CN102680452A - Dual-detection biochemical sensing detector integrated with optofluidics - Google Patents
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Abstract
本发明公开了一种集成微流控光学的双探测生化传感检测仪。本发明的传感检测仪包括：光源系统、光路整形系统、传感芯片、三维控制系统、表面增强拉曼SERS探测系统、局域表面等离子体共振LSPR探测系统；以及自动进样控制系统。本发明的传感芯片同时采用LSPR和SERS两种光学检测手段，并与微流控技术相结合形成了新型的微流控光学传感芯片，这一技术为扩展了传统LSPR或SERS的应用，这种基于双等离子体结构的传感芯片在基于液体样品的生化检测中有很大吸引力，将会加快微流控光学传感系统的研发与应用。利用LSPR和SERS两种互补模式检测样品，在同一基底上实现LSPR与SERS分析技术的组合。
The invention discloses a dual-detection biochemical sensor detector integrated with microfluidic optics. The sensing detector of the present invention includes: a light source system, an optical path shaping system, a sensor chip, a three-dimensional control system, a surface-enhanced Raman SERS detection system, a localized surface plasmon resonance LSPR detection system; and an automatic sample feeding control system. The sensor chip of the present invention adopts two optical detection methods of LSPR and SERS at the same time, and combines with microfluidic technology to form a new type of microfluidic optical sensor chip. This technology expands the application of traditional LSPR or SERS, The sensor chip based on the dual plasmon structure is very attractive in the biochemical detection based on liquid samples, and will accelerate the development and application of microfluidic optical sensing systems. Two complementary modes of LSPR and SERS are used to detect samples, and the combination of LSPR and SERS analysis techniques is realized on the same substrate.
Description
技术领域 technical field
本发明属于生化传感检测领域，涉及一种集成微流控光学的表面增强拉曼光谱—局域表面等离子体共振双探测生化传感检测仪。The invention belongs to the field of biochemical sensing and detection, and relates to a surface-enhanced Raman spectrum-localized surface plasmon resonance dual-detection biochemical sensing detector integrated with microfluidic optics.
背景技术 Background technique
微流控光学等离子体是将微流体、光子学和等离子体集成在一起，是近年逐步形成的一个新的研究方向，将等离子体光学最前沿的研究成果应用于此领域中，因此微流控光学等离子体在生物、化学、生物化学、医学、工程等领域会产生无穷多的新应用。等离子体具有独特的物理属性，局域纳米尺度的光学性质会增强光与具有自由电子的物质相互作用。其中一个重要应用就是基于等离子体的生物传感器。在医学、卫生、食品、环境科学领域有着广泛的应用和明确的产业化前景。Microfluidic optical plasmonics is the integration of microfluidics, photonics and plasmas. It is a new research direction gradually formed in recent years. Optical plasmons will generate infinitely many new applications in the fields of biology, chemistry, biochemistry, medicine, engineering, etc. Plasmons have unique physical properties, localized nanoscale optical properties that enhance the interaction of light with matter with free electrons. One of the important applications is plasmon-based biosensors. It has a wide range of applications and clear industrialization prospects in the fields of medicine, hygiene, food, and environmental science.
目前主要利用光学属性的微流控光学生化分析或传感有：折射率检测，荧光检测，表面增拉曼散射检测，光学捕获及光学操控。无标记生化检测方法得到迅速发展，特别是基于局域表面等离子体共振LSPR传感技术，特别是在探测单分子与金属纳米颗粒表面结合的过程中，局域表面等离子体共振LSPR光谱的变化和表面增强拉曼散射SERS“指纹”谱是重要的手段。单分子与金属表面结合时，LSPR光谱变化分析是可操控的；而此时，SERS可探测识别分子并获取金属表面吸附分子的取向。然而，SERS的产生由单波长的激光激发，LSPR则由白光光源激发，因此，由于激发光源不同，所以这两种检测手段不能实现原位同步检测。At present, microfluidic optical biochemical analysis or sensing mainly using optical properties includes: refractive index detection, fluorescence detection, surface enhanced Raman scattering detection, optical capture and optical manipulation. Label-free biochemical detection methods have been developed rapidly, especially based on localized surface plasmon resonance (LSPR) sensing technology, especially in the process of detecting the binding of single molecules to the surface of metal nanoparticles, the changes of localized surface plasmon resonance LSPR spectra and Surface-enhanced Raman scattering SERS "fingerprint" spectrum is an important means. When a single molecule binds to a metal surface, LSPR spectral change analysis is manipulable; at this time, SERS can detect the recognition molecule and obtain the orientation of the adsorbed molecule on the metal surface. However, the generation of SERS is excited by a single-wavelength laser, while LSPR is excited by a white light source. Therefore, due to the different excitation light sources, these two detection methods cannot achieve in-situ simultaneous detection.
发明内容 Contents of the invention
为了解决传统单一测试技术LSPR或SERS技术中的不足，利用局域表面等离子体共振LSRP技术、微流控技术和微纳米加工技术，实现高灵敏度、免标记的纳米阵列生化检测仪，并利用LSPR探测生物分子结合过程，用SERS技术来判定所吸咐的分子。In order to solve the deficiencies in the traditional single testing technology LSPR or SERS technology, the local surface plasmon resonance LSRP technology, microfluidic technology and micro-nano processing technology are used to realize a high-sensitivity, label-free nano-array biochemical detector, and use LSPR Detect the binding process of biomolecules, and use SERS technology to determine the adsorbed molecules.
本发明的目的在于提供一种集成微流控光学的表面增强拉曼光谱—局域表面等离子体共振双探测生化传感检测仪。The object of the present invention is to provide a surface-enhanced Raman spectroscopy-localized surface plasmon resonance dual-detection biochemical sensor detector integrated with microfluidic optics.
本发明的双探测生化传感检测仪包括：光源系统、光路整形系统、传感芯片、三维控制系统、表面增强拉曼SERS探测系统、局域表面等离子体共振LSPR探测系统；以及自动进样控制系统；其中，传感芯片安装在三维控制系统上；自动进样控制系统向传感芯片注射样品；由光源系统提供激光或白光光源；经光路整形系统垂直入射到传感芯片；经传感芯片反射的激光进入SERS探测系统，并进行相应的SERS数据处理与分析；从传感芯片投射的白光进入LSPR探测系统，并进行相应的LSPR数据处理与分析。The dual-detection biochemical sensor detection instrument of the present invention includes: a light source system, an optical path shaping system, a sensor chip, a three-dimensional control system, a surface-enhanced Raman SERS detection system, a localized surface plasmon resonance LSPR detection system; and an automatic sample feeding control system; wherein, the sensor chip is installed on the three-dimensional control system; the automatic sample injection control system injects the sample into the sensor chip; the laser or white light source is provided by the light source system; the optical path shaping system is vertically incident on the sensor chip; The reflected laser light enters the SERS detection system for corresponding SERS data processing and analysis; the white light projected from the sensor chip enters the LSPR detection system for corresponding LSPR data processing and analysis.
光源系统包括：光源；安装在光源上的切换设备；与光源相连接用以控制光源功率的功率控制器。从而，光源系统可以通过切换设备提供激发SERS的单波长的激光，以及激发LSPR的白光。The light source system includes: a light source; a switching device installed on the light source; a power controller connected with the light source to control the power of the light source. Therefore, the light source system can provide single-wavelength laser light for exciting SERS and white light for exciting LSPR through a switching device.
光源采用的白光光源为LED、卤素灯、钠灯及汞灯中的一种，其光谱范围在200~2000nm之间。The white light source used for the light source is one of LED, halogen lamp, sodium lamp and mercury lamp, and its spectral range is between 200 and 2000nm.
传感芯片固定在三维控制系统中，可以实现三维精确移动，其精度可达到10微米。The sensor chip is fixed in the three-dimensional control system, which can realize three-dimensional precise movement, and its precision can reach 10 microns.
SERS探测系统包括：SERS探测设备；SERS探测设备得到的数据传送至一号计算机，由设置在一号计算机中的SERS数据采集与处理软件对数据进行分析。The SERS detection system includes: SERS detection equipment; the data obtained by the SERS detection equipment is transmitted to the No. 1 computer, and the SERS data acquisition and processing software installed in the No. 1 computer analyzes the data.
LSPR探测系统包括：LSPR探测器；LSPR探测器收集信号，传输至光谱仪；光谱仪得到的数据传送至二号计算机，由设置在二号计算机中的LSPR数据采集与处理软件对数据进行分析。The LSPR detection system includes: LSPR detector; the LSPR detector collects signals and transmits them to the spectrometer; the data obtained by the spectrometer is sent to the No. 2 computer, and the LSPR data acquisition and processing software installed in the No. 2 computer analyzes the data.
自动进样控制系统包括两路以上注射泵，可以程序化设置参数，从而实现对样品进行精确控制。The automatic sample injection control system includes more than two syringe pumps, and the parameters can be programmed to achieve precise control of the sample.
传感芯片包括K*L陈列的传感芯片单元，传感芯片单元包括：衬底以及与其键合在一起的上片；形成在上片内构成微流通道的进口、出口及微腔；形成在衬底上并处于微腔内的m*n阵列的纳米颗粒；其中，m、n、K和L为自然数。The sensor chip includes a K*L sensor chip unit, and the sensor chip unit includes: a substrate and an upper sheet bonded to it; an inlet, an outlet, and a microcavity formed in the upper sheet to form a microfluidic channel; An m*n array of nanoparticles on a substrate and in a microcavity; wherein m, n, K and L are natural numbers.
传感芯片单元的尺寸范围在几十微米到几微米之间。纳米颗粒的尺寸范围在几十纳米到几百纳米之间，纳米颗粒之间的间距范围在几十纳米到几百纳米之间。The size of the sensor chip unit ranges from tens of microns to several microns. The size of nanoparticles ranges from tens of nanometers to hundreds of nanometers, and the distance between nanoparticles ranges from tens of nanometers to hundreds of nanometers.
纳米颗粒为金或银等贵重金属颗粒，形状为球形、椭球形、纳米缝以及纳米孔中的一种。衬底的材料为K9玻璃、石英玻璃、聚二甲硅氧烷PDMS、聚甲基丙烯酸甲酯PMMA、聚苯乙烯PS等透明材料中的一种。上片的材料为聚二甲硅氧烷PDMS或聚甲基丙烯酸甲酯PMMA。Nanoparticles are precious metal particles such as gold or silver, and the shape is one of spherical, ellipsoidal, nanoslit and nanopore. The material of the substrate is one of transparent materials such as K9 glass, quartz glass, polydimethylsiloxane PDMS, polymethyl methacrylate PMMA, and polystyrene PS. The material of the upper sheet is polydimethylsiloxane PDMS or polymethyl methacrylate PMMA.
微腔的体积范围在几微升到几十微升之间，其形状可以是球形或立方形。微流通道的宽度范围在10微米到200微米之间，深度范围在50微米到200微米之间。The volume of the microcavity ranges from a few microliters to tens of microliters, and its shape can be spherical or cubic. The width of the microfluidic channels ranges from 10 microns to 200 microns, and the depth ranges from 50 microns to 200 microns.
本发明的传感芯片同时采用LSPR和SERS两种光学检测手段，并与微流控技术相结合形成了新型的微流控光学传感芯片，这一技术为扩展了传统LSPR或SERS的应用，这种基于双等离子体结构的传感芯片在基于液体样品的生化检测中有很大吸引力，将会加快微流控光学传感系统的研发与应用。利用LSPR和SERS两种互补模式检测样品，在同一基底上实现LSPR与SERS分析技术的组合。The sensor chip of the present invention adopts two optical detection methods of LSPR and SERS at the same time, and combines with microfluidic technology to form a new type of microfluidic optical sensor chip. This technology expands the application of traditional LSPR or SERS, The sensor chip based on the dual plasmon structure is very attractive in the biochemical detection based on liquid samples, and will accelerate the development and application of microfluidic optical sensing systems. Two complementary modes of LSPR and SERS are used to detect samples, and the combination of LSPR and SERS analysis techniques is realized on the same substrate.
本发明的优点：Advantages of the present invention:
（1）设备结构简单、无需标记可进行直接、实时探测的设备；(1) The equipment has a simple structure and can perform direct and real-time detection without marking;
（2）实现了SERS与LSPR的优势互补；(2) Realized the complementary advantages of SERS and LSPR;
（3）可以实现多通道、高通量、并行检测，提高了探测的效率；(3) Multi-channel, high-throughput, parallel detection can be realized, which improves the detection efficiency;
（4）操作方便、智能化程度高；(4) Convenient operation and high degree of intelligence;
（5）进样系统具有高精度、易控制的特点；(5) The sampling system has the characteristics of high precision and easy control;
（6）多单元、阵列结构；(6) Multi-unit, array structure;
（7）采用低成本的纳米加工技术，可实现大面积加工；(7) Using low-cost nano-processing technology, large-area processing can be realized;
（8）与微流控技术相结合，实现微流控光学传感系统的集成。(8) Combining with microfluidic technology to realize the integration of microfluidic optical sensing system.
附图说明 Description of drawings
图1为本发明的双探测生化传感检测仪的结构示意图；Fig. 1 is the structural representation of double detecting biochemical sensing detector of the present invention;
图2为本发明的传感芯片的结构示意图；Fig. 2 is the structural representation of sensor chip of the present invention;
图3是本发明的实施例中的LSPR测试结果；Fig. 3 is the LSPR test result in the embodiment of the present invention;
图4是本发明的实施例中的SERS测试结果。Fig. 4 is the SERS test result in the embodiment of the present invention.
具体实施方式 Detailed ways
下面结合附图，通过具体实施例对本发明的具体实施方式作进一步的详细描述。Below in conjunction with the accompanying drawings, the specific embodiments of the present invention will be further described in detail through specific examples.
如图1所示，本发明的双探测生化传感检测仪包括：光源系统1、光路整形系统2、传感芯片3、三维控制系统4、表面增强拉曼SERS探测系统5、局域表面等离子体共振LSPR探测系统6；以及自动进样控制系统7；其中，传感芯片3安装在三维控制系统4上；自动进样控制系统7向传感芯片3注射样品；由光源系统1提供激光或白光光源；经光路整形系统2垂直入射到传感芯片3；经传感芯片3反射的激光进入SERS探测系统5，并进行相应的SERS数据处理与分析；从传感芯片3透射的白光进入LSPR探测系统6，并进行相应的LSPR数据处理与分析。As shown in Figure 1, the dual-detection biochemical sensor detector of the present invention includes: a
光源系统1中激光光源波长为488nm、632.8nm和850nm。The wavelengths of the laser light sources in the
如图2所示，本发明的传感芯片包括K*L陈列的传感芯片单元，传感芯片单元包括：衬底31以及与其键合在一起的上片32；形成在上片32内构成微流通道的进口、出口33及微腔34；形成在衬底上并处于微腔内的m*n阵列的纳米颗粒35；其中，m、n、K和L为自然数。As shown in Fig. 2, the sensing chip of the present invention comprises a sensing chip unit arranged in K*L, and the sensing chip unit comprises: a
本实施例利用本发明一种集成微流控光学的表面增强拉曼光谱---局域表面等离子体共振双探测生化传感检测仪，用于检测不同折射率的多种样品。SERS测试中所选用的SERS探测系统是HORIBAJOBIN YVON公司的HR800SERS探测设备，光源为HeNe激光器，其功率为20mW，波长为632.8nm。LSPR测试中所选择的光源系统中的光源是光谱范围在200~1100nm，电流500mA，电压12VDC，输出功率6.5瓦的卤素灯，经光纤和反射式探头（Ocean Optics，QR400-7-UV-VIS）后出射光束经透镜后，光照射在传感系统中的采用石英作为基底的金纳米结构传感单元，将其固定在一个三维高精度固定架上。光谱仪选用OceanOptics公司的QE65000型光谱分析仪，光透过芯片后，由探测器收集信号，再被后端的光谱仪所探测，所采集的数据通过USB2.0接口与计算机系统进行数据通信，数据处理软件可以包括用户界面、探测、控制和数据处理及输出显示几个功能模块。In this embodiment, a surface-enhanced Raman spectroscopy with integrated microfluidic optics—localized surface plasmon resonance dual-detection biochemical sensor detector of the present invention is used to detect various samples with different refractive indices. The SERS detection system selected in the SERS test is the HR800SERS detection equipment of HORIBAJOBIN YVON Company, the light source is a HeNe laser with a power of 20mW and a wavelength of 632.8nm. The light source in the light source system selected in the LSPR test is a halogen lamp with a spectral range of 200-1100nm, a current of 500mA, a voltage of 12VDC, and an output power of 6.5 watts. ) After the outgoing beam passes through the lens, the light irradiates the gold nanostructure sensing unit using quartz as the substrate in the sensing system, and fixes it on a three-dimensional high-precision fixing frame. The spectrometer uses OceanOptics’ QE65000 spectrum analyzer. After the light passes through the chip, the detector collects the signal, and then is detected by the back-end spectrometer. The collected data communicates with the computer system through the USB2.0 interface, and the data processing software It can include user interface, detection, control and data processing and output display several functional modules.
利用所加工的集成微流控传感单元与本发明公布的生化传感系统进行测试，LSPR测试样品为：空气、NaCl（20%）、丙三醇。经分析软件处理得到的数据结果如图3所示，由于空气NaCl（20%）、丙三醇三种测试样品的折射率分别为1、1.3684和1.473，因测试样品折射率不同，通过本设备测试获得的LSPR消光光谱的最大值的位置分别在533.52nm、577.97nm和587.37nm，折射率增加，相应的谱线发生了红移。SERS测试样品为：若丹明R6G，经分析软件处理得到的数据结果如图4所示，其特征峰的波数分别为1197、1277、1366、1509和1647cm-1。The processed integrated microfluidic sensing unit and the biochemical sensing system announced by the present invention are used for testing, and the LSPR test samples are: air, NaCl (20%), and glycerin. The results of the data processed by the analysis software are shown in Figure 3. Since the refractive indices of the three test samples of air NaCl (20%) and glycerin are 1, 1.3684 and 1.473 respectively, due to the different refractive indices of the test samples, the The positions of the maximum values of the LSPR extinction spectra obtained by the test are respectively at 533.52nm, 577.97nm and 587.37nm, the refractive index increases, and the corresponding spectral lines red shift. The SERS test sample is: rhodamine R6G, and the data obtained by processing the analysis software is shown in Figure 4. The wavenumbers of the characteristic peaks are 1197, 1277, 1366, 1509 and 1647cm -1 respectively.
最后需要注意的是，公布实施方式的目的在于帮助进一步理解本发明，但是本领域的技术人员可以理解：在不脱离本发明及所附的权利要求的精神和范围内，各种替换和修改都是可能的。因此，本发明不应局限于实施例所公开的内容，本发明要求保护的范围以权利要求书界定的范围为准。Finally, it should be noted that the purpose of publishing the implementation is to help further understand the present invention, but those skilled in the art can understand that various replacements and modifications can be made without departing from the spirit and scope of the present invention and the appended claims. It is possible. Therefore, the present invention should not be limited to the content disclosed in the embodiments, and the protection scope of the present invention is subject to the scope defined in the claims.
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