兰州大学机构库 >化学化工学院
Alternative TitlePerformance Optimization of Miniaturized Laser Induced Fluorescence Detector and Its On-site Analysis Application
Thesis Advisor蒲巧生
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学硕士
Degree Discipline分析化学
Keyword激光诱导荧光检测器 LIF detector 微型化 miniaturization 低成本 low-cost 芯片电泳 MCE 现场检测 on-site detection 有机磷农药 organophosphorus pesticides

激光诱导荧光(Laser induced fluorescence, LIF)检测器是目前灵敏度最高的检测器之一,兼具响应速度快、选择性好等优点,是微流控分析技术中常用的检测器。目前商品化LIF检测器价格较高、体积较大,不能很好满足现场检测的需求。采用廉价易得的元器件,搭建低成本、高灵敏的微型化LIF检测器,是解决这个问题的重要途径之一,而且容易推广。结合芯片电泳(Microchip capillary electrophoresis, MCE)分离速度快、样品消耗少、污染小及便携等优势,MCE-LIF检测设备可实现多种待测物的现场高灵敏检测。本工作的目的是对影响LIF检测器灵敏度的因素进行探究,以低成本的方式实现微型化,拓展其在现场检测中的应用。



第二章:为了降低聚合物芯片荧光背景对检测的干扰,课题组前期搭建了发射光波长在红色光区(635 nm)的LIF检测器,但与蓝光激发的LIF相比,其灵敏度还有提升空间。本工作针对几种可能影响灵敏度的因素进行了探究。在不改变原LIF检测器光路结构及元件配置的基础上,对光路重新进行校准,考察了雪崩光电二极管(Avalanche photon diode, APD)光敏面在荧光光斑不同位置时仪器的检出限(Limit of detection, LOD)。结果表明,当其落在荧光光斑最亮部分的中心时LOD最低为60 pM。以AD630为核心芯片,自制了廉价的小型锁相放大器,并将其引入该LIF检测器的电路系统,确定低通滤波器截止频率为0.2 Hz,激光调制频率为200 Hz,参考信号和待测信号之间的相位差为30°。在此条件下,仪器的LOD进一步降低至15 pM,并探究了不同量子产率的荧光染料对测定仪器灵敏度的影响。

第三章:采用低成本、高精度且打印速度快的液晶成像(Liquid crystal display, LCD)光固化3D打印技术,实现了对基于共聚焦光路的微型化LIF检测模块的高效、低成本3D打印。选用普适性更好的455 nm激光二极管为光源,小尺寸的APD为光电检测器,以及国产的滤光片和微型透镜,完成了4个LIF模块中光学元件的组装。对其光路进行了校准,测试了相关器件的性能及每个LIF模块的检出限及模块之间的重复性。测试结果证明,所提出的方式成本低(2000元)、体积小(光学模块尺寸为4 × 4 × 1 cm3),灵敏度高(LOD 37–46 pM),所采用的整体模块化设计,使其仅需要更换激发光波长与适配的滤光片,就可轻松搭建出不同激发/发射波长组合的LIF检测器,适用范围广。

第四章:利用搭建的微型化LIF检测器,结合3D打印所制作的微型化MCE电泳仪,实现了对湖水样品中草铵膦(GLUF)、草甘膦(GLYP)及其代谢产物氨甲基膦酸(AMPA)的现场快速分离分析。考虑到户外环境的复杂性,对微型化LIF检测器的户外稳定性、检出限及太阳光对信号的影响进行了考察。在所制作的MCE仪上,实现了5种氨基酸的分离检测,证明该微型MCE-LIF仪器具备现场检测的能力。在选定的分离条件下,GLUF、GLYP及AMPA可基线分离,3种分析物的检出限分别为0.10 nM、0.39 nM、0.11 nM,方法的重现性良好,有望实现对更多种类农药残留的现场快速分离分析。

Other Abstract

Laser induced fluorescence (LIF) is one of the most sensitive detection modes, with the advantages of fast response and high selectivity, and widely used in microfluidics. At present, due to the relatively high prices and sizes of commercially available LIF detectors they cannot fit the requirements for the on-site detection very well. It is one of the important ways to solve this problem by building low-cost and highly sensitive miniaturized LIF detectors with cheap and easily available components, which make them more easily to be widely adopted in normal detections. Combining the advantages of fast separation speed, low sample consumption, low pollution and portability of microchip capillary electrophoresis (MCE), MCE-LIF devices can realize the highly sensitive on-site detection of multiple analytes. The purpose of this work is to explore the factors that affect the sensitivity of the LIF detectors, miniaturize them in a low-cost way, and expand their application for on-site detection.

The dissertation contains four chapters:

Chapter 1, the introduction, which provides a detailed description of the main components, optical structure, miniaturization and multi-channel detection of the LIF detectors, and the application of 3D printing in the development of LIF detectors. The microfluidics and its requirements for detectors are also introduced. Various detectors used in microfluidics are summarized. Finally, the applications of microfluidics using LIF detectors for the environmental monitoring, food analysis, pharmaceutical analysis and disease diagnosis are briefly described.

Chapter 2, to reduce the interference of the fluorescent background of polymeric microchips on detection, our research group has built a LIF detector with an excitation wavelength in the red region (635 nm), but comparing with the blue light excitation LIF detector, its sensitivity still has room to improve. In this work, several factors that may affect the sensitivity of the LIF detector were explored. Without changing the original optical structure and component configuration, the optical path was re-calibrated, and the limits of detection (LODs) of the LIF detector were examined for the different positions of the photo-sensing spot of the avalanche photon diode (APD). The result shows that the lowest LOD is 60 pM when it was in the center of the brightest part of the fluorescence spot. Using integrated circuit AD630, a cheap and miniaturized lock-in amplifier was built for the LIF detector. The cut-off frequency of the low-pass filter was 0.2 Hz, the modulation frequency of the laser was 200 Hz, and the phase difference between the reference and the signal was 30°. Under this condition, the LOD of the LIF detector was further reduced to 15 pM. The influence of fluorescent dyes with different quantum yields on the determined sensitivity of the LIF detector was investigated.

Chapter 3, efficient and low-cost 3D printing of the miniaturized LIF detection module with confocal optical structure was achieved by using low-cost, high precision and fast printing speed LCD photo-curing 3D printing technology. The optical components of the four LIF modules were assembled by selecting more universal 455 nm laser diodes as the light source, and small-sized APDs as the photodetector, as well as domestic filters and micro-lenses. The optical path of the four LIF modules were calibrated, and the performances, including LODs and repeatabilities were also tested. The results demonstrated that the proposed module has the advantages of low cost (¥2000), small size (optical module size is 4 × 4 × 1 cm3), and high sensitivity (LODs are 37–46 pM). The overall design of the module makes it easy to build LIF detectors with different excitation/emission wavelengths, simply through changing the excitation wavelength of the light source and corresponding filters, which implies a broad range of applications.

Chapter 4, using the miniaturized LIF module, a portable MCE device was made with the assistance of 3D printing, which was used for the on-site rapid separation and analysis of glufosinate (GLUF), glyphosate (GLYP) and its metabolites aminomethylphosphonic acid (AMPA) in a lake water sample. Considering the complexity of the outdoor environment, the outdoor stability, LOD and the influence of sunlight on the signal of the miniaturized LIF detector were investigated. The outdoor separation and detection of five amino acids were realized by using the portable MCE device, which proved the ability of on-site detection of the built MCE-LIF device. Under the selected separation condition, GLUF, GLYP and AMPA could be baseline separated, and the LODs of the three analytes were 0.10 nM, 0.39 nM and 0.11 nM, respectively. The reproducibility was good, and it is expected to be used for the rapid on-site separation and determination of more pesticide residues.

MOST Discipline Catalogue理学 - 化学 - 分析化学
Other Code262010_220200923950
Document Type学位论文
Recommended Citation
GB/T 7714
余洁. 微型化激光诱导荧光检测器的性能优化及其现场分析应用[D]. 兰州. 兰州大学,2023.
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