CN1385690A - Biochip analysis instrument - Google Patents

Abstract

The present invention adopts the light emitted by two light sources with different wavelengths to be respectively incident on the vibrating mirror which swings back and forth around the axis through the reflecting mirror, and the angle-changing light reflected by the vibrating mirror is converted into linear displacement light by the scanning objective lens, and is diverted to the living organism through the prism. The chip realizes the one-dimensional scanning of the biological chip, and the other one-dimensional scanning is a mechanical scanning driven by a motor. The fluorescence emitted by the biochip is diverted to the scanning objective through the prism, then through the vibrating mirror, reflector, and condenser, and then through the aperture of the confocal diaphragm to reach the optical filter. After filtering out the stray light, it is converted into an electrical signal by the photodetector. In the present invention, an aperture is set on the optical path emitted by the light source, and a synchronously switched aperture and a color filter are used to allow only one beam of excitation light with a specific wavelength to pass through in one scanning process, thereby realizing the dual detection of the biological chip in a time-division multiplexing manner. Wavelength fluorescence scanning detection. The invention overcomes the disadvantages of large mechanical scanning inertia, long reciprocating stroke, large vibration, low scanning frequency, etc., and can effectively suppress crosstalk in dual-wavelength simultaneous scanning.

Description

Biochip Analyzer

Technical field

The invention relates to a biochip analyzer.

Background technique

Biochips are widely used in gene research, drug research, disease diagnosis and other fields. It uses the principle of molecular hybridization to label the samples to be detected with fluorescent molecules, and then performs hybridization reactions with biochips of known structures, and uses biochip analyzers to detect the occurrence of hybridization. The fluorescent signal at the reaction site is displayed graphically.

The laser confocal biochip analyzer currently in use uses fully mechanical two-dimensional scanning, dual light sources, and dual photodetectors to achieve dual-wavelength fluorescence detection. It includes dual light sources and dual photodetectors that are staggered at a certain angle. The laser beam emitted by the light source reaches a certain point on the biochip through lenses, beam splitters and prisms, and excites the fluorescent dye molecules at the point to emit fluorescence, and the fluorescence passes through the prism. , reflected by the beam splitter to the reflector, and then converged by the condenser to reach the photodetector. The two-dimensional scanning of the chip is to use the linear driver to drive the prism to move in the X direction, and the stepper motor to drive the biochip to move in the Y direction. This biochip analyzer has the following disadvantages: 1) its scanning speed is limited by the operating frequency (up to about 20 Hz) of the linear drive, the analysis time of the biochip is long, and the vibration of the linear drive is large, the scanning inertia is large, and the reciprocating The stroke is long, resulting in a long stroke of the corresponding prism, which makes the focus spot of the laser beam change greatly, which affects the resolution of the instrument; 2) The numerical aperture NA of the condenser is limited by the mechanical structure, and the value is not very large, which affects the analysis of the instrument Sensitivity; 3) It uses dual light sources and dual detectors to realize simultaneous scanning and imaging of dual-wavelength fluorescence, which makes the structure of the entire instrument complex, and there is crosstalk in the scanning image, which affects the performance of the instrument.

Contents of Invention

The object of the present invention is to provide a biochip analyzer with simple structure, low cost and good performance.

The biochip analyzer of the present invention comprises first and second light sources of different wavelengths, a reflector and a total reflection mirror of parallel devices, the light emitted by the second light source is reflected by the reflector, and passes through the holes of the perforated reflector to be incident to the vibrating mirror, the light emitted by the first light source is reflected by the total reflection mirror, passes through the reflector and then enters the vibrating mirror through the hole of the perforated mirror. The vibrating mirror is installed on the rotating shaft and swings back and forth around the axis. On the optical path of the chip, the device has a scanning objective lens that converts the reflected light from the vibrating mirror into linear displacement light and a prism that turns the light emitted by the scanning objective lens onto the biochip, forming light scanning in the X direction of the biochip, and biological The chip is installed on a linear guide rail, driven by a stepping motor to move along the Y direction of the two-dimensional plane formed by X and Y. The fluorescence emitted by the biochip is incident on the prism, and then transformed into a parallel beam incident on the vibrator by the prism steering and scanning objective lens. The mirror is reflected from the oscillating mirror to the perforated mirror. On the optical path reflected by the perforated mirror, the condenser lens is sequentially installed along the reflected light, and the confocal diaphragm is perpendicular to the optical path and in the same plane. Two pieces are switched on the optical path. The color filter and the photodetector are provided with a first diaphragm corresponding to the first color filter and switched synchronously between the first light source and the total reflection mirror, and a second diaphragm with the second filter is provided between the second light source and the reflection mirror. The color film corresponds to the diaphragm that is switched synchronously. When the first diaphragm is opened, the second diaphragm is closed, and the first color filter is on the optical path. When the first diaphragm is closed, the second diaphragm is opened, and the second filter is on the optical path. film on the light path.

When working, the excitation light emitted by the light source is reflected by the mirror to the vibrating mirror, and then the incident angle-changing excitation beam is converted into a linear displacement in a certain direction through the scanning objective lens, and the direction is changed by the prism to reach a certain point on the biochip . The target molecules marked with fluorescent dyes generate fluorescence under the excitation of excitation light, and the fluorescence is diverted to the scanning objective lens through the prism, collected by the scanning objective lens and transformed into parallel light. Aperture: Fluorescence passing through the aperture of the confocal aperture passes through a filter to filter out stray light of other wavelengths, and then is converted into an electrical signal by a photodetector for subsequent processing and imaging analysis. The scanning in one dimension of the biochip analyzer adopts optical scanning combined with a vibrating mirror and a scanning objective lens, and scanning in another dimension is mechanical scanning driven by a stepping motor. At the same time, the aperture and color filter that are switched synchronously allow only one beam of excitation light with a specific wavelength to pass through in one scanning process. In a time-division multiplexing manner, only one photodetector is used to realize the dual-wavelength fluorescence scanning of the biochip. detection.

Typically, the scanning objective adopts a telecentric f-theta scanning objective. In order to maintain the uniformity of fluorescence detection during the optical scanning process, the chief ray of the excitation beam incident on the biochip through the scanning objective lens is always perpendicular to the biochip.

The present invention adopts two-dimensional scanning combining optical scanning and mechanical scanning, so it overcomes the shortcomings of the linear drive in full mechanical scanning, such as large scanning inertia, long reciprocating stroke, large vibration, and low scanning frequency;

The invention realizes the dual-wavelength fluorescence scanning of the biological chip in a time-division multiplexing manner, which can effectively suppress the crosstalk in the dual-wavelength simultaneous scanning, and at the same time make the structure of the whole instrument simple and reduce the cost;

Because the galvanometer has high rotation angle accuracy and repeatability, and the rotation angle step size is very small, high-resolution biochip scanning images can be obtained. Moreover, the scanning frequency of the vibrating mirror is much higher than that of the linear drive, which greatly improves the scanning efficiency of the biochip;

In addition, the f-theta scanning objective lens can make the direction of the main fluorescence light emitted from each point on the biochip be the normal direction of the biochip, which can ensure the consistency of the fluorescence radiation angle of each measured point.

Description of drawings

Accompanying drawing is a schematic diagram of the present invention.

Detailed ways

With reference to accompanying drawing, the biochip analyzer of invention comprises the light source 1,2 of the first and second two different wavelengths, the reflection mirror 6 and total reflection mirror 5 of parallel device, two light sources are different laser sources of wavelength, wavelength The range is from 400nm to 650nm. For example, the first light source 1 uses a 635nm red laser, and the second light source 2 uses a 532nm green laser. The light emitted by the second light source 2 is reflected by the reflector 6, passes through the hole of the perforated reflector 7, and enters the vibrating mirror 8, and the light emitted by the first light source 1 is reflected by the total reflection mirror 5, passes through the reflector 6 and passes through the hole The hole of the reflector 7 is incident on the oscillating mirror 8, and the oscillating mirror 8 is installed on the rotating shaft 8' and swings back and forth around the axis (as shown by the dotted line in the figure). On the optical path from the oscillating mirror 8 to the biochip 12, a scanning objective lens 9 and prism 10, the vibrating mirror 8 swings around the axis, so that the incident angle of the excitation beam relative to the vibrating mirror is constantly changing, therefore, the vibrating mirror reflects the exciting beam to the scanning objective lens 9 at different angles, and the scanning objective lens 9 changes the angle of the exciting beam The change is converted into a displacement change along a certain direction, and the direction is changed by the prism 10 to be incident on the biochip 12 to realize the optical scanning of the biochip 12 in a one-dimensional direction (such as the X direction). The biochip is installed on the linear guide rail 11, and is driven by a stepping motor to move along the Y direction of the two-dimensional plane formed by X and Y, so as to realize the scanning of the biochip in another dimension. The excitation beam excites the fluorescent dye molecules at the corresponding positions on the biochip to emit fluorescence, and the emitted fluorescence is changed by the prism 10 to reach the scanning objective lens 9, and after passing through the scanning objective lens 9, it becomes a parallel beam and reaches the vibrating mirror 8. The size ratio of the parallel beam is The size of the excitation beam is much larger, therefore, most of the energy of the fluorescent light is reflected after reaching the perforated mirror 7, and only a small part of the fluorescent light is lost through the small hole of the perforated mirror 7. On the optical path reflected by the perforated reflector 7, the condenser lens 13 is installed sequentially along the reflected light, the confocal diaphragm 14 is perpendicular to the optical path and is on the same plane, and two color filters 15, 16 and photodetectors are switched on the optical path. 17, the photodetector 17 can be a photomultiplier tube or an avalanche diode or a PIN photodiode. The first color filter 15 and the corresponding synchronous switching of the first diaphragm 3 arranged between the first light source 1 and the total reflection mirror 5, the second color filter 16 and the first diaphragm 3 arranged between the second light source 2 and the reflector 6 The second aperture 4 corresponds to synchronous switching. The fluorescent light reflected by the hole mirror 7 is converged by the condenser lens 13, passes through the small hole of the confocal diaphragm 14, and reaches the color filter corresponding to the wavelength of the excitation beam. When the first diaphragm 3 is opened, the second diaphragm 4 is closed , the first color filter 15 is on the optical path. At this time, the first light source 1 realizes single-wavelength scanning of a certain wavelength on the biochip. When the first aperture 3 is closed, the second aperture 4 is opened, and the second color filter 16 is on the optical path, at this time, the second light source 2 scans the biochip with a single wavelength of another wavelength, and the light beam after the stray light is filtered by the color filter is converted into an electrical signal by the photodetector 17 for subsequent processing and imaging analysis .

Claims (4)

1. biochip analysis instrument, comprise first, the light source (1) of the second two different wave lengths, (2), catoptron of parallel device (6) and completely reflecting mirror (5), the light of secondary light source (2) emission is reflected by catoptron (6), galvanometer (8) is incided in the hole of passing through catoptron with holes (7), the light of first light source (1) emission is reflected by completely reflecting mirror (5), galvanometer (8) is incided in the hole of passing through catoptron with holes (7) behind the penetration mirror (6), galvanometer (8) device is in rotating shaft 8 ', around the axle reciprocating swing, at galvanometer (8) to the light path of biochip (12), device has the reflected light that will change from the angle of galvanometer (8) to be transformed into the scanning objective (9) of linear displacement light and the light of scanning objective outgoing is redirect to prism (10) on the biochip (12), forming light scans at the biochip directions X, biochip is installed on the line slideway (11), by step motor drive along X, the Y direction of the two dimensional surface that Y constitutes moves, the biochip emitted fluorescence, incide prism (10), turn to and scanning objective (9) is transformed into parallel beam and incides galvanometer (8) through prism (10), reflex to catoptron with holes (7) by galvanometer (8), on the light path of catoptron with holes (7) reflection, install condenser (13) successively along reflected light, confocal diaphragm (14), perpendicular to light path and be in same plane, two color filters (15) that on light path, switch mutually, (16) and photodetector (17), between first light source (1) and completely reflecting mirror (5), be provided with corresponding first diaphragm (3) that switches synchronously with first color filter (15), between secondary light source (2) and catoptron (6), be provided with the corresponding diaphragm (4) that switches synchronously with second color filter (16), when first diaphragm (3) when opening, second diaphragm (4) is closed, first color filter (1 5) is on light path, when first diaphragm (3) when closing, second diaphragm (4) is opened, and second color filter (16) is on light path.

2. by the described biochip analysis instrument of claim 1, it is characterized in that said scanning objective (9) is a heart f-θ scanning objective far away.

3. by the described biochip analysis instrument of claim 1, it is characterized in that two light sources (1) and (2) are the different lasing light emitters of wavelength, wavelength coverage is at 400nm～650nm.

4. by the described biochip analysis instrument of claim 1, it is characterized in that photodetector (17) is photomultiplier or avalanche diode or PIN photodiode.