阅读说明：本技术 一种基因芯片阅读仪 (Gene chip reader ) 是由 华子昂 钭理强 刘宝全 竹添 张建 朱美瑛 万君兴 李娜 于 2021-11-09 设计创作，主要内容包括：本发明涉及一种基因芯片阅读仪,所述基因芯片阅读仪包括电路单元、数据处理分析单元、激发光源、芯片阅读单元和定位支持装置,其中激发光源采用LED光源交叉激发,芯片阅读单元选用百万级别像素的数码相机,LED光源和数码相机替换了传统基因芯片阅读仪的激光器与共聚焦成像装置,有效地降低基因芯片阅读仪的成本,其他优化措施使其可以适用于绝大多数荧光物质的激发,此外,不仅可以避免激发光线对基因芯片成像的背景干扰,还可以最大程度地保证了基因芯片阅读仪输出结果的灵敏度,非常适合将基因芯片向POCT领域的应用、推广和普及。(The invention relates to a gene chip reader, which comprises a circuit unit, a data processing and analyzing unit, an excitation light source, a chip reading unit and a positioning support device, wherein the excitation light source is excited by adopting an LED light source in a crossed manner, the chip reading unit adopts a digital camera with million-level pixels, the LED light source and the digital camera replace a laser and a confocal imaging device of the traditional gene chip reader, the cost of the gene chip reader is effectively reduced, and other optimization measures ensure that the gene chip reader can be suitable for excitation of most fluorescent substances.)

1. A gene chip reader comprising: a circuit unit, a data processing and analyzing unit, an excitation light source, a chip reading unit and a positioning and supporting device, wherein,

the excitation light source comprises an LED light source and is used for exciting a fluorescent substance on the gene chip probe to emit fluorescence;

the chip reading unit comprises a digital camera and is used for capturing and identifying fluorescent signals excited on the gene chip probe at one time;

the circuit unit is used for providing power and transmitting fluorescence signal data;

the data processing and analyzing unit is used for receiving the fluorescence signal data, processing and analyzing the fluorescence signal data and outputting a result;

the positioning support device is used for adjusting and fixing the positions of the gene chip, the excitation light source and the chip reading unit.

2. The gene chip reader according to claim 1, wherein the LED light source is disposed above and to the side of the same side of the probe on the gene chip, and the incident angle of the emitted exciting light is greater than the critical angle and less than 90 degrees.

3. The gene chip reader according to claim 2, wherein the LED light source comprises at least two symmetrically arranged LED light sources and the two LED light sources cross-excite the probes on the gene chip.

4. The gene chip reader according to claim 1, wherein the wavelength of the LED light source is a fixed wavelength selected according to the type of fluorescent substance labeled on the probe of the gene chip to be detected.

5. The gene chip reader of claim 4, wherein the LED light source has a light intensity of greater than 1000 Lux.

6. The gene chip reader according to claim 1, wherein the digital camera is a CMOS camera or a CCD camera, and the number of pixels is more than 200 ten thousand.

7. The gene chip reader according to claim 6, wherein said digital camera is equipped with a filter.

8. The gene chip reader according to claim 6, wherein said digital camera uses a fixed focus lens.

9. The gene chip reader according to any one of claims 1 to 8, wherein the positioning support device comprises a support structure, the position adjustment comprises a height adjustment and an angle adjustment, and the position fixing device comprises a slot structure for fixedly mounting the gene chip and/or the excitation light source.

10. Use of the gene chip reader according to any one of claims 1 to 9 in the field of detection of gene chips of low and medium density.

Technical Field

The invention relates to the field of detection instruments and equipment, in particular to a gene chip reader which is particularly suitable for reading the real-time detection result of a gene chip with medium and low density.

Background

The gene chip is prepared by planting a series of probes with known sequences on the substrate of the gene chip, can be used for hybridization detection of specific labeled nucleic acid, and can report the nucleic acid information in a detection object by carrying out identification detection and information processing on the hybridization result of the gene chip through a specific gene chip reader.

The civil health industry urgently needs a point-of-care testing (POCT) technology which is low in cost and can be carried to thousands of households, which is the development direction of medical testing instruments in the future, the POCT and the like move a central testing room of a hospital to the bedside of a patient, and the POCT and the like are used in sampling sites, ICU monitoring rooms, operating rooms, emergency rooms and the like. The gene chip is particularly suitable for POCT identification of multi-factor screening such as infectious disease detection, epidemic situation monitoring, tumor marker detection, drug abuse and the like, wherein the number of probes of the medium-low density gene chip is between dozens and thousands, and the gene chip is different from a high-density gene chip (the probes are tens of thousands to millions) used in the fields of large-scale gene expression profiling, drug research and molecular research and the like, so the medium-low density gene chip is more suitable for the fields of medical diagnosis, clinical scientific research and POCT.

However, in the current market, the detection instrument for the hybridization result of the gene chip is mainly a confocal laser reading instrument, and most of the devices use laser with good monochromaticity, high light intensity and good directivity as an excitation light source, and detect the fluorescence label of the specific binding nucleic acid sample on the positive hybridization probe of the gene chip by a confocal imaging method, thereby forming the detection result of the positive hybridization probe on the gene chip. Correspondingly, the laser and the confocal imaging device also lead the gene chip reader to face the problems of complexity, large size and high price, are mainly purchased and used in scientific research institutions and large hospitals, and limit the application of the gene chip in the POCT field to a certain extent.

In order to meet the demand of POCT market development and be more suitable for pushing the medium-low density gene chip to the POCT field, a gene chip reader which is miniaturized, simple to operate, low in cost and suitable for the medium-low density gene chip is developed, the problem of instant detection in the fields of early quick disease screening, health management, virus detection, food detection, environment monitoring and the like is solved, and the health protection and navigation of the large health industry is promoted and developed.

Disclosure of Invention

The invention aims to provide a gene chip reader which is miniaturized, simple to operate, low in cost and suitable for medium-low density gene chips.

In order to achieve one of the above purposes, the invention provides the following technical scheme:

a gene chip reader comprising: the device comprises a circuit unit, a data processing and analyzing unit, an excitation light source, a chip reading unit and a positioning support device, wherein the excitation light source comprises an LED light source and is used for exciting a fluorescent substance on a gene chip probe to emit fluorescence; the chip reading unit comprises a digital camera and is used for capturing and identifying fluorescent signals excited on the gene chip probe at one time; the circuit unit is used for providing power supply and transmitting data; the data processing and analyzing unit is used for receiving the fluorescence signals collected by the chip reading unit, processing and analyzing the data and outputting results; the circuit unit is used for providing power and transmitting fluorescence signal data; the data processing and analyzing unit is used for receiving the fluorescence signal data, processing and analyzing the fluorescence signal data and outputting a result; and the positioning support device is used for adjusting and fixing the positions of the gene chip, the excitation light source and the chip reading unit.

According to a preferred embodiment of the invention, the LED light source is arranged above the side of the same side of the probe on the genechip, emitting exciting light rays with an angle of incidence greater than the critical angle and less than 90 degrees.

According to a preferred embodiment of the present invention, the LED light source comprises at least two symmetrically arranged LED light sources and the two LED light sources cross-excite the probes on the gene chip.

According to a preferred embodiment of the present invention, the wavelength of the LED light source is a fixed wavelength selected according to the type of fluorescent substance labeled on the probe of the gene chip to be detected.

According to a preferred embodiment of the invention, the LED light source has a light intensity of more than 1000 Lux.

According to a preferred embodiment of the present invention, the digital camera is a CMOS camera or a CCD camera, and the number of pixels is more than 200 ten thousand.

According to a preferred embodiment of the invention, the digital camera is equipped with a filter, preferably a narrow band pass filter.

According to a preferred embodiment of the present invention, the digital camera uses a fixed focus lens.

According to a preferred embodiment of the present invention, the positioning and supporting device comprises a support structure, the position adjusting function comprises height adjustment and angle adjustment, and the position fixing device comprises a slot structure for fixing and placing the gene chip and/or the excitation light source.

According to a preferred embodiment of the present invention, the gene chip reader is applied to the field of gene chip detection of medium and low density.

In the invention, the fluorescence signal excited on the gene chip probe specifically refers to: after the probe on the gene chip is hybridized, the fluorescent substance marked by the marked nucleic acid combined with the probe generates a fluorescent signal after being excited by an excitation light source, and the fluorescent signal is a fluorescent positive signal, and the position of the probe combined with the unmarked nucleic acid has no fluorescence, namely a fluorescent negative signal.

According to the concept of the present invention, the laser light source is replaced by an LED light source, preferably two LED light sources are provided, and the two LED light sources are set above the same side of the probe on the gene chip for cross excitation, that is: each LED light source only excites one half area of the gene chip and one half area far away from the LED light source, and the excitation light and the reflected light thereof can not enter the digital camera by cross excitation and ensuring that the incidence angle of the excitation light emitted by the LED light source is larger than a critical angle (theta) and smaller than 90 degrees, so that background interference of the excitation light and the reflected light thereof on imaging is avoided; on the other hand, the LED light source of the gene chip reader and the digital camera are both arranged on the same side of the probe on the gene chip, so that the excitation light can directly excite the fluorescent substance on the labeled nucleic acid hybridized on the gene chip without penetrating through the substrate of the gene chip, and the energy loss of the excitation light can be reduced; the emitted light generated by the excited fluorescent substance combined with the label of the nucleic acid on the hybridized probe can also directly enter a digital camera for imaging, thereby improving the imaging effect.

Influence factors and setting process of the setting of the position of the excitation light source: the present application mainly adopts an LED light source, wherein the LED light source position setting needs to ensure that the incident angle of the emitted excitation light needs to be larger than a critical angle (θ) and smaller than 90 degrees, wherein the critical angle (θ) has a relationship of tg θ = L1/H1, (L1 is half of the sum of the array width and the lens diameter, and H1 is the working distance of the lens), and therefore, the LED light source position is influenced by L1 and H1. The cross excitation is very easy to realize by increasing the working distance H1 of the lens, and as long as the incident angle is larger than the critical angle (theta) and smaller than 90 degrees, all the excitation light and the reflection light thereof cannot enter the lens of the digital camera in the cross excitation process, so that the interference of the excitation light and the reflection light thereof in the information acquisition and imaging process is also ensured.

Specific explanation on the size of the light source position: l1 is half of the sum of the array width and the lens diameter, the array is the gene chip of the invention, therefore, in the application, L1 is half of the sum of the gene chip to be detected and the lens outer diameter, the gene chip length is generally less than 75mm, the lens outer diameter is generally less than 50mm, so L1 is generally less than 62.5 mm; h1 is the working distance of the lens, and the lens with the working distance of about 350mm can meet the lowest configuration requirement of the gene chip reader; particularly, the H1 is limited by the lens selected by the digital camera, because the products of the gene chip reader are generally sold in series, the imaging effect and the resolution are closely related to the cost price of the lens configured by the series products, relatively speaking, the lens with the working distance of about 350mm is cheaper, the lens within 100mm is more expensive, and correspondingly, the numerical imaging effect of the lens with the smaller working distance is relatively more ideal, therefore, under comprehensive consideration, the cost performance of the lens with the working distance of between 150 mm and 250mm selected by the H1 is higher.

The reading process of the gene chip detection result needs to use a gene chip reader, however, the existing laser confocal gene chip reader has the defects of complex operation, large size and high price, is not suitable for the application and popularization of the gene chip in a timely detection field, and needs to be improved urgently.

The invention has the beneficial effects that:

in order to meet the popularization of POCT, the key point is to reduce the cost of the gene chip reader on the premise of ensuring the quality, the invention is improved from two aspects of a light source and a chip reading unit, an LED light source is used for replacing a common laser at present to excite a fluorescent dye for marking nucleic acid on a hybridized gene chip probe, a digital camera is used for replacing an expensive confocal imaging detection device, a stepping device is removed, the manufacturing cost is saved, the time required by stepping scanning is shortened, the detection of the hybridization result of the whole gene chip is finished at one time, the cost of the gene chip reader is effectively reduced, and the detection speed is effectively improved.

The excitation light source used by the invention comprises an LED light source, and has the advantages of low cost and volume, long service life, high efficiency and the like compared with laser emitted by a laser; the method comprises the following steps of selecting an LED light source with fixed wavelength, namely satisfying the fluorescent excitation of a marked fluorescent substance on a gene chip, wherein the light emitting wavelength of the LED light source depends on a semiconductor material used for manufacturing a light emitting diode, and expanding the wavelength coverage range of the LED light source by configuring the LED light source comprising the light emitting diodes with different colors, preferably selecting an LED lamp bead group; in addition, the LED light source with fixed wavelength can be further matched with different optical filters, so that the excitation of the fluorescence of the probe on the gene chip marked by different fluorescent substances can be met, and the fluorescent probe is suitable for reading the result of the gene chip hybridized by different fluorescent substance marked nucleic acid samples; the digital camera uses a fixed-focus lens, and imaging detection is directly carried out without focusing after adjustment, so that the operation is simpler and more convenient; a scanning imaging component based on a stepping device is removed, and a one-time imaging technology is adopted, so that the cost is reduced and the imaging speed is improved; the LED light source is used for replacing expensive precision accessories with similar functions such as a laser, a laser beam expander and the like; therefore, the gene chip reader can effectively reduce the cost of the gene chip reader, effectively reduce the appearance size of the gene chip reader and meet the basic requirements of miniaturization, simplification and low cost for detecting and applying the gene chip to the POCT field.

In addition, after the laser light source is replaced by an LED light source, the LED light source position is further defined, and the incident angle of the emitted excitation light needs to be greater than the critical angle (θ) and less than 90 degrees; and the cross excitation is carried out, so that the background interference of a laser light source on imaging is avoided, and finally the specificity and the sensitivity of the detection result of the gene chip reader can be improved.

Furthermore, the LED light source is matched with the LED light source and utilizes the light intensity of more than 1000Lux and the fixed wavelength, and the preferred scheme is that the LED light source adopts the integration of an LED lamp bead plate, so that the condition that the light intensity is more than 1000Lux can be met, and fluorescent substances which are combined with the nucleic acid sample marks on the hybridized gene chip probe can be collected by a digital camera after being excited so as to clearly image; on the other hand, two symmetrically arranged LED light sources are used in a matched manner, and the two LED light sources cross-excite the fluorescent substance which is combined with the labeled nucleic acid on the hybridization gene chip probe, so that the influence of excitation light and reflected light thereof on imaging is further removed; meanwhile, the LED light source is arranged above the side of the same side of the gene chip probe, and the fluorescent substance labeled by the nucleic acid sample combined on the probe is directly excited by the LED light source, so that the energy loss of excitation light in the process of passing through the substrate can be effectively reduced.

The chip reading unit is a digital camera, pixels can meet the million level, and preferably more than 500 ten thousand pixels can meet the requirement of one-time capture and identification of gene chip detection results of different fluorescence. The current commercially available digital cameras at thousand yuan level can basically reach 500 ten thousand pixels, and the digital cameras replace the expensive confocal imaging detection device adopted by the existing large gene chip reader to realize one-time imaging, namely: the digital camera can complete the imaging of all the probe fluorescence results on the gene chip at one time, so that the cost of the gene chip reader is further reduced, and compared with a gene chip reader which needs a stepping device to perform partitioned scanning, the digital camera can obviously accelerate the reading speed of the detection result.

The digital camera of the invention can select a CMOS camera and a CCD camera, only the requirement of million-level pixels is required, and the conventional digital camera of thousand-yuan level can meet the condition; furthermore, in order to enhance the imaging definition, a filter, preferably a narrow band pass filter, is optionally added between the gene chip and the digital camera, and more particularly, a band pass adaptive to the labeled fluorescence used for binding labeled nucleic acid on the probe is selected, so that the requirements of different labeled fluorescence can be met, the types of detectable labeled fluorescent substances can be expanded, and the interference of background fluorescence outside the selection range of the narrow band pass filter can be further prevented; in addition, the digital camera uses the fixed focus lens, which not only has small volume and light weight, but also has large light flux because the focal length of the fixed focus lens is relatively fixed, is convenient for shooting under the condition of low illumination, and is suitable for the use of the fixed shooting object type of reading gene chips only, and the distance determination of H1 is easy to control relative to the zoom lens, so that the imaging definition is better.

The price of the high-density gene chip readers currently on the market is millions, for example: affymetrix GCS 3000Dx v.2, the price of the medium-low density gene chip reader is also in the range of 30-90 ten thousand yuan, such as: the price of gene chip readers such as RPAS710G, GenePix 4300A, InnoScan 710, Senssopot FL RG, SCI READWE FL2, etc., and domestic confocal laser readers is also in the order of hundreds of thousands. According to the concept of the invention, after the LED light source and the digital camera are replaced, the selling price of the gene chip reader is greatly reduced, so that the selling price of the gene chip reader can be reduced to the ten thousand yuan level, and the gene chip reader can get rid of the limitation of high selling price, really walk to the user and enter thousands of households, effectively serve the common people, and ensure the application of the gene chip technology in the large health industry.

In addition, in cooperation with the replacement of core components of the gene chip reader, in order to ensure the sensitivity of detection results, a series of optimization designs are also carried out, such as the optimization settings of light intensity, wavelength and cross excitation of an LED light source and the optimization selection of pixels and optical filters of a digital camera, so that the sensitivity of output results of the gene chip reader is ensured to the greatest extent on the basis of effectively reducing the cost of the gene chip reader, and the gene chip reader is very suitable for application, popularization and application of the gene chip to the POCT field.

Drawings

FIG. 1 is a block diagram showing the connection relationship between the main components of the gene chip reader of the present invention;

FIG. 2 is a schematic view of the operation of the gene chip reader of the present invention;

FIG. 3 is a schematic view of the cross-excitation working principle of the gene chip reader of the present invention;

FIG. 4 is a photograph showing the results of several readings using the gene chip reader of the present invention;

in the figure: 1. gene chip; 2. a probe; 3. a chip reading unit; 4. a data link; 5. a data processing and analyzing unit; 6. exciting light; 7. emitting light; 8. an optical filter; 9. a critical angle; 10. half of the sum of the array width and the lens diameter; 11. the working distance of the lens; 12. the light is reflected.

Detailed Description

Exemplary embodiments of the present invention will hereinafter be described in detail with reference to the accompanying drawings, wherein like or similar reference numerals denote like or similar elements. Furthermore, in the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the disclosure. It may be evident, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are shown in schematic form in order to simplify the drawing.

The invention provides a gene chip reader and application of the gene chip reader in the field of gene chip detection of medium and low density.

According to the concept of the invention, the LED light source and the digital camera are used for replacing an expensive laser and confocal scanning imaging, the LED light source and the chip reading unit are arranged above the side of the same side of the gene chip probe and are subjected to cross excitation, so that the excitation light 6 and the reflection light 12 thereof are prevented from entering the chip reading unit 3, and the background noise of the imaging result is favorably reduced; in addition, the LED lamp bead group is used, the improvement of light intensity, fixed wavelength and cross excitation is optimized, and the optimization matching of pixels, focal length, narrow band pass filters and the like of a digital camera is realized, so that the excitation of fluorescent substances of different labeling types on labeled nucleic acid after hybridization of the gene chip probe is finally realized, an imaging picture of gene chip hybridization is obtained at one time, and a gene chip detection result can be obtained after the automatic data analysis.

As shown in FIG. 1-2, the gene chip reader of the present invention comprises the following components: the device comprises a circuit unit, a data processing and analyzing unit 5, an excitation light source, a chip reading unit 3 and a positioning support device, and is characterized in that the circuit unit is used for providing power for the excitation light source and the chip reading unit 3 and transmitting a fluorescence signal data diagram collected by the chip reading unit 3 to the data processing and analyzing unit 5 through a data connecting line 4; the data processing and analyzing unit 5 is used for receiving the fluorescent signal pictures acquired by the chip reading unit 3, or a software analysis system matched with a computer terminal and connected with the data connecting line 4, or a singlechip approach, processing and analyzing data and outputting corresponding results or printing a detection report according to actual requirements; the excitation light source comprises an LED light source, the LED light source is used for exciting fluorescent substances on labeled nucleic acid combined after hybridization of the probes 2 of the gene chip 1 to emit fluorescence, at least 1 group of two LED light sources which are bilaterally symmetrical are arranged on one side of the surface of the gene chip 1 where the probes 2 are located, the two LED light sources complete cross excitation of the gene chip 1, excitation light rays 6 and reflection light rays 12 of the excitation light rays are effectively prevented from entering the chip reading unit 3 while exciting the fluorescent substances on the labeled nucleic acid combined after hybridization of the probes 2 on the gene chip 1, and background noise of imaging is reduced; the chip reading unit 3 is a digital camera, is positioned right above the gene chip 1 and is arranged at one side of the gene chip 1 where the probe 2 is positioned, the working distance 11(H1) of a lens of the digital camera ensures that the lens can cover the visual field range of the whole gene chip 1 where the probe 2 is fixed, and is used for capturing and identifying the emission light 7 generated by the stimulated emission after the hybridization of the probe 2 of the gene chip 1 at one time and forming corresponding fluorescence signal data, an optical filter 8 is required to be added and matched during specific imaging reading, preferably a narrow band pass optical filter 8 is selected, and the image data of the fluorescence signal is fed back to the data processing and analyzing unit 5 after the imaging reading; the positioning support device is used for adjusting and fixing the positions of the gene chip 1, the excitation light source and the chip reading unit 3, is preferably a support structure, can be used for adjusting the heights and angles of the gene chip 1, the excitation light source and the chip reading unit 3, and is provided with a clamping groove structure for fixing the gene chip 1 and the excitation light source.

The working principle of the gene chip reader of the present invention will be described with reference to FIGS. 2-4 and the specific examples.

Specific example 1: the gene chip reader is used for detecting the gene chip 1 with the CY3 mark and medium and low density, and the specific detection process is as follows:

1) the gene chip 1 with the medium-low density to be detected is a gene chip 1 (with the length of 60mm and the width of 18 mm) with 10 x 10 array repetition, the gene chip 1 to be detected and a nucleic acid sample marked by CY3 are subjected to hybridization reaction under the optimized hybridization condition, and the gene chip 1 to be detected is taken out after the hybridization is finished;

2) putting the gene chip 1 to be detected after hybridization into a gene chip clamping groove configured on a positioning and supporting device of a bracket structure in a gene chip reader, and ensuring that one surface with the probe 2 is upwards placed;

3) a group of LED lamp bead groups (products of CREE company) with the wavelength of 535nm of an LED light source of a gene chip reader are respectively arranged in excitation light source clamping grooves (partial structures of a positioning and supporting device of a bracket structure) of a positioning and supporting device (the left side and the right side above a gene chip 1);

4) a chip reading unit 3 of the gene chip reader adopts a CMOS camera (PYTHON 5000 sensor, pixel 4.8 μm multiplied by 4.8 μm, lens diameter =38 mm) of a global shutter and is fixed on a positioning support device of a support structure, and a narrow band pass filter 8 (Shenzhen laser Elett photoelectricity Limited company) with a band pass of 560nm +/-5 nm is fixedly installed below the CMOS camera;

5) the LED light source and the CMOS camera are connected by the circuit unit for power supply, the CMOS camera is connected with a computer (the data processing and analyzing unit 5 of the embodiment adopts a computer and is provided with image acquisition and analysis software in a matching way) through a data line of the circuit unit, and the power supply is respectively switched on and the CMOS camera and the computer are switched on;

6) the working distance of the lens 11(H1=236mm), the critical angle 9 (θ) =11.7 degrees is automatically calculated by the information input image acquisition and analysis software (the calculation is based on: l1= (60mm +38mm)/2=49mm, H1=236mm, tg θ = L1/H1=49mm/236mm = 0.2076), so that the LED light source satisfies that in the optional range of 11.7 degrees < incident angle <90 degrees, the incident angle of the LED light source is adjusted by the support arm on the support structure, to which the excitation light source card slot is fixed, so that the incident angles of the left and right sides of the group of LED light sources are both 50 degrees;

7) and opening image acquisition and analysis software at the computer end, acquiring and analyzing image data of the fluorescence signal on the gene chip 1 acquired by the CMOS camera, comparing and analyzing the image data with the sequence of the probe 2 fixed on the gene chip 1, and finally grouping and outputting a positive result and a negative result.

Remarking: in this embodiment, the incident angle of the LED light source on each side is 50 degrees, which can cross-excite the fluorescence excitation of the fluorescent dye on the gene chip 1, and neither the excitation light 6 nor the reflection light 12 thereof can enter the chip reading unit 3, so that the imaging background interference of the positive fluorescence emission light 7 at the probe 2 position is not formed, and the specific excitation of the specific fluorescent substance is also realized, and the interference of the background fluorescence outside the selection range of the narrow band pass filter 8 is further prevented by using a CMOS camera (pixel 4.8 μm × 4.8 μm) with 500 ten thousand pixels and a global shutter in cooperation with the 560nm ± 5nm narrow band pass filter 8, which can preferentially let the emission light 7 with the specific wavelength enter the digital camera, so that the specificity and accuracy of the reading result of the gene chip 1 are further increased.

Specific example 2: the gene chip reader is used for detecting the gene chip 1 with the CY5 mark and medium and low density, and the specific detection process is as follows:

1) the gene chip 1 with the medium-low density to be detected is a gene chip 1 (with the length of 60mm and the width of 18 mm) with 10 x 10 array repetition, the gene chip 1 to be detected and a nucleic acid sample marked by CY5 are subjected to hybridization reaction under the optimized hybridization condition, and the gene chip 1 to be detected is taken out after the hybridization is finished;

2) putting the gene chip 1 to be detected after hybridization into a gene chip clamping groove in a gene chip reader, and ensuring that the side with the probe 2 is upwards placed;

3) a group of LED lamp bead groups (CREE products) with the wavelength of 630nm of an LED light source of a gene chip reader are respectively arranged in excitation light source clamping grooves (part of a structure of a positioning and supporting device of a bracket structure) on two sides;

4) a chip reading unit 3 of the gene chip reader adopts a CMOS camera (PYTHON 5000 sensor, pixel 4.8 mu m multiplied by 4.8 mu m, lens diameter =38 mm) of a global shutter and is fixed on a positioning support device of a support structure, and a narrow band pass filter 8 (Shenzhen laser Elett photoelectricity Limited company) with a band pass of 670nm +/-5 nm is fixedly installed below the CMOS camera;

5) the LED light source and the CMOS camera are connected by the circuit unit for power supply, the CMOS camera is connected with a computer (the data processing and analyzing unit 5 of the embodiment adopts a computer and is provided with image acquisition and analysis software in a matching way) through a data line of the circuit unit, and the power supply is respectively switched on and the CMOS camera and the computer are switched on;

6) the working distance of the lens is 11(H1=236mm), the information is input into image acquisition and analysis software, the critical angle 9 (θ) =11.7 degrees is automatically calculated, and the incident angle of the LED light source is adjusted by a support arm fixed with an excitation light source clamping groove on the support structure, so that the incident angles of the left and right sides of the group of LED light sources are both 50 degrees (the LED light source satisfies 11.7 degrees < incident angle <90 degrees, and the excitation light 6 and the reflection light 12 thereof do not enter the chip reading unit 3);

7) and opening image acquisition and analysis software at the computer end, acquiring and analyzing the image data of the fluorescence signal of the gene chip 1 acquired by the CMOS camera, comparing and analyzing the image data with a probe 2 sequence fixed on the gene chip 1, and finally grouping and outputting a positive result and a negative result.

In order to reduce the cost of the instrument, the gene chip reader utilizes the LED light source to replace a laser light source and also serves as a solid light source, the LED light source has the advantages of small volume and low cost, but compared with the laser light source, the LED light source has the biggest defects of unreal and natural color, low light intensity and brightness and relatively high energy consumption. The gene chip reader does not need to enrich natural colors, but in order to overcome the defects of light intensity and energy consumption, the invention adopts the following measures:

aiming at the light intensity, the single lamp bead of the LED light source cannot meet the minimum requirement of the light intensity, so the invention adopts different integration forms of LED lamp strips, LED lamp bead groups, LED surface light sources and the like to be combined through research and development, and unexpectedly discovers that: the excitation of the fluorescent material can be completed as long as the light intensity of the LED light source is adjusted to be at least more than 1000Lux, so that the defect of weak light intensity of the LED light source can be overcome to a certain extent by limiting the light intensity of the LED light sources in different combination forms. In addition, at least one group of LED light sources, that is, 2 symmetrical LED light sources are arranged for cross excitation, that is, the LED light source on the side close to the gene chip 1 mainly completes excitation of the fluorescent substance on 1/2 gene chip 1, and the 1/2 gene chip 1 completing excitation of the fluorescent substance is far away from the position of the emitted LED light source, specifically, each LED light source mainly completes excitation of the fluorescent substance on the 1/2 gene chip 1 far away from the end, so that the illumination direction (cross excitation) and the number (at least 1 symmetrical group, that is, 2) of the LED light sources are increased, the brightness defect of the LED light sources is weakened to a certain extent, the light intensity of the excitation light 6 emitted by the LED light sources is further enhanced, and further contribution is made to completely replacing the laser excited by the laser.

Aiming at energy consumption, in the gene chip reader, the LED light source is excited at one side of the gene chip 1 where the probe 2 is, as shown in detail in FIG. 3, the exciting light 6, the probe 2, the emitting light 7 and the chip reading unit 3 are all positioned at the same side of the gene chip 1, so that the exciting light 6 and the emitting light 7 are effectively prevented from entering the chip reading unit 3 only by penetrating through the substrate of the gene chip 1, the utilization efficiency of the exciting light 6 is improved, and the imaging effect of the gene chip reader is improved.

For the application of cross excitation in the gene chip reader of the present invention, the following relationship is required, as shown in FIG. 3: tg θ = L1/H1, where L1 is half the sum of the array width and the lens diameter 10 and H1 is the working distance of the lens 11. In practical studies, we mainly perform statistical analysis by adjusting the distance of the chip reading unit 3 (digital camera) and the angle of the excitation light source, and finally find that if the working distance 11(H1) of the lens is increased, it is easier to satisfy the condition of cross excitation, such as the excitation light 6 from the left side to the right side of the LED light source, as long as the incident angle is greater than the critical angle 9 (θ), it can be ensured that the excitation light 6 and the reflected light 12 thereof will not enter the lens of the digital camera, and will not be an interfering background of the emission light 7 of the fluorescent substance labeled after hybridization of the probe 2 on the gene chip 1 in the information collection and imaging process, but since we preferably employ cross excitation, the incident angle of each symmetric direction is further limited to be less than 90 degrees, i.e., the critical angle 9 (θ) <90 degrees.

Further studies on the location of the light source found: the array is 10 (L1) half of the sum of the array width and the lens diameter, the array is the gene chip 1 of the invention, therefore, in the application, the 10 (L1) half of the sum of the array width and the lens diameter is half of the sum of the gene chip 1 to be detected and the lens outer diameter, the length of the gene chip 1 is generally less than 75mm, the lens outer diameter is generally less than 50mm, and the L1 is generally less than 62.5 mm; the working distance 11(H1) of the lens is related to the working distance of the chip reading unit 3 (i.e. the digital camera), and the working distance can meet the minimum configuration requirement of the gene chip reader when the working distance is about 350 mm. First, H1 is limited by the lens selected by the digital camera, and the positioning range of the LED light source can be enlarged by increasing the working distance 11 of the lens (H1). Because gene chip reader products are generally sold in series, imaging effect and resolution are closely related to the cost price of lenses configured by series products, and comparatively speaking, digital cameras with working distances of about 350mm are cheaper, digital cameras with working distances of 100mm are more expensive, and correspondingly, the imaging effect of selecting lenses with smaller working distance values is more ideal, so that after the price is comprehensively considered, the cost performance of H1 with the preferred working distance of 150 plus 250mm is higher.

Specific example 3: the imaging results of different medium-low density gene chips 1 detected by using different parameter gene chip readers are shown in fig. 4, wherein fig. 4-a and fig. 4-B are respectively imaging result graphs of specific example 1 and specific example 2, fig. 4-C and fig. 4-D are imaging result graphs of a plurality of digital cameras which are randomly selected and respectively debug LED light source types, light intensities, wavelengths, angles and more than 200 ten thousand pixels in the instrument research and development debugging process, in the imaging result graph shown in fig. 4, it is obvious that the positive probe 2 result is light spots with smooth boundaries and clear images, the negative result is obviously distinguished from the positive result, meanwhile, the contrast imaging result is consistent with the detection result of the hybridization reaction of the gene chip 1 to be detected and a nucleic acid substance, and the detection result coincidence rate is over 99%.

It should be noted that: the image acquisition and analysis software installed at the computer end and matched with the gene chip reader can also be installed in mobile equipment as an application program in system optimization debugging at present. Further planning is: the data processing and analyzing unit 5 is optimized and upgraded by a single chip microcomputer, so that the automatic identification capability and the sensitivity of an image output by the chip reading unit 3 can be improved, the integrated rapid detection capability of sample input and result output of a hybridization result of the gene chip 1 can be really realized by utilizing the gene chip reader, the automatic identification and analysis of an imaging result of the gene chip 1 with the medium-low density and the number of the probes 2 within thousands can be expanded, and the accuracy of image result analysis can be ensured.