拉曼光谱编码的荧光液相生物芯片检测系统研究

Raman Spectroscopy-Encoded Fluorescence Suspension Array Detection System

随着医疗诊断需求的增加,生物分子检测技术越来越受到人们的重视,液相生物芯片技术作为一种高通量,多通道的分子检测手段在近几年得到了飞速发展。通过层层自组装方法制备以微片为载体的拉曼光谱编码液相生物芯片,并利用自行搭建的一套高灵敏度、高分辨率的光学系统,实现对液相生物芯片的定性与定量分析。光学系统由拉曼光谱检测系统与荧光显微成像系统耦合而成。在拉曼光谱检测系统中激光器发射出785nm波长的激光,通过二向色镜,带反反射镜与物镜汇聚到样品上,样品产生的拉曼散射光,经物镜,带反反射镜,二向色镜与拉曼滤波片,最后通过凹透镜聚焦到光谱仪的狭缝上,光谱仪色散实现在线阵CCD上拉曼光谱的获取。荧光显微成像系统应用光学成像原理,通过调节凹透镜与405nm的激发光之间的距离,使激发光通过物镜均匀的照射到样品之上,样品激发出的荧光,通过物镜,带反反射镜,二向色镜,滤波片与相应的凹透镜,最后成像到面阵CCD上。改进传统便携式拉曼光谱检测系统光路并选用相应波段的带反反射镜与焦距20倍的物镜完成拉曼光谱检测系统与荧光显微成像系统的耦合。为了减少两路系统之间的相互影响选用合适的二向色镜以及滤波片,在提高耦合系统获取数据的准确性中有着重要的作用。该系统通过对反应之后的液相生物芯片进行拉曼光谱检测,以完成对每个编码玻片的定性识别,即解码;同时激发反应后液相生物芯片的荧光并采集荧光强度图,根据每个解码玻片上的荧光强度值完成对目标检测物的定量分析。区别于传统荧光编码液相生物芯片,拉曼光谱编码具有稳定性更强,光谱分辨率更高等优点。该光学系统集拉曼光谱检测系统与荧光显微成像系统于一体,解决了目前未有基于拉曼编码的液相生物芯片的检测系统的问题,并且可同时对多种目标物进行识别和定量分析,提升了实验结果的准确性。

With the increasing demands for medical diagnosis,more and more attention has been paid to the technology of biomolecular detection.As a high throughput and multiplexed molecular detection method,suspension array has developed rapidly in recent years.In this study,a Raman spectra-encoded suspension array with micro-quartz pieces as the carrier was prepared by the layer-by-layer self-assembly method,and a high sensitivity and high resolution optical system was built to realize the qualitative and quantitative analysis of the suspension array.The home-built optical system was obtained by coupling Raman spectroscope with a fluorescence microscope.For the Raman spectroscope,a 785 nm laser was converged on the sample through dichroic mirrors,reflector and object lens.Then the Raman scattering light produced by the sample passed through the objective lens,anti-reflection mirror,dichroic mirror and Raman filter,and focused on the slit of the spectrometer via the concave lens.And finally,Raman spectra can be obtained by the dispersion effect of the spectrometer.For the fluorescence microscope,which used the optical imaging principle,the excitation light could irradiate the sample uniformly through the objective lens by adjusting the distance between the concave lens and the excited light of 405 nm.Then,the emitted fluorescence passed through an objective lens,an anti-reflection mirror,dichroic mirror,a filter and a concave lens,and finally imaged on the matrix CCD.The coupling of the Raman spectroscope and the fluorescence microscope was completed by improving the optical path of the conventional portable Raman spectroscope and selecting the anti-reflecting mirror with the specific band and the objective lens with a focal length of 20×.In order to reduce the interaction between the Raman spectroscope and the fluorescence microscope,the appropriate dichroic mirror and filter were selected to improve the coupling system.The Raman spectra of the suspension array were detected by home-built system to accomplish the qualitative identification of each encoded micro-quartz pieces.At the same time,the fluorescence of the encoded micro-quartz pieces was excited and the fluorescence signal was collected to complete the quantitative analysis of the target analyst according to the fluorescence intensity value on each encoded micro-quartz pieces.Compared with traditional fluorescence-encoded suspension arrays,Raman spectra encoding method has the advantages of stronger stability and higher spectral resolution.This optical system integrates Raman spectroscope and fluorescence microscope,which solves the problem that there is no suspension array detection system based on Raman encoding method at present and can qualitatively and quantitatively analyze a variety of target molecules at the same time,improving the accuracy of the experimental results.