CN101464411A - Capillary array analyzer - Google Patents

Abstract

The invention belongs to the technical field of biological analysis, and relates to a capillary array analyzer. The excitation light emitted by a light source is incident on a vibrating mirror by a reflector and a fluorescent separation mirror, and the vibrating mirror swings back and forth around a rotation axis, and the excitation light also swings back and forth; The objective lens, the scanning objective lens gathers the excitation light to the focal plane where the capillary array is placed, and is transformed into a one-dimensional translational scanning on the focal plane through the scanning objective lens. The fluorescence emitted by the labeled fluorescent substance is collected by the scanning objective lens, and converted into a parallel beam that is coaxial with the excitation light and propagates in the opposite direction. The parallel beam is reflected and separated from the excitation beam, and then enters the converging lens. The background light is filtered out by the fluorescent filter on the color wheel, passed through the confocal aperture, and detected by the photodetector. The capillary array analyzer of the invention has the advantages of simple structure, small volume, low cost and excellent performance.

Description

Capillary Array Analyzer

technical field

The invention belongs to the technical field of biological analysis, in particular to a capillary array analyzer.

Background technique

Capillary electrophoresis is to make the sample migrate in the capillary through the action of the electric field, and different components in the sample have different migration speeds, so as to achieve separation, and detect at the detection window in the capillary, and finally detect the sample through the obtained detection signal for analysis. This technology is widely used in biochemical analysis, such as DNA and protein separation analysis, which has high separation efficiency, low sample consumption and high degree of automation. It is widely used in DNA sequencing, biological and clinical research, forensic identification, environmental protection, analytical chemistry and Food testing and other fields have a wide range of applications. The detection of electrophoretic samples mainly includes isotope labeling, chemiluminescence methods, surface plasmon resonance, and laser-induced fluorescence. Optical detection is usually used, and laser-induced fluorescence detection is the most sensitive and widely used.

In order to achieve high-throughput analysis, multiple capillaries can be electrophoresed at the same time, and the detection windows can be arranged in an array for parallel detection, usually capillary array electrophoresis and fluorescence detection. The current capillary array electrophoresis detection methods mainly include the following: (1) The capillaries are arranged in parallel, and the imaging objective lens is imaged onto the CCD for non-scanning non-confocal detection, but the detection sensitivity of the non-confocal method is limited. (2) The capillaries are arranged in parallel and placed on a scanning platform, and the scanning confocal detection is realized through the movement of the scanning platform, or the scanning of the capillary array is realized by moving the lens relative to the capillaries. The scanning speed of this scanning method is limited by the drive, and the vibration is large. (3) The capillaries are arranged on a cylinder, the lens is arranged in the cylinder perpendicular to the central axis of the cylinder, and the scanning is realized through the circular movement of the lens around the central axis. This method requires capillaries to be arranged in a circle, which brings inconvenience to some analyzes and applications. (4) The radial array of capillaries or microchannels is arranged on a disk plane, and the lens is arranged perpendicular to the disk and rotates around the center of the disk to realize scanning. However, the disk arrangement of the capillary will also bring inconvenience to the analysis and application, and the lens rotation mechanism is complicated and difficult to miniaturize.

In the current confocal detection, the multi-color fluorescence detection technology adopts the color filter spectroscopic method. For example, the 4-color fluorescence detection uses a color filter to first separate the fluorescence of the four wavelength bands into two light paths according to the long-wave and short-wave, and then each The first optical path is separated into two optical paths by a color filter, and each optical path is detected by a photodetector. This spectroscopic detection method firstly requires many photodetectors, which increases the production cost and the volume of the detector; secondly, due to the large number of color filters and the loss of fluorescence, the detection sensitivity is limited; finally, due to the multi-color filter configuration in The optical path, therefore, requires large space, further increasing the volume of the detector.

Contents of the invention

The invention provides a capillary array analyzer with simple structure, small volume, low cost and excellent performance.

The technical scheme adopted in the present invention is as follows: a capillary array analyzer, comprising a light source, a reflector, a vibrating mirror, a scanning objective lens, a fluorescent separation mirror, a converging lens, a color wheel, a confocal aperture, a light detector, and the excitation light emitted by the light source It is reflected by the reflector, and enters the vibrating mirror through the fluorescent separation mirror. The vibrating mirror swings back and forth around the rotation axis, so that the excitation light also swings back and forth; the excitation light is incident on the scanning objective lens, and the scanning objective lens focuses the excitation light to the focal point where the capillary array is placed. On the surface, the oscillation of the excitation light is converted into a one-dimensional translational scanning on the focal plane through the scanning objective lens. During the scanning process, the excitation light excites the samples marked with fluorescent substances in the capillary in turn, and the fluorescence emitted by the fluorescent substances marked by the samples is obtained by The scanning objective lens collects and converts the fluorescent parallel beam coaxial with the excitation light and propagates in the opposite direction. The fluorescent parallel beam is reflected at the fluorescent separation mirror and separated from the excitation beam, and then enters the converging lens. The converged fluorescent parallel beam The background light is filtered out by the fluorescent filter on the color wheel, focused through the confocal aperture, and detected by the photodetector.

In the capillary array analyzer, the capillaries of the capillary array are arranged in parallel on one plane, or the capillary array is a microchannel array etched in the chip.

In the capillary array analyzer, the capillary array is sandwiched between two substrates.

In the capillary array analyzer, the light source is a laser or a light emitting diode.

In the capillary array analyzer, the light source, the capillary array, and the confocal aperture are in a conjugate position to form a confocal detection.

In the capillary array analyzer, the scanning objective adopts an image square telecentric f-theta objective lens.

In the capillary array analyzer, the fluorescence separation mirror is at an angle of 45° to the optical axis of the excitation light.

In the capillary array analyzer, the fluorescence separation mirror is a dichroic mirror or a total reflection mirror with a hole in the center.

In the capillary array analyzer, the light detector adopts a photomultiplier tube, or an avalanche photodiode, or an intrinsic photodiode.

In the capillary array analyzer, the fluorescent color filter is a color filter group installed on the color wheel, and the rotation of the color wheel makes the color filters with different spectral characteristics in the optical path to detect the fluorescence of different wavelengths.

During the working process, the two ends of the capillary are inserted into the buffer pool and connected to the positive and negative poles of the power supply respectively. The capillary is filled with sieving medium, and the positive and negative poles apply voltage to the capillary for pre-electrophoresis. After the current is stable, according to the charge of the sample properties, sample injection at the positive or negative electrode. After sample injection, the sample in the capillary is separated by electrophoresis. When the sample with fluorescent substance migrates to the detection window, it is excited by the excitation spot scanned to the capillary, and the emitted fluorescence is collected by the scanning objective lens, vibrating mirror and fluorescent separation mirror. The reflections, converged on the converging objective, are filtered through a fluorescent filter on the color wheel and detected by a photodetector.

In order to ensure the uniformity of each capillary during scanning, the scanning objective lens adopts a telecentric f-theta objective lens, so that the chief ray of each scanning position is parallel to the optical axis, and the system has no vignetting, so as to ensure that each scanning position collects Consistency of fluorescence. The f-theta lens converts the angular change of the beam reflected from the galvanometer to the scanning objective lens into a linear displacement change on the surface where the capillary array is located during the scanning process, ensuring the uniformity of the scanning speed. In the present invention, the method of capillary array laser-induced fluorescence detection with a vibrating mirror combined with a telecentric f-theta objective lens has the advantages of small scanning inertia, few moving parts, stable performance of the detector, high detection uniformity, and compact structure.

The present invention adopts color wheel rotation to make multiple color filters time-division multiplexed for multi-color fluorescence detection, which reduces the loss of fluorescence energy and the number of photodetectors used, thus reducing both the manufacturing cost and the volume.

Description of drawings

Fig. 1 is a structural schematic diagram of the present invention.

Fig. 2 is a schematic diagram of capillary array arrangement.

Fig. 3 is a schematic diagram of multi-color fluorescence detection by color wheel rotation.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings.

Fig. 1 is a structural schematic diagram of the present invention. The light source 1 adopts a laser, and a reflector 3 is installed in the light emitting direction of the light source 1, and the excitation light 2 emitted by the light source 1 is reflected by the reflector 3, and the reflector 3 can be used for adjusting the optical path of the excitation light 2. A fluorescence separation mirror 4 is installed in the direction of the outgoing light of the reflection mirror 3 , and the fluorescence separation mirror 4 forms an angle of 45° with the optical axis of the excitation light 2 . The fluorescence separating mirror 4 is a total reflection mirror with a hole in the center, the center hole is used to pass the excitation light 2, and the total reflection mirror is used to reflect fluorescence. The reflected excitation light 2 passes through the central hole of the fluorescent separation mirror 4 and is incident on the vibrating mirror 5 . The vibrating mirror 5 is installed on the rotating shaft 501 and can swing back and forth around the shaft. The galvanometer lens is also a total reflection mirror. The excitation light 2 is reflected by the oscillating mirror 5. As the oscillating mirror 5 swings around the rotation axis 501, the reflected excitation light 2 is also oscillating, and the oscillating excitation light 2 is incident on the scanning objective lens 6 again. , the scanning objective lens 6 adopts an image space telecentric f-theta objective lens, and the scanning objective lens 6 converges the excitation light onto the focal plane of the objective lens. The capillary array 7 composed of capillaries is placed on the focal plane of the scanning objective lens 6, and the oscillation of the excitation light 2 is transformed into a one-dimensional translational scanning on the focal plane through the scanning objective lens 6. During the scanning process, the excitation light 2 sequentially excites the samples marked with fluorescent substances in each capillary, and the fluorescence 8 emitted by the fluorescent substances of the marked samples is collected by the scanning objective lens 6, and transformed into a parallel beam that is coaxial with the excitation light and propagates in the opposite direction. Light, fluorescent parallel light beam 8 is reflected at fluorescent separation mirror 4 and separated from excitation beam 2, and then enters converging lens 9, and the converged fluorescent light passes through fluorescent color filter 101 on color wheel 10 to filter out background light, and is focused to pass through The confocal aperture 11 is detected by a photodetector 12, and the photodetector 12 adopts a photomultiplier tube. The light source 1 , the capillary array 7 , and the confocal aperture 11 in front of the photodetector 12 are in a conjugate position to form a confocal detection.

2 is a schematic diagram of the arrangement of the capillary array 7. A plurality of capillaries 71 are arranged in parallel on a plane. These capillaries are positioned on a capillary fixing substrate 72 engraved with a V-shaped groove. There is a hole in the middle of the substrate 72 for transmitting excitation light and fluorescence. The substrate 73 is fixed to the substrate 72 by screws 74, and the capillary 71 is fixed between the two substrates 72, 73, and the contact surface between the substrate 73 and the capillary 7 is a plane.

3 is a schematic diagram of color wheel rotation time-division multiplexing detection of multicolor fluorescence. A plurality of color filters 101 with different transmission spectra are fixed on the color wheel 10. The color filter surface of the color filter 101 is perpendicular to the central axis of the fluorescent beam 8. Moreover, the rotation axis center 102 of the color wheel deviates from the center of the fluorescent beam 8 , so that during the rotation of the color wheel 10 , the central axis of each color filter 101 can just coincide with the central axis of the fluorescent beam 8 . By rotating the color wheel 10, when each color filter 101 filters the fluorescent beam 8, different spectral components, that is, different colors of fluorescent light pass through the color filter 101 and reach the photodetector 12, realizing time-division multiplexed multicolor fluorescent signal detection .

Of course, those of ordinary skill in the art should recognize that the above is only an explanation of a specific embodiment of the present invention, but not a limitation of the present invention. For example, a light emitting diode can be used as a light source, and the capillary array can be a microchannel array etched in a chip. Fluorescence splitting mirror can adopt dichroic mirror, photodetector can adopt avalanche photodiode or intrinsic photodiode etc., all will fall within the scope of the present invention to the change of above embodiment, modification all will fall into within the scope of protection.

Claims (10)

1, capillary array analyzer, comprise light source, catoptron, galvanometer, scanning objective, it is characterized in that: also comprise fluorescence splitting mirror, convergent lens, colour wheel, confocal aperture, photo-detector, the exciting light that light source sends is by mirror reflects, see through the fluorescence splitting mirror and incide galvanometer, galvanometer is reciprocating swing around the shaft, and makes also reciprocally swinging of exciting light; Back exciting light incides scanning objective, scanning objective is poly-to the focal plane of placing capillary array with exciting light, the swing of exciting light scans through the one-dimensional translation that scanning objective is converted on the focal plane, in scanning process, exciting light excites the sample that is marked with fluorescent material in the kapillary successively, the fluorescence that fluorescent material sent of sample mark is collected by scanning objective, and be converted to the fluorescence parallel beam of and backpropagation coaxial with exciting light, the fluorescence parallel beam is reflected and is separated with excitation beam at fluorescence splitting mirror place, after incide convergent lens, fluorescence parallel beam after the convergence is through the fluorescence color filter wiping out background light on the colour wheel, focus on by confocal aperture, survey by photo-detector.

2, by the described capillary array analyzer of claim 1, it is characterized in that: the kapillary of described capillary array is parallel to a plane, or capillary array is the micro channel array that is etched in the chip.

3, by the described capillary array analyzer of claim 2, it is characterized in that: described capillary array is sandwiched between two substrates.

4, by the described capillary array analyzer of claim 1, it is characterized in that: described light source adopts laser or light emitting diode.

5, by each described capillary array analyzer of claim 1-4, it is characterized in that: light source, capillary array, confocal aperture three are in conjugate position, form confocal detection.

6, by the described capillary array analyzer of claim 1, it is characterized in that: described scanning objective adopts picture side's heart f-theta object lens far away.

7, by the described capillary array analyzer of claim 1, it is characterized in that: the optical axis of described fluorescence splitting mirror and exciting light is at 45.

8, by the described capillary array analyzer of claim 7, it is characterized in that: described fluorescence splitting mirror adopts dichroic mirror or center total reflective mirror with holes.

9, by the described capillary array analyzer of claim 1, it is characterized in that: described photo-detector adopts photomultiplier, or Avalanche Photo Diode, or the intrinsic photodiode.

10, by the described capillary array analyzer of claim 1, it is characterized in that: described fluorescence color filter is mounted in the color filter group on the colour wheel, and colour wheel rotates the color filter of different spectral characteristics is in the light path, surveys the fluorescence of different wave length.

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