基于光子晶体的生物分子编码载体
摘要
多元分析技术是一种同时分析大量种类的生物分子相互作用的技术。这种技术的原理一般是基于把不同的生物分子与不同的载体或者标记连接来进行分子标识，也就是所谓的生物分子编码。与传统的一元分析相比，多元分析技术可以使多种分子同时被分析，从而节省了分析时间、成本和样品的消耗量。现在的编码方法已经从基因微阵列式的固定载体编码发展到了多种多样的流动载体编码。光学编码是各种编码中操作、检测最简单，应用最广的一种编码方式。但是目前光学编码中最常用的编码元素还是荧光染料，由于荧光染料所产生的编码颜色来自于分子的化学结构，因此稳定性不高，还有用于编码时遇到的检测以及编码量等问题都对其应用造成了一定的限制。因此本文提出了一种利用光子晶体来编码生物分子载体的新方法，试图避免这些问题，并对编码载体的制备和应用做了如下相应的研究：
1．开发了一套连续制备单分散编码微球的装置。探讨了影响水相溶液流速、油相溶液流速，锥形玻璃滴头到导流管距离，锥形玻璃管内径等因素对微球大小，单分散度等的影响和变化舰律。还探讨了溶液的配比对成球稳定性和球形度等的影响。
2．利用该微球制备装置，制备了光子晶体珠光颜料编码聚苯乙烯微球。控制影响微球与生物分子偶联以及生物检测的因素，制备的微球粒径大小均匀，具有均质性良好的表面，同时编码颜色着色均匀，编码稳定。通过对微球的超声洗涤处理，可以得到适合吸附或者共价固定生物分子如蛋白质的编码微球载体。试验结果标明，该编码聚苯乙烯町以利用物理吸附法和共价偶联法实现与生物分子的连接，同时适用于荧光法和酶联显色法等标记检测方法。其检测不需要复杂的仪器设备，具有很大的潜在应用价值，有望成为96孔板的替代选择。
3．利用该微球制备装置，将单分散纳米粒子的胶体溶液注入到油相溶液，以胶体溶液液滴为模板制备了大小可控的胶体光子晶体编码微球。这种微球比表面积大，编码颜色稳定。并将其用于多元免疫检测分析，探讨了生物分子的固定方法，编码稳定性、准确性与编码量以及检测的灵敏度等问题。通过多元免疫检测证明了其做为编码载体的可靠性与优越性。
本文制备的两种光子晶体编码载体与文献报道的编码载体相比具有以下优点：
1．编码来源与光子晶体的物理结构，非常稳定，没有荧光染料编码的光漂白、光淬灭等现象，易于储存。
2．编码载体的解码，只要普通白光光源即可，不需要专门的激发光与滤光片，也不需要高分辩率的光学系统，所以解码系统非常简单。
Multiplex technology can analysis the interactions of thousands of kinds of biomolecules in parallel. The principle lies in that different kinds of biomolecules are marked by different carriers or labels, which is called biomolecular encoding. Compared with traditional single analysis technology, multiplex technology can save the time, sample consumption and the cost by analyzing lots of kinds of targets within the same sample. Up to now, the encoding strategies developed from fixed carriers such as glass slides used in gene chip to kinds of fiuidic carriers such as beads and barcodes, among which the optical encoding strategies are the mostly used methods because of their simplicity in operation and decoding. But the mostly used encoding element in optical encoding is fluorescence, which comes from the material chemical structure and is not stable. The encoding capacity and decoding of fluorescence limit their applications. Hence, in this dissertation a new encoding method for biomolecular carriers based on photonic crystal nanomaterial is proposed, of which the fabrication and application are studied as below.
1 An apparatus were developed to fabricate monodispersed encoded polymer beads based on the emulsion droplets. The factors which will affect the sizes and the dispersities of the beads such as the velocity of the water phase and oil phase solution, the diameter of the glass orifice, and the distance between the orifice and the glass tube are discussed. The mixing ratio of the solution is optimized for the best sphere structure and stability of the droplets.
2 Polystyrene beads encoded with biomimic pearl pigment were prepared with the apparatus. The factors affect the bioanalysis, the robust conjugation between biomolecules, and the encoded carriers were discussed and well controlled. The result beads were uniform in color and sizes and have homogeneous surfaces. After ultrasonic washing the beads were ready for binding with biomolecules such as proteins. Our experiments show that the beads are suitable for protein immobilization techniques such as physical adsorption and covalent binding. At the same time, kinds of lable detection technique can be applied based on the encoded beads such as fluorescent labels and enzyme lables. Since the signal detection of the beads is very simple and can use conventional equipments in bioanalysis, the encoded beads can be alternatives for commonly use 96-well microplates.
1．开发了一套连续制备单分散编码微球的装置。探讨了影响水相溶液流速、油相溶液流速，锥形玻璃滴头到导流管距离，锥形玻璃管内径等因素对微球大小，单分散度等的影响和变化舰律。还探讨了溶液的配比对成球稳定性和球形度等的影响。
2．利用该微球制备装置，制备了光子晶体珠光颜料编码聚苯乙烯微球。控制影响微球与生物分子偶联以及生物检测的因素，制备的微球粒径大小均匀，具有均质性良好的表面，同时编码颜色着色均匀，编码稳定。通过对微球的超声洗涤处理，可以得到适合吸附或者共价固定生物分子如蛋白质的编码微球载体。试验结果标明，该编码聚苯乙烯町以利用物理吸附法和共价偶联法实现与生物分子的连接，同时适用于荧光法和酶联显色法等标记检测方法。其检测不需要复杂的仪器设备，具有很大的潜在应用价值，有望成为96孔板的替代选择。
3．利用该微球制备装置，将单分散纳米粒子的胶体溶液注入到油相溶液，以胶体溶液液滴为模板制备了大小可控的胶体光子晶体编码微球。这种微球比表面积大，编码颜色稳定。并将其用于多元免疫检测分析，探讨了生物分子的固定方法，编码稳定性、准确性与编码量以及检测的灵敏度等问题。通过多元免疫检测证明了其做为编码载体的可靠性与优越性。
本文制备的两种光子晶体编码载体与文献报道的编码载体相比具有以下优点：
1．编码来源与光子晶体的物理结构，非常稳定，没有荧光染料编码的光漂白、光淬灭等现象，易于储存。
2．编码载体的解码，只要普通白光光源即可，不需要专门的激发光与滤光片，也不需要高分辩率的光学系统，所以解码系统非常简单。
Multiplex technology can analysis the interactions of thousands of kinds of biomolecules in parallel. The principle lies in that different kinds of biomolecules are marked by different carriers or labels, which is called biomolecular encoding. Compared with traditional single analysis technology, multiplex technology can save the time, sample consumption and the cost by analyzing lots of kinds of targets within the same sample. Up to now, the encoding strategies developed from fixed carriers such as glass slides used in gene chip to kinds of fiuidic carriers such as beads and barcodes, among which the optical encoding strategies are the mostly used methods because of their simplicity in operation and decoding. But the mostly used encoding element in optical encoding is fluorescence, which comes from the material chemical structure and is not stable. The encoding capacity and decoding of fluorescence limit their applications. Hence, in this dissertation a new encoding method for biomolecular carriers based on photonic crystal nanomaterial is proposed, of which the fabrication and application are studied as below.
1 An apparatus were developed to fabricate monodispersed encoded polymer beads based on the emulsion droplets. The factors which will affect the sizes and the dispersities of the beads such as the velocity of the water phase and oil phase solution, the diameter of the glass orifice, and the distance between the orifice and the glass tube are discussed. The mixing ratio of the solution is optimized for the best sphere structure and stability of the droplets.
2 Polystyrene beads encoded with biomimic pearl pigment were prepared with the apparatus. The factors affect the bioanalysis, the robust conjugation between biomolecules, and the encoded carriers were discussed and well controlled. The result beads were uniform in color and sizes and have homogeneous surfaces. After ultrasonic washing the beads were ready for binding with biomolecules such as proteins. Our experiments show that the beads are suitable for protein immobilization techniques such as physical adsorption and covalent binding. At the same time, kinds of lable detection technique can be applied based on the encoded beads such as fluorescent labels and enzyme lables. Since the signal detection of the beads is very simple and can use conventional equipments in bioanalysis, the encoded beads can be alternatives for commonly use 96-well microplates.
引文
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