CN101587075A - Capillary electrophoresis-column laser induced fluorescence polarization detection device - Google Patents

Abstract

The invention relates to a capillary electrophoresis-on-column laser-induced fluorescence polarization detection device, which mainly includes a capillary electrophoresis separation unit, a fluorescence excitation unit, an optical path adjustment and calibration unit, a fluorescence polarization detection unit, a signal conversion and recording unit and a data processing unit. In the device, the excitation light adopts a linearly polarized laser light source, so that the fluorescence emitted by the excited phosphor retains the polarization characteristics. The excited fluorescence is divided into vertically polarized light and horizontally polarized light by the polarization splitting optical device, and the two parts of polarized light are respectively collected by two fluorescence polarization detection elements, and recorded in the computer through a signal converter. The relative size of the two polarized lights can be used to calculate the size of the fluorescence polarization response, and the fluorescence polarization can be used to study the interaction between biological macromolecules. Therefore, this device not only has the advantages of high efficiency, rapid separation and high sensitivity detection, but also provides a new instrument platform for the study of the interaction between biological macromolecules.

Description

Capillary electrophoresis-on-column laser-induced fluorescence polarization detection device

technical field

The invention relates to a capillary electrophoresis-on-column laser-induced fluorescence polarization detection device, which combines capillary electrophoresis-on-column laser-induced fluorescence detection and fluorescence polarization detection technology into one, not only has the advantages of high-efficiency separation and high-sensitivity detection, but also It provides fluorescence polarization information for studying the interaction between molecules.

Background technique

Capillary electrophoresis is an efficient and fast analysis and separation method nowadays, and it is widely used in many fields such as chemistry, biology, environment, medicine, etc.: including detection of organic and inorganic small molecules, separation of peptides and proteins, DNA sequence and chiral separation etc. The laser-induced fluorescence detection method (LIF) is a highly sensitive detection method, and its detection sensitivity can even reach the single-molecule level. The analytical instrument produced by the combination of the two, capillary electrophoresis-laser-induced fluorescence detection and analysis instrument (CE-LIF), is currently one of the separation and detection technologies with high sensitivity and rapid development. And it has the advantages of high sensitivity, high resolution, high speed, less sample consumption, low cost and easy automation.

Fluorescence polarization means that when the fluorescent molecules are excited by plane polarized light, the fluorescent molecules emit polarized light. When the molecular weight of the fluorescent marker is relatively small, the rotational movement in the solution is fast, and the fluorescence emission generated when the molecule is excited by polarized light is almost depolarized; when it is combined with another macromolecular substance, its volume emits Large changes, the rotational motion of the phosphor is suppressed, and the fluorescence polarization increases significantly. Therefore, the determination of fluorescence polarization utilizes the characteristic that the rotation speed of fluorescent substance molecules in solution is inversely proportional to the molecular size, and is a method especially suitable for studying intermolecular interactions (such as protein-protein interaction, protein-DNA bonding, antigen- Antibody immune response, etc.) technology.

The present invention combines capillary electrophoresis-on-column laser-induced fluorescence detection with fluorescence polarization detection technology, which not only has the advantages of high-efficiency separation and high-sensitivity detection, but also obtains the information of fluorescence polarization at the same time, which provides a basis for studying the interaction between biological macromolecules. A new analytical method and instrument platform were developed.

Contents of the invention

The invention relates to a capillary electrophoresis-on-column laser-induced fluorescence polarization detection device. The device combines the advantages of high-efficiency, fast-separated capillary electrophoresis and high-sensitivity laser-induced fluorescence detection. Fluorescence polarization can also be used for the study of biological macromolecules (proteins) -The interaction between protein, protein-DNA, antigen-antibody) provides a new analytical method and instrument platform.

The present invention proposes a capillary electrophoresis-on-column laser-induced fluorescence polarization detection device, which is mainly composed of a capillary electrophoresis separation unit, a fluorescence excitation unit, an optical path adjustment and calibration unit, a fluorescence polarization detection unit, a signal conversion and recording unit and a data processing unit. The unit consists of six parts. The key components in the device mainly include: polarized laser light source, optical path adjustment lens, buffer bottle, Pt electrode, quartz capillary, high voltage power supply, pinhole, fluorescence polarization spectroscopic optical device, filter, fluorescence detection element and signal converter (see Figure 1). Its basic working principle is: the quartz capillary and the Pt electrode are respectively inserted into the buffer bottles at both ends, and the high voltage required for electrophoretic separation is provided by a high voltage power supply. After the polarized laser is focused by the optical path adjustment system, it is irradiated onto the detection window of the quartz capillary to excite the fluorescent substances in the capillary. The excited fluorescence is focused by the optical lens and enters the detection system. The polarized fluorescence is divided into vertically polarized light and horizontally polarized light by the polarizer. After passing through the filter respectively, the fluorescence signal is collected by two fluorescence detection elements and recorded on the computer through the signal converter. , for data analysis and processing.

In the device, a linearly polarized laser light source is used, so that the fluorescence emitted by the excited phosphor will retain the polarization characteristics. The fluorescence emitted by the phosphor is separated into two beams of light by the fluorescence polarization spectroscopic optical device, which are vertically polarized light and horizontally polarized light (see Figure 1). The two beams of polarized light pass through the two fluorescence detection elements and are recorded respectively, and thus the fluorescence polarization information of the fluorescent substance is obtained. The fluorescent detection element used in the device can be a photomultiplier tube or a charge-coupled digital imaging device.

In the device, the detection window can be the detection window on the column by removing the surface coating of the capillary, or a quartz sheath flow cell can be connected behind the capillary column. The laser light source is passed through the optical path adjustment lens (2), so that the excitation light is focused on the capillary detection window, and the spot diameter is less than 20 μm, thereby effectively reducing light scattering and improving the sensitivity of the instrument. The excitation light and the fluorescence detection system are vertically arranged at an angle of 90°, which effectively avoids the interference caused by excitation light scattering on the detection of weak fluorescence, and significantly improves the detection sensitivity. The distance between the optical lens (7) of the fluorescence detection unit and the detection window of the capillary is less than 0.5mm, which can effectively prevent stray light in the environment from entering the detection system and reduce the interference to the fluorescence detection.

In this device, the high-voltage power supply can provide positive and negative high voltages. The electrode near the detection window is grounded, so the experiment is safer. By changing the polarity of the output voltage of the power supply, the positive and negative separation modes of capillary electrophoresis can be realized.

The invention combines capillary electrophoresis-on-column laser-induced fluorescence detection with fluorescence polarization technology, so that it not only has the advantages of high efficiency, rapid separation and high sensitivity detection, but also provides a fluorescence polarization method for studying the interaction between biological macromolecules. information.

Description of drawings

Fig. 1 is capillary electrophoresis-on-column laser-induced fluorescence polarization detection device structural schematic diagram;

Figure 2 is a capillary electrophoresis diagram of highly sensitive detection of DNA;

Figure 3 is a capillary electrophoresis fluorescence polarization diagram of the interaction between the DNA adduct fluorescent probe and the antibody;

Fig. 4 is a capillary electrophoresis fluorescence polarization diagram of the interaction between thrombin and thrombin aptamer.

Detailed ways

The basic structure of the capillary electrophoresis-on-column laser-induced fluorescence polarization detection device involved in the present invention is shown in FIG. 1 . 1 in the figure is a polarized laser light source, which can be argon ion laser, He-Ne laser, dye laser, semiconductor laser, or other light sources, which can be selected according to needs; 2 is an optical path adjustment lens; 3 is a buffer bottle; 4 is a Pt electrode ; 5 is a quartz capillary; 6 is a high voltage power supply; 7 is an optical lens; 8 is a pinhole; 9 is a fluorescence polarization spectroscopic optical device; 10 is a filter; 11 is a fluorescence detection element, which can be a photomultiplier tube (PMT), It can also be a charge-coupled digital imaging device (CCD); 12 is a signal converter; 13 is a computer.

Its working principle: the quartz capillary (5) and the Pt electrode (4) are respectively inserted into the buffer solution bottle (3) at both ends, and the high voltage required for electrophoretic separation is provided by the high-voltage power supply (6), thus forming a capillary electrophoresis separation unit . The high-intensity laser (1) is focused to the detection window of the quartz capillary through the optical path adjustment lens (2), and the diameter of the laser spot is less than 20 μm, thereby constituting a fluorescence excitation unit. In this part, the excitation light source and the fluorescence detection system are distributed at an angle of 90 degrees, which can effectively avoid the interference of the excitation light scattering on the fluorescence detection. Driven by high voltage, fluorescent molecules pass through the capillary detection window and are excited by laser light, and the emitted fluorescence can be received by the fluorescence detection unit. This part includes an optical lens (7); a pinhole (8); a fluorescence polarization spectroscopic optical device ( 9); optical filter (10); fluorescent detection element (11). Wherein, the distance between the optical lens (7) and the detection window of the capillary is less than 0.5mm, which can effectively prevent stray light in the environment from entering the detection system and interfere with the detection of weak fluorescence. After the fluorescence signal is detected by the fluorescence detection element (11), it is recorded in the computer (13) through the signal converter (12).

In this device, a linearly polarized laser light source is used, and the fluorescence emitted by the excited fluorescent molecules will retain the polarization characteristics. The polarization response of the fluorescence is related to the volume of the fluorescent molecule: when the molecular weight of the fluorescent marker is relatively small, the rotational movement in the solution is fast, and the fluorescence emission generated when the molecule is excited by polarized light is almost depolarized; After being combined with another macromolecular substance, its volume emission changes greatly, the rotational motion of the phosphor is suppressed, and the fluorescence polarization increases significantly. The fluorescence is focused into the detection system by the optical lens (7), and is divided into two beams of light by the fluorescence polarization spectroscopic optical device (9), which are vertically polarized light (I _v ) and horizontally polarized light (I _h ). The ratio of the intensities allows the calculation of the magnitude of the polarization (FP) response of the fluorescence.

$\mathrm{<span\; class="notranslate">\; FP</span>}<span\; class="notranslate">\; =</span>\frac{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; v</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; h</span>}}}{{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; v</span>}}<span\; class="notranslate">\; +</span>{\mathrm{<span\; class="notranslate">\; I</span>}}_{\mathrm{<span\; class="notranslate">\; h</span>}}}$

After the two beams of polarized light pass through the optical filter (10) respectively, the fluorescence signals are respectively detected by the two fluorescence detection elements (11), and are recorded in the computer (13) through the signal converter (12) for data analysis and processing.

Example 1:

Fig. 2 is a capillary electrophoresis diagram of the present invention for highly sensitive detection of DNA. Experimental conditions: a polyacrylamide-coated capillary with an inner diameter of 50 microns, a total length of 40 cm, and an effective length of 33 cm. Tris-glycine buffer (Tris 30mM, glycine 160mM, pH=8.5), separation voltage is 20kV. As shown, dsDNA is double-stranded calf thymus DNA.

The device of the present invention can detect 22×10 ^-15 mol/L of calf thymus DNA labeled with fluorescent light within 5 minutes, reflecting the advantages of high efficiency, rapid separation and high sensitivity detection of the device.

Example 2:

Fig. 3 is a capillary electrophoresis fluorescence polarization diagram for studying the interaction between DNA adduct fluorescent probes and antibodies of the present invention. Experimental conditions: a fused silica capillary with an inner diameter of 25 microns, a total length of 30 cm, and an effective length of 24 cm. Tris-glycine buffer (Tris 25mM, glycine 192mM, pH=8.3), separation voltage is 15kV. By using the device of the invention, the fluorescence polarization values of different peaks can be obtained at the same time of highly sensitive detection, which provides favorable data for studying the interaction between the DNA adduct fluorescent probe and the antibody. As shown in the figure, the fluorescence polarization values of the three peaks are 0.006, 0.109, and 0.131, respectively. According to the difference in the fluorescence polarization of the three peaks, the above three peaks can be easily determined as follows: peak 1 is the TMR fluorescent internal standard, peak 2 is the DNA adduct fluorescent probe, and peak 3 is the DNA adduct fluorescent probe and antibody compound.

Example 3:

Fig. 4 is a capillary electrophoresis fluorescence polarization diagram for studying the interaction between thrombin and thrombin aptamer (aptamer) in the present invention. Experimental conditions: a fused silica capillary with an inner diameter of 25 microns, a total length of 30 cm, and an effective length of 24 cm. Tris-glycine buffer (Tris 25mM, glycine 192mM, pH=8.3), separation voltage is 20kV. By applying the device of the present invention, the fluorescence polarization values of different peaks can be obtained while detecting the thrombin with high sensitivity, which provides favorable data for studying the interaction between the thrombin and the thrombin aptamer. As shown, the fluorescence polarization values of the two peaks are 0.304 and 0.196, respectively. According to the difference in the fluorescence polarization of the two peaks, the above two peaks can be conveniently determined as follows: peak 1 is the complex of thrombin and thrombin aptamer, and peak 2 is the fluorescently labeled thrombin aptamer.

Claims (10)

1. Capillary electrophoresis-on-column laser-induced fluorescence polarization detection device, mainly composed of six parts: capillary electrophoresis separation unit, fluorescence excitation unit, optical path adjustment and calibration unit, fluorescence polarization detection unit, signal conversion and recording unit, and data processing unit. The key components mainly include: polarized laser light source, optical path adjustment lens, buffer bottle, Pt electrode, quartz capillary, high voltage power supply, pinhole, fluorescence polarization spectroscopic optical device, filter, fluorescence detection element and signal converter. Its basic working principle is: the quartz capillary and Pt electrodes are respectively inserted into the buffer bottles at both ends, and the high voltage required for electrophoretic separation is provided by a high-voltage power supply to form a capillary electrophoresis separation unit. When the fluorescent substance is separated by capillary electrophoresis and reaches the detection window, it is excited by the high-intensity laser emitted by the fluorescence excitation unit. The emitted fluorescence enters the fluorescence polarization detection system through the optical lens, and the polarized fluorescence is divided into vertically polarized light and horizontally polarized light by the fluorescence polarization spectroscopic optical device. Recorded in a computer for data analysis and processing. 2. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the device combines capillary electrophoresis-on-column laser-induced fluorescence detection with fluorescence polarization technology, making it not only efficient, The advantages of fast separation and high-sensitivity detection, while also providing information on fluorescence polarization, provide a new analytical method and instrument platform for studying the interaction between biomacromolecules. 3. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that the excitation light source is a linearly polarized laser light source, so that the fluorescence emitted by the excited phosphor will retain the polarization characteristics. 4. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the polarized fluorescence is separated into two beams of light by the fluorescence polarization spectroscopic optical device, which are vertically polarized light and horizontally polarized light respectively, and the two The beam of polarized light is collected and recorded through two fluorescent detection elements respectively, and then the fluorescence polarization information of the fluorescent substance is obtained. 5. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the high-voltage power supply can provide positive and negative high voltages, and by changing the polarity of the voltage output by the power supply, the positive polarity of capillary electrophoresis can be realized. , Negative separation mode. 6. Capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the detection window can pass through the on-column detection window with the capillary surface coating removed, or connect the quartz sheath flow behind the capillary column pool. 7. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the laser light source passes through the optical path adjustment lens to focus the excitation light on the capillary detection window, and the spot diameter is less than 20 μm, thereby effectively Light scattering is reduced and the sensitivity of the instrument is improved. 8. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the laser and the fluorescence detection system are vertically arranged at an angle of 90°, effectively avoiding the interference caused by excitation light scattering, thereby Significantly improved the detection sensitivity. 9. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that: the distance between the optical lens of the fluorescence detection unit and the detection window of the capillary is less than 0.5mm, which can effectively avoid the entry of stray light in the environment The detection system reduces interference to fluorescence detection. 10. The capillary electrophoresis-on-column laser-induced fluorescence polarization detection device according to claim 1, characterized in that the fluorescence detection element can be a photomultiplier tube or a charge-coupled digital imaging device.

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