CN117701740A - Portable intelligent detection micro-platform and method for accurately and rapidly quantifying target concentration through fluorescence - Google Patents
Portable intelligent detection micro-platform and method for accurately and rapidly quantifying target concentration through fluorescence Download PDFInfo
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Abstract
The invention discloses a portable intelligent detection micro-platform and a method for accurately and rapidly quantifying the concentration of a target by fluorescence, wherein the micro-platform comprises a micro-channel chip, a nucleic acid fluorescence quantitative reaction system and a quantitative detection system; the quantitative detection system comprises an image acquisition module, an image optimization module, a micro-reaction chamber identification module and a quantitative module, wherein the image acquisition module is used for acquiring a micro-reaction chamber fluorescence image after the fluorescent reaction of the target nucleic acid is completed based on the nucleic acid fluorescence quantitative reaction system, the micro-reaction chamber identification module is used for identifying the micro-reaction chamber in the micro-reaction chamber fluorescence image and acquiring the RGB intensity of the micro-reaction chamber, and the quantitative module is used for quantitatively detecting the target nucleic acid according to the RGB intensity of the micro-reaction chamber and the linear relation between the RGB and the target concentration, which are constructed in advance. According to the invention, only the mobile terminal is used for acquiring the micro-reaction fluorescent image, and the image analysis is carried out on the micro-reaction fluorescent image to obtain the detection result, so that the burden of expensive and non-portable instruments is greatly reduced, the experimental requirements are simplified, the detection process is accelerated, and the micro-reaction fluorescent image detection device is used for rapid screening and accurate quantification in clinical practice.
Description
Technical Field
The invention belongs to the technical field of cell biological detection, and particularly relates to a portable intelligent detection micro-platform and method for accurately and rapidly quantifying the concentration of a target through fluorescence.
Background
Bacterial infections are responsible for a variety of diseases in humans, animals and plants, and serious pathogenic bacterial infections can lead to a variety of symptoms, especially threatening patients with immunodeficiency or antibiotic drugs. Since bacteria are numerous and different bacteria may contain multiple subtypes, causing different symptoms, each of which needs to be diagnosed and identified to formulate an appropriate/personalized treatment strategy, this places a lot of burden on clinical diagnosis and treatment. The rapid identification of bacterial subtype infections and accurate quantification of their thallus content is an important means for timely treatment and control of infections. Traditional detection techniques rely heavily on expensive specialized equipment, which greatly reduces their applicability in large-scale population screening. Traditional bacterial infection analysis methods, exemplified by cryptococcus, are generally required to be highly specific based on real-time quantitative polymerase chain reaction (RT-qPCR). However, this technique relies heavily on expensive qPCR instruments/procedures for accurate temperature control and result readout, and is therefore limited in large-scale clinical screening applications. The novel CRISPR-Cas system can provide endonucleases, probes, etc. for specific detection of different bacteria to detect bacteria. Such isothermal amplification-based probes avoid reliance on expensive temperature control instrumentation. However, in the current technology, a special instrument or procedure, such as a fluorescence spectrometer, is still required for quantifying the fluorescence signal, and the popularization and wide application of the technology are also restricted.
Disclosure of Invention
The invention aims to provide a portable intelligent detection micro-platform and a method for accurately and rapidly quantifying the concentration of a target by fluorescence, which can rapidly and accurately quantify bacterial infection without depending on professional instruments (such as high-pixel image acquisition equipment such as a microscope or an electron microscope, and the like) and are convenient to carry.
In order to solve the technical problems, the invention adopts the following technical scheme: a portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a target by fluorescence comprises a micro-channel chip, a nucleic acid fluorescence quantitative reaction system and a quantitative detection system; the micro-channel chip comprises a substrate and a central liquid inlet hole arranged on the substrate; the device also comprises a positive control area and a negative control area which are arranged around the central liquid inlet hole, and a plurality of detection areas; each zone is provided with a micro-channel communicated with the central liquid inlet, and a plurality of micro-reaction chambers communicated with the micro-channel are arranged in each zone; the tail end of the micro-channel is provided with a liquid outlet hole; the quantitative detection system comprises an image acquisition module, an image optimization module, a micro reaction chamber identification module and a quantitative detection module, wherein the image acquisition module is used for acquiring a micro reaction chamber fluorescence image after target nucleic acid fluorescence reaction is completed based on the nucleic acid fluorescence quantitative reaction system, the image optimization module is used for optimizing the micro reaction chamber fluorescence image, the micro reaction chamber identification module is used for identifying each micro reaction chamber in the micro reaction chamber fluorescence image and acquiring RGB intensity of each micro reaction chamber, and the quantitative detection module is used for calculating RGB intensity average values according to RGB intensity of all micro reaction chambers in a current detection area, establishing a linear relation between the RGB intensity average values and corresponding known target concentrations, and quantitatively detecting target nucleic acid based on the linear relation;
The micro-reaction chamber identification module comprises: the gray threshold setting module is used for setting threshold ranges 0-N and threshold step length N so as to obtain a plurality of gray thresholds; the binarization image acquisition module is used for comparing the gray values of the pixel points with a plurality of gray threshold values respectively, setting the pixel points with the gray values larger than the gray threshold values as white and setting the pixel points smaller than or equal to the gray threshold values as black, so as to obtain a plurality of binarization images; the spot combining module is used for respectively extracting the white pixels communicated in each binarized image as spots, and combining the spots overlapped in the geometric centers in all the binarized images into a spot group; the spot group selecting module is used for selecting spot groups according to the number of pixels, the convexity, the inertia ratio and the roundness so as to select the spot groups representing the micro-reaction chamber; (i.e., one set of spots corresponds to one micro-reaction chamber); the micro-reaction chamber position marking module is used for recording the positions of the spot groups and marking the positions of the corresponding micro-reaction chambers on the optimized micro-reaction chamber fluorescent images according to the positions of the spot groups; and the RGB intensity acquisition module is used for weighting and averaging the RGB values of the pixel points in the micro-reaction chamber in the optimized micro-reaction chamber fluorescent image to acquire the RGB intensity of each representative micro-reaction chamber.
As an improvement, the detection zone comprises a subtype I detection zone and a subtype II detection zone; the subtype I detection area, the subtype II detection area, the positive control area and the negative control area are symmetrically arranged along the center of the central liquid inlet hole.
As an improvement, the nucleic acid fluorescent quantitative reaction system is a CRISPR system comprising one of CRISPR-Cas9, CRISPR-Cas12 or CRISPR-Cas13 systems.
As an improvement, the CRISPR system comprises Cas-12a protein, primer crRNA, fluorescent reporter and reaction buffer; the fluorescent Reporter (Reporter) has a quenching group (BHQ-1) and carboxyfluorescein (Fluorescein Amidite, FAM) attached thereto.
As an improvement, each reagent in the nucleic acid fluorescence quantitative reaction system is placed in the micro flow channel chip in a freeze-dried form.
As an improvement, the image acquisition module comprises an ultraviolet lamp and an intelligent mobile terminal integrated with a camera.
The invention also provides a method for accurately and rapidly quantifying the concentration of the target by fluorescence, which is applied to the portable detection micro-platform for accurately and rapidly quantifying the concentration of the target by fluorescence, wherein the target is a bacterial target gene and comprises the following steps:
S01: the method comprises the steps of reacting a preset freeze-dried nucleic acid fluorescence quantitative reaction system with nucleic acid of a known sample in a micro-channel chip; the target concentration calculated for the known sample is known; s02: irradiating the micro-channel chip subjected to nucleic acid fluorescence quantitative reaction by using an ultraviolet lamp, and collecting a fluorescence image of a micro-reaction chamber by using an intelligent mobile terminal; s03: optimizing the acquired fluorescence image of the micro-reaction chamber; s04: identifying a micro-reaction chamber in the micro-reaction chamber fluorescence image and acquiring RGB intensity corresponding to the micro-reaction chamber in the micro-reaction chamber fluorescence image; s05: calculating RGB average values of all micro-reaction chambers in a current detection area, and establishing a linear equation between the RGB average values and the known target concentration of the known sample nucleic acid according to the RGB intensity average values; s06: adding a sample to be detected into the micro-channel chip to react with a preset freeze-dried nucleic acid fluorescent quantitative reaction system; the target concentration of the sample to be detected is unknown; s07: irradiating the micro-channel chip subjected to nucleic acid fluorescence quantitative reaction by using an ultraviolet lamp, and collecting a fluorescence image of a micro-reaction chamber by using an intelligent mobile terminal; s08: optimizing the acquired fluorescence image of the micro-reaction chamber; s09: identifying a micro-reaction chamber in the micro-reaction chamber fluorescence image, acquiring the RGB intensity corresponding to the micro-reaction chamber in the micro-reaction chamber fluorescence image, and calculating the RGB average value of all the micro-reaction chambers in the current detection area; s10: and carrying the obtained RGB intensity mean value of the sample to be detected into the linear equation to calculate and obtain the nucleic acid content of the sample to be detected.
As an improvement, the step of optimizing the acquired image of the micro-reaction chamber includes: expanding the pixels of the acquired micro-reaction chamber fluorescent image to a preset value by adopting a bilinear interpolation algorithm; the difference in brightness between each pixel and surrounding pixels is reduced using gaussian filtering.
As an improvement, the step of identifying the micro-reaction chamber in the micro-reaction chamber fluorescence image and taking the micro-reaction chamber RGB intensity in the micro-reaction chamber fluorescence image comprises: setting a threshold range of 0-N and a threshold step length of N, thereby obtaining a plurality of gray thresholds; respectively comparing the gray values of the pixel points with a plurality of gray threshold values, setting the pixel points with gray values larger than the threshold values as white, and setting the pixel points smaller than or equal to the threshold values as black, so as to obtain a plurality of binarized images; respectively extracting white pixels communicated in each binarized image as spots, and merging the spots overlapped in the geometric centers in all the binarized images into a spot group; selecting a spot group representing the micro-reaction chamber by screening the spot group according to the number of pixels, the convexity, the inertia ratio and the roundness; recording the positions of the spot groups, and marking the positions of the micro-reaction chambers on the optimized micro-reaction chamber fluorescent images according to the positions of the spot groups; and carrying out RGB value weighting and average on pixel points in the micro-reaction chamber in the optimized micro-reaction chamber fluorescent image to obtain the RGB intensity of each representative micro-reaction chamber.
As an improvement, the specific steps of the reaction between the preset freeze-dried nucleic acid fluorescence quantitative reaction system and the sample nucleic acid are as follows: s01: taking a sample to be detected, and lysing cells to expose DNA, wherein the sample is cryptococcus; s02: adding a sample solution containing exposed DNA from a central liquid inlet hole of the chip, and simultaneously applying negative pressure and vacuumizing outside the chip to enable the sample solution containing exposed DNA to fully enter the chip and react with a preset freeze-dried nucleic acid fluorescence quantitative reaction system; s03: after Cas12a protein in the nucleic acid fluorescence quantitative reaction system is combined with target sample specific crRNA, releasing nonspecific endonuclease activity, cutting off a quenching group and carboxyfluorescein on a fluorescence reporter molecule, and releasing fluorescence; the sequence of the target sample specific crRNA is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.
The crRNA sequences involved in the methods of the invention are shown in the following table.
TABLE 1
The invention has the advantages that: according to the invention, the sample is detected through the micro-channel chip and a nucleic acid fluorescence quantitative reaction system (such as a CRISPR system) preset in the micro-channel chip, then a fluorescence image is acquired through non-professional equipment such as a mobile intelligent terminal, and a detection result is rapidly and quantitatively acquired in a mode of carrying out image analysis processing by utilizing a quantitative detection system, and the micro-platform is convenient to carry. When target DNA is present in the sample solution, cas12a-crRNA conjugate is activated by reporter cleavage at 37 ℃, resulting in separation between the quencher group (BHQ-1) and the reporter carboxyfluorescein (Fluorescein Amidite, FAM) and emission of amplified fluorescent signal fluorescein. In contrast, no fluorescent signal is generated in the negative region or in the microreactor without target DNA. The specific subtype of cryptococcus may be identified by a hand-held ultraviolet lamp (480 nm) and the corresponding fluorescence image captured by a smart mobile terminal, e.g. a smart phone.
The invention integrates portability of the micro-channel array biochip, high specificity of CRISPSR-Cas 12a technology and accuracy of intelligent imaging program. The detection result is detected only by acquiring and processing images through pixels of a personal smart phone and the like instead of expensive professional equipment such as a professional camera or a microscope, so that the burden of expensive and non-portable instruments is greatly reduced, the experiment requirement is simplified, the detection process is accelerated, and the method is used for rapid screening and accurate quantification in clinical practice.
According to the invention, the number of detection areas can be adjusted according to the requirements, and various bacterial subtypes can be detected at the same time, so that the detection efficiency is improved.
The nucleic acid fluorescence quantitative detection system is preset in the micro-channel chip in a freeze-dried mode, so that the portability and the use convenience of the product are improved, the result can be obtained only by injecting a sample, and the nucleic acid fluorescence quantitative detection system is not required to be configured on site. During transportation, the nucleic acid fluorescence quantitative detection system in a freeze-dried form can be kept at a preset position due to lack of fluidity, and cannot flow around to cause failure.
The detection areas and the negative and positive control areas are symmetrically arranged along the center of the central liquid inlet hole, so that the consistency of solution flow in each area is ensured, and the accuracy of the whole detection result is improved.
In the process of quantitative detection, the invention optimizes the image, adopts bilinear interpolation algorithm to expand the pixels of the acquired image to a preset value such as 1000 pixels, and aims to eliminate the interference of low pixels and inconsistent pixels of different images. The difference in brightness between each pixel point and surrounding pixel points is reduced by gaussian filtering, and the influence of high-frequency noise is minimized. In addition, in the process of identifying the micro-reaction chamber in the image and acquiring the RGB intensity of the micro-reaction chamber in the image, the method firstly carries out binarization processing to acquire a binarized image, and then identifies spots and split spots from the image so as to identify the position of the micro-reaction chamber. Because the micro-channel chip is small in size, the shooting precision of the existing smart phone camera is limited, and the micro-reaction chamber is difficult to accurately divide from the image by adopting the existing modes such as example division. Therefore, in order to improve the robustness of the invention, the micro-reaction chamber is subjected to the segmentation processing through the steps, so that the requirement on image shooting is greatly reduced.
Finally, a database containing the linear relationship between the various known different target concentrations of the samples and the RGB intensities is constructed by constructing in advance a linear equation between the different target concentrations of the respective samples and the RGB intensities, i.e., by a large number of preliminary experiments, wherein the RGB intensities are taken as independent variables and the target concentrations are taken as dependent variables. After the RGB intensity of each micro-reaction chamber in the detection area corresponding to the sample to be detected is obtained, the RGB mean value of the detection area (namely the RGB intensity corresponding to the sample) is calculated, and the value is brought into a linear equation to obtain the target concentration corresponding to the sample, so that the use convenience of the invention is further improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 is a schematic diagram of a detection micro-platform according to an exemplary embodiment of the present invention;
fig. 2A is a schematic diagram of the dimensions of a chip system constructed on a micro flow channel chip according to an exemplary embodiment of the present invention;
FIG. 2B is a schematic size diagram of the single micro flow channel chip shown in FIG. 2A;
FIG. 2C is a schematic illustration of the partial microreactor of FIG. 2B;
FIG. 3 is a flow chart of a detection method according to an exemplary embodiment of the present invention;
FIG. 4 is a graph of experimental results of the detection results according to the embodiment of the present invention;
FIG. 5A is a schematic diagram showing a micro flow channel chip according to an embodiment of the present invention;
FIG. 5B is a fluorescence image of a microreactor for detecting different bacterial subtypes and corresponding RBG intensities;
FIG. 5C is a graph of RGB values corresponding to different target concentrations reflecting the same bacterial subtype;
FIG. 5D is a linear relationship constructed based on different target concentrations and corresponding RGB intensities for the same bacterial subtype;
FIG. 6 is a schematic diagram of a quantitative determination process of the quantitative determination system according to the present invention;
FIG. 7 shows the corresponding time and sensitivity results of detection of cryptococcus target DNA by the CRISPR system in an embodiment of the invention;
FIG. 8 is a reaction time of a CRISPR-Cas12a system detecting a cryptococcus subtype DNA sample in an embodiment of the present invention;
FIG. 9 is a representation of the purity of targets used in the examples of the present invention (each target is not contaminated), each target is not contaminated;
FIG. 10 is a graph showing the measurement using a target of known concentration in an embodiment of the present invention, which establishes a linear relationship between the target concentration and the average RGB intensity of the microreactor.
The marks in the figure: the micro-fluidic chip comprises a central liquid inlet 1, a micro-fluidic channel 2, a liquid outlet 3, a micro-reaction chamber 4, a negative control area 5, a subtype I detection area 6, a subtype II detection area 7, a positive control area 8, an ultraviolet lamp 9 and a smart phone 10.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In this document, suffixes such as "module", "component", or "unit" used to represent elements are used only for facilitating the description of the present invention, and have no particular meaning in themselves. Thus, "module," "component," or "unit" may be used in combination. The terms "upper," "lower," "inner," "outer," "front," "rear," "one end," "the other end," and the like herein refer to an orientation or positional relationship based on that shown in the drawings, merely for convenience of description and to simplify the description, and do not denote or imply that the devices or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be construed as limiting the invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. The terms "mounted," "provided," "connected," and the like, herein, are to be construed broadly as, for example, "connected," either permanently connected or removably connected, unless otherwise expressly specified and defined Connected, or integrally connected; the two components can be mechanically connected, can be directly connected or can be indirectly connected through an intermediate medium, and can be communicated with each other. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art. Herein, "and/or" includes any and all combinations of one or more of the associated listed items. Herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc. As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value. In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a range The description of (c) should be taken as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
Example 1: as shown in FIG. 1, the invention provides a portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a target by fluorescence, which comprises a micro-channel chip, a nucleic acid fluorescence quantitative reaction system and a quantitative detection system. The micro-channel chip comprises a substrate and a central liquid inlet hole 1 arranged on the substrate; the device also comprises a positive control area 8, a negative control area 5 and a plurality of detection areas which are arranged around the central liquid inlet hole 1; each zone is provided with a micro-channel 2 which is communicated with the central liquid inlet 1, and a plurality of micro-reaction chambers 4 which are communicated with the micro-channel 2 are arranged in each zone; the tail end of the micro-channel 2 is provided with a liquid outlet 3. The nucleic acid fluorescence quantitative reaction system is a CRISPR system, in particular one of CRISPR-Cas9, CRISPR-Cas12 or CRISPR-Cas13 systems; the CRISPR system contains a fluorescent reporter molecule connected with a fluorescent group and a fluorescence quenching group. In the invention, the CRISPR system is a freeze-dried reagent system preset in a micro-reaction chamber, and specifically comprises Cas-12a protein, a primer crRNA, a fluorescent reporter molecule and a reaction buffer solution. The quantitative detection system comprises an image acquisition module for acquiring a fluorescence image of the micro-reaction chamber, an image optimization module for optimizing the fluorescence image of the micro-reaction chamber, a micro-reaction chamber identification module for identifying the micro-reaction chamber in the fluorescence image of the micro-reaction chamber and acquiring RGB intensity of the micro-reaction chamber in the fluorescence image of the micro-reaction chamber, and a quantitative module for establishing a linear relation between the average value of RGB intensity of all the micro-reaction chambers in each region and target concentration and quantifying target nucleic acid (for example, bacterial infection amount) based on the linear relation.
According to the invention, a sample is detected through the micro-channel chip and a CRISPR system preset in the micro-channel chip, and then a detection result is rapidly and quantitatively obtained by a quantitative detection system in an image analysis processing mode. When target DNA is present in the sample solution, cas12a-crRNA conjugate is activated by reporter cleavage at 37 ℃, resulting in separation between the quencher group (BHQ-1) and the reporter carboxyfluorescein (Fluorescein Amidite, FAM) and emission of amplified fluorescent signal fluorescein. In contrast, no fluorescent signal is generated in the negative region or in the microreactor without target DNA. The specific subtype of cryptococcus can be identified by a hand-held ultraviolet lamp (480 nm), and corresponding fluorescent images are captured by intelligent mobile terminals such as smart phones. Namely, the invention integrates portability of the micro-channel array biochip, high specificity of CRISPSR-Cas 12a technology and accuracy of intelligent imaging program. The detection result is read out only by a personal smart phone, so that the burden of expensive and non-portable instruments is greatly reduced, the experimental requirements are simplified, the detection process is accelerated, and the method is used for rapid screening and accurate quantification in clinical practice.
The detection zone comprises a subtype I detection zone 6 and a subtype II detection zone 7; the subtype I detection zone 6, the subtype II detection zone 7, the positive control zone 8 and the negative control zone 5 are arranged in a central symmetry manner along the central liquid inlet 1, so that the consistency of solution flow in each area is ensured, and the accuracy of the whole detection result is improved. In this example, two assays were used to pair the two most common cryptococcus subtypes, namely Cryptococcus Neoformans (NEO) and Cryptococcus Gatus (GAT), respectively. Of course, more subtypes can be paired, and the number of detection areas can be specifically adjusted according to needs, which is not limited in the present invention. In order to eliminate the signal difference between the devices, a positive control area and a negative control area are specifically provided in the present invention for normalizing the fluorescence intensity from the detection area. For example, the positive control zone microreactor chamber contains 5nM of target DNA and a CRISPR-Cas12a system, and the negative zone microreactor chamber contains Cas12a, reporters and crRNA with disordered DNA sequences.
As shown in fig. 2A to 2C, more specifically, the micro-reaction chamber 4 is circular, and has a diameter D3 of 780 micrometers to 820 micrometers and a depth of 78 micrometers to 82 micrometers; the width W1 of the micro-channel 2 is 95-105 micrometers, and the depth H1 is 38-42 micrometers; the micro flow channel 2 is arranged in a serpentine bending mode, each area comprises 26-34 micro reaction chambers, and the micro reaction chambers are symmetrically arranged on two sides of the micro flow channel 2. The depth of the micro-reaction chamber 4 is about twice that of the micro-flow channel, so that the sample solution is fully filled in the micro-reaction chamber. The four areas are connected with a central liquid inlet hole 1 through a micro flow channel 2, and the diameter D1 of the central liquid inlet hole 1 is 1900-2100 micrometers; the micro flow channels 2 of each area are respectively ended at the liquid outlet holes 3, and the diameter D2 of the liquid outlet holes 3 is 1400-1600 microns. Each zone has a length L3 of 6.5 microns to 7 mm and a width L4 of 5.4 microns to 6.0 microns. In addition, the micro flow channel chip can be a plurality of blocks integrated together, thereby providing high use efficiency. Wherein the length L2 of the single chip is 16 micrometers to 20 millimeters, and the length L1 of the system formed by integration is 56 millimeters to 60 millimeters.
The image acquisition module for acquiring the fluorescence image of the micro-reaction chamber comprises an ultraviolet lamp 9 and a smart phone 10 integrated with a camera. The ultraviolet lamp 9 and the camera are used for acquiring fluorescent images of the micro-channel chip, the micro-reaction chamber identification module and the quantitative module can be built in the smart phone 10 as a mobile phone APP, and the functions of micro-reaction chamber identification and infection quantification can be realized through the operation of a processor of the smart phone 10.
More specifically, the micro-reaction chamber identification module comprises: the gray threshold setting module is used for setting threshold ranges 0-N and threshold step length N so as to obtain a plurality of gray thresholds; the binarization image acquisition module is used for comparing the gray values of the pixel points with a plurality of gray threshold values respectively, setting the pixel points with the gray values larger than the threshold values as white, and setting the pixel points with the gray values smaller than or equal to the threshold values as black, so that a plurality of binarization images are obtained; the spot combining module is used for respectively extracting the white pixels communicated in each binarized image as spots, and combining the spots overlapped in the geometric centers in all the binarized images into a spot group; the spot group selecting module is used for selecting spot groups according to the number of pixels, the convexity, the inertia ratio and the roundness so as to select the spot groups representing the micro-reaction chamber; the micro-reaction chamber position marking module is used for recording the positions of the spot groups and marking the positions of all the micro-reaction chambers in the detection area on the fluorescence image after the optimization treatment according to the positions of the spot groups; and the RGB intensity acquisition module is used for weighting and averaging the RGB values of the pixel points in the micro-reaction chamber in the optimized fluorescent image to acquire the RGB intensity of each representative micro-reaction chamber.
As shown in fig. 3, the present invention further provides a method for precisely and rapidly quantifying the target concentration by fluorescence, which is applied to the portable intelligent detection micro-platform for precisely and rapidly quantifying the target concentration by fluorescence, and the present embodiment still uses quantitative cryptococcus subtypes, i.e., cryptococcus Neoformans (NEO) and cryptococcus Gartersii (GAT), as an example, but the present invention is not limited thereto. The method specifically comprises the following steps:
s11, carrying out vacuum treatment on the micro-channel chip, and respectively injecting 4 mixtures of Cas12a, crRNA, reporter and 1 x reaction buffer solution from liquid outlet holes of 4 areas. The purpose of the vacuum treatment is to enable the micro flow channel on the micro flow channel chip and the micro reaction chamber to be in a vacuum state, and the solution filling and no bubble generation are ensured when the CRISPR-Cas12a system is filled. In this embodiment, the time for the vacuum treatment is 25min to 35min. Four areas on the micro-channel chip, namely a subtype I detection area, a subtype II detection area, a positive control area and a negative control area, need to be provided with corresponding CRISPR-Cas12a systems, namely Cas12a, crRNA, reporter and 1 x reaction buffer solution respectively.
S12, freezing the micro-channel chip until the mixture becomes dry powder, and carrying out vacuum treatment on the micro-channel chip again. The purpose of carrying out vacuum treatment again on the micro-channel chip is to make the interior of the micro-channel chip in a vacuum state, the treatment time is 25-35 min, and the micro-channel chip is sealed and stored for use after the treatment.
S13, extracting a DNA sample from a bacterial sample and identifying the DNA sample by qPCR.
S14, injecting a mixture of a single sample and at least two samples into the central liquid inlet hole, and then incubating at 37 ℃ for 25-35 min.
S15, irradiating the micro flow channel chip by using an ultraviolet lamp, and collecting fluorescent images of the micro reaction chambers in 4 areas (or detection areas). Specifically, the images of each region may be employed separately, or the images including all the regions may be acquired at once.
As shown in fig. 6, the following steps are specific steps for quantitative detection by the quantitative detection system.
And S16, optimizing the acquired micro-reaction chamber fluorescence image. In this embodiment, the specific method for optimizing the image includes: s161 uses bilinear interpolation algorithm to expand the pixels of the acquired micro-chamber fluorescence image to a preset value, e.g. a width of 1000 pixels, with the purpose of excluding the interference of low and non-uniform pixels of different images. S162 reduces the brightness difference between each pixel point and surrounding pixel points using gaussian filtering, minimizing the effect of high frequency noise.
S17, identifying the micro-reaction chambers in the micro-reaction chamber fluorescence image and acquiring RGB intensity of each micro-reaction chamber in the micro-reaction chamber fluorescence image. The micro-reaction chamber is cylindrical, and is shown as a 'spot' in a top view photo, and the position of the micro-reaction chamber is identified by finding the boundary of the spot, which specifically comprises: s171 setting threshold ranges 0 to N and threshold step sizes n, thereby obtaining several gray thresholds. In order to ensure the accuracy of the final result, in this embodiment, the gray level threshold is set to 0 to 255, and the threshold step is set to 1, so that 256 gray level thresholds in total from 0 to 255 can be obtained. S172 compares the pixel gray values with several gray thresholds, respectively, and sets the pixel having a gray value greater than the threshold to 1 (white) and the pixel having a gray value less than or equal to the threshold to 0 (black), thereby obtaining several binarized images. The purpose of this step is to independently binarize the image. The image independent binarization (Independent Binary Segmentation) is a process of dividing one gray-scale image into two parts of black and white. A certain threshold is typically used as a black-and-white demarcation point, and pixels are classified as black or white according to the magnitude of the pixel gray value versus the threshold. The purpose of independent binarization is to distinguish between subjects and backgrounds in images and to facilitate subsequent image processing and analysis. That is, all pixels in the image are non-black, i.e., white, after the independent binarization process. Since 256 gradation thresholds are set in the present embodiment, 256 binarized images will be obtained eventually. Compared with the traditional method that one image to be detected is directly converted into a gray value image of 0 or 255, the method is characterized in that a plurality of gray thresholds are set to obtain more data, so that logics which are not likely to be misplaced are optimized, and a large amount of data are prevented from being missed; of course, the large amount of data obtained may lead to the presence of duplicate or redundant spots in the subsequently identified spots, and thus, the appropriate spots are screened therefrom by the corresponding screening conditions (see later section for specific screening patterns). S173, respectively extracting connected white pixels in each binary image as spots, and merging the spots with overlapped geometric centers in all binary images into a spot group. The white pixels connected in 256 binarized images are respectively extracted through the findContours function to form spots, and the positions and the number of the black and white pixels in each binarized image are different, so that the shapes of the extracted spots in each binarized image are different. After the spots are extracted, the spots are fused to form a set of spots representing the microreactor. The principle of the split is to make the geometry in The overlapping spots are pieced together into a spot group. Since the shapes of the spots representing the same micro-chamber in each binarized image are different, it is possible that the same micro-chamber may form several spot groups. To select a most representative group of blobs, it is also necessary to screen the group of blobs. S174, screening the spot groups by the number of pixels, convexity, inertia ratio and roundness to select the spot groups representing the micro-reaction chambers. In the present invention, the 4 aspects are used to screen the spot groups to select a most suitable spot group. Specifically, the first, the number of pixels is screened, for example 9000 to 13000, and a group of spots of a suitable size can be screened by normalizing the number of pixels. Secondly, screening the concave-convex propertyGroups of spots can be screened that are smoother, more continuous, and have no concave features and sharp corners. Thirdly, screening the inertia ratio, namely +.>The stretched groups of blobs may be rejected. Fourth, screening the roundness to obtain +.>Groups of spots that are approximately circular can be screened out. S175, recording the positions of the spot groups, and marking the positions of the micro-reaction chambers on the original image after the optimization treatment according to the positions of the spot groups. After the most suitable spot group is selected to represent a certain micro-reaction chamber, the position of the spot group is recorded, and then the position of the micro-reaction chamber is marked on the corresponding position in the original image after the optimization treatment. Through the processing of the steps, the position of the micro-reaction chamber in the image can be accurately found, so that the subsequent detection accuracy is improved. Because the micro-channel chip is small in size, the shooting precision of the existing smart phone camera is limited, and the micro-reaction chamber is difficult to accurately divide from the image by adopting the existing modes such as example division. Therefore, in order to improve the robustness of the present invention, the micro-reaction chamber is subjected to the steps The segmentation processing is carried out, and the shooting requirement on the image is greatly reduced. And S176, carrying out RGB value weighting and average on pixel points in the micro-reaction chamber in the optimized micro-reaction chamber fluorescent image to obtain the RGB intensity of each representative micro-reaction chamber. After the group of spots representing the micro-chamber is selected, the RGB values of the pixels within the group of spots are weighted and averaged to obtain RGB intensities for subsequent use.
S18, calculating the average value of RGB intensity of all the micro-reaction chambers in each detection area, and establishing a linear equation between the average value of RGB intensity and the corresponding target concentration so as to quantify cryptococcus infection (namely target nucleic acid). The RGB intensity of the micro-reaction chamber and the target concentration have a certain linear relation, a plurality of pre-experiments can be used for constructing a linear equation of the RGB intensity and the target concentration in advance, and then the RGB intensity of the micro-reaction chamber is taken as an independent variable, and the target concentration is taken as a dependent variable. After the RGB intensity of a certain micro-reaction chamber is obtained, the RGB average value of all the micro-reaction chambers in the same detection area is calculated, and the value is brought into a linear equation to obtain the corresponding target concentration.
Fig. 5A to 5D illustrate the above-described embodiment of the present invention. Wherein, fig. 5A shows a physical micro flow channel chip. In fig. 5B, a fluorescence photograph of the chip after filling the sample NEO with a known target concentration, a fluorescence photograph of the chip after filling the sample GAT with a known target concentration, and a fluorescence photograph of the chip after filling the mixed sample with a known target concentration are shown, wherein the upper left side of the chip is a negative control area, the upper right side is a positive control area, the lower left side is a subtype I detection area, and the lower right side is a subtype II detection area; the lower row in FIG. 5B shows the corresponding RGB intensities of the fluorograms (the thin solid line in each figure shows the RGB mean of the corresponding region, where the RGB mean of each sample NEO is 148.27 when detected alone, 153.97 when detected alone, and 136.60 when detected alone. Fig. 5C schematically shows the RGB intensities of the respective micro-reaction chambers for known different target concentrations of the sample NEO. Fig. 5D shows a linear relationship between RGB intensities (i.e., RGB averages of a plurality of micro-reaction chambers in the corresponding detection regions) and target concentrations, which are constructed in advance according to different target concentrations of the sample NEO and RGB averages obtained by image processing.
In addition, in order to further improve the convenience of use, in practice, the linear relationship between the RGB intensity and the target concentration is established in advance, as shown in fig. 10, by detecting several groups of samples with known target concentration, the relationship between the fluorescence value and the RGB value is obtained in advance, and the fluorescence value actually reflects the target concentration, so that when detecting the sample to be detected, the linear equation between the RGB value and the target concentration can be constructed by and implementation, and when detecting the sample to be detected, the target concentration of the sample to be detected can be calculated by directly taking the linear equation between the RGB value and the target concentration into the linear equation between the RGB value and the target concentration.
Chinese patent application CN202211064842.4 discloses a portable uranyl ion fluorescence detection method based on RGB analysis, comprising: mixing a uranyl ion in-situ monitoring probe of corn polypeptide sensitized curcumin fluorescence with solutions containing different concentrations of uranyl ions to obtain a mixed solution; irradiating the mixed solution with a purple flashlight under a dark condition, and shooting an image with a camera; reading R, G, B values of the images by using a color picker, and drawing three different standard curves; irradiating the uranyl ion solution to be detected with a purple flashlight under a light-shielding condition, and shooting an image of the uranyl ion solution to be detected by using a camera; and (3) reading R, G, B values of the images by using a color extractor, respectively taking the R, G, B values into the standard curves corresponding to the fourth step, and calculating to obtain the concentration of the uranyl ion solution to be detected. In the above prior art, although the physicochemical values are also measured by RGB values of the solution fluorescence image, it is required "the camera parameters are adjusted to 4000 ISO, the shutter speed is 1/125 seconds, the aperture f/7.1, and the focal length is 22 mm" according to its original description. The requirement on the shot image in the prior art is higher, and the camera with a special function is required, and the better identification effect can be achieved only by specific parameters. In the invention, through the series of processing steps, the requirement on image quality is greatly reduced in detection, and the universality of the invention is improved.
Example 2: the embodiment provides a method for preparing a micro-channel chip, which specifically comprises the following steps: step 1: and (3) preparing a silicon-based mold. Step 1.1: and drawing a photoetching mask plate model by using AutoCAD, and manufacturing the mask plate. The specific dimensions are as follows: the diameter of the central liquid inlet hole is 2000 mu m; the width of the micro-channel is 100 mu m; the length of the side branch flow passage is 200 mu m, and the width is 200 mu m; the diameter of the reaction micropore is 800 mu m; the diameter of the liquid outlet hole is 1500 μm. As in fig. 8. In order to prevent the liquid from flowing out after the liquid adding, a deep reaction chamber structure is designed: the chip consists of two layers of PDMS, and mask plates are respectively designed. Becomes a mask plate I and a mask plate II. The first layer has a flow channel structure and a reaction chamber structure, and the second layer has only a reaction chamber structure. The two layers of PDMS are bonded together to form the morphological characteristics of shallow flow channel and deep reaction chamber for storing liquid. Two-layer structure, each layer structure thickness 40 (+ -3) μm. Step 1.2: washing the silicon wafer, firstly wiping the silicon wafer with acetone, and washing with acetone, alcohol and ultrapure water in sequence to remove organic impurities and particulate impurities. And after the cleaning is finished, drying by using nitrogen, and then putting the dried product into an oven for baking to remove water vapor adsorption. Step 1.3: and (5) oxygen is injected. The silicon wafer is further cleaned in a plasma cleaner to ashe the organic impurities. And oxygen is injected to temporarily form free-OH groups on the surface of the silicon wafer, so that the adhesiveness between the silicon wafer and the photoresist is enhanced. Step 1.4: and (5) whirling glue 1. The photoresist is coated by spin coating (also called as spin coating), and a photoresist coating condition can be obtained by referring to a spin coating curve. The silicon wafer is placed on a spin coater, the silicon wafer is fixed by vacuumizing, and photoresist SU-8 2025 is spin-coated at the rotating speed of 1700rpm, so that the photoresist with the thickness of 40 μm is obtained. Step 1.5: and (5) pre-baking 1. In the pre-baking (pre-rake or soft-rake) process, placing the silicon wafer on a hot plate at the right side of the spin coater, heating to 65 ℃ for 3min, heating to 80 ℃ for 3min, heating to 95 ℃ for 6min, cooling to room temperature, and taking out. Step 1.6: and (5) photoetching. The exposure is completed through an exposure mask and an exposure system. And (3) placing the silicon wafer on an ultraviolet photoetching machine, sucking the silicon wafer under negative pressure, placing a mask plate above the silicon wafer, enabling the silicon wafer and the mask plate to be attached as much as possible but not extruded, sucking the mask plate under negative pressure, and exposing the silicon wafer for 14s. Step 1.7: and (5) post-baking 1. Placing the silicon wafer on a hot plate, baking and fixing, heating to 65deg.C for 2min, heating to 80deg.C for 1min, heating to 95deg.C for 5.5min, cooling to room temperature, and taking out. Step 1.8: and (5) whirling glue 2. And (3) repeating the step of spin coating 1, placing the silicon wafer on a spin coater, vacuumizing to fix the silicon wafer, and spin coating photoresist SU-8 2025 at the rotating speed of 1700rpm to obtain a second layer of photoresist with the thickness of 40 mu m. Step 1.9: and (5) pre-baking 2. The step of pre-baking 1 is repeated. Placing the silicon wafer on a hot plate on the right side of a spin coater, heating to 65 ℃ for 3min, heating to 80 ℃ for 3min, heating to 95 ℃ for 6min, cooling to room temperature, and taking out. Step 1.10: and (5) overlay. An exposure is performed on the second layer of photoresist. And (3) placing the silicon wafer on an ultraviolet photoetching machine, sucking under negative pressure, placing a mask plate above the silicon wafer, sucking the mask plate under negative pressure, and lifting the silicon wafer to enable the silicon wafer and the mask plate to be relatively close to each other. And then adjusting the deflection angle micro-rule and the transverse and longitudinal spiral micro-rule to coincide the positioning mark of the mask plate of the second layer with the positioning mark of the first layer, and then lifting the silicon wafer to make the two layers be attached as much as possible but not extruded. Exposure 14s, taking care not to look straight. Step 1.11: and (5) post-baking 2. After the second exposure is completed, the operation of post-baking 1 is repeated. Placing the silicon wafer on a hot plate, baking and fixing, heating to 65deg.C for 2min, heating to 80deg.C for 1min, heating to 95deg.C for 5.5min, cooling to room temperature, and taking out. Step 1.12: and (5) developing. The silicon wafer is immersed in a developing solution which takes propylene glycol monomethyl ether acetate as a developing solution, and the reaction is slowly and fully shaken and developed for 3min. After that, the mixture was rinsed with ultra pure water for 30 seconds and dried with nitrogen, and then baked and fixed in an oven at 120 ℃. Step 1.13: and (5) surface modification. The silicon wafer film was silanized with 3. Mu.L of trichloro (1H, 2H) -perfluorooctylsilane in a vacuum desiccator at a pressure of 100mbar overnight to form a hydrophobic surface. Step 2: and (5) performing reverse molding. Step 2.1: a matrix of Polydimethylsiloxane (PDMS) and a curing agent (abbreviated as hybrid glue) were uniformly mixed in a mass ratio of 10:1. Placing into a vacuum dish, and vacuumizing to remove bubbles. Step 2.2: and 2.1, pouring the mixed glue into a flat mold, and vacuumizing again to ensure no bubbles. Step 2.3: and (3) putting the PDMS in the step (2.2) into a high-temperature oven at 80 ℃ for heating, crosslinking and curing. Step 2.4: the PDMS layer cured in step 2.3 was peeled off the mold. Step 2.5: liquid inlet and outlet holes were made by punching holes in the cured PDMS layer. Step 3: and assembling the micro-channel chip. Step 3.1: PDMS with microchannels and slides were treated separately with plasma. Step 3.2: and (3) taking out the two in the step 3.1 to align and bond. And firm Si0 bond combination is formed between the glass sheet and the silicon alkyl of the PDMS, so that irreversible bonding between the glass sheet and the silicon alkyl of the PDMS is completed, and the micro-channel chip is formed.
Example 3; the embodiment provides a rapid, accurate, stable and sensitive investigation for the typing detection of each subtype of cryptococcus.
S01: the DNA sample was incubated with the CRISPR-Cas12a system at 37 ℃. The fluorescence intensities of each group were measured every 1min (fig. 4 a). In the presence of the target sequence, these three CRISPR-Cas12a systems significantly enhanced the fluorescence signal, reaching saturation within 20 minutes (fig. 4b-c, fig. 7, fig. 8 a-c).
S02: the high specificity of the CRISPR-Cas12a system was demonstrated by the addition of disordered DNA, which resulted in a lower fluorescent signal line, comparable to the No Template Control (NTC) group (fig. 4 d).
S03: sensitivity of the CRISPR-Cas12a system to DNA sample detection is further explored. The calibration curve results show that the 3 detection systems are linear in the range of 0.1nM to 4nM with a detection Limit (LOD) of 0.1nM (FIGS. 4 e-f). Considering that the concentration of the sample in clinic is often higher than 0.1nM, the LOD results detected are satisfactory.
Accuracy evaluation: CRISPR typing detection is carried out on alternating combinations of specific DNA sequences of each subtype of cryptococcus and DNA sequences shared by cryptococcus, three groups of parallel experiments are set, and the accuracy of the method is evaluated by comparing the real-time fluorescence PCR detection result with the chip detection result. As shown in FIG. 4d, only when the CRISPR system type corresponds to the table type, the detection signal exists, which shows that the invention has good specificity, the accuracy is 100%, and the experiment time is 30min.
Stability evaluation: CRISPR typing detection is carried out on each subtype specific DNA sequence of cryptococcus and the DNA sequence shared by cryptococcus, 3 detection is carried out on each sample, the consistency among 3 results is compared, and the stability of the kit is evaluated. The experimental results are shown in FIGS. 4b-f and 7-8. The error limit in the result shows that the system has stronger stability, and the experimental time is 37 minutes.
Sensitivity evaluation: and (3) carrying out CRISPR typing detection on the DNA sequences specific to each subtype of cryptococcus and the DNA sequences shared by cryptococcus by gradient dilution to 0.1 nM. Recording the detection result and evaluating the sensitivity of the kit. The results of the experiment are shown in FIGS. 4e-f and Table 2, and the detection sensitivity is high, the detection limit is 0.1nM, and the detection time is 42min.
Rapid evaluation: the time from sample addition to detection completion of the above experiment was recorded, and the results showed that detection could be completed within 50 minutes, which was a rapid detection.
Table 2 sensitivity evaluation detection structure
| Detecting concentration | Repeat 1 | Repeat 2 | Repeat 3 |
| 0.05nM | - | - | - |
| 0.1nM | + | + | + |
| 0.25nM | + | + | + |
| 0.5nM | + | + | + |
| 1nM | + | + | + |
| 2nM | + | + | + |
| 5nM | + | + | + |
"+" represents a detectable type; "-" represents no significant difference.
In this example, samples of DNA sequences specific for each subtype of Cryptococcus and DNA sequences common to Cryptococcus were used, and were diluted in concentration gradient. And crRNA and PCR primers designed for each DNA sequence. The method is used for testing the rapidity, the accuracy, the stability and the sensitivity of the detection system.
The nucleotide sequences of the crRNA probe, target dnas, reporters and PCR referred to in this example are shown in table 3.
TABLE 3 nucleotide sequences of crRNA probes, target dnas, reporters and PCRs
The target recognition region in the crRNA sequence is bolded. CRY, cryptococcus.NEO Cryptococcus neoformans. GAT, cryptococcus gattii. Neg, negative control. F, forward. R, reverse.
Through pre-experimental exploration, the optimal reaction system of CRISPR-Cas12a respectively designed by determining the specific DNA sequences of each subtype of cryptococcus and the DNA sequences shared by cryptococcus is shown in table 4.
TABLE 4CRISPR-Cas12a optimal reaction System
It will be appreciated that the sequences of the crRNA probes, target dnas, reporters and nucleotides of PCR in the present invention and the reaction conditions in the present invention are not limited to the above list.
FIG. 9 is a representation of the purity of targets used in the examples of the present invention (neither target was contaminated). The Ct value represents the number of cycles that the fluorescent signal undergoes when reaching a set threshold (the concentration of the "amplified subject" reaches the threshold). When the ct value is greater than 40, 40 is taken and the "amplified object" is considered to be absent in the solution.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
From the above description of the embodiments, it will be clear to those skilled in the art that the above-described embodiment method may be implemented by means of software plus a necessary general hardware platform, but of course may also be implemented by means of hardware, but in many cases the former is a preferred embodiment. Based on such understanding, the technical solution of the present invention may be embodied essentially or in a part contributing to the prior art in the form of a software product stored in a storage medium (e.g. ROM/RAM, magnetic disk, optical disk) comprising several instructions for causing a computer terminal (which may be a mobile phone, a computer, a server, or a network device, etc.) to perform the method according to the embodiments of the present invention. The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (10)
1. A portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a target by fluorescence is characterized in that: comprises a micro-flow channel chip, a nucleic acid fluorescence quantitative reaction system and a quantitative detection system;
the micro-channel chip comprises a substrate and a central liquid inlet hole arranged on the substrate; the device also comprises a positive control area, a negative control area and a plurality of detection areas, wherein the positive control area, the negative control area and the detection areas are arranged around the central liquid inlet hole; each zone is provided with a micro-channel communicated with the central liquid inlet, and a plurality of micro-reaction chambers communicated with the micro-channel are arranged in each zone; the tail end of the micro-channel is provided with a liquid outlet hole;
the quantitative detection system comprises an image acquisition module, an image optimization module, a micro reaction chamber identification module and a quantitative determination module, wherein the image acquisition module is used for acquiring a micro reaction chamber fluorescence image after the fluorescent reaction of the target nucleic acid is completed based on the nucleic acid fluorescent quantitative reaction system, the image optimization module is used for optimizing the micro reaction chamber fluorescence image, the micro reaction chamber identification module is used for identifying each micro reaction chamber in the micro reaction chamber fluorescence image and acquiring RGB intensity of the micro reaction chamber in the micro reaction fluorescence image, and the quantitative determination module is used for calculating RGB intensity average values according to RGB intensity of all the micro reaction chambers in a current detection area, and establishing a linear relation between the RGB intensity average values and target concentration so as to quantitatively detect the target nucleic acid based on the linear relation;
The micro-reaction chamber identification module comprises:
the gray threshold setting module is used for setting threshold ranges 0-N and threshold step length N so as to obtain a plurality of gray thresholds;
the binarization image acquisition module is used for comparing the gray values of the pixel points with a plurality of gray threshold values respectively, setting the pixel points with the gray values larger than the gray threshold values as white and setting the pixel points smaller than or equal to the gray threshold values as black, so as to obtain a plurality of binarization images;
the spot combining module is used for respectively extracting the white pixels communicated in each binarized image as spots, and combining the spots overlapped in the geometric centers in all the binarized images into a spot group;
the spot group selecting module is used for selecting spot groups according to the number of pixels, the convexity, the inertia ratio and the roundness so as to select the spot groups representing the micro-reaction chamber;
the micro-reaction chamber position marking module is used for recording the positions of the spot groups and marking the positions of the corresponding micro-reaction chambers on the optimized micro-reaction chamber fluorescent images according to the positions of the spot groups; and the RGB intensity acquisition module is used for weighting RGB values of pixel points in the micro-reaction chamber fluorescent image and averaging to acquire the RGB intensity corresponding to each micro-reaction chamber.
2. The portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a fluorescent target according to claim 1, wherein the portable intelligent detection micro-platform is characterized in that: the detection zone comprises a subtype I detection zone and a subtype II detection zone; the subtype I detection area, the subtype II detection area, the positive control area and the negative control area are symmetrically arranged along the center of the central liquid inlet hole.
3. The portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a fluorescent target according to claim 1, wherein the portable intelligent detection micro-platform is characterized in that: the nucleic acid fluorescent quantitative reaction system is a CRISPR system, comprising CRISPR-Cas9, CRISPR-Cas12 or CRISPR-Cas13.
4. The portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a fluorescent target according to claim 3, wherein the CRISPR system comprises Cas-12a protein, primer crRNA, fluorescent reporter and reaction buffer; the fluorescent reporter molecule is connected with a quenching group and carboxyfluorescein.
5. The portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a fluorescent target according to claim 4, wherein the portable intelligent detection micro-platform is characterized in that: each reagent in the nucleic acid fluorescence quantitative reaction system is pre-arranged in the micro-channel chip in a freeze-dried mode.
6. The portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of a fluorescent target according to claim 1, wherein the portable intelligent detection micro-platform is characterized in that: the image acquisition module comprises an ultraviolet lamp and an intelligent mobile terminal integrated with a camera.
7. A method for accurately and rapidly quantifying the concentration of a fluorescent quantitative target, which is applied to a portable intelligent detection micro-platform for accurately and rapidly quantifying the concentration of the fluorescent quantitative target according to any one of claims 1 to 6, wherein the target is a bacterial target gene, and is characterized by comprising the following steps:
s01: the method comprises the steps of reacting a preset freeze-dried nucleic acid fluorescence quantitative reaction system with nucleic acid of a known sample in a micro-channel chip; the target concentration of the known sample nucleic acid is known;
s02: irradiating the micro-channel chip after nucleic acid fluorescence quantitative reaction by using an ultraviolet lamp, and collecting a fluorescence image of a micro-reaction chamber by using an intelligent mobile terminal;
s03: optimizing the acquired fluorescence image of the micro-reaction chamber;
s04: identifying the micro-reaction chambers in the optimized micro-reaction chamber fluorescent image, acquiring the RGB intensities corresponding to each micro-reaction chamber in the micro-reaction chamber fluorescent image, and calculating the RGB average value of all the micro-reaction chambers in the current detection area;
S05: establishing a linear equation according to the RGB intensity mean value and the known target concentration of the known sample nucleic acid;
s06: adding a sample to be detected into the micro-channel chip to react with a preset freeze-dried nucleic acid fluorescent quantitative reaction system;
s07: irradiating the micro-channel chip subjected to nucleic acid fluorescence quantitative reaction by using an ultraviolet lamp, and collecting a fluorescence image of a micro-reaction chamber by using an intelligent mobile terminal;
s08: optimizing the acquired fluorescence image of the micro-reaction chamber;
s09: identifying micro-reaction chambers in a micro-reaction chamber fluorescence image, acquiring RGB intensity corresponding to each micro-reaction chamber in the micro-reaction chamber fluorescence image, and calculating RGB average values of all the micro-reaction chambers in a current detection area;
s10: and carrying the RGB intensity mean value of the sample to be detected into the linear equation so as to calculate the nucleic acid content of the sample to be detected.
8. The method for accurate and rapid fluorescence quantification of a target concentration of claim 7, wherein the step of optimizing the acquired micro-chamber fluorescence image comprises:
expanding the pixels of the acquired micro-reaction chamber fluorescent image to a preset value by adopting a bilinear interpolation algorithm;
The difference in brightness between each pixel and surrounding pixels is reduced using gaussian filtering.
9. The method for precisely and rapidly quantifying the target concentration by fluorescence according to claim 7, wherein the steps of identifying the micro-reaction chambers in the micro-reaction chamber fluorescence image and obtaining the corresponding RGB intensities of each micro-reaction chamber in the micro-reaction chamber fluorescence image comprise:
setting a threshold range of 0-N and a threshold step length of N, thereby obtaining a plurality of gray thresholds;
respectively comparing the gray values of the pixel points with a plurality of gray threshold values, and setting the pixel points with the gray values larger than the threshold value as white and the pixel points smaller than or equal to the threshold value as black so as to obtain a plurality of binarized images;
respectively extracting white pixels communicated in each binarized image as spots, and merging the spots overlapped in the geometric centers in all the binarized images into a spot group;
selecting a spot group representing the micro-reaction chamber by screening the spot group according to the number of pixels, the convexity, the inertia ratio and the roundness;
recording the positions of the spot groups, and marking the positions of the corresponding micro-reaction chambers on the optimized micro-reaction chamber fluorescent images according to the positions of the spot groups;
And carrying out RGB value weighting and average on pixel points in the micro-reaction chamber in the optimized micro-reaction chamber fluorescent image to obtain the RGB intensity of each representative micro-reaction chamber.
10. The method for accurate and rapid fluorescence quantification of a target concentration of claim 7, wherein: the specific steps of reacting the sample nucleic acid with the preset freeze-dried nucleic acid fluorescence quantitative reaction system are as follows:
s01: taking a sample to be detected, and lysing cells to expose DNA, wherein the sample is cryptococcus;
s02: adding a sample solution containing exposed DNA from a central liquid inlet hole of the micro-channel chip, and simultaneously applying negative pressure and vacuumizing outside the micro-channel chip to enable the sample solution containing exposed DNA to fully enter the micro-channel chip and react with a preset freeze-dried nucleic acid fluorescence quantitative reaction system;
s03: after Cas12a protein in the nucleic acid fluorescence quantitative reaction system is combined with target sample specific crRNA, releasing nonspecific endonuclease activity, cutting off a quenching group and carboxyfluorescein on a fluorescence reporter molecule, and releasing fluorescence; the sequence of the target sample specific crRNA is shown as SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO. 3.
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