptamer was immobilized on CNBr-activated Sepharos and then packed in acolumn to remove low molecule weight polluntants in drinking water. The aptamercan effectively purity water and regenerate. In this section, the interactions of aptamerand pollutant were firstly studied. The adsorption data were fit well by the pseudo-second kinetics with an exothermic reaction. The reaction involved three steps:activation, adsorption and incubation. The whole reaction also could be divided intochemical kinetics control period, thermodynamic control period and transition period.The section provided a new technology to purity contaminated water and explored theinteraction mechanisms between aptamer and low molecule weight polluntants.
(2) Aptamer was immobilized on graphene oxide by the reaction of aminogroups and carboxyl groups. The complex improved the water solubility and thespecificity of graphene oxide as well as the protection of aptamer from nuclease.Furthermore, the effects of pH, temperature, ionic strength and natural organic matteron the activity of aptamer were studied.The affinity would reduce in extreme pH andhigh natural organic matter. The removal of pollutants slightly reduced with theincrease of temperature. A certain concentration of ionic strength enhanced theremoval of pollutants due to the decrease of electrostatic repel forces and development of connection bridge. The section discussed the influence ofenvironmental factors and provided a stable method to purify the pollutants incontaminated water.
(3) Aptamer enhanced the photocatalytic activities of graphene oxide forbiological contaminantion-bacteriophage under broad visible-light spectrum.Moreover, the photocatalytic mechanisms were explored. The protein coating wasbroke, and then enveloped nucleic acid released. Subsequently, the proteins posedcarbonylation and the nucleic acid bases received oxidized modification. Finaly, thegraphene oxide was reduced. The active species generated during the photocatalyticprocess. The photocatalysis paths involved energy transfer (generation of singletoxygen), electron transfer (generation of anion radicals) and water dissociation(generation of the precursor to the hydrated electron). The section showed a new ideato enhance the photocatalytic activities of graphene oxide for biologic contaminantionunder broad visible-light spectrum and studied the possible mechanisms.
(4) Aptamer was immobilized on nanoparticle as well as the antidote of mercurywas packed. The bioconjugates can diminish the toxicity of mercury in vivo. Thenanoparticle-aptamer markedly reduced the accumulation of mercury in both thebrain and kidney, and enhanced the excretion of urinary mercury. Thenanoparticle-aptamer ameliorated the neurotoxicity and improved the learning andmemory of animals. The toxicology of nanoparticle-aptamer was also studied byhematologic tests, and exhibited excellent biocompatibility. The section estabilished anew method to diminish the toxicity of heavy metals in vivo and expounded themechanisms.
(5) Aptamer was immobilized on solid phase micro-extraction by sol-gel methodto quickly and accurately detect a biomarker-adenosine in water solution and plasma.The desorption time and desorption solution were optimized, and then the effects oftemperature and ionic strength were discussed. Finally, the storage and regenerationof aptamer were studied. Given the special assay of solid phase micro-extraction invivo and metabolomics, aptamer/solid phase micro-extraction can be a device todiagnose environmental contamination and health.
The above results suggest aptamer can be a novel material for the separation, accumulation and purification of pollutants, and the development the toxicity ofheavy metals in vivo, even the diagnosis of environmental contamination and health.Although there are some challenges for the application of aptamer, such as the lowstability and adverse effect, it is worth to conduct the related research of aptamerfurthermore.
(1)适配体固定到琼脂糖颗粒上，然后装配到小柱内，用于饮用水中小分子污染物的净化。适配体不但可以高效净化污水，而且容易再生，达到重复利用的目的。在这部分内容中，我们首次探讨了适配体与小分子污染物的作用机理。适配体吸附小分子污染物符合假二级动力学，且为自发放热反应。适配体与小分子污染物的作用过程可以分为三个步骤，即活化步骤、吸附步骤和杂交步骤；又可以将整个反应过程分为三个阶段，即前期的化学动力学控速阶段，后期的热动力学控速阶段和中间的过渡阶段。这部分研究内容提供了一种净化废水中小分子污染物的新技术，并首次阐述了适配体作用小分子污染物的机理。
(2)适配体通过氨基和羧基作用固定到氧化石墨烯上，增强了氧化石墨烯的水溶性和特异性亲和污染物的能力，并提高了适配体自身抵抗核酸酶降解的能力。我们进一步探讨了pH、温度、离子强度和天然有机质对适配体活性的影响。极端的pH和高浓度的天然有机质会导致适配体失活，从而降低适配体吸附污染物的能力。适配体在低温下具有较强的亲和污染物能力。随着温度升高，适配体吸附污染物的能力出现了轻微的降低。一定范围的离子强度，通过缓解静电排斥和连接架桥作用，增强适配体吸附污染物的能力。这部分研究内容首次详细地分析了环境因素对适配体净化污染物的影响，建立了一种稳定净化污染物的新方法。
(3)适配体在固定到氧化石墨烯上之后，在可见光下促进了氧化石墨烯光催化降解生物型污染物-噬菌体的能力。课题深入地研究了适配体提高石墨烯催化降解生物型污染物的机制。首先噬菌体表面蛋白衣壳被破坏，然后包裹的核酸释放出来。随后，蛋白发生羰基化，核酸发生氧化修饰，氧化石墨烯被还原。在催化过程中，产生的可能活性物种包括羟基自由基、氧自由基和水解离前体物质等。催化过程包含了能量的转化和电子的转移。这部分研究内容提出了一种在可见光下催化降解生物型污染物的新方法，并探索了可能的反应机理。
(4)适配体固定到纳米颗粒上，装载重金属的螯合剂，用来有效地缓解重金属在生物体内的毒性。实验以汞为例，研究了适配体对生物体内汞毒性的影响。适配体纳米颗粒减少了汞在动物肾和脑中的积累，增强了尿液中汞的排泄。行为试验表明了适配体可以缓解受汞污染动物的神经毒性，增强动物的学习和记忆功能。通过血液学研究，实验进一步证实了适配体纳米颗粒具有很好的生物兼容性。适配体纳米颗粒缓解汞毒性的机制包括氧化损伤的降低和关键酶活性的升高。这部分研究内容提出了一种缓解重金属在生物体内毒性的新方法，并探讨了可能的毒性机制。
(5)实验通过溶胶-凝胶的方法将适配体固定到固相微萃取表面，用来快速准确地分析水溶液和和血浆中的生物标志物腺苷。实验首先优化了适配体–固相微萃取的解吸时间和解吸溶剂，然后探讨了温度和离子强度对适配体吸附靶分子的影响，进一步研究了适配体–固相微萃取的存贮和再生能力，最后用适配体固相微萃取成功地分析了血浆中低浓度的腺苷。考虑到固相微萃取具有in vivo取样的能力和在代谢物分析上的优势，生物兼容性的适配体–固相微萃取有望成为一种灵敏诊断环境污染与健康的新装置。
上述的研究结果表明了适配体可以作为一种新型的生物材料用于污染物的分离、富集、净化以及缓解体内污染物毒性，甚至用于环境污染与健康的早期诊断。尽管适配体在环境污染净化与健康上的应用存在一些问题，例如适配体的不稳定性和轻微的毒副作用，但是它的应用潜力值得进一步深入研究。
Aptamers are a new class of single-stranded DNA/RNA molecules selected fromsynthetic nucleic acid libraries for molecular recognition. These single-strandDNA/RNA folded into their specific secondary or tertiary structures are mainlyresponsible for binding target molecules, leading to the separation and accumulationof target molecules (pollutants). Compared to other function materials, aptamer hasunique merits including high affinity, low molecule weight, easy to modification, lowtoxicity and excellent biocompatibility. Given the advantages, aptamer is attractive inchemical, medical, and biological fields, but the related research is rare inenvironmental science. In the present work, the applications and mechanisms ofaptamer were explored in environmental purification and health. The methods andresults are presented as following:
(1) Aptamer was immobilized on CNBr-activated Sepharos and then packed in acolumn to remove low molecule weight polluntants in drinking water. The aptamercan effectively purity water and regenerate. In this section, the interactions of aptamerand pollutant were firstly studied. The adsorption data were fit well by the pseudo-second kinetics with an exothermic reaction. The reaction involved three steps:activation, adsorption and incubation. The whole reaction also could be divided intochemical kinetics control period, thermodynamic control period and transition period.The section provided a new technology to purity contaminated water and explored theinteraction mechanisms between aptamer and low molecule weight polluntants.
(2) Aptamer was immobilized on graphene oxide by the reaction of aminogroups and carboxyl groups. The complex improved the water solubility and thespecificity of graphene oxide as well as the protection of aptamer from nuclease.Furthermore, the effects of pH, temperature, ionic strength and natural organic matteron the activity of aptamer were studied.The affinity would reduce in extreme pH andhigh natural organic matter. The removal of pollutants slightly reduced with theincrease of temperature. A certain concentration of ionic strength enhanced theremoval of pollutants due to the decrease of electrostatic repel forces and development of connection bridge. The section discussed the influence ofenvironmental factors and provided a stable method to purify the pollutants incontaminated water.
(3) Aptamer enhanced the photocatalytic activities of graphene oxide forbiological contaminantion-bacteriophage under broad visible-light spectrum.Moreover, the photocatalytic mechanisms were explored. The protein coating wasbroke, and then enveloped nucleic acid released. Subsequently, the proteins posedcarbonylation and the nucleic acid bases received oxidized modification. Finaly, thegraphene oxide was reduced. The active species generated during the photocatalyticprocess. The photocatalysis paths involved energy transfer (generation of singletoxygen), electron transfer (generation of anion radicals) and water dissociation(generation of the precursor to the hydrated electron). The section showed a new ideato enhance the photocatalytic activities of graphene oxide for biologic contaminantionunder broad visible-light spectrum and studied the possible mechanisms.
(4) Aptamer was immobilized on nanoparticle as well as the antidote of mercurywas packed. The bioconjugates can diminish the toxicity of mercury in vivo. Thenanoparticle-aptamer markedly reduced the accumulation of mercury in both thebrain and kidney, and enhanced the excretion of urinary mercury. Thenanoparticle-aptamer ameliorated the neurotoxicity and improved the learning andmemory of animals. The toxicology of nanoparticle-aptamer was also studied byhematologic tests, and exhibited excellent biocompatibility. The section estabilished anew method to diminish the toxicity of heavy metals in vivo and expounded themechanisms.
(5) Aptamer was immobilized on solid phase micro-extraction by sol-gel methodto quickly and accurately detect a biomarker-adenosine in water solution and plasma.The desorption time and desorption solution were optimized, and then the effects oftemperature and ionic strength were discussed. Finally, the storage and regenerationof aptamer were studied. Given the special assay of solid phase micro-extraction invivo and metabolomics, aptamer/solid phase micro-extraction can be a device todiagnose environmental contamination and health.
The above results suggest aptamer can be a novel material for the separation, accumulation and purification of pollutants, and the development the toxicity ofheavy metals in vivo, even the diagnosis of environmental contamination and health.Although there are some challenges for the application of aptamer, such as the lowstability and adverse effect, it is worth to conduct the related research of aptamerfurthermore.
引文
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