CN102331415B - Method for positioning capillary tube array by using raman spectral imaging - Google Patents

Abstract

Translated fromChinese

本发明提供一种利用拉曼光谱定位毛细管阵列在电荷耦合元件上成像位置的方法，所述方法包括：(1)用激光束照射毛细管束发出拉曼光，(2)通过拉曼成像仪成像在电荷耦合元件CCD上，(3)分析电荷耦合元件CCD上的光谱图对毛细管束中各毛细管进行定位。本发明采用双束激光激发石英或水的拉曼光谱实现光谱成像，实现了毛细管在CCD上的位置定位和平行度调整，可消除毛细管阵列进行微量试剂分析时的误差，提高分析准确度。

The invention provides a method for using Raman spectroscopy to locate the imaging position of a capillary array on a charge-coupled device, the method comprising: (1) irradiating the capillary bundle with a laser beam to emit Raman light, (2) imaging with a Raman imager On the charge-coupled device CCD, (3) analyzing the spectrum on the charge-coupled device CCD to locate each capillary in the capillary bundle. The invention adopts double-beam lasers to excite the Raman spectrum of quartz or water to realize spectral imaging, realizes the position positioning and parallelism adjustment of the capillary on the CCD, can eliminate the error when the capillary array conducts trace reagent analysis, and improves the analysis accuracy.

Description

Translated fromChinese

一种利用拉曼光谱成像定位毛细管阵列的方法A Method for Locating Capillary Arrays Using Raman Spectroscopy Imaging

技术领域technical field

本发明涉及一种定位毛细管阵列在电荷耦合元件上成像位置的方法，属于高通量微量分析技术领域。The invention relates to a method for locating the imaging position of a capillary array on a charge-coupled element, and belongs to the technical field of high-throughput microanalysis.

背景技术Background technique

由于毛细管具有良好的散热效能，且承受电压高、进样量小，具有仪器分析所要求的高效，快速，样品用量少等最基本和最优异的特点。毛细管两端所加电压可高至30kV，分离毛细管的纵向电场强度可达到400V/cm以上，因而分离操作可以在很短的时间内，达到非常高的分离效率(理论塔板数达到400000/m以上，最高达107/m数量级)。因为毛细管的内径很小(一般＜100μm)，对内径50μm，长度为50cm的毛细管，其容积不足1μL，进样体积在nL级，样品浓度可低于10～4mol/L。Because the capillary has good heat dissipation performance, high withstand voltage and small injection volume, it has the most basic and excellent characteristics such as high efficiency, fast speed and less sample consumption required by instrumental analysis. The voltage applied to both ends of the capillary can be as high as 30kV, and the longitudinal electric field strength of the separation capillary can reach more than 400V/cm, so the separation operation can achieve a very high separation efficiency in a short period of time (the number of theoretical plates can reach 400,000/m above, up to 107/m order of magnitude). Because the inner diameter of the capillary is very small (generally <100 μm), for a capillary with an inner diameter of 50 μm and a length of 50 cm, the volume is less than 1 μL, the injection volume is at the nL level, and the sample concentration can be lower than 10-4 mol/L.

此外，毛细管电泳技术还有容易自动化，操作简便，溶剂消耗少，环境污染小等优点。毛细管电泳技术的应用首先集中在氨基酸，糖类，核酸和蛋白质等生物分子的分离分析上，随着此项技术的不断发展和完善，其应用已逐渐的向医药卫生，食品化工，环境等领域渗透。毛细管电泳技术还应用于DNA的高速测序，蛋白质的高效分离，糖类分析，细胞分析，手性拆分，物理化学常数的测定，生产工程控制等。In addition, capillary electrophoresis technology has the advantages of easy automation, simple operation, less solvent consumption, and less environmental pollution. The application of capillary electrophoresis technology first focuses on the separation and analysis of biomolecules such as amino acids, sugars, nucleic acids and proteins. With the continuous development and improvement of this technology, its application has gradually spread to the fields of medicine and health, food chemical industry, environment and so on. penetration. Capillary electrophoresis technology is also used in high-speed sequencing of DNA, high-efficiency separation of proteins, carbohydrate analysis, cell analysis, chiral resolution, determination of physical and chemical constants, production engineering control, etc.

为了提高毛细管检测的通量，毛细管阵列作为高通量电泳分析手段被广泛应用于生物化学分析中。在医疗、化学化工、生物制药、法医分析等领域广泛应用毛细管阵列进行微量试剂的定性和定量分析。毛细管阵列可以同时实现批量试剂的分析。但由于毛细管之间的位置关系存在不确定性，使得毛细管阵列进行微量试剂的定性和定量分析时存在误差。目前尚没有消除这种误差的成熟技术。In order to improve the throughput of capillary detection, capillary array is widely used in biochemical analysis as a high-throughput electrophoresis analysis method. Capillary arrays are widely used in the fields of medical, chemical, biopharmaceutical, and forensic analysis for qualitative and quantitative analysis of trace reagents. Capillary arrays enable simultaneous analysis of bulk reagents. However, due to the uncertainty of the positional relationship between the capillaries, there are errors in the qualitative and quantitative analysis of micro reagents by the capillary array. At present, there is no mature technology to eliminate this error.

发明内容Contents of the invention

为了同时对多根毛细管进行分析，需要对毛细管在电荷耦合元件(CCD)上的成像位置进行定位，以消除毛细管阵列进行微量试剂定性或定量分析时的误差。In order to analyze multiple capillaries at the same time, the imaging position of the capillary on the charge-coupled device (CCD) needs to be positioned to eliminate errors when the capillary array is used for qualitative or quantitative analysis of trace reagents.

本发明提供一种利用拉曼光谱成像定位毛细管阵列的方法，所述方法包括：(1)用激光束照射毛细管束发出拉曼光，(2)通过拉曼成像仪成像在电荷耦合元件(CCD)上，(3)分析电荷耦合元件CCD上的光谱图对毛细管束中各毛细管进行定位。The invention provides a method for positioning a capillary array by using Raman spectrum imaging, the method comprising: (1) irradiating the capillary bundle with a laser beam to emit Raman light, (2) imaging the capillary array on a charge-coupled device (CCD) by a Raman imager ), and (3) analyze the spectrogram on the charge-coupled device CCD to locate each capillary in the capillary bundle.

进一步，所述方法步骤(1)用激光束照射毛细管束发出拉曼光是指激光束被毛细管的石英或管内溶液散射发出拉曼光。Furthermore, the method step (1) irradiating the capillary bundle with a laser beam to emit Raman light means that the laser beam is scattered by the quartz of the capillary or the solution in the tube to emit Raman light.

进一步，所述拉曼成像仪通过取光狭缝、光栅分光及聚焦透镜使拉曼光成像在CCD上。Further, the Raman imager makes the Raman light image on the CCD through the light-taking slit, the grating beam splitter and the focusing lens.

进一步，所述方法步骤(1)用激光束照射毛细管束内的水散射发出拉曼光，所用激光束为双激光束，所述双激光束的波长分别为488nm和514.5nm。Further, the method step (1) irradiates the water in the capillary bundle with a laser beam to scatter and emit Raman light, the laser beams used are dual laser beams, and the wavelengths of the dual laser beams are 488nm and 514.5nm respectively.

作为优选方式，所述单数毛细管采集488nm激光器激发的拉曼光，双数毛细管采集514.5nm激光器激发的拉曼光。As a preferred manner, the odd-numbered capillaries collect Raman light excited by a 488nm laser, and the even-numbered capillaries collect Raman light excited by a 514.5nm laser.

作为另一优选方式，所述双数毛细管采集488nm激光器激发的拉曼光，单数毛细管采集514.5nm激光器激发的拉曼光。As another preferred manner, the even-numbered capillaries collect Raman light excited by a 488nm laser, and the odd-numbered capillaries collect Raman light excited by a 514.5nm laser.

进一步，所述方法步骤(3)中以像素数为横轴，光谱强度为纵轴得到空间校正谱图，通过峰识别得到每根毛细管中心位置和每根毛细管之间的有效宽度。Further, in step (3) of the method, the horizontal axis is the number of pixels, and the vertical axis is the spectral intensity to obtain a spatially corrected spectrum, and the center position of each capillary and the effective width between each capillary are obtained through peak identification.

本发明的位置定位技术采用双激光束，分别为488nm和514.5nm。水的特征拉曼峰位于3400cm^-1处。对于488nm(20491.8cm^-1)，水散射的拉曼特征峰位于17091.8cm^-1，对应的波长为585nm。对于514.5nm(19436.3cm^-1)，水散射的拉曼特征峰位于16036.3cm^-1，对应的波长为623.6nm。图2是水散射的特征拉曼峰。从图中可以看出，其谱主要分布在2800cm^-1到3800cm^-1波数之间。The position positioning technology of the present invention adopts double laser beams, respectively 488nm and 514.5nm. The characteristic Raman peak of water is located at 3400 cm⁻¹ . For 488nm (20491.8cm^-1 ), the characteristic Raman peak of water scattering is located at 17091.8cm^-1 , corresponding to a wavelength of 585nm. For 514.5nm (19436.3cm^-1 ), the characteristic Raman peak of water scattering is located at 16036.3cm^-1 , corresponding to a wavelength of 623.6nm. Figure 2 is the characteristic Raman peak of water scattering. It can be seen from the figure that its spectrum is mainly distributed between 2800cm^-1 and 3800cm^-1 wavenumbers.

488nm和514.5nm激光被水散射的特征拉曼峰投影到数据采集CCD上可以得到图3。蓝光(左侧)是采集488nm激光被水散射的拉曼光强度，红光(右侧)对应的是采集514.5nm激光被水散射的拉曼荧光强度，每一个峰对应一根毛细管，采用相邻毛细管间隔采集不同波长激光被散射的拉曼光的特点，即单数毛细管采集488nm激光器激发的拉曼光，双数毛细管采集514.5nm激光器激发的拉曼光，反之亦可。从图中我们可以看到，488nm和514.5nm激光被水散射的拉曼峰恰好能够分开，且正好分布在CCD检测范围之内：500～660nm。这恰好符合了空间校正中采集两组数据进行校正的需求。图4给出了空间校正CCD上的一帧光谱图，从图4上可以明显看到每根毛细管上对应两个亮点(拉曼峰值)。Figure 3 can be obtained by projecting the characteristic Raman peaks of 488nm and 514.5nm lasers scattered by water onto the data acquisition CCD. The blue light (left) is the collected Raman light intensity of 488nm laser scattered by water, the red light (right) corresponds to the collected Raman fluorescence intensity of 514.5nm laser scattered by water, each peak corresponds to a capillary, using phase The characteristics of adjacent capillary intervals to collect Raman light scattered by different wavelengths of laser light, that is, an odd-numbered capillary collects Raman light excited by a 488nm laser, and an even-numbered capillary collects Raman light excited by a 514.5nm laser, and vice versa. We can see from the figure that the Raman peaks of 488nm and 514.5nm lasers scattered by water can just be separated, and they are just distributed within the detection range of CCD: 500-660nm. This just meets the requirement of collecting two sets of data for correction in spatial correction. Figure 4 shows a frame of spectrograms on the spatially corrected CCD, and it can be clearly seen from Figure 4 that there are two bright spots (Raman peaks) corresponding to each capillary.

对图4进行横轴数据求和作为光谱强度，以纵轴象素为横坐标作图给出空间校正谱图如图5所示。通过简单的峰识别可以给出每根毛细管中心位置(如图6所示)和有效宽度(一般左右各取一个或两个象素)。The summation of the data on the horizontal axis in Figure 4 is used as the spectral intensity, and the vertical axis pixels are plotted on the horizontal axis to give a spatially corrected spectrum as shown in Figure 5. The center position of each capillary (as shown in Figure 6) and the effective width (generally one or two pixels are taken from the left and right sides) can be given through simple peak identification.

本发明的有益效果：Beneficial effects of the present invention:

(1)采用激光激发石英或水的拉曼光谱实现光谱成像；(1) Using laser to excite the Raman spectrum of quartz or water to realize spectral imaging;

(2)可采用单一或多激光器激发；(2) Single or multiple lasers can be used for excitation;

(3)实现毛细管在CCD上的位置定位和平行度调整；(3) Realize the position positioning and parallelism adjustment of the capillary on the CCD;

(4)方法实现装置结构简单，易操作。(4) The method realizes that the device has a simple structure and is easy to operate.

(5)可消除毛细管阵列进行微量试剂分析时的误差，提高分析准确度。(5) It can eliminate the error when the micro reagent is analyzed by the capillary array, and improve the analysis accuracy.

附图说明Description of drawings

图1为本发明定位方法操作示意图；Fig. 1 is the operation schematic diagram of positioning method of the present invention;

图2为水的拉曼光谱图：水散射的特征拉曼峰；Figure 2 is the Raman spectrum of water: the characteristic Raman peak of water scattering;

图3为水的拉曼光谱图：488nm和514.5nm激光被水散射的特征拉曼峰投影到数据采集CCD上Figure 3 is the Raman spectrum of water: the characteristic Raman peaks of 488nm and 514.5nm lasers scattered by water are projected onto the data acquisition CCD

图4为毛细管阵列拉曼光在CCD上的成像图；Fig. 4 is the imaging diagram of the capillary array Raman light on the CCD;

图5为空间位置定位分析示意图。横轴为象素数，纵轴为光谱强度作图Fig. 5 is a schematic diagram of spatial position positioning analysis. The horizontal axis is the number of pixels, and the vertical axis is the spectral intensity.

图6为空间定位分析数据Figure 6 shows the spatial positioning analysis data

其中：1-激光束，2-毛细管阵列，3-单根毛细管，4-拉曼光，Among them: 1-laser beam, 2-capillary array, 3-single capillary, 4-Raman light,

5-拉曼光谱成像CCD5- Raman Spectroscopy Imaging CCD

具体实施方式Detailed ways

现结合附图及实施例对本发明方法作进一步详细说明。The method of the present invention will now be described in further detail in conjunction with the accompanying drawings and embodiments.

图1是本发明方法的操作示意图。用激光束1照在毛细管束2的检测窗口处，激光被单根毛细管3的石英或管内溶液中的水散射，发出拉曼光4，通过取光狭缝，光栅分光，透镜聚焦后成像在CCD 5上，分析CCD 5上的光谱图对毛细管束进行位置定位。Figure 1 is a schematic diagram of the operation of the method of the present invention. Use thelaser beam 1 to shine on the detection window of thecapillary bundle 2, the laser is scattered by the quartz of thesingle capillary 3 or the water in the tube solution, and emits Ramanlight 4, which passes through the light-taking slit, the grating splits the light, and the lens is focused and imaged on theCCD 5, analyze the spectrogram on theCCD 5 to locate the position of the capillary bundle.

位置定位技术采用双激光束，分别为488nm和514.5nm。488nm和514.5nm激光被水散射的特征拉曼峰投影到数据采集CCD上可以得到图3。蓝光(左侧)是采集488nm激光被水散射的拉曼光强度，红光(右侧)对应的是采集514.5nm激光被水散射的拉曼荧光强度，每一个峰对应一根毛细管，采用相邻毛细管间隔采集不同波长激光被散射的拉曼光的特点，即单数毛细管采集488nm激光器激发的拉曼光，双数毛细管采集514.5nm激光器激发的拉曼光，反之亦可。488nm和514.5nm激光被水散射的拉曼峰恰好能够分开，且正好分布在CCD检测范围之内：500～660nm。这恰好符合了空间校正中采集两组数据进行校正的需求。图4给出了空间校正CCD上的一帧光谱图，从图4上可以明显看到每根毛细管上对应两个亮点(拉曼峰值)。Position positioning technology uses dual laser beams, 488nm and 514.5nm respectively. Figure 3 can be obtained by projecting the characteristic Raman peaks of 488nm and 514.5nm lasers scattered by water onto the data acquisition CCD. The blue light (left) is the collected Raman light intensity of 488nm laser scattered by water, the red light (right) corresponds to the collected Raman fluorescence intensity of 514.5nm laser scattered by water, each peak corresponds to a capillary, using phase The characteristics of adjacent capillary intervals to collect Raman light scattered by different wavelengths of laser light, that is, an odd-numbered capillary collects Raman light excited by a 488nm laser, and an even-numbered capillary collects Raman light excited by a 514.5nm laser, and vice versa. The Raman peaks of the 488nm and 514.5nm lasers scattered by water can just be separated, and they are just distributed within the detection range of the CCD: 500-660nm. This just meets the requirement of collecting two sets of data for correction in spatial correction. Figure 4 shows a frame of spectrograms on the spatially corrected CCD, and it can be clearly seen from Figure 4 that there are two bright spots (Raman peaks) corresponding to each capillary.

对图4进行横轴数据求和作为光谱强度，以纵轴象素为横坐标作图给出空间校正谱图如图5所示。通过简单的峰识别可以给出每根毛细管中心位置(如图6所示)和有效宽度(一般左右各取一个或两个象素)。The summation of the data on the horizontal axis in Figure 4 is used as the spectral intensity, and the vertical axis pixels are plotted on the horizontal axis to give a spatially corrected spectrum as shown in Figure 5. The center position of each capillary (as shown in Figure 6) and the effective width (generally one or two pixels are taken from the left and right sides) can be given through simple peak identification.

尽管通过参照发明的某些优选实施例，已经对发明进行了描述，但本领域的普通技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离所附权利要求书所限定的本发明的精神和范围。Although the invention has been described with reference to certain preferred embodiments thereof, workers skilled in the art will understand that various changes in form and details may be made therein without departing from the appended The spirit and scope of the present invention are defined by the claims.

Claims (1)

1. one kind is utilized the method for Raman spectrum imaging locating capillary array, it is characterized in that, described method comprises: (1) sends Raman light with laser beam irradiation capillary bundle, refer to laser beam by quartz capillaceous or manage interior solution scattering and send Raman light, Raman light is sent in aqueous solvent scattering in described laser beam irradiation capillary bundle, laser beam used is double laser beam, the wavelength of described double laser beam is respectively 488nm and 514.5nm, described odd number kapillary is gathered to the Raman light that 488nm laser instrument excites, even numbers kapillary gathers the Raman light that 514.5nm laser instrument excites, or described even numbers kapillary is gathered to the Raman light that 488nm laser instrument excites, odd number kapillary gathers the Raman light that 514.5nm laser instrument excites, (2) be imaged on charge coupled cell CCD by Raman image instrument, described Raman image instrument is by getting optical slits, grating beam splitting and condenser lens are imaged on CCD Raman light, (3) spectrogram of analyzing on charge coupled cell CCD positions each kapillary in capillary bundle, take pixel count as transverse axis, spectral intensity is that the longitudinal axis obtains free-air correction spectrogram, identify the effective width obtaining between every capillary center and every capillary by peak.

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