Chemical reaction chip, preparation method and application thereof, and method for detecting amino acid and identifying amino acid sample based on image vision

Abstract

The invention provides a chemical reaction chip, a preparation method and application thereof, and a method for detecting amino acid and identifying an amino acid sample based on image vision, and belongs to the technical field of analysis and detection. The chemical reaction chip provided by the invention comprises a substrate and reaction units distributed on the substrate in an array, the reaction units are loaded with chemical reaction reagents and a color developing agent, and the chemical reaction reagents comprise at least two of CuCl2, Na2S2O3 and tartaric acid. Specific chemical reaction reagents are adopted in the chemical reaction chip provided by the invention and have a good interaction effect with the amino acid, and on the basis, the chemical reaction chip is combined with an image visual analysis technology to develop a novel high-throughput amino acid analysis method; all information of the amino acid sample can be rapidly obtained at a time, and a new strategy is provided for low-cost, rapid, accurate and nondestructive analysis of the amino acid.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical detection, and particularly to a chemical reaction chip, a preparation method and an application thereof, and a method for detecting amino acids and identifying amino acid samples based on image vision. Background Art

[0002] Amino acids (AAs) play an important role in the nutritional characteristics of fruits and vegetables, are important indicators for measuring the nutritional value of fruits and vegetables, and have a variety of health functions. Although traditional amino acid analysis techniques, such as capillary electrophoresis, gas-liquid ion exchange chromatography (GLC), electronic sensors, high-resolution mass spectrometry, etc., have achieved outstanding results in amino acid analysis, these methods usually involve separation techniques and complex pretreatment steps, which are relatively cumbersome and costly. Especially for some samples with complex matrices, it is difficult to directly obtain non-destructive detection of multi-data parallel output of the samples. Summary of the Invention

[0003] The purpose of the present invention is to provide a chemical reaction chip, a preparation method and an application thereof, and a method for detecting amino acids and identifying amino acid samples based on image vision. The chemical reaction chip provided by the present invention can realize the detection of amino acids and the identification of amino acid samples based on image vision, providing a new strategy for the low-cost, rapid, accurate and non-destructive analysis of amino acids.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a chemical reaction chip, which includes a substrate and reaction units arrayed on the substrate. The reaction units are loaded with a chemical reaction reagent and a chromogenic agent. The chemical reaction reagent includes at least two of CuCl 2 , Na 2 S 2 O 3 and tartaric acid.

[0006] Preferably, the chemical reaction reagent is CuCl 2 and tartaric acid, or CuCl 2 and Na 2 S 2 O 3 , or Na 2 S 2 O 3 and tartaric acid, or CuCl 2 , Na 2 S 2 O 3 and tartaric acid.

[0007] Preferably, when CuCl is loaded in the reaction units of the chemical reaction chip 2 , the loading amount of CuCl in each row of the reaction units is the same, and from top to bottom, the loading amount of CuCl in each column of the reaction units decreases; 2 2

[0008] When Na 2 S 2 O 3 is loaded in the reaction units of the chemical reaction chip, the loading amount of Na 2 S 2 O 3 in each column of the reaction units is the same, and from left to right, the loading amount of Na 2 S 2 O 3 in each row of the reaction units decreases;

[0009] When tartaric acid is loaded in the reaction units of the chemical reaction chip, the loading amount of tartaric acid in each column of the reaction units is the same, and from left to right, the loading amount of tartaric acid in each row of the reaction units decreases; or, the loading amount of tartaric acid in each row of the reaction units is the same, and from top to bottom, the loading amount of tartaric acid in each column of the reaction units first decreases and then increases symmetrically.

[0010] Preferably, the chemical reaction reagent is CuCl 2 and tartaric acid; wherein the loading amount of CuCl in each row of the reaction units is the same, and from top to bottom, the loading amount of CuCl in each column of the reaction units decreases; the loading amount of tartaric acid in each column of the reaction units is the same, and from left to right, the loading amount of tartaric acid in each row of the reaction units decreases. 2 2

[0011] Preferably, the chromogenic agent includes ninhydrin, sodium fluorescein or coomassie brilliant blue; the substrate includes a PET substrate, an organic filter membrane or art paper.

[0012] The present invention provides a preparation method of the chemical reaction chip described in the above technical solution, including the following steps:

[0013] Print the chemical reaction reagent solution and the chromogenic agent solution on the surface of the reaction units of the substrate by a chemical printing method to obtain the chemical reaction chip.

[0014] The present invention provides an application of the chemical reaction chip described in the above technical solution or the chemical reaction chip prepared by the preparation method described in the above technical solution in detecting amino acids or identifying amino acid samples based on image vision.

[0015] ​​​​The present invention provides a method for detecting amino acids based on image vision, comprising the following steps:

[0016] Pretreat the sample to be detected to obtain a test solution;

[0017] Print a chemical reaction reagent solution, a color developer solution and the test solution on the surface of the reaction unit of the substrate by a chemical printing method, and after a color reaction, obtain a color-developed substrate; the chemical reaction reagents in the chemical reaction reagent solution include CuCl 2 , Na 2 S 2 O 3 and at least two of tartaric acid;

[0018] Obtain the gray value of the reaction unit according to the color development result of the reaction unit on the color-developed substrate;

[0019] Qualitatively or quantitatively detect amino acids in the sample to be detected according to the gray value of the reaction unit.

[0020] The present invention provides a method for identifying an amino acid sample based on image vision, comprising the following steps:

[0021] Pretreat the sample to be identified to obtain a test solution;

[0022] Print a chemical reaction reagent solution, a color developer solution and the test solution on the surface of the reaction unit of the substrate by a chemical printing method, and after a color reaction, obtain a color-developed substrate; the chemical reaction reagents in the chemical reaction reagent solution include CuCl 2 , Na 2 S 2 O 3 and at least two of tartaric acid;

[0023] Obtain the gray value of the reaction unit according to the color development result of the reaction unit on the color-developed substrate;

[0024] Identify the sample to be identified according to the gray value of the reaction unit.

[0025] Preferably, the temperature of the color reaction is 105±2°C and the time is 2-15 min.

[0026] The present invention provides a chemical reaction chip, comprising a substrate and reaction units arrayed on the substrate, wherein the reaction units are loaded with chemical reaction reagents and a color developer, and the chemical reaction reagents include CuCl 2 , Na 2 S 2 O 3and at least two of tartaric acid. The chemical reaction chip provided by the present invention uses specific types of chemical reaction reagents, which have good interaction with amino acids. On this basis, the present invention combines the chemical reaction chip with image vision analysis technology to develop a new high-throughput amino acid analysis method, which can quickly obtain all information of amino acid samples at one time, providing a new strategy for the low-cost, rapid, accurate and non-destructive analysis of amino acids, promoting the application of high-throughput chemistry and image processing technology in agriculture and food, and laying a foundation for more extensive sample analysis in combination with machine learning in the future. Description of the Drawings

[0027] Figure 1 It is a diagram showing the screening results of the chemical reaction reagents in Example 1;

[0028] Figure 2 It is a schematic diagram of the AC template, AB template, BC template and ABC template in the examples;

[0029] Figure 3 It is a contour map obtained by using a 17-amino acid mixed standard product for experiments based on different printing templates in the examples;

[0030] Figure 4 It is a grid distribution diagram corresponding to the highest Gray values (GVs) of 4 different amino acid samples on the chemical reaction chip in the examples;

[0031] Figure 5 It is a visualization analysis diagram of 4 amino acid samples;

[0032] Figure 6 It is a schematic diagram of the corresponding printing method in the case of a combination of two amino acid samples or a combination of three amino acid samples in the examples;

[0033] Figure 7 It is a diagram showing the color reaction results of 12 amino acid samples under the action of a chemical chip (the printing template used is the AC template);

[0034] Figure 8 It is a trend diagram of the gray values of parallel samples of 4 amino acid samples in Test Example 1. Detailed Embodiments

[0035] The present invention provides a chemical reaction chip, which includes a substrate and reaction units arrayed on the substrate. The reaction units are loaded with a chemical reaction reagent and a color developer. The chemical reaction reagent includes CuCl 2 、Na 2 S 2 O 3 and at least two of tartaric acid.

[0036] Traditional amino acid analysis techniques, such as capillary electrophoresis, gas-liquid ion exchange chromatography, electronic sensors, high-resolution mass spectrometry, etc., have achieved outstanding results in amino acid analysis. However, these methods usually involve separation techniques and complex pre-treatments, which are relatively cumbersome and costly. Especially for some samples with complex matrices, it is difficult to directly obtain non-destructive detection of multi-data parallel output of the samples. Therefore, it is crucial to develop a method with simple operation, economy, and no need for complex pre-treatment to achieve the detection of amino acids. In recent years, with the progress of microreaction and high-throughput technologies, amino acid analysis methods based on chemical chips have received extensive attention. But in current related technologies, standard substances of amino acids are usually selected as the research objects, lacking effective analysis of actual samples, and relatively few effective correlation analyses are carried out between samples and chemical chips. In terms of chemical chip technology, the construction of chemical chips is the key to the accuracy of detection results. The more complex the reaction of the chemical chip to the sample detection results, the higher the accuracy of the detection results. The chemical chip can reconstruct various substances in the sample (specifically, reconstruct according to the designed chemical chip model), so that the sample changes from a chemical equilibrium in an initial state to a new chemical equilibrium reconstructed by the chemical chip. The present invention provides a chemical reaction chip, which uses specific types of chemical reaction reagents and has good interaction with amino acids. On this basis, by combining the chemical reaction chip with image vision analysis technology, a new high-throughput amino acid analysis method is developed, which can quickly obtain all information of amino acid samples at one time, providing a new strategy for low-cost, fast, accurate, and non-destructive analysis of amino acids, promoting the application of high-throughput chemistry and image processing technologies in agriculture and food, and laying a foundation for more extensive sample analysis in combination with machine learning in the future. First, the chemical reaction chip provided by the present invention will be described in detail below.

[0037] The chemical reaction chip of the present invention includes a substrate. As an embodiment of the present invention, the substrate may include a polyethylene terephthalate (PET) substrate, an organic filter membrane, or art paper, specifically, it may be a PET substrate. The size of the PET substrate used in the embodiments of the present invention may be 210 mm × 297 mm.

[0038] The chemical reaction chip of the present invention includes reaction units arrayed on the substrate, and chemical reaction reagents and color developers are loaded on the reaction units.

[0039] As an embodiment of the present invention, the developer may include ninhydrin, sodium fluorescein or Coomassie Brilliant Blue, specifically based on the ability to effectively act on the sample reaction to visualize the research substance in the sample or the complex information inside the sample. In the embodiments of the present invention, ninhydrin is used as the developer, which can effectively carry out a color reaction with amino acids, and the results show different color reactions due to differences in the types, concentrations, and amino acid components of the amino acid samples, which is conducive to the differential analysis of the color reaction results of amino acid samples and the significant result analysis of single indicators of different contents of amino acids.

[0040] The chemical reaction reagent of the present invention includes CuCl 2 , Na 2 S 2 O 3 and at least two of tartaric acid. As an embodiment of the present invention, the chemical reaction reagent may be CuCl 2 and tartaric acid, or may be CuCl 2 and Na 2 S 2 O 3 , or may be Na 2 S 2 O 3 and tartaric acid, or may be CuCl 2 , Na 2 S 2 O 3 and tartaric acid. By using the above combinations of chemical reaction reagents in the present invention, the internal information of amino acid samples can be presented comprehensively and visually as much as possible, and the full information of the samples can be better achieved from one chemical equilibrium to another chemical equilibrium state without complex pretreatment. Thus, during the chemical reaction process to complete the full information of the sample, due to the known design of the chemical reaction chip, accurate analysis after the chemical reaction equilibrium can be achieved. As an embodiment of the present invention, the loading amount of each chemical reaction reagent on the chemical reaction chip can be 4.99 - 5.33 μmol / 100 cm 2 (loading amount at 100% printing).

[0041] As an embodiment of the present invention, when CuCl 2 is loaded in the reaction unit of the chemical reaction chip, the loading amount of CuCl 2 in each row of the reaction unit is the same, and from top to bottom, the loading amount of CuCl 2 in each column of the reaction unit decreases; when Na 2 S 2 O 3 is loaded in the reaction unit of the chemical reaction chip, the loading amount of Na 2 S2 O 3 has the same loading amount, and from left to right, the loading amount of Na in each row of the reaction units 2 S 2 O 3 decreases; when tartaric acid is loaded in the reaction units of the chemical reaction chip, the loading amount of tartaric acid in each column of the reaction units is the same, and from left to right, the loading amount of tartaric acid in each row of the reaction units decreases; or, the loading amount of tartaric acid in each row of the reaction units is the same, and from top to bottom, the loading amount of tartaric acid in each column of the reaction units first decreases and then increases in a symmetric setting.

[0042] As an embodiment of the present invention, the chemical reaction reagent can be CuCl 2 and tartaric acid; wherein the loading amount of CuCl in each row of the reaction units 2 is the same, and from top to bottom, the loading amount of CuCl in each column of the reaction units 2 decreases; the loading amount of tartaric acid in each column of the reaction units is the same, and from left to right, the loading amount of tartaric acid in each row of the reaction units decreases. In the embodiment of the present invention, this situation is denoted as the AC template (as shown in Figure 2 ).

[0043] As an embodiment of the present invention, the chemical reaction reagent can be CuCl 2 and Na 2 S 2 O 3 ; wherein the loading amount of CuCl in each row of the reaction units 2 is the same, and from top to bottom, the loading amount of CuCl in each column of the reaction units 2 decreases; the loading amount of Na in each column of the reaction units 2 S 2 O 3 is the same, and from left to right, the loading amount of Na in each row of the reaction units 2 S 2 O 3 decreases. In the embodiment of the present invention, this situation is denoted as the AB template (as shown in Figure 2 ).

[0044] As an embodiment of the present invention, the chemical reaction reagent can be Na 2 S 2 O 3 and tartaric acid; wherein the loading amount of Na in each column of the reaction units 2 S 2 O 3 is the same, and from left to right, the loading amount of Na in each row of the reaction units 2 S 2 O3 The loading amount decreases; the loading amount of tartaric acid in each column of the reaction units is the same, and from left to right, the loading amount of tartaric acid in each row of the reaction units decreases. In the embodiments of the present invention, this situation is denoted as the BC template (as shown in Figure 2 ).

[0045] As an embodiment of the present invention, the chemical reaction reagent may be CuCl 2 , Na 2 S 2 O 3 and tartaric acid; wherein the loading amount of CuCl 2 in each row of the reaction units is the same, and from top to bottom, the loading amount of CuCl 2 in each column of the reaction units decreases; the loading amount of Na 2 S 2 O 3 in each column of the reaction units is the same, and from left to right, the loading amount of Na 2 S 2 O 3 in each row of the reaction units decreases; the loading amount of tartaric acid in each row of the reaction units is the same, and from top to bottom, the loading amount of tartaric acid in each column of the reaction units first decreases and then increases symmetrically. In the embodiments of the present invention, this situation is denoted as the ABC template (as shown in Figure 2 ).

[0046] As an embodiment of the present invention, the loading amount of the color developing agent on each reaction unit of the chemical reaction chip is the same. As an embodiment of the present invention, the loading amount of the color developing agent on the chemical reaction chip may be 49.9 - 53.5 μL / 100 cm 2 (loading amount at 100% printing).

[0047] The present invention provides a preparation method of the chemical reaction chip described in the above technical solution, including the following steps:

[0048] Print the chemical reaction reagent solution and the color developing agent solution on the surface of the reaction units of the substrate by a chemical printing method to obtain the chemical reaction chip.

[0049] As an embodiment of the present invention, the chemical printing method may specifically be a piezoelectric inkjet printing method, and the piezoelectric inkjet printer used in the embodiments of the present invention is specifically an Epson inkjet printer.

[0050] The present invention has no special limitation on the printing order of the chemical reaction reagent solution and the color developing agent solution, and the chemical reaction reagent solution may be printed first, or the color developing agent solution may be printed first.

[0051] As an embodiment of the present invention, when printing the chemical reaction reagent solution, specifically, single - type chemical reaction reagent solutions are printed separately. For example, when the chemical reaction reagent is CuCl 2 , Na 2 S 2 O 3 and tartaric acid, specifically, CuCl 2 solution, Na 2 S 2 O 3 solution and tartaric acid solution are printed separately, and there is no special limitation on the printing order of the three; the concentrations of the CuCl 2 solution, Na 2 S 2 O 3 solution and tartaric acid solution can specifically be 0.1 mol / L; among them, the loading amount of the CuCl 2 solution at 100% loading can be 6.7091 - 7.1662 ng / 100 cm 2 , specifically can be 6.9377 ng / 100 cm 2 ; the loading amount of the Na 2 S 2 O 3 solution at 100% loading can be 7.8897 - 8.4273 ng / 100 cm 2 , specifically can be 8.1585 ng / 100 cm 2 ; the loading amount of the tartaric acid solution at 100% loading can be 7.4895 - 7.9998 ng / 100 cm 2 , specifically can be 7.7447 ng / 100 cm 2 . In the present invention, when the chemical reaction reagent is in other combination situations, refer to the above method, and details will not be elaborated here.

[0052] As an embodiment of the present invention, when printing the CuCl 2 solution on the substrate, specifically, based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from top to bottom; when printing the Na 2 S 2 O 3 solution on the substrate, specifically, based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from left to right; when printing the tartaric acid solution, specifically, based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from left to right, or based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 2% and then uniformly increased from 0% to 100% at a gradient of 2%.

[0053] As an embodiment of the present invention, print the CuCl 2 solution, Na 2 S 2 O 3 solution and tartaric acid solution can also be printed on the entire substrate according to a fixed printing amount, such as 100% of the printing amount, and can be selected according to actual needs.

[0054] As an embodiment of the present invention, the concentration of the printing chromogenic agent solution can be 1.5-2.5 wt%, specifically 2 wt%. As an embodiment of the present invention, when printing the printing chromogenic agent solution on the substrate, specifically, the entire substrate can be printed according to a printing amount of 100%.

[0055] The present invention provides the application of the chemical reaction chip described in the above technical solution or the chemical reaction chip prepared by the preparation method described in the above technical solution in detecting amino acids or identifying amino acid samples based on image vision. The chemical reaction chip of the present invention can be used to detect amino acids for non-diagnostic purposes based on image vision technology, specifically, it can qualitatively detect amino acids or quantitatively detect amino acids. Specifically, the chemical reaction chip of the present invention can analyze the interaction relationship between the sample and the chemical reaction reagent, and perform data analysis through the interaction relationship (such as the size of the gray value, the distribution of strong and weak characteristic regions, the color change of the complex reaction of the amino acid content and the chemical reaction reagent, etc.), establish a large model, and further realize the qualitative analysis of the sample (such as according to the characteristic distribution of each graph, different perceptual hash values, etc.); in addition, the present invention can construct a database according to the chromatographic detection results and realize the quantification of amino acids on this basis. The chemical reaction chip of the present invention can also be used to identify amino acid samples based on image vision, specifically, it can identify the types of single amino acid samples or the types of amino acid samples contained in mixed amino acid samples.

[0056] Traditional amino acid analysis usually requires the use of large and expensive analytical instruments (such as amino acid analyzers, liquid chromatography instruments, liquid chromatography-mass spectrometry equipment, etc.). The pretreatment operation is complex and the analysis and detection time is relatively long. Taking the amino acid analysis using an amino acid analyzer as an example, first, the amino acid sample needs to be weighed to ensure the accurate measurement of the starting material. Then, hydrochloric acid with a concentration of 6M is added to the amino acid sample, and a hydrolysis reaction is carried out at 110 ± 1 °C for 22 h. During this process, the hydrolysis tube is sealed under reduced pressure to remove any oxygen and prevent the sample from being oxidized. After the hydrolysis reaction, the resulting product system is dried under reduced pressure to remove hydrochloric acid and any other volatile components. The dried sample is dissolved in hydrochloric acid with a concentration of 0.02M, and then the sample is filtered using a 0.22 μm organic filter membrane to remove any particulate matter or undissolved substances that may interfere with subsequent machine analysis. Finally, the filtered sample is analyzed using an amino acid analyzer. Using this method, the time required for one sample to be tested on the machine is 1 h, and the detection time is relatively long. The present invention prepares the chemical reaction chip by using high-throughput chemical printing technology. Through the chemical reaction chip and image vision analysis, the python image analysis has the characteristics of fast speed and stability, and can obtain all the information of the amino acid sample at one time, providing a new strategy for the low-cost, fast, accurate, and non-destructive analysis of amino acids, promoting the application of high-throughput chemistry and image processing technology in agriculture and food, and laying a foundation for more extensive sample analysis by combining machine learning in the future.

[0057] The present invention provides a method for detecting amino acids based on image vision, comprising the following steps:

[0058] Pretreat the sample to be tested to obtain a test solution;

[0059] Adopt a chemical printing method to print a chemical reaction reagent solution, a color developing agent solution, and the test solution on the surface of the reaction unit of the substrate. After a color development reaction, a color-developed substrate is obtained. The chemical reaction reagents in the chemical reaction reagent solution include at least two of CuCl 2 , Na 2 S 2 O 3 and tartaric acid;

[0060] Obtain the gray value of the reaction unit according to the color development result of the reaction unit on the color-developed substrate;

[0061] Qualitatively or quantitatively detect the amino acids in the sample to be tested according to the gray value of the reaction unit.

[0062] The sample to be tested in the present invention is pretreated to obtain a test solution. As an embodiment of the present invention, the sample to be tested may include fruit samples, vegetable samples, traditional Chinese medicine samples or biological samples; the fruit samples may include one or more of red grapes, pepino, Red Fuji apples and golden pears; the vegetable samples may include one or more of cucumbers, Shanghai greens, chayote and Pleurotus eryngii; the traditional Chinese medicine samples may include one or more of Scutellaria barbata, Forsythia suspensa, Polygonatum sibiricum, Polygala tenuifolia and Liuwei Dihuang; the biological samples may include human urine samples. As an embodiment of the present invention, the corresponding pretreatment method can be selected according to the type of the sample to be tested. Specifically, since the fruit samples and vegetable samples have a high water content, they can be juiced respectively and then centrifuged at 5000 rpm for 10 min, and the supernatant is collected and filtered through a membrane to obtain a fruit sample solution and a vegetable sample solution as the test solution respectively; the traditional Chinese medicine samples are soaked in hot water at 70 °C for 2 h, the material-liquid ratio of the traditional Chinese medicine samples to the hot water is 1 g:20 mL, and then centrifuged at 5000 rpm for 10 min, and the supernatant is collected and filtered through a membrane to obtain a traditional Chinese medicine sample solution as the test solution; the human urine sample is directly centrifuged at 5000 rpm for 10 min, the supernatant is collected and filtered through a membrane, and the obtained filtrate is used as the test solution.

[0063] The present invention uses a chemical printing method to print a chemical reaction reagent solution, a color developer solution and a test solution on the surface of the reaction unit of the substrate. After a color reaction, a colored substrate is obtained; the chemical reaction reagents in the chemical reaction reagent solution include CuCl 2 , Na 2 S 2 O 3 and at least two of tartaric acid. The selection of the chemical printing method, the substrate, the chemical reaction reagents and the color developer in the present invention can refer to the above technical solutions and will not be elaborated here. The present invention has no special limitation on the printing order of the chemical reaction reagent solution, the color developer solution and the test solution. The chemical reaction reagent solution can be printed first, or the color developer solution can be printed first, or the test solution can be printed first, and then the other two solutions are printed continuously. Moreover, the present invention has no special limitation on the printing order of the other two solutions, and any order is acceptable. As an embodiment of the present invention, the printing method of the test solution can be selected according to actual needs. For example, it can be printed according to the gradient change of the printing amount with reference to the above technical solution, or it can be printed on the entire substrate according to a fixed printing amount such as 100% of the printing amount with reference to the above technical solution, and it can be selected according to actual needs.

[0064] As an embodiment of the present invention, the temperature of the color reaction can be 105 ± 2 °C; the time can be 2 to 15 min, specifically 5 min.

[0065] After obtaining the color-developing substrate, the present invention obtains the gray value of the reaction unit according to the color-developing result of the reaction unit on the color-developing substrate. As an implementation manner of the present invention, a flatbed scanner can be specifically used to simultaneously scan the color-developing result of the entire color-developing substrate to simultaneously obtain the color-developing result on the color-developing substrate, which is convenient for unified calibration, so as to obtain a series of image information; then the image information is converted into a gray value and read through Python, and finally the gray value of each reaction unit on the color-developing substrate is obtained (specifically, the maximum value, minimum value or average value can be taken).

[0066] After obtaining the gray value of the reaction unit, the present invention realizes the qualitative or quantitative detection of amino acids in the sample to be measured according to the gray value of the reaction unit. The present invention can reflect the color development situation between the chemical reaction reagent and the amino acid sample through the gray value, and comprehensively judge according to the best reaction conditions and the pattern information presented after the reaction on the entire chemical reaction chip to realize the qualitative determination of amino acids; according to the qualitative result, the content of the relevant sample obtained previously and the result of the corresponding gray value at the same position are used to realize the quantitative determination of amino acids; among them, for the quantitative determination of amino acids, in the present invention, the sample to be measured can be subjected to chromatographic detection, and a database is constructed according to the chromatographic detection result, and the quantitative determination of amino acids is realized according to the gray value of the reaction unit and the database. When the sample amount increases, the quantitative determination of amino acids can be realized based on large model machine learning and the like without relying on the chromatographic detection result.

[0067] The present invention provides a method for identifying amino acid samples based on image vision, including the following steps:

[0068] Pretreat the sample to be identified to obtain the sample to be measured;

[0069] Use a chemical printing method to print a chemical reaction reagent solution, a color developer solution and the sample to be measured on the surface of the reaction unit of the substrate. After a color development reaction, a color-developing substrate is obtained; the chemical reaction reagents in the chemical reaction reagent solution include CuCl 2 , Na 2 S 2 O 3 and at least two of tartaric acid;

[0070] Obtain the gray value of the reaction unit according to the color-developing result of the reaction unit on the color-developing substrate;

[0071] Identify the sample to be identified according to the gray value of the reaction unit.

[0072] As an embodiment of the present invention, the sample to be identified can be a single sample or a mixed sample. As an embodiment of the present invention, for the pretreatment method, the selection of the chemical printing method, chemical reaction reagents and color developers, the conditions of the color reaction, the acquisition of gray values, etc., reference can be made to the above technical solutions and will not be elaborated here.

[0073] As an embodiment of the present invention, after obtaining the gray value of the reaction unit, the sample to be identified can be specifically identified according to the characteristic distribution and key characteristic values presented by the gray value of the reaction unit sample. As an embodiment of the present invention, after obtaining the gray value of the reaction unit, it can be specifically compared with the established database, and the reaction results can be identified, compared and analyzed through a semi-supervised mode (such as setting corresponding image feature thresholds, perceptual hash values, gray value sizes, etc.) to realize the identification of the sample to be identified.

[0074] The present invention combines high-throughput screening and color space to construct a combination of chemical reaction reagents suitable for the color reaction of amino acids with ninhydrin; on this basis, using chemical printing technology and combining computer-guided generation of preset templates, a series of chemical reaction chips are prepared, thus building a medium and environment for the color reaction of amino acids. Through the integration of CCD (a fine scanner containing an inductive coupling device; V370, Seiko Epson Corporation) and Python technology, accurate recording and computational analysis of experimental results are achieved. With the help of image analysis technology, the characteristic information of experimental results is deeply explored, the interaction relationship between amino acid samples and the combination of chemical reaction reagents is explored, the most valuable information and laws revealed by the full-factor analysis of amino acid samples by chemical reaction chips are further analyzed, and the influence of matrix difference and amino acid type specificity on the color reaction results is discussed. Therefore, the present invention provides a new strategy for the low-cost, rapid, accurate and non-destructive analysis of amino acids, and provides a new perspective for the integration of chemical chips and image analysis technology. Specifically, the present invention has at least the following beneficial effects:

[0075] 1. Innovative integration of chemical chips and image analysis

[0076] The present invention combines a chemical chip (CCC) with image analysis technology to construct a high-throughput detection system without complex pretreatment. The chip is prepared by using chemical printing technology on a PET substrate, and through the extraction and processing of gray information, principal component analysis (PCA) and heat map analysis, etc., rapid and non-destructive detection of amino acids is achieved. This method breaks through the dependence of traditional chromatography or mass spectrometry technology on sample pretreatment, significantly improves the analysis efficiency (error < 2%), and reduces costs, providing a new paradigm for real-time and in-situ detection.

[0077] 2. Intelligent design of multi-reagent gradient chips

[0078] The present invention has developed a multi-reagent combination chip (CCC@PET) based on CuCl 2 , Na 2 S 2 O 3 and tartaric acid, that is, the chemical reaction chip described in the present invention. By precisely regulating the reagent concentration gradient (0% - 100%, step size 1%), sensitive discrimination of amino acids is achieved. The optimal AC template (gray-scale difference up to 97) is screened out through research, which can clearly analyze the compositional differences of amino acids (such as the concentration gradients of glutamic acid and proline) in different samples (such as red grapes and cucumbers), and the sensitivity is significantly improved compared with traditional methods. This design reveals the dynamic interaction between amino acids and reagents through the mechanisms of metal ion coordination and competitive reactions, providing a new tool for the analysis of complex matrices.

[0079] 3. Scalable application of high-throughput chemical printing technology

[0080] The present invention adopts an improved inkjet printing technology to achieve the rapid preparation of chemical chips (10 4 data points / sample). By controlling reagent deposition through the CMYK color model and combining Python image processing and machine learning potential, a scalable analysis platform is constructed. This method not only supports the precise generation of complex chemical gradients (such as the design of the cyclic concentration of tartaric acid), but also verifies high repeatability (Pearson correlation coefficient > 0.99) through error analysis, laying a technical foundation for large-scale sample screening and precision nutrition research in agriculture and food science.

[0081] Therefore, through interdisciplinary technology integration (chemistry, image processing, engineering), the present invention solves the bottlenecks of high cost and low throughput in traditional amino acid analysis, and demonstrates significant advantages in detection sensitivity, sample adaptability, and scalability, providing an important reference for the development of future intelligent analysis systems.

[0082] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0083] In the embodiments of the present invention, the substrate used for preparing the chemical reaction chip is a PET substrate. The size of each PET substrate is 210mm × 297mm, and 2000 (40 × 50) reaction units distributed in an array can be printed on each PET substrate, and the size of each reaction unit is 100 × 100 pixels (0.42cm × 0.42cm).

[0084] In the embodiment of the present invention, the preparation of the chemical reaction chip is completed by high-throughput chemical printing technology. Specifically, the piezoelectric inkjet printing (Hydraulic inkjet printer) method is adopted. Different pulsed voltages are generated according to the incoming image information and act on the voltage components inside the tube wall. The voltage components deform under the action of the voltage, and the chemical ink inside the tube wall is extruded onto the substrate. The voltage material of the piezoelectric inkjet printer outputs voltage under different electric fields. Compared with the bubble inkjet printing method, the piezoelectric inkjet printing method has higher printing accuracy. And because the chemical ink is directly extruded by the deformation of the conductive sponge material, there are fewer intermediate links and fewer factors to be controlled, so the error is smaller compared with the bubble inkjet printing method.

[0085] In the embodiment of the present invention, the piezoelectric inkjet printer used to prepare the chemical reaction chip is an Epson inkjet printer (the printer is originally equipped with ink cartridges representing different colors). And the paper feeding device of the traditional printer is set as a flat fixed stage. At the same time, the print head is set as a movable print head that can slide left and right and move back and forth. During the printing process of this printer, the position of the PET substrate is fixed, only the print head moves. Therefore, when ensuring that the starting position of the print head printing is fixed, it can be ensured that each reagent or sample can be loaded on the PET substrate completely according to the designed pattern during printing. The specific steps are as follows:

[0086] (1) Use an ultrasonic oscillator to clean a glass plate (25mm×75mm×5mm) as the stage, and then rinse it again with distilled water and air-dry it naturally at room temperature;

[0087] (2) Fix the PET substrate on the glass plate with double-sided tape;

[0088] (3) Transfer the glass plate with the PET substrate to the positioning system of the printer and accurately position it;

[0089] (4) Use distilled water to clean the ink cartridge and the sponge (the role of the sponge is to adsorb the chemical printing pool), and rinse the sponge of the ink cartridge with the reagent solution or sample solution to be printed to ensure the accuracy of the experiment;

[0090] (5) Use a clean syringe to suck 3 mL of the reagent solution or sample solution to be printed, and then inject it into the ink cartridge;

[0091] (6) Use Aero RIP Colorver8.2 software to control the operation of the printer.

[0092] In the embodiments of the present invention, through a chemical printing method, each reagent or sample is delivered to a PET substrate according to a certain concentration gradient (by controlling the printing amount of the printer, outputting between 0% and 100% according to the designed printing template, and the adjacent gradient change is 1% or 2%). Among them, three chemical reaction reagent solutions (CuCl 2 solution, Na 2 S 2 O 3 solution, tartaric acid solution) are delivered to the PET substrate according to the printing rules based on the preset printing template, and finally different combinations of the three chemical reaction reagents are presented on the PET substrate.

[0093] When conducting experiments on actual samples in the embodiments of the present invention, the actual samples used include fruit samples (such as red dates and ginseng fruits), vegetable samples (such as cucumbers and Shanghai greens), traditional Chinese medicine samples (such as Scutellaria barbata, Forsythia suspensa, Polygonatum sibiricum, and Polygala tenuifolia), and human urine samples. They are pretreated before printing. Among them, fruit samples and vegetable samples have a high water content. After juicing respectively, they are centrifuged at 5000 rpm for 10 min, and the supernatant is collected and filtered through a membrane to obtain fruit sample solution and vegetable sample solution respectively; the traditional Chinese medicine samples are soaked in hot water at 70 °C for 2 h, and the material-liquid ratio of the traditional Chinese medicine samples to hot water is 1 g:20 mL. Then they are centrifuged at 5000 rpm for 10 min, and the supernatant is collected and filtered through a membrane to obtain traditional Chinese medicine sample solution; the human urine samples are directly centrifuged at 5000 rpm for 10 min, and the supernatant is collected and filtered through a membrane.

[0094] Example 1

[0095] In this example, chemical reaction reagents are screened. Specifically, compounds used to induce mild chemical reactions (such as acid-base reactions, complexation reactions, precipitation-dissolution reactions, reduction-oxidation reactions) with different amino acids are selected. They can participate in the reaction directly (linear reaction), that is, the chemical reaction directly occurs between the chemical reaction reagent and the amino acid in the sample, or they can participate in the reaction indirectly (non-linear coupling reaction), that is, the interaction reaction between the sample matrix, amino acid concentration, and environmental medium, etc., and the chemical reaction reagent.

[0096] In this example, 15 chemical reaction reagents are used for screening, specifically NiSO 4 、CuCl 2 、AlCl 3 、MgSO 4 、MnSO 4 、ZnCl 2 、CdCl 2 、hexamethylenetetramine (HMTA), KH 2 PO 4, Calcium benzoate, Bicine-Na, Na 2 S 2 O 3 , tartaric acid, citric acid, diethylenetriaminepentaacetic acid (DTPA); An aqueous solution of each of the above chemical reaction reagents (concentration 0.1 mol / L, volume 2 μL) was placed in different microreactors, and a color reagent solution (specifically, a ninhydrin solution with a concentration of 2 wt%, volume 10 μL) was added. The microreactors were placed in a drying cabinet and treated at 105 ± 2 °C for 5 min; A fine scanner (V370, Seiko Epson Corporation) was placed above the reaction area to capture color changes in all reaction areas. Using the RGB color space, based on the hue theory and color difference formula, mathematical methods were used to represent colors. The following calculation method was used to evaluate the color difference:

[0097] D(X i , X j ) = [(R i - R j ) 2 + (G i - G j ) 2 + (B i - B j ) 2 1 / 2 ;

[0098] X i = (R i , G i , B i );

[0099] X j = (R j , G j , B j ).

[0100] Where the color distance D is the difference between the RGB values of the test sample and the blank sample; X i and X j represent the RGB values of different samples.

[0101] The screening results of the chemical reaction reagents were further verified by orthogonal experiments. The specific screening results are shown in Table 1 and Figure 1 as shown, where Figure 1 ​In Figure A, a single-factor color reaction result graph of 15 chemical reagents with amino acids and ninhydrin is established. In Figure B, a color reaction result graph of multi-factor interaction within the group is shown. In Figure C, a color reaction result graph of multi-factor between groups is presented. From this, it can be seen that among the three types of combined chemical reagents, by comparing with the blank group, the color distances of various chemical reagents are obtained. The results show that the metal ion competition reagent A2 (CuCl 2 ), the acid-base adjustment chemical reagent B5 (Na 2 S 2 O 3 ), and the complexing reagent C1 (tartaric acid) have the most prominent influence on the color reaction results of amino acids in the interactive color reaction experiment. Therefore, in the experiment, the metal ion competition reagent A2, the acid-base adjustment chemical reagent B5, and the complexing reagent C1 are selected as three combined reagents to prepare a chemical reaction chip, further realizing the construction of a color system with rich amino acid information. This screening method based on color distance can effectively reflect the interference degree of reagents on the color reaction, providing a scientific basis for the subsequent construction of chemical chips.

[0102] Table 1 Screening Results of Chemical Reaction Reagents

[0103]

[0104]

[0105] Example 2

[0106] In this example, the printing template is screened. Contour maps can analyze the mutual relationships between different data through statistical methods and present the patterns and properties between data in the form of charts. In scientific research and data analysis, researchers can compare the effects of different clustering analysis or classification methods through contour maps. By observing the data distribution patterns on the contour map, researchers can select the most appropriate analysis method. Therefore, contour maps have important scientific value. In this example, specifically, the results of the mixed amino acid color reaction are used to extract grayscale values through Python, and Origin 2021 is used for contour processing and contour map drawing. For the convenience of standardized analysis, the color card value range of the contour map is normalized during the drawing process. According to the complexity of the pattern and the differences between different regions, the best printing template is selected for the amino acid sample color reaction experiment.

[0107] The chemical reaction chip images in the experiment (i.e., the images obtained by scanning after color development) are first converted into digital information available for analysis through digital image processing technology. The first step in the image processing flow is to standardize the image, unifying the size to 400×400 pixels, with each pixel corresponding to an independent element in the matrix. The size of the processed matrix is 400×400, and each element contains one or more values, depending on the color channel type of the image. Specifically, in terms of the color mode of the image, two types of images are used in this embodiment, namely single-channel grayscale images and three-channel RGB images. Each pixel in the grayscale image contains only one value, representing the grayscale intensity of that pixel; while each pixel in the RGB image consists of three numerical values, corresponding to the intensities of the red (R), green (G), and blue (B) channels respectively, and these numerical values together determine the hue of that pixel. Since the main hue of the chemical reaction image in the experiment is red, the RGB image needs to be converted into a grayscale image to more effectively extract the brightness information. The image processing steps are as follows: First, remove the color channels irrelevant to the reaction. Considering that the main color in the reaction image is red, replace the cyan (C) and yellow (Y) channels with white (i.e., set the corresponding pixel values to 255), and then copy the magenta (M) channel to the black (K) channel. This step can be achieved by setting pixel values to convert the image into grayscale mode. During the conversion process, the RGB values of each pixel will be converted into grayscale values according to the weighted average method, and this grayscale value is unique. Use Python to extract digital information. Through the PIL (Pillow) library in Python, the value of each pixel in the image can be extracted and converted into digital information. The grayscale value of each pixel will be stored in a matrix of 400×400 pixels for analysis as experimental data.

[0108] Based on the richness of internal information of amino acids, the screening of printing templates includes using the contour image as an independent analytical and statistical representation, and clarifying the complex relationships between each data point through charts. In order to comprehensively analyze the complex information inside amino acids, rich color information must be obtained from amino acids. Therefore, in this embodiment, the best printing template is screened from 4 different printing templates (AC template, AB template, BC template, and ABC template, as specifically shown in Figure 2 ). The chemical reaction reagents and printing methods used are as follows:

[0109] Reagent A (CuCl 2 solution, with a concentration of 0.1 mol / L), during printing, based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from top to bottom;

[0110] Reagent B (Na 2 S 2 O 3Solution (concentration: 0.1 mol / L). During printing, based on each reaction unit, the printing amount is uniformly decreased from 100% to 0% at a gradient of 1% from left to right.

[0111] Reagent C (tartaric acid solution, concentration: 0.1 mol / L). During printing, based on each reaction unit, the printing amount is uniformly decreased from 100% to 0% and then increased to 100% at a gradient of 2% from top to bottom (corresponding to the ABC template), or the printing amount is uniformly decreased from 100% to 0% at a gradient of 1% from left to right (corresponding to the AC template and BC template).

[0112] In this example, 17 kinds of amino acid mixed standards were used for experiments to screen the best printing template. Threshold and gray-scale contour techniques were used to process the chemical reaction chip images obtained after the color reaction and scanning of the printed 17 kinds of amino acid mixed standards. The 17 kinds of amino acids are specifically L-aspartic acid, L-threonine, L-serine, L-glutamic acid, glycine, L-alanine, L-cystine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-lysine, L-histidine, L-arginine, and L-proline. Specifically, a 17-amino acid mixed standard solution with a concentration of 100 nmol / mL was prepared (the solvent used is ultrapure water). Reagent A, Reagent B, and Reagent C were printed in the aforementioned manner, the 17-amino acid mixed standard solution was printed with a printing amount of 100%, and then the color developer (2 wt% ninhydrin solution) was printed with a printing amount of 100%. The color reaction was carried out at a temperature of 105 ± 2 °C for 5 min.

[0113] Figure 3 It is the contour map obtained from the experiments using 17 kinds of amino acid mixed standards based on different printing templates in Example 2. The results show that there are obvious differences in the color distribution range and pattern changes of the contour maps corresponding to different printing templates. The color band corresponding to the AB template is 135 - 202, with a difference of 67; the color band corresponding to the AC template is 108 - 205, with a difference of 97; in contrast, the color band corresponding to the BC template shows a narrower range, which is 116 - 176, with a difference of only 60; the color band corresponding to the ABC template is 115 - 190, with a difference of 75. In summary, the order of the color distribution ranges of each printing template is as follows: AC > ABC > AB > BC. Therefore, the AC template was used to construct the chemical reaction chip in the subsequent experiments of the present invention.

[0114] Example 3

[0115] In this embodiment, the interaction between the amino acid sample and the chemical reaction chip is analyzed. The color reaction of amino acids is often related to the type of sample, the concentration of amino acids in the sample, the content ratio or concentration of 17 amino acid components in the sample, and the reaction conditions, etc. Therefore, by analyzing their interaction, the variation law of the amino acid color reaction is explored and the key influencing factors are analyzed to improve the specific recognition effect of the chemical reaction chip on amino acids.

[0116] In this embodiment, red grapes, cucumbers, pepinos, and Shanghai greens are used as amino acid samples for experiments, and the specific description is as follows.

[0117] In this embodiment, an Epson inkjet printer is used for printing. First, open the pre-designed pattern (specifically the pattern corresponding to the AC template) in the software, select the "CMYK" mode, and start printing after ensuring that all working indicators are stable. The steps are as follows:

[0118] (1) The first pass of printing is to print two chemical reaction reagents represented by cyan and yellow in Layer-CY on the PET substrate in sequence. The chemical reaction reagent represented by cyan is reagent A (CuCl 2 solution, with a concentration of 0.1 mol / L; based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from top to bottom). The chemical reaction reagent represented by yellow is reagent C (tartaric acid solution, with a concentration of 0.1 mol / L; based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from left to right).

[0119] (2) The second pass of printing is to print the developer on the PET substrate. The developer is specifically ninhydrin solution (with a concentration of 2 wt%; the entire PET substrate is printed with a printing amount of 100%).

[0120] (3) The third pass of printing is to select Layer-C to print the amino acid sample onto the PET substrate. Based on each reaction unit, according to printing template C, the printing amount is uniformly reduced from 100% to 1% at a gradient of 1% from top to bottom, which is equivalent to loading amino acid samples with different amino acid contents on 100 reaction units of the PET substrate, and the amino acid content in the amino acid sample on each reaction unit is different.

[0121] After the above printing is completed, the ink cartridge of the printer is cleaned, and the obtained PET substrate loaded with the amino acid sample is placed in an oven and subjected to a color reaction at a temperature of 105 ± 2 °C for 5 min to obtain a colored PET substrate.

[0122] A flatbed scanner is used to simultaneously scan the color development results of the entire color-developed PET substrate, so as to simultaneously obtain the color development results on the color-developed PET substrate, which is convenient for unified calibration, and a series of image information is obtained. The image information is converted into grayscale values and read through Python, and finally the grayscale values (the maximum values are taken in this embodiment) of each reaction unit on the color-developed PET substrate are obtained.

[0123] In this embodiment, the chemical reaction reagent combinations of each reaction unit on the chemical reaction chip for 4 amino acid samples are in one-to-one correspondence and consistent; secondly, the types and proportions of amino acids in the amino acid samples loaded on each independent reaction unit on the chemical reaction chip are determined. Therefore, the specific chemical reaction reagent components on each independent reaction unit can be known through the chemical reaction chip. The types and contents of amino acids in each amino acid sample and the percentages of different amino acids can be obtained through chromatographic analysis. The fixed grayscale values on each independent reaction unit can be obtained through color development reaction (105±2°C, 5 min) and extraction of color development results, and thus the amino acid samples, chemical reaction reagent combinations, and amino acids can be connected.

[0124] To simplify the analysis, in this embodiment, 10% of the experimental data with the printing amount is selected. According to the grayscale values from high to low, the top fifteen grayscale values are selected from each amino acid sample to draw a grid diagram (specifically, it is drawn after an equal-proportion scaling of 10×10 according to the chemical reaction chip), and the distribution of amino acid color development results in different amino acid samples is represented in the form of color coding and grid layout.

[0125] Figure 4 It is the grid distribution diagram corresponding to the highest Grayvalues (GVs) values of 4 different amino acid samples on the chemical reaction chip. Among them, A is the grape sample, B is the cucumber sample, C is the pepino sample, and D is the Shanghai green sample. The results show that by comparing the grayscale value distributions of different amino acid samples, the diversity of their color development reactions can be evaluated. For example, the grape sample and the cucumber sample are mainly distributed in the upper left of the grid, the pepino sample is mainly distributed on the left side of the grid, and the Shanghai green sample is mainly distributed in the middle and lower positions of the grid. In this embodiment, the size of the bubble is used to evaluate the size of the grayscale value. Among them, the larger the grayscale value, the larger the bubble, indicating a stronger color development reaction. Different amino acid samples, different amino acid types and percentages are distinguished by colors. The same position indicates that under the condition of the same chemical reaction reagent combination, the amino acid samples have relatively similar color development reactions at this position. In addition, for the convenience of description, according to the amino acid content (g / 100g) from high to low, the top three amino acids in the amino acid sample are selected as the main analysis objects, and the remaining 14 amino acids are used as auxiliary analysis objects. Figure 5 It is the visualization analysis diagram of 4 amino acid samples, where Figure 5In it, A is the chemical color reaction result diagram, B is the principal component analysis PCA diagram obtained by 4 samples through GVs, and C is the heat map analysis result diagram of the samples. The results show that except for the Shanghaiqing sample, the high gray values of the other three amino acid samples are mainly distributed on the right side of the chemical reaction chip, which is consistent with Figure 4 the results; from Figure 4 the bubble size in it, it can be obtained that the gray value of the grape sample is the highest and the gray value of the cucumber sample is the lowest.

[0126] Table 2 shows the chromatographic detection results of the amino acid content in different samples. This result can be used as the first amino acid database for the amino acid content (unit: g / 100g) in each sample. After combining it with the chemical reaction chip, the information database obtained on the chemical reaction chip can be used as the second amino acid database. After the two databases are combined, deep learning is carried out, from semi-supervised learning to unsupervised learning, and finally an active recognition and result giving in the artificial intelligence mode are formed.

[0127] Table 2 Chromatographic detection results of amino acid content in different samples

[0128] Sample Red grape Cucumber Solanum muricatum Shanghaiqing (Brassica rapa subsp. chinensis) Total content (g / 100g) <![CDATA[0.706±0.264 d > <![CDATA[0.764±0.257 c > <![CDATA[0.889±0.525 b > <![CDATA[1.669±0.935 a > Asp <![CDATA[0.048±0.278 d > <![CDATA[0.057±0.716 c > <![CDATA[0.509±0.435 a > <![CDATA[0.183±0.531 b > Thr <![CDATA[0.025±0.239 c > <![CDATA[0.028±1.260 b > <![CDATA[0.016±0.330 d > <![CDATA[0.077±0.422 a > Ser <![CDATA[0.025±0.214 c > <![CDATA[0.037±1.455 b > <![CDATA[0.022±0.555 d > <![CDATA[0.080±0.237 a > Glu <![CDATA[0.099±0.068 d > <![CDATA[0.295±1.295 b > <![CDATA[0.115±0.376 c > <![CDATA[0.318±1.020 a > Gly <![CDATA[0.029±0.510 c > <![CDATA[0.044±1.526 b > <![CDATA[0.022±0.812 d > <![CDATA[0.088±1.700 a > Ala <![CDATA[0.037±0.541 b > <![CDATA[0.029±0.714 c > <![CDATA[0.019±1.201 d > <![CDATA[0.117±0.212 a > Cys <![CDATA[0.002±0.292 c > <![CDATA[0.002±0.213 b > <![CDATA[0.003±3.175 a > <![CDATA[0±0.000 d > Val <![CDATA[0.029±0.205 c > <![CDATA[0.036±0.850 b > <![CDATA[0.020±0.614 d > <![CDATA[0.101±0.401 a > Met <![CDATA[0.003±2.557 c > <![CDATA[0.009±4.401 a > <![CDATA[0.001±3.163 d > <![CDATA[0.006±0.352 b > Ile <![CDATA[0.019±0.871 c > <![CDATA[0.028±0.253 b > <![CDATA[0.016±0.325 d > <![CDATA[0.075±0.410 a > Leu <![CDATA[0.033±0.299 c > <![CDATA[0.047±0.262 b > <![CDATA[0.026±0.542 d > <![CDATA[0.138±0.115 a > Tyr <![CDATA[0.017±1.063 c > <![CDATA[0.020±1.261 b > <![CDATA[0.016±0.239 d > <![CDATA[0.057±0.209 a > Phe <![CDATA[0.023±0.183 c > <![CDATA[0.027±1.033 b > <![CDATA[0.015±0.161 d > <![CDATA[0.094±0.306 a > Lys <![CDATA[0.041±0.264 b > <![CDATA[0.041±0.266 c > <![CDATA[0.028±0.713 d > <![CDATA[0.120±0.231 a > His <![CDATA[0.023±0.170 b > <![CDATA[0.015±0.192 c > <![CDATA[0.014±0.362 d > <![CDATA[0.035±0.301 a > Arg <![CDATA[0.115±0.076 a > <![CDATA[0.028±0.101 c > <![CDATA[0.022±0.382 d > <![CDATA[0.105±0.112 b > Pro <![CDATA[0.140±0.371 a > <![CDATA[0.020±0.953 d > <![CDATA[0.024±1.593 c > <![CDATA[0.074±0.718 b >

[0129] In addition, according to Table 2 and Figure 4 it can be known that the total amino acid content of the grape sample is 0.706%, and the amino acid percentage is Glu 0.047 Arg 0.155 Pro 0.140 , and its corresponding best chemical reaction reagent combination is CuCl 2 and tartaric acid (the volume ratio of the CuCl 2 solution to the tartaric acid solution is 80:30), that is, the gray value is the largest at this time; the amino acid content of the cucumber sample is 0.764%, and the amino acid percentage is Asp 0.057 Glu 0.295 Leu 0.047 , and its corresponding best chemical reaction reagent combination is CuCl 2 and tartaric acid (the volume ratio of the CuCl 2 solution to the tartaric acid solution is 90:10); the amino acid content of the pepino sample is 0.889%, and the amino acid percentage is Asp 0.509 Glu 0.115 Lys 0.028 , and its corresponding best chemical reaction reagent combination is CuCl 2 and tartaric acid (the volume ratio of the CuCl 2 solution to the tartaric acid solution is 50:10); the amino acid content of the Shanghaiqing sample is 1.669%, and the amino acid percentage is Asp 0.183 Glu 0.318 Leu 0.138, and its corresponding optimal combination of chemical reaction reagents is CuCl 2 and tartaric acid (the volume ratio of the CuCl 2 solution to the tartaric acid solution is 40 - 50:30).

[0130] In addition, the bubbles of the red grape samples are significantly larger than those of the other three amino acid samples, indicating that the amino groups and carboxyl groups of different amino acids have different affinities for Cu 2+ , resulting in different complexation degrees of amino acids with Cu 2+ , and ultimately affecting the color reaction results. For example, the side chain with an amino group in arginine (Arg) may form a more stable complex with Cu 2+ , thus enhancing its contribution in the ninhydrin color reaction. Moreover, due to the different numbers, positions, and electronic environments of the carboxyl groups of different amino acids, the color reaction results will also vary. For example, aspartic acid (Asp) has two acidic functional groups (-COOH), so it can more easily form a complex with Cu 2+ , thereby enhancing the color reaction. And the Asp content in the cucumber, pepino, and Shanghai green samples may be the main factor affecting the bubble size. This shows that through multivariate analysis and multi-dimensional composite processing, the present invention can further achieve information interpretability and identifiability, and complete the dimensionality reduction analysis and accurate output of complex information.

[0131] Example 4

[0132] In this example, the printing amount of the amino acid samples is gradually changed. Specifically, the printing amount is reduced from 100% to 0% at a gradient of 10% to study the color reaction between the chemical reaction chip and the amino acids in the amino acid samples, aiming to clarify the patterns and color development presented by the samples with different amino acid contents loaded on the chemical reaction chip. By comparing the patterns and color development of the samples with different amino acid contents, the differences between the same samples and different samples under different amino acid content conditions are further explored. On the basis of distinguishing the types of amino acid samples, the quantitative analysis of amino acids in the amino acid samples is further realized. The following is a specific description.

[0133] In this example, an Epson inkjet printer is used for printing. First, open the pre-designed pattern (specifically the pattern corresponding to the AC template) in the software, select the "CMYK" mode, and start printing after ensuring that all working indicators are stable. The steps are as follows:

[0134] (1) The first pass of printing is to print the two chemical reaction reagents represented by cyan and yellow in Layer-CY on the PET substrate in sequence. The chemical reaction reagent represented by cyan is Reagent A (CuCl 2Solution, with a concentration of 0.1 mol / L; based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from top to bottom), and the chemical reaction reagent represented by yellow is Reagent C (tartaric acid solution, with a concentration of 0.1 mol / L; based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from left to right).

[0135] (2) The second printing is specifically to print a developer on the PET substrate. The developer is specifically ninhydrin solution (with a concentration of 2 wt%; the entire PET substrate is printed with a printing amount of 100%).

[0136] (3) The third printing is specifically to select Layer-C to print an amino acid sample (specifically taking cucumber as an example) onto the PET substrate. Based on each reaction unit, according to printing template C, the printing amount is uniformly reduced from 100% to 1% at a gradient of 1% from top to bottom, which is equivalent to loading amino acid samples with different amino acid contents in 100 amino acids onto 100 reaction units of the PET substrate, and the amino acid content in the amino acid sample on each reaction unit is different.

[0137] After the above printing is completed, the ink cartridges of the printer are cleaned, and the obtained PET substrate loaded with amino acid samples is placed in an oven and subjected to a color development reaction at a temperature of 105 ± 2 °C for 5 min to obtain a color-developed PET substrate.

[0138] A flatbed scanner is used to simultaneously scan the color development results of the entire color-developed PET substrate to simultaneously obtain the color development results on the color-developed PET substrate, which is convenient for unified calibration, so as to obtain a series of image information. The image information is converted and read for gray values through Python, and finally the gray value (taking the maximum value in this embodiment) of each reaction unit on the color-developed PET substrate is obtained. Through the difference in gray values, the color development situation between the chemical reaction reagent and amino acid samples with different concentrations is reflected. Through the color development situation, the characteristic regions and the optimal chemical reaction reagent combination ratio of amino acid samples under different concentration conditions are obtained. The amino acid samples can be qualitatively analyzed through the characteristic regions, and the amino acid content in the amino acid samples can be quantitatively analyzed through the optimal chemical reaction reagent combination ratio. Through Python, the optimal chemical reaction reagent combination and gray value corresponding to the same amino acid sample under different concentration conditions can be calculated, and the distribution map of characteristic regions between different amino acid samples can also be obtained.

[0139] Example 5

[0140] The amino acid samples used in this embodiment include fruit samples, vegetable samples, traditional Chinese medicine samples, and human urine samples. Different types of amino acid samples are identified, and on this basis, the amino acid concentration in the amino acid samples can also be quantified to further determine the universality and stability of the method of the present invention. The following is a specific description.

[0141] Prepare the color-developing PET substrate according to the method of Example 4, except that the sample solutions used in this embodiment are the sample solutions obtained after pretreatment of fruit samples, vegetable samples, traditional Chinese medicine samples, and human urine samples respectively; then, according to the method of Example 4, use a flatbed scanner to simultaneously scan the color-developing results to extract the color-developing results. Compare different types of amino acid samples according to the color-developing results to obtain a differential analysis method, and further explore the differences between different types of amino acid samples. On the basis of realizing qualitative identification of amino acid sample types, refer to the method of Example 4 to quantify the amino acid concentration in the amino acid samples.

[0142] Example 6

[0143] In this embodiment, the reaction mode between the chemical reaction chip and the amino acid sample is changed. The reaction mode includes printing, modification, or ultramicro reaction, and the specific reactions of different chemical reaction reagent combinations with different types of amino acid samples are studied, so as to further establish a differential analysis of fine amino acid samples. The following is a specific description.

[0144] Operate according to the method of Example 4, except that in this embodiment, printing is carried out according to the following steps:

[0145] (1) The first printing is specifically to print amino acid samples on the PET substrate, including the following situations:

[0146] ① Single-type amino acid samples: Based on each chemical chip, for the amino acid sample represented by blue in Layer-C (specifically the red grape sample solution), the printing amount is uniformly reduced from 100% to 0% (100%-0%) at a gradient of 1% from top to bottom; or for the amino acid sample represented by red in Layer-M (specifically the Shanghai green sample solution), the printing amount is uniformly reduced from 100% to 0% (100%-0%) at a gradient of 1% from left to right; or for the amino acid sample represented by yellow in Layer-Y (specifically the cucumber sample solution), the printing amount is uniformly reduced from 100% to 0% at a gradient of 2% from top to bottom and then uniformly increased from 0% to 100% at a gradient of 2% (100%-0%-100%);

[0147] ② Combinations of two amino acid samples (such as Figure 6As shown in the figure: Referring to the printing method in ①, successively print the amino acid samples represented by red in Layer-M and the amino acid samples represented by yellow in Layer-Y; or successively print the amino acid samples represented by blue in Layer-C and the amino acid samples represented by red in Layer-M; or successively print the amino acid samples represented by blue in Layer-C and the amino acid samples represented by yellow in Layer-Y;

[0148] ③ Three combinations of amino acid samples (such as Figure 6 As shown in the figure: Referring to the printing method in ①, successively print the amino acid samples represented by blue in Layer-C, the amino acid samples represented by red in Layer-M, and the amino acid samples represented by yellow in Layer-Y.

[0149] (2) The second printing is specifically to print the developer represented by red in Layer-M on the PET substrate. The developer is specifically ninhydrin solution (concentration: 2 wt%; the entire PET substrate is printed according to a printing amount of 100%).

[0150] (3) The third printing is specifically to successively print two chemical reaction reagents represented by cyan and yellow in Layer-CY on the PET substrate. The chemical reaction reagent represented by cyan is Reagent A (CuCl 2 solution, concentration: 0.1 mol / L; based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from top to bottom), and the chemical reaction reagent represented by yellow is Reagent C (tartaric acid solution, concentration: 0.1 mol / L; based on each reaction unit, the printing amount is uniformly reduced from 100% to 0% at a gradient of 1% from left to right).

[0151] In this example, PET substrates with different concentration combinations of the same amino acid sample, or PET substrates with the same concentration combination of different amino acid samples, or PET substrates with different concentration combinations of different amino acid samples are obtained through the above printing. Then, the color reaction is carried out according to the method of Example 4, and the color reaction results are simultaneously scanned using a flatbed scanner to extract the color reaction results.

[0152] Figure 7 It is a color reaction result diagram of 12 amino acid samples under the action of a chemical chip (the printing template used is the AC template). Among them, the samples corresponding to numbers 1 to 12 are Red Fuji apples, Huangjin pears, chayote, Pleurotus eryngii, Scutellaria barbata - flower, Scutellaria barbata - root, Forsythia suspensa - old fruit, Forsythia suspensa - young fruit, Liuwei Dihuang Pills, Polygala tenuifolia, Polygonatum cyrtonema, and Polygonatum kingianum. The results show that the color reaction results of the 12 amino acid samples have significant differences, indicating the universality of this chemical chip.

[0153] Example 7

[0154] In this embodiment, different types of amino acid samples are mixed, and the obtained mixed amino acid sample is printed. The color development results on each reaction unit are obtained through Python, so as to further extract the corresponding color reaction characteristics. According to the characteristic reaction result information of CuCl 2 and tartaric acid for a single sample, the color development rule of amino acids in the mixed amino acid sample is further clarified, and the image visual separation and analysis of the mixed amino acid sample are realized. The following is a specific description.

[0155] The mixed amino acid sample used in this embodiment is prepared from grape, cucumber, Shanghaiqing and pepino. Specifically, the grape with a total amino acid content of 0.706 g / 100 g, the cucumber with a total amino acid content of 0.764 g / 100 g, the pepino with a total amino acid content of 0.889 g / 100 g, and the Shanghaiqing with a total amino acid content of 1.669 g / 100 g are juiced respectively, centrifuged at 5000 rpm for 10 min, the supernatant is collected and filtered through a membrane, and the supernatants are mixed according to an equal volume ratio to obtain a mixed amino acid sample solution.

[0156] The method of Example 4 is referred to for operation, with the difference that in this embodiment, printing is carried out according to the following steps:

[0157] (1) The first pass of printing is specifically to print the chemical reagents (CuCl 2 and tartaric acid) on the PET substrate at a printing amount of 100%. Among them, the volume ratios of the CuCl 2 solution (concentration of 0.1 mol / L) to the tartaric acid solution (concentration of 0.1 mol / L) are 80:30, 90:10, 50:10, and 40:10 respectively. And the volume ratio of the chemical reaction reagent solution is changed every 25 prints, that is, in the order from left to right and from top to bottom. Each group forms a 5×5 square array, and finally a 10×10 square array is formed. Specifically, the volume ratio of the CuCl 2 solution to the tartaric acid solution on the 1st to 25th reaction units is 80:30, the volume ratio of the CuCl 2 solution to the tartaric acid solution on the 26th to 50th reaction units is 90:10, the volume ratio of the CuCl 2 solution to the tartaric acid solution on the 51st to 75th reaction units is 50:10, and the volume ratio of the CuCl 2 solution to the tartaric acid solution on the 76th to 100th reaction units is 40:10.

[0158] (2) The second pass of printing is specifically to print the color developer on the PET substrate at a printing amount of 100%. The color developer is specifically a ninhydrin solution (concentration of 2 wt%).

[0159] (3) The third printing is specifically to print the mixed amino acid sample solution on the PET substrate at a printing amount of 100%.

[0160] In this embodiment, PET substrates with the same concentration combination of different amino acid samples or PET substrates with different concentration combinations of different amino acid samples are obtained through the above printing. Then, the color reaction is carried out according to the method of Example 4, and the color development results are simultaneously scanned by a flatbed scanner to extract the color development results. Specifically, the image information obtained by scanning can be converted into grayscale values and read through Python, and finally the grayscale values of each reaction unit on the color-developed PET substrate are obtained; according to the database constructed from the color development results of the PET substrates with the same concentration and different concentration combinations of the same amino acid sample obtained in Example 3 and Example 4, through the difference in grayscale values, the color development situation between the chemical reaction reagent and the mixed amino acid sample is reflected, and the characteristic region of the mixed amino acid sample is obtained through the color development situation. The types of amino acid samples in the mixed amino acid sample are qualitatively determined through the characteristic region, and further separation of the mixed amino acid sample in the color development image is realized.

[0161] Test Example 1

[0162] To verify the repeatability and accuracy of the method of the present invention, the error of the color development results of parallel samples was analyzed in this test example. First, two groups of parallel samples were randomly selected, and each group of samples contained 10 4 data points. For each sample, the relevant data of the corresponding grayscale values were calculated, and the results were statistically analyzed. On this basis, the grayscale value trend diagrams of the parallel samples of four amino acid samples were drawn, and the error changes under different displacements were studied. Figure 8 It is the grayscale value trend diagram of the parallel samples of the 4 amino acid samples in Test Example 1, where A is the grape sample, B is the pepino sample, C is the cucumber sample, and D is the Shanghai green sample. The results show that the accuracy of the method of the present invention is that the errors of the parallel samples of the 4 amino acid samples are all less than 2%. In addition, the Pearson correlation coefficient (Pearson's r) of the parallel samples is greater than 0.99, showing a high degree of consistency of the pixel information between the parallel samples. This indicates that the information overlap degree of the parallel images from the same sample is relatively high at the pixel level, further verifying the high precision and high accuracy of the method of the present invention.

[0163] From the above results, it can be seen that the present invention has developed an innovative analysis method based on high-throughput combinatorial chemistry chips and digital image information analysis technology, which can be used for qualitative and quantitative studies of amino acids in samples such as fruits and vegetables. By combining chemical chip technology, ninhydrin color reaction, and image processing technology, the present invention provides an efficient, economical, and analysis approach without complex sample pretreatment, with significant advantages. Compared with traditional analysis methods, the combinatorial chemistry chip provided by the present invention has the characteristics of high throughput, complete information, and significant differences among samples in amino acid sample analysis. The present invention is based on AC template-CuCl 2 -tartaric acid to construct a chemical chip that can obtain richer amino acid color development information, making different amino acid samples have obvious differences on this combinatorial chemistry chip. It can be obtained through image analysis that the color development result is related to the image gray value, and the higher the gray value, the more significant the color development result. On this basis, the sample information, amino acid content, and amino acid type percentage corresponding to the best color development results of 4 amino acid samples can be obtained, where the combinatorial chemistry conditions are as follows: Red grape-T Amin-acid =0.706%, Glu 0.047 Arg 0.155 Pro 0.140 , the volume ratio of CuCl 2 solution to tartaric acid solution is 80:30; Cucumber-T Amin-acid =0.764%, Asp 0.057 Glu 0.295 Leu 0.047 , the volume ratio of CuCl 2 solution to tartaric acid solution is 90:10; Pepino-T Amin-acid =0.889%, Asp 0.509 Glu 0.115 Lys 0.028 , the volume ratio of CuCl 2 solution to tartaric acid solution is 50:10; Shanghaiqing-T Amin-acid =1.669%, Asp 0.183 Glu 0.318 Leu 0.138 , the volume ratio of CuCl 2The volume ratio of the solution to the tartaric acid solution is 40:10. The above results indicate that the color reaction of amino acids in fruit and vegetable samples is a complex process affected by multiple factors, mainly including sample matrix, amino acid concentration, amino acid type, competitive interaction between ions, and complexation, etc. In addition, the coordination reaction that may form between the amino and carboxyl groups in amino acids and the ions on the chemical reaction chip will also have a significant impact on the color reaction results. The combined effect of these factors makes the chemical reaction chip unique for each amino acid sample. By using image analysis technology to quickly and efficiently extract all the information on the chemical reaction chip and fully analyze the differential characteristics between amino acid samples, it becomes possible to conduct a comprehensive factor analysis of amino acids. Therefore, the high-throughput technology and image analysis technology provided by the present invention enable the chemical reaction chip not only to be limited to the analysis of a few standard samples, but also to be applied to the complex analysis of multiple samples and multiple variables.

[0164] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A chemical reaction chip, comprising a substrate and reaction units arrayed on the substrate, wherein the reaction units are loaded with chemical reaction reagents and color developers, and the chemical reaction reagents include at least two of CuCl2, Na2S2O3 and tartaric acid.

2. The chemical reaction chip according to claim 1, characterized in that: The chemical reaction reagents are CuCl2 and tartaric acid, or CuCl2 and Na2S2O3, or Na2S2O3 and tartaric acid, or CuCl2, Na2S2O3 and tartaric acid.

3. The chemical reaction chip according to claim 1 or 2, characterized in that: When the reaction units of the chemical reaction chip are loaded with CuCl2, the loading amount of CuCl2 in the reaction units in each row is the same, and the loading amount of CuCl2 in the reaction units in each column decreases from top to bottom; When Na2S2O3 is loaded in the reaction units of the chemical reaction chip, the loading amount of Na2S2O3 in the reaction units in each column is the same, and the loading amount of Na2S2O3 in the reaction units in each row decreases from left to right; When tartaric acid is loaded in the reaction units of the chemical reaction chip, the loading amount of tartaric acid in each column of the reaction units is the same, and the loading amount of tartaric acid in each row of the reaction units decreases from left to right; or, the loading amount of tartaric acid in each row of the reaction units is the same, and the loading amount of tartaric acid in each column of the reaction units first decreases and then increases from top to bottom, forming a symmetrical arrangement.

4. The chemical reaction chip according to claim 3, characterized in that: The chemical reaction reagents are CuCl2 and tartaric acid; the loading amount of CuCl2 in the reaction units in each row is the same, and from top to bottom, the loading amount of CuCl2 in the reaction units in each column decreases; the loading amount of tartaric acid in the reaction units in each column is the same, and from left to right, the loading amount of tartaric acid in the reaction units in each row decreases.

5. The chemical reaction chip according to claim 1 or 2, characterized in that: The color developer includes ninhydrin, sodium fluorescein or Coomassie brilliant blue; the substrate includes PET substrate, organic filter membrane or coated paper.

6. The method for preparing the chemical reaction chip according to any one of claims 1 to 5, comprising the following steps: The chemical reaction chip is obtained by printing a chemical reaction reagent solution and a color developer solution on the reaction unit surface of the substrate using a chemical printing method.

7. Use of the chemical reaction chip according to any one of claims 1 to 5 or the chemical reaction chip prepared by the preparation method according to claim 6 in image-based visual detection of amino acids or identification of amino acid samples.

8. A method for detecting amino acids based on image vision, comprising the following steps: Pre-treating the sample to be tested to obtain a test solution; A chemical reaction reagent solution, a color developer solution and a test solution are printed on the reaction unit surface of the substrate by a chemical printing method, and a color development substrate is obtained through a color development reaction; The chemical reaction reagent solution contains at least two of CuCl2, Na2S2O3 and tartaric acid; Obtaining a grayscale value of the reaction unit according to the color development result of the reaction unit on the color development substrate; Qualitative or quantitative detection of amino acids in the sample to be tested is achieved according to the gray value of the reaction unit.

9. A method for identifying amino acid samples based on image vision, comprising the following steps: Pre-treating the sample to be identified to obtain a test solution; A chemical reaction reagent solution, a color developer solution and a test solution are printed on the reaction unit surface of the substrate by a chemical printing method, and a color development substrate is obtained through a color development reaction; The chemical reaction reagent solution contains at least two of CuCl2, Na2S2O3 and tartaric acid; Obtaining a grayscale value of the reaction unit according to the color development result of the reaction unit on the color development substrate; The identification of the sample to be identified is achieved according to the gray value of the reaction unit.

10. The method according to claim 8 or 9, characterized in that: The temperature of the color development reaction is 105±2° C. and the time is 2 to 15 minutes.

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