CN112964688B - Method for detecting gene chip hybridization result by total internal reflection - Google Patents
Method for detecting gene chip hybridization result by total internal reflection Download PDFInfo
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Abstract
The invention discloses a method for detecting gene chip hybridization result by total internal reflection, which comprises the following steps: constructing a gene chip detector, wherein a dark box (1) of the gene chip detector is divided into a light source dark box (3) and an imaging dark box (16) by using a quartz glass plate (2); adding an isorefractive index solution (9) having the same refractive index as that of the quartz glass onto the quartz glass plate (2); putting the quartz glass substrate of the gene chip on the isorefractive index solution (9); adjusting the position and the angle of the plane parallel light source generator (6) to ensure that the emitted plane parallel light forms total internal reflection; the total internal reflection evanescent wave (11) excites fluorescent substances on a probe (12) of the gene chip to emit labeled fluorescence (13); the marker fluorescence (13) is captured by a high-resolution camera (15), and the detection result is finally obtained. The method of the invention avoids background interference in the fluorescent imaging process, and improves the signal-to-noise ratio and the detection sensitivity of the gene chip hybridization result.
Description
Technical Field
The invention relates to the technical field of nucleic acid analysis, in particular to a method for detecting a hybridization result of a gene chip by total internal reflection, which is a method for detecting nucleic acid and adopts a product for nucleic acid analysis. The invention also relates to an in-vitro diagnosis and detection instrument, belongs to a high-flux detection and analysis instrument in the in-vitro diagnosis and detection instrument, and also relates to a molecular biological information analysis and processing system in the molecular diagnosis and detection instrument.
Background
The biochip technology has been widely applied to clinical disease diagnosis, health management, drug research and development, animal and plant quarantine, food detection, environmental monitoring, scientific research, forensic detection and other fields, and has wide application prospect and great market demand.
The gene chip is one kind of biochip, which is prepared through planting serial probes with known sequence on the chip substrate, and may be used in the hybridization detection of specific mark nucleic acid and reporting the nucleic acid information in the detected object through identification and information processing.
The current methods for detecting the hybridization result of the gene chip mainly include two major types, one is to scan and determine the hybridization state of each probe position one by using a laser confocal scanner, and the other is to image at one time by using a CCD (charge coupled device) camera or a CMOS (Complementary Metal-Oxide-Semiconductor) camera and then analyze the image and obtain the hybridization result of the gene chip. No matter which kind of fluorescence imaging, all use band pass filter to incite light refraction to the mark fluorescent substance on the probe and arouse fluorescent substance and send the mark fluorescence, mark fluorescence rethread band pass filter gets into the detector and accomplishes fluorescence imaging, because the incite light exists on the probe with the mark fluorescence simultaneously, must have the background interference of incite light to the fluorescence imaging result. In fluorescence imaging, too high background interference can result in false negatives or false positives; false negatives can result in delayed treatment of the disease and can also result in irreparable loss of life. Effectively reduces the background interference during the detection of the gene chip, improves the accuracy of the detection result of the gene chip and has great significance for the disease screening based on the gene chip.
Disclosure of Invention
The present disclosure is directed to overcoming, at least in part, the deficiencies of the prior art and providing a novel method for detecting gene chip hybridization results by total internal reflection.
The present disclosure is also directed to a method for detecting hybridization results of a gene chip by total internal reflection, which overcomes the problem of false negative or false positive caused by background interference of excitation light on fluorescence imaging results.
In order to achieve one of the above purposes, the present disclosure provides the following technical solutions:
a method for detecting gene chip hybridization results by total internal reflection, the method comprising: constructing a gene chip detector, wherein a quartz glass plate is arranged in the gene chip detector, and a dark box of the gene chip detector is divided into a light source dark box and an imaging dark box by the quartz glass plate; adding an isorefractive index solution having the same refractive index as that of the quartz glass to the quartz glass plate; the quartz glass substrate of the gene chip is placed on the equal-refractive-index solution, and the quartz glass substrate of the gene chip and the quartz glass plate are bonded together by the equal-refractive-index solution, so that the quartz glass substrate of the gene chip, the equal-refractive-index solution and the quartz glass plate become optical media with continuous and consistent refractive indexes; adjusting the position and angle of the plane parallel light source generator to make the emitted plane parallel light irradiate the quartz glass substrate of the gene chip to form total internal reflection, and forming total internal reflection evanescent wave on the surface of the quartz glass substrate of the gene chip while the light source exciting light is totally reflected to form light source reflecting light; exciting fluorescent substances which are hybridized and fluorescently labeled on probes on a quartz glass substrate of the gene chip by the evanescent waves of total internal reflection to emit labeled fluorescence; the marked fluorescence is captured by a high-resolution camera to form a fluorescence imaging picture, and finally the detection result of gene chip hybridization is obtained after data analysis and arrangement.
According to a preferred embodiment of the invention, the plane-parallel light forms an evanescent wave of total internal reflection in the range of 100nm of the surface of the quartz glass substrate.
According to a preferred embodiment of the present invention, the excitation light of the light source of which the plane-parallel light is irradiated onto the silica glass substrate can cover the silica glass substrate.
According to a preferred embodiment of the present invention, the light blocking reflective material is laid on the inner surface of the camera bellows such that the light blocking reflective material on the inner surface of the camera bellows prevents light impinging on the inner surface of the camera bellows from being reflected.
According to a preferred embodiment of the present invention, the plane-parallel light is emitted by a plane-parallel light source generator including a laser light source and a laser beam expander.
According to a preferred embodiment of the present invention, the adjustment of the position and the angle of the plane-parallel light source generator is performed by adjusting the position of a servo motor connected to the plane-parallel light source generator on the laser light source support and the rotation angle of the plane-parallel light source generator on the servo motor.
According to a preferred embodiment of the invention, the method detects hybridization results without scanning.
According to a preferred embodiment of the invention, the light source excitation light, the light source reflection light and the evanescent wave formed by the method cannot be captured by a high-resolution camera, only the marked fluorescence is captured by the high-resolution camera, the image captured by the high-resolution camera is analyzed to obtain the hybridization result of the gene chip, the background interference of fluorescence imaging is eliminated in the detection process, and the signal-to-noise ratio of the hybridization signal is improved. Compared with a common Resolution camera (equivalent to a civil camera), the high Resolution camera has the main difference of Resolution (Resolution), wherein the Resolution refers to the number of pixel points (Pixels) of an image acquired by the camera each time, the pixel distinguishing indexes are different with different ages, and the most popular high Resolution camera after 2018 is 1800 to 2400 ten thousand Pixels.
According to a preferred embodiment of the present invention, the high resolution camera is a charge coupled device camera or a complementary metal oxide semiconductor camera.
According to a preferred embodiment of the invention, the marker fluorescence is captured by a high resolution camera after selective filtering by a band pass filter.
According to a preferred embodiment of the invention, the bandpass filter is selective and label-fluorescent adaptive.
According to a preferred embodiment of the present invention, the iso-refractive index solution is a glycol solution.
According to a preferred embodiment of the present invention, the imaging camera bellows is located above the light source camera bellows, and a dimension of the imaging camera bellows in the horizontal first direction is smaller than a dimension of the light source camera bellows in the horizontal first direction.
According to a preferred embodiment of the invention, the band-pass filter is selected according to the wavelength of the fluorescence of the label emitted by the probe.
The invention provides a method for detecting gene chip hybridization results by total internal reflection, which applies the total internal reflection imaging principle to the detection of gene chip hybridization results, and leads light source exciting light of plane parallel light to form total internal reflection in a detector by light path design. The quartz glass substrate and the quartz glass plate are bonded together through the equal-refractive-index solution, the quartz glass plate of the detector, the equal-refractive-index solution and the quartz glass substrate are guaranteed to have the same refractive index and be equal to one piece of quartz glass, the total internal reflection evanescent wave is transmitted on the surface of the quartz glass substrate, the marked fluorescence is excited, the light source exciting light, the light source reflected light and the evanescent wave cannot be captured by the high-resolution camera, the light irradiated to the inner surface of the camera bellows is prevented from being reflected again by the blocking light reflecting material, and finally only the marked fluorescence is captured by the high-resolution camera through the selection of the band-pass filter.
The most core technology of the invention is to use the solution with equal refractive index, and the quartz glass substrate of the gene chip is bonded on the quartz glass plate through the solution with equal refractive index. The intrinsic characteristic of equal refractive index solution 'equal refractive index' is utilized, the characteristic that the equal refractive index solution has certain 'bonding effect' is utilized, so that a quartz glass plate, the equal refractive index solution and a quartz glass substrate of a gene chip are bonded together, the quartz glass plate, the equal refractive index solution and the quartz glass substrate of the gene chip are equal to one piece of quartz glass, the refractive index is consistent, a light source exciting light emitted by a plane parallel light source generator is ensured to be totally internally reflected to form an evanescent wave of total internal reflection, the evanescent wave of the total internal reflection is only transmitted along the surface of the quartz glass substrate of the gene chip, a marking fluorescent substance on a probe which is hybridized and fluorescently marked on the gene chip in a thin layer within 100nm of the quartz glass substrate of the gene chip is excited to emit marking fluorescence, and the marking fluorescent substance and the evanescent wave of the plane parallel light exciting light and the total internal reflection cannot enter a camera for imaging through a camera of a detector, thereby solving the problem of background interference on fluorescence imaging.
According to the detector with the structure, the dark box is divided into the light source dark box and the imaging dark box through the quartz glass plate, light source excitation light and light source reflection light in the light source dark box do not enter the imaging dark box, and the problem that fluorescence imaging is interfered by excitation light background is further solved. The detector adjusts the position and the angle of the surface parallel light source generator through the servo motor arranged on the laser light source bracket, ensures that light source exciting light emitted by the surface parallel light source generator forms total internal reflection on a quartz glass plate, and light source reflecting light does not enter an imaging dark box, and further solves the problem of background interference on fluorescence imaging. In addition, the light reflection blocking material is arranged on the inner surface of the dark box, so that all light on the inner surface of the dark box can not be reflected again, and background interference caused by light reflection pollution in the dark box is blocked again.
According to the detector with the structure, the band-pass filter which can be selectively replaced is designed, the band-pass which is adaptive to the labeled fluorescence emitted by the probe is selected in the using process, and the interference of background fluorescence outside the selection range of the band-pass filter is further prevented.
The method for detecting the gene chip hybridization result by utilizing the surface light source total internal reflection has the advantages of avoiding background interference in the fluorescent imaging process to the maximum extent, improving the signal-to-noise ratio of the gene chip hybridization result and the detection sensitivity of the gene chip hybridization result and having good use effect.
Drawings
FIG. 1 is a method for detecting gene chip hybridization results by total internal reflection according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a detector constructed by the method for detecting hybridization results of gene chips by total internal reflection according to an embodiment of the present invention;
in the figure: 1. a dark box; 2. a quartz glass plate; 3. a light source dark box; 4. a laser light source bracket; 5. a servo motor; 6. a plane parallel light source generator; 7. exciting light by a light source; 8. the light source reflects light; 9. an isorefractive index solution; 10. a quartz glass substrate; 11. a totally internally reflected evanescent wave; 12. a probe; 13. labeling fluorescence; 14. a band-pass filter; 15. a high resolution camera; 16. an imaging dark box; 17. blocking the light reflective material.
Detailed Description
Exemplary embodiments of the present disclosure are described in detail below with reference to the 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 method for detecting a gene chip hybridization result by total internal reflection and a total internal reflection type gene chip detector constructed by the method.
As shown in FIG. 1-2, the method for detecting the hybridization result of the gene chip by total internal reflection comprises the following steps: constructing a gene chip detector, wherein a quartz glass plate 2 is arranged in the gene chip detector, and a dark box 1 of the gene chip detector is divided into a light source dark box 3 and an imaging dark box 16 by the quartz glass plate 2; adding an equal-refractive-index solution 9 having the same refractive index as that of the silica glass to the silica glass plate 2; the quartz glass substrate 10 of the gene chip is placed on the equal-refractive-index solution 9, and the quartz glass substrate 10 of the gene chip and the quartz glass plate 2 are bonded together by the equal-refractive-index solution 9, so that the quartz glass substrate 10 of the gene chip, the equal-refractive-index solution 9 and the quartz glass plate 2 become optical media with continuous and consistent refractive indexes; adjusting the position and the angle of the surface parallel light source generator 6 to enable the emitted surface parallel light to irradiate the quartz glass substrate 10 of the gene chip to form total internal reflection, wherein the total internal reflection evanescent wave 11 is formed into light source reflection light 8 by total reflection, and simultaneously the total internal reflection evanescent wave 11 is formed on the surface of the quartz glass substrate 10 of the gene chip; the light source exciting light 7 excites the fluorescent substance on the probe 12 which has completed the hybridization and the fluorescent labeling to emit labeled fluorescence 13; the labeled fluorescence 13 is captured by a high-resolution camera 15 to form a fluorescence imaging picture, and the detection result of gene chip hybridization is finally obtained after data analysis and arrangement.
The detection method comprises the steps of enabling light source exciting light of a surface parallel light source to form surface light source total internal reflection in a detector through light path design, specifically, bonding a quartz glass substrate of a gene chip on a quartz glass plate through an equal-refractive-index solution, enabling light emitted by the surface parallel light source to be emitted to an integrated structure formed by the quartz glass plate, the equal-refractive-index solution and the quartz glass substrate, and enabling the light to be subjected to total internal reflection on the surface of the quartz glass substrate.
Firstly, a plane parallel light source is utilized, wherein the plane parallel light source is emitted by a plane parallel light source generator, and the plane parallel light source generator comprises a laser light source and a laser beam expander; the position and the angle of the surface parallel light source generator are adjusted by adjusting the position of a servo motor connected with the surface parallel light source generator on a laser light source bracket and the rotation angle of the surface parallel light source generator on the servo motor; the light source exciting light of the surface parallel light source irradiating the surface of the quartz glass substrate can cover the quartz glass substrate, and forms total internal reflection evanescent wave in the surface 100nm range of the quartz glass substrate, the fluorescent substance which has finished hybridization and fluorescence labeling on the total internal reflection evanescent wave exciting probe emits labeled fluorescence, and simultaneously the light source exciting light is ensured to generate total reflection on the quartz glass plate.
Secondly, the detection method also utilizes two characteristics of the equal-refractive-index solution, firstly utilizes the intrinsic characteristic of the equal-refractive-index solution of equal refractive index, secondly utilizes the characteristic that the equal-refractive-index solution has certain bonding effect, thereby ensuring that a quartz glass plate, the equal-refractive-index solution and the quartz glass substrate of the gene chip are bonded together, is equal to a piece of quartz glass, has consistent refractive index, forms an integrated structure of the quartz glass plate, the equal-refractive-index solution and the quartz glass substrate, ensures that light source exciting light emitted by a plane parallel light source generator is totally internally reflected and forms an evanescent wave of the total internal reflection, the evanescent wave of the total internal reflection only propagates along the surface of the quartz glass substrate of the gene chip, and leads the mark on the probe which is hybridized and fluorescently marked on the quartz glass substrate in a thin layer within 100nm of the quartz glass substrate of the gene chip to be excited to emit mark fluorescence, and through the camera imaging of the detector, the plane parallel light exciting light and the total internal reflection evanescent wave cannot enter the camera for imaging, so that the background interference problem of fluorescence imaging is solved.
FIG. 2 is a schematic diagram showing the construction of a total internal reflection type GeneChip detector constructed by the method according to the embodiment of the present invention, which places a quartz glass plate 2 in a dark box 1, thereby dividing the dark box 1 into a light source dark box 3 and an imaging dark box 16; the quartz glass plate 2 is embedded in the dark box 1, and the thickness of the quartz glass plate 2 is smaller than that of the dark box 1 in the transition area of the light source dark box 3 and the imaging dark box 16; the length of the quartz glass plate 2 is longer than that of the quartz glass substrate 10 of the gene chip. The quartz glass plate 2 separates the dark box 1, so that the light source exciting light 7 and the light source reflected light 8 in the light source dark box 3 do not enter the imaging dark box 16 in the imaging process of the imaging dark box 16, the interference of background light can be avoided to a certain extent, and the reason for the effect is that the light source exciting light 7 generates total reflection on the quartz glass plate 2.
Further, the imaging camera chamber 16 is located above the light source camera chamber 3, and the dimension of the imaging camera chamber 16 in the horizontal first direction is smaller than the dimension of the light source camera chamber 3 in the horizontal first direction. The quartz glass plate 2 has a dimension in the horizontal first direction which is greater than the dimension of the imaging dark box 16 in the horizontal first direction.
The inner wall surface of the camera bellows 1 is also provided with a light blocking reflection material 17, wherein the camera bellows 1 comprises the light source camera bellows 3 and the imaging camera bellows 16, namely, the inner surfaces of the light source camera bellows 3 and the imaging camera bellows 16 are both provided with the light blocking reflection material 17, and particularly, the surface of the imaging camera bellows 16 is provided with the light blocking reflection material 17, so that all light on the inner surface of the camera bellows 1 can not be reflected again, and the background interference caused by light reflection pollution in the camera bellows 1, particularly the imaging camera bellows 16 is blocked.
A servo motor 5 is arranged on a laser light source bracket 4, and a plane parallel light source generator 6 is also arranged and connected with the servo motor 5. The plane parallel light source generator 6 includes a laser light source and a laser beam expander. By adjusting the up-down position of a servo motor 5 arranged on a laser light source bracket 4 and adjusting the rotation angle of a plane parallel light source generator 6 on the servo motor 5, the incidence angle of the light source excitation light 7 emitted by the plane-parallel light source generator 6 on the quartz glass plate 2 can be adjusted, when the incident angle is adjusted to be larger than the critical angle of 'quartz glass plate-isorefractive index solution-quartz glass substrate', meanwhile, when the light source exciting light 7 emitted by the plane parallel light source can irradiate and cover the whole quartz glass substrate 10, the total internal reflection light source reflecting light 8 is generated, and the total internal reflection evanescent wave 11 is formed in a thin layer within the range of 100nm of the quartz glass substrate 10, and the light source reflected light 8 is absorbed by the light blocking reflection material 17 attached to the inner wall surface of the light source dark box 3, so that reflection or scattering is not formed, and the light does not enter the imaging dark box 16. Thus, the position and angle of the excitation light of the plane parallel light source are adjusted and the secondary absorption of the light reflecting material 17 is blocked, and the background interference is further avoided.
Specific example 1: after the detector is constructed according to the illustration in fig. 2, an isorefractive index solution 9 is dropped on the quartz glass plate 2, where the isorefractive index solution 9 is selected to be an ethylene glycol solution with a consistent refractive index with the quartz glass, a blank quartz glass substrate 10 is placed above the isorefractive index solution 9 to form an integrated structure of "quartz glass plate-ethylene glycol solution-blank quartz glass substrate" with a consistent refractive index, then a nucleic acid solution marked by Alexa Fluor 488 (prepared by introducing an EdU (thymidine) analogue, 5-ethyl-2' -deoxyuridine) by a PCR method) is dropped on the blank quartz glass substrate 10, and a plane parallel light source generator 6 is opened, where the plane parallel light source generator 6 includes a laser light source and a laser beam expander, and the product is selected from MDL-XS-488 model of new optical technology ltd, the incident angle of the plane-parallel light source is adjusted, and the bandpass filter 14 corresponding to Alexa Fluor 488 (absorption peak 495nm, emission peak 519 nm) is selected, and may be, for example, a filter with a Bandpass (BP) of 525/30; after the filtering and selection of the band-pass filter 14, the high-resolution camera 15 can only observe green fluorescence, and blue excitation light cannot be detected in the imaging dark box 16 outside the light source dark box 3, so that the light source excitation light 7 is verified to be totally reflected to form light source reflection light 8 and total internal reflection evanescent wave 11, the debugging of a light path system is completed, and the fluorescence excitation is completed by the total internal reflection evanescent wave of the excitation light.
Specific example 2: after the optical path system is debugged according to the specific embodiment 1, taking out the blank quartz glass substrate 10, ensuring that the ethylene glycol solution of the equal refractive index solution 9 is still in a full state, and placing the quartz glass substrate 10 which is hybridized and fluorescently labeled on the upper part of the equal refractive index solution 9 in the imaging dark box 16; and opening the plane parallel light source generator 6, acquiring a fluorescence image of the gene chip hybridization result through the high-resolution camera 15, and uploading the acquired fluorescence imaging image to a data center for gene chip hybridization detection result analysis.
By the specific embodiment 1, the position and the angle of the plane parallel light source generator 6 are adjusted, the light source exciting light 7 is ensured to form the total internal reflection evanescent wave 11, the total internal reflection evanescent wave 11 only propagates along the surface of the detector on the quartz glass substrate 10 of the gene chip, the light source exciting light 7 cannot enter the imaging dark box 16, the background interference of the light source exciting light 7 on fluorescence imaging is eliminated, and the signal-to-noise ratio of the gene chip hybridization result is improved. Meanwhile, the total internal reflection evanescent wave 11 can excite the probe 12 which has completed hybridization and fluorescent labeling on the gene chip, and emit labeled fluorescence 13, and the labeled fluorescence 13 passes through the band-pass filter 14 to enter the high-resolution camera 15 and complete fluorescence imaging. In the later process of constructing the detector, optimization can be carried out, and an intelligent system is introduced, so that automatic debugging of the front total reflection optical path system is realized. In addition, a luminance meter may be incorporated into the imaging camera 16 to detect light entering the imaging camera 16 to further ensure that blue excitation light from the light source camera 3 does not enter the imaging camera 16.
The high-resolution camera 15 is arranged in the imaging camera bellows 16, and is an imaging device of the detector, the high-resolution camera 15 is embedded at the side of the imaging camera bellows 16 opposite to the quartz glass plate, the high-resolution camera 15 is a high-quantum efficiency and low-noise spectral imaging camera, the camera resolution is high, the high-resolution camera is more suitable for the precise analysis of the gene chip hybridization result, and the detection accuracy is enhanced, and here, the high-resolution camera 15 can be a CCD camera (PCO. pixelfly usb series, PCO, Germany). The high-resolution camera 15 is also provided with a band-pass filter 14 at one end inside the imaging dark box 16, and the band-pass filter 14 mainly functions to perform spectrum selection on the marked fluorescence 13 emitted by the probe 12 and select the band-pass filter 14 with a proper band-pass, so that the marked fluorescence 13 emitted by the probe 12 enters the high-resolution camera 15, and light with an unnecessary spectrum is prevented from passing through, thereby being more suitable for the detector to perform accurate imaging analysis.
Specific example 3: in the process of constructing the total internal reflection type gene chip detector, in order to ensure that the light source exciting light irradiated on the substrate of the gene chip by the plane parallel light can irradiate the substrate covering the whole gene chip, for example, the substrate of the gene chip is selected from a quartz glass substrate 10, and possibly, part of the light source exciting light 7 can pass through two ends of the quartz glass substrate 10, in order to ensure that the light source exciting light 7 does not enter an imaging dark box 16, two ends of the placing position of the quartz glass substrate 10 are provided with movable blocking light reflecting materials 17, after the quartz glass substrate 10 is placed, the movable blocking light reflecting materials 17 are adjusted, and two ends of the quartz glass substrate 10 are ensured to be closely connected with the movable blocking light reflecting materials 17, so that the background interference of the light source exciting light 7 on imaging is further.
Specific example 4: after the optical path system is debugged according to the specific embodiment 1, an intelligent system is introduced into the detector to calculate the optical path of the light source excitation light 7 emitted by the surface parallel light source and the irradiation range data of the lower part of the quartz glass plate 2, so as to calculate the length of the blocking light reflecting material 17 required to be arranged at the lower part of the quartz glass plate 2, introduce the movable blocking light reflecting material 17 below the quartz glass plate 2, set the length data of the blocking light reflecting material 17 according to the calculated requirement, and movably adjust the covering surface of the movable blocking light reflecting material 17 below the quartz glass plate 2, so as to ensure that only the required light source excitation light 7 in the light source dark box 3 is irradiated onto the quartz glass plate 2, and other background light is blocked by the blocking light reflecting material 17 and does not enter the imaging dark box 16 through the gaps at the two ends of the quartz glass plate 2.
The core of the invention is that total reflection is formed by constructing a structure integrating a quartz glass plate, an equal refractive index solution and a quartz glass substrate, wherein the equal refractive index solution 9 is the key for integrating the quartz glass plate 2 and the quartz glass substrate 10, and the key step for constructing the total internal reflection type gene chip detector is to drip the equal refractive index solution 9, so that a space capable of bearing the equal refractive index solution 9 is reserved at the upper end of the quartz glass plate 2, and the space is dripped and filled with the equal refractive index solution 9 through a liquid transfer device during detection; when the quartz glass substrate 10 is replaced, whether the equal-refractive-index solution 9 is in a full state needs to be checked; removing all the solution 9 with equal refractive index by a liquid moving machine after use; when the solution 9 with different equal refractive index is replaced, the original solution 9 with equal refractive index needs to be removed, and then the solution 9 with new equal refractive index needs to be rinsed for at least 1 time and then dripped to be filled.
According to the general concept of the invention, a method for detecting gene chip hybridization results by total internal reflection is provided, firstly, a plane parallel light source is adjusted by light path design, namely, the angle of light source exciting light 7 of the plane parallel light source emitted by a plane parallel light source generator 6 is adjusted, so that the light source exciting light 7 emitted by the plane parallel light source generator generates total reflection on a quartz glass plate 2; secondly, the quartz glass substrate 10 and the quartz glass plate 2 are bonded together through the equal-refractive-index solution 9 to form an optical system with continuous and consistent refractive index, so that the total-internal-reflection evanescent wave is transmitted on the surface of the quartz glass substrate 10, the light source exciting light 7 and the total-internal-reflection evanescent wave 11 are ensured not to enter a high-resolution camera, and background interference of fluorescence imaging is eliminated.
According to the general concept of the invention, a total internal reflection type gene chip detector is constructed, a dark box 1 is divided into a light source dark box 3 and an imaging dark box 16 by a quartz glass plate 2, and light source exciting light 7 and light source reflecting light 8 in the light source dark box 3 are ensured to be in the light source dark box 3 and not enter the imaging dark box 16; at the same time, the light reflection blocking material 17 is attached to the inner wall surface of the camera 1 to ensure that all light on the inner surface of the camera 1 (the camera 1 described herein includes both the light source camera 3 and the imaging camera 16) is not reflected again, thereby further blocking light reflection contamination in the imaging camera 16. A quartz glass substrate 10 of the gene chip is bonded with a quartz glass plate 2 through an equal refractive index solution 9 to form a light path system with a consistent refractive index, so that a total internal reflection evanescent wave 11 is formed on the quartz glass substrate 10, the total internal reflection evanescent wave 11 excites a probe 12 which is hybridized and fluorescently labeled on the gene chip, labeled fluorescence 13 emitted by the probe 12 passes through a band-pass filter 14 to complete fluorescence imaging on a high-resolution camera 15, and the fluorescence imaging result is subjected to data analysis and arrangement to finally obtain a detection result of gene chip hybridization. By the separation of the independent imaging dark box 16 and the layout of the blocking light reflection material 17, the incident light total internal reflection system with the same refractive index of the quartz glass material and the adjustment of the laser position and the angle of the surface light source, the background interference of background light such as an excitation light source, a reflection light source, a scattering light source and the like on a fluorescence imaging result is progressively blocked layer by layer.
In the process of detecting the gene chip hybridization result, fluorescence imaging is required, the excitation light is refracted to the labeled fluorescent substance on the probe and excites the fluorescent substance to emit labeled fluorescence, and the labeled fluorescence enters the detector to complete fluorescence imaging. In the imaging process of the conventional detector for gene chip hybridization results, excitation light and labeled fluorescence exist on the probe at the same time, so that the existing excitation light can cause background interference on the fluorescence imaging results, and false negative or false positive can be formed due to overhigh background interference; false negatives can result in delayed treatment of the disease and can also result in irreparable loss of life.
According to the method for detecting the gene chip hybridization result by the total internal reflection provided by the invention, the principle of forming the total internal reflection imaging by using the plane parallel light source is applied to the detection of the gene chip hybridization result, the background interference of fluorescence imaging is eliminated, and the signal to noise ratio of a hybridization signal is improved.
The total internal reflection type gene chip detector made by the method for detecting the gene chip hybridization result by total internal reflection divides the dark box into a light source dark box and an imaging dark box by a quartz glass plate, and ensures that light in the light source dark box does not enter the imaging dark box; the light reflection blocking material on the inner wall of the dark box ensures that all light on the inner surface of the dark box cannot be reflected again, so that light reflection pollution is further blocked; the quartz glass substrate and the quartz glass plate are bonded together through the equal-refractive-index solution to form a light path system with the same refractive index, so that the excitation light is ensured to form an evanescent wave of total internal reflection on the surface of the quartz glass substrate of the gene chip and cannot enter an imaging device to cause background interference.
Therefore, the total internal reflection type gene chip detector is constructed by optimizing the method for detecting the gene chip hybridization result through total internal reflection, the background interference in the fluorescence imaging process is avoided to the maximum extent by using the method and the instrument, the signal-to-noise ratio of the gene chip hybridization result and the detection sensitivity of the gene chip hybridization result are improved, the using effect is good, and the detection sensitivity and the specificity are higher.
Although embodiments of the present disclosure have been shown and described, it would be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the disclosure. The scope of applicability of the present disclosure is defined by the appended claims and their equivalents.
Claims (10)
1. A method for detecting gene chip hybridization results by total internal reflection, the method comprising:
constructing a gene chip detector, wherein a quartz glass plate (2) is arranged in the gene chip detector, and a dark box (1) of the gene chip detector is divided into a light source dark box (3) and an imaging dark box (16) by the quartz glass plate (2);
adding an isorefractive index solution (9) having the same refractive index as that of the quartz glass onto the quartz glass plate (2);
the quartz glass substrate (10) of the gene chip is placed on the equal-refractive-index solution (9), and the quartz glass substrate (10) of the gene chip and the quartz glass plate (2) are bonded together by the equal-refractive-index solution (9), so that the quartz glass substrate (10), the equal-refractive-index solution (9) and the quartz glass plate (2) of the gene chip become optical media with continuous and consistent refractive indexes;
the position and the angle of a plane parallel light source generator (6) are adjusted, so that the emitted plane parallel light can irradiate a quartz glass substrate (10) of the gene chip to form total internal reflection, and light source exciting light (7) is totally reflected to form light source reflecting light (8) and simultaneously form a total internal reflection evanescent wave (11) on the surface of the quartz glass substrate (10) of the gene chip;
the total internal reflection evanescent wave (11) excites a fluorescent substance which is hybridized and fluorescently labeled on a probe (12) on a quartz glass substrate (10) of the gene chip to emit labeled fluorescence (13);
the marked fluorescence (13) is captured by a high-resolution camera (15) to form a fluorescence imaging picture, and finally the detection result of gene chip hybridization is obtained after data analysis and arrangement.
2. The method for detecting gene chip hybridization results according to claim 1, wherein the plane-parallel light forms an evanescent wave (11) of total internal reflection within 100nm of the surface of the quartz glass substrate (10).
3. The method for detecting hybridization results of gene chips by total internal reflection according to claim 2, wherein the light blocking reflective material (17) is laid on the inner surface of the dark box (1) so that the light blocking reflective material (17) on the inner surface of the dark box (1) prevents the light irradiated on the inner surface of the dark box (1) from being reflected.
4. The method for detecting gene chip hybridization results according to claim 1, wherein the plane-parallel light is emitted from a plane-parallel light source generator (6), and the plane-parallel light source generator (6) comprises a laser light source and a laser beam expander.
5. The method for detecting hybridization results of gene chip by total internal reflection according to claim 4, wherein the adjustment of the position and angle of the plane-parallel light source generator (6) is performed by adjusting the position of the servo motor (5) connected to the plane-parallel light source generator (6) on the laser light source holder (4) and the rotation angle of the plane-parallel light source generator (6) on the servo motor (5).
6. The method for detecting gene chip hybridization results according to claim 1, wherein said high resolution camera (15) is a CCD camera or a CMOS camera.
7. The method for detecting hybridization results of gene chip by total internal reflection according to claim 1, wherein the labeled fluorescence (13) is captured by a high resolution camera (15) after being selectively filtered by a band pass filter (14).
8. The method for detecting hybridization results of gene chip by total internal reflection according to any of claims 1 to 7, wherein said isorefractive index solution (9) is a glycol solution.
9. The method for detecting gene chip hybridization results according to any of claims 1-7, wherein the imaging chamber (16) is located above the light source chamber (3), and the dimension of the imaging chamber (16) along the horizontal first direction is smaller than the dimension of the light source chamber (3) along the horizontal first direction.
10. The method for detecting hybridization results of gene chip by total internal reflection according to claim 7, wherein said band-pass filter (14) is selected according to the wavelength of the labeled fluorescence (13) emitted from the probe (12).
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