An in vitro multiplexed detection method and related devices

Abstract

The application relates to the technical field of light-emitting materials, and discloses an in-vitro multiplex detection method and related equipment. The in-vitro multiplex detection method comprises the following steps: obtaining a first photoluminescence spectrum corresponding to a plurality of up-conversion luminescence coding materials and a second photoluminescence spectrum corresponding to a magnetic separation product, obtaining a color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in a LAB color space, and comparing each color difference with a preset threshold value, so that the target type of a sample to be detected can be determined. The first photoluminescence spectra do not need to have different luminescence peak wavelengths, and obvious photoluminescence color differences do not need to be generated. In the multiplex detection application, the type of the up-conversion luminescence coding materials that can be used is greatly increased, and errors caused by human eye judgment and equipment limitations can be effectively avoided, so that the accuracy of the in-vitro multiplex detection is improved.

Description

Technical Field

[0001] This application relates to the field of luminescent materials technology, and more specifically, to an in vitro multiplex detection method and related equipment. Background Technology

[0002] In conventional in vitro diagnostic methods, if multiple targets are involved, the sample needs to be divided into several portions, and each target needs to be tested individually. This significantly increases manpower, time, and reagent costs. Therefore, multiplexing is an inevitable trend in the development of in vitro diagnostics. Multiplexing, by simultaneously performing qualitative or quantitative analysis of multiple proteins or nucleic acids in a single sample, is widely used in disease diagnosis, treatment, monitoring, and prognosis observation. It can effectively help doctors quickly obtain more diagnostic information, thereby enabling precise treatment for patients.

[0003] Currently, there are two main methods for determining the simultaneous detection of multiple indicators using upconversion luminescent materials: (1) Distinguishing by the position of the emission peak: Commonly used rare earth doping methods include Yb / Er, Yb / Tm, and Yb / Ho. These three doping methods will produce three photoluminescence spectra of 550 nm / 660 nm, 480 nm / 650 nm / 700 nm / 800 nm, and 544 nm / 648 nm, respectively. Different indicators can be distinguished according to the different peak shapes and positions. However, since upconversion luminescent materials usually exhibit multi-peak emission, in order to avoid interference between emission peaks, the two doping methods of Yb / Er and Yb / Ho with similar emission peaks are usually not used at the same time. Some researchers have optimized the material preparation process to make upconversion luminescent materials exhibit single-peak emission. For example, existing technologies have adjusted the two rare earth-doped upconversion luminescent materials, Yb / Tm and Yb / Er, to three single-peak emission at 480 nm, 540 nm and 650 nm by coating the outer layer of upconversion luminescent nanoparticles with silica and loading fluorescent dyes, so that they can be applied to the triple detection of intracellular tumor markers; (2) Distinguish by photoluminescence color: Since different rare earth doping types and doping ratios affect the energy transfer process of photons, upconversion luminescent materials exhibit different colors of light emission under near-infrared light excitation, and different indicators can be distinguished in in vitro detection by fluorescence imaging or naked eye observation. For example, existing technology loads three upconversion luminescent materials, NaYF4:Yb / Tm@NaYF4:Er@NaYF4:Yb / Tm, NaYF4:Yb / Er, and NaYF4:Yb / Tm, which can produce red, green, and blue colors under near-infrared light irradiation, onto polystyrene microspheres, conjugates them with antibodies, and applies them to the simultaneous detection of three toxins, AFB1, OTA, and ZEN. The indicators can be distinguished by taking a photoluminescence imaging photo with a mobile phone.

[0004] Since the most efficient and commonly used luminescent center ions in upconversion luminescent materials are only Er3+, Ho3+, and Tm3+, the positions of the emission peaks are fixed and limited. At the same time, due to the influence of material surface defects and test conditions, multiple emission peaks in close proximity may be generated, making peak position determination difficult and resulting in low accuracy of the results. In addition, due to the limitations of the human eye's color discrimination threshold or the influence of image processing methods, obvious color differences are required to be applied to the detection of multiple indicators simultaneously. However, the types of obvious color differences that can be used determine the number of codes. Therefore, it is impossible to verify multiple indicators in the same test.

[0005] There is currently no effective technical solution to the above problems. Summary of the Invention

[0006] The purpose of this application is to provide an in vitro multiplex detection method and related equipment, which can improve the accuracy of detection results and enable upconversion luminescent coding materials to expand the number of codes when applied in multiplex in vitro diagnostics.

[0007] Firstly, this application provides an in vitro multiplex detection method, comprising the following steps:

[0008] A1: Obtain the photoluminescence spectra of various upconversion luminescent coding materials. The photoluminescence spectra of each of the various upconversion luminescent coding materials are different. The photoluminescence spectrum of the upconversion luminescent coding material is denoted as the first photoluminescence spectrum.

[0009] A2: Obtain the photoluminescence spectrum of the magnetic separation product, denoted as the second photoluminescence spectrum. The magnetic separation product is the product extracted from the upconversion luminescent coding material, magnetic particles, and the sample to be tested using a double antibody sandwich method or a sandwich method.

[0010] A3: Obtain the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space;

[0011] A4: By comparing the color differences and preset thresholds, the target type of the sample to be tested is determined.

[0012] This application quantifies the differences between different first photoluminescence spectra by obtaining the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. It does not require the different emission peaks of various first photoluminescence spectra at different wavelengths, nor does it require the generation of obvious differences in photoluminescence colors. In multiplex detection applications, the types of upconversion luminescent coding materials that can be used are greatly increased. Moreover, the Lab color space is widely used in computer vision and is device-independent. Therefore, calculating and comparing the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space can effectively avoid errors caused by human judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0013] Preferably, the magnetic separation product is extracted using a double-antibody sandwich method, specifically including the following steps:

[0014] Different first antibodies were labeled on various upconversion luminescent coding materials;

[0015] Different second antibodies were labeled on each of the magnetic particles;

[0016] A first detection solution is prepared by mixing all the upconversion luminescent coding materials labeled with different first antibodies, all the magnetic particles labeled with different second antibodies, and the sample to be tested. After operating according to the antigen-antibody binding steps, the first detection solution is subjected to magnetic separation to obtain the magnetic separation product containing the magnetic particles.

[0017] Preferably, the magnetic separation product is extracted using a sandwich method, the specific steps of which include:

[0018] Different first nucleic acid probes were labeled on various upconversion luminescent coding materials;

[0019] Different second nucleic acid probes are labeled on each of the magnetic particles;

[0020] A second detection solution is prepared by mixing all the upconversion luminescent coding materials labeled with different first nucleic acid probes, all the magnetic particles labeled with different second nucleic acid probes, and the sample to be tested. After operating according to the nucleic acid hybridization steps, the second detection solution is subjected to magnetic separation to obtain the magnetic separation product containing the magnetic particles.

[0021] Preferably, step A3 includes:

[0022] A301. Convert the various first photoluminescence spectra and second photoluminescence spectra into points on the CIE chromaticity diagram to obtain the corresponding first RGB values ​​and second RGB values;

[0023] A302. Convert each of the first RGB values ​​and the second RGB values ​​into Lab values ​​in the Lab color space, and obtain the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra based on the Lab values.

[0024] Preferably, step A302 includes:

[0025] Convert each of the first RGB values ​​and the second RGB values ​​to XYZ values ​​in the XYZ color space using the following formula:

[0026] ;

[0027] In the formula, R is the R value in the first RGB value or the second RGB value, G is the G value in the first RGB value or the second RGB value, B is the B value in the first RGB value or the second RGB value, X is the X value in the XYZ value, Y is the Y value in the XYZ value, and Z is the Z value in the XYZ value.

[0028] According to the following formula, the XYZ values ​​of each first RGB value and the second RGB value are converted to Lab values ​​in the Lab color space to obtain the corresponding values. value, Value and value:

[0029] ;

[0030] ;

[0031] , , ;

[0032] In the formula, L is the L value in the Lab value. For the Lab value value, For the Lab value value, This is a reference value for the X value in the XYZ values. This is the reference value for the Y value in the XYZ values. t is the reference value of Z in the XYZ numerical values, f is the transformation function, and t is the independent variable of the transformation function;

[0033] According to the L value, the Value and the stated The color difference between the second photoluminescence spectrum and various first photoluminescence spectra is calculated.

[0034] The Lab value is calculated using the above method, thereby calculating the color difference between the second photoluminescence spectrum and various first photoluminescence spectra. This eliminates the need for human eyes or equipment to make judgments, which helps improve the accuracy of in vitro multiplex detection.

[0035] Optionally, the step of basing the L value on the L value ... Value and the stated The steps for calculating the color difference between the second photoluminescence spectrum and various first photoluminescence spectra include:

[0036] Calculate the absolute value deviation of the L value between the second photoluminescence spectrum and each of the first photoluminescence spectra. absolute value deviation and Absolute value deviation;

[0037] Based on the absolute value deviation of the L value, the... absolute value deviation and the aforementioned The absolute value deviation of the color difference is calculated using the following formula:

[0038] ;

[0039] In the formula, The color difference between the second photoluminescence spectrum and the first photoluminescence spectrum. The absolute value deviation of the L value. For the absolute value deviation For the Absolute value deviation.

[0040] The color difference between the second photoluminescence spectrum and various first photoluminescence spectra calculated using the above method does not require the different emission peaks of various first photoluminescence spectra with different wavelengths, nor does it require the generation of obvious differences in photoluminescence color. It can effectively avoid errors caused by human eye judgment and equipment limitations, and is conducive to improving the accuracy of in vitro multiplex detection.

[0041] Optionally, step A1 includes:

[0042] Various upconversion luminescent coding materials are irradiated with illumination light in the wavelength range of 380nm-780nm to obtain the corresponding first photoluminescence spectrum.

[0043] Secondly, this application provides an in vitro multiplex detection device, comprising:

[0044] The first acquisition module is used to acquire the photoluminescence spectra of various upconversion luminescent coding materials. The photoluminescence spectra of each upconversion luminescent coding material are different. The photoluminescence spectrum of the upconversion luminescent coding material is denoted as the first photoluminescence spectrum.

[0045] The second acquisition module is used to acquire the photoluminescence spectrum of the magnetic separation product, denoted as the second photoluminescence spectrum. The magnetic separation product is the product extracted from the upconversion luminescent coding material, magnetic particles and the sample to be tested by a double antibody sandwich method or a sandwich method.

[0046] The third acquisition module is used to acquire the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space;

[0047] The judgment module is used to compare the color differences of each sample with a preset threshold to determine the target type of the sample to be tested.

[0048] Thirdly, this application provides an electronic device including a processor and a memory, the memory storing a computer program executable by the processor, wherein when the processor executes the computer program, it performs the steps of the in vitro multiplex detection method described above.

[0049] Fourthly, this application provides a computer storage medium having a computer program stored thereon, which, when executed by a processor, performs the steps of the in vitro multiplex detection method described above. Beneficial effects

[0050] The in vitro multiplex detection method and related equipment provided in this application acquire the first photoluminescence spectra corresponding to various upconversion luminescent coding materials and the second photoluminescence spectra corresponding to magnetic separation products. The method then acquires the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. By comparing each color difference with a preset threshold, the target type of the sample can be determined. This application quantifies the differences between different first photoluminescence spectra by acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. It eliminates the need for different wavelengths of emission peaks in various first photoluminescence spectra and avoids obvious differences in photoluminescence color. In multiplex detection applications, this significantly increases the types of upconversion luminescent coding materials that can be used. Furthermore, the Lab color space is widely used in computer vision and is device-independent. Therefore, calculating and comparing the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space effectively avoids errors caused by human judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection. Attached Figure Description

[0051] Figure 1 A flowchart of the in vitro multiplex detection method provided in this application.

[0052] Figure 2 This is a schematic diagram of the in vitro multiplex detection device provided in this application.

[0053] Figure 3 A schematic diagram of the structure of the electronic device provided in this application.

[0054] Figure 4 This is a schematic diagram showing the positions of the five first photoluminescence spectra in Example 1 on the CIE 1931 chromaticity diagram.

[0055] Figure 5 This is a schematic diagram comparing the parameters of the five upconversion luminescent coding materials in Example 1.

[0056] Figure 6 This is a schematic diagram of the color difference statistics results in Example 1.

[0057] Figure 7 This is a schematic diagram of the photoluminescence spectrum of the upconversion luminescent coding material in Example 2.

[0058] Figure 8 This is a schematic diagram showing the position of the first photoluminescence spectrum in the CIE1931 chromaticity diagram in Example 2.

[0059] Figure 9 This is a schematic diagram of the color difference statistics results in Example 2.

[0060] Figure 10 This is a schematic diagram showing the comparison of test results in Example 2.

[0061] Labeling Explanation: 1. First Acquisition Module; 2. Second Acquisition Module; 3. Third Acquisition Module; 4. Judgment Module; 301. Processor; 302. Memory; 303. Communication Bus. Detailed Implementation

[0062] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely represents selected embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0063] It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures. Furthermore, in the description of this application, terms such as "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0064] Please refer to Figures 1-10 , Figure 1 This is a flowchart of an in vitro multiplex detection method in the embodiments of this application, which can improve the accuracy of detection results and enable upconversion luminescent coding materials to expand the number of codes when applied in multiplex in vitro diagnostics.

[0065] Firstly, this application provides an in vitro multiplex detection method, comprising the following steps:

[0066] A1: Obtain the photoluminescence spectra of various upconversion luminescent coding materials. The photoluminescence spectra of various upconversion luminescent coding materials are different. The photoluminescence spectrum of the upconversion luminescent coding material is denoted as the first photoluminescence spectrum.

[0067] A2: Obtain the photoluminescence spectrum of the magnetically separated product, denoted as the second photoluminescence spectrum. The magnetically separated product is the product extracted from the upconversion luminescent coding material, magnetic particles, and the sample to be tested using a double antibody sandwich method or a sandwich method.

[0068] A3: Obtain the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space;

[0069] A4: Compare the various color differences and preset thresholds to determine the target type of the sample to be tested.

[0070] The preset threshold can be set according to actual needs.

[0071] Specifically, by acquiring the first photoluminescence spectra corresponding to various upconversion luminescent coding materials and the second photoluminescence spectra corresponding to magnetic separation products, and by acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space, and comparing each color difference with a preset threshold, the target type of the sample to be tested can be determined. This application quantifies the differences between different first photoluminescence spectra by acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. It does not require the difference of different wavelength emission peaks of various first photoluminescence spectra, nor does it require the generation of obvious differences in photoluminescence colors. In multiplex detection applications, the types of upconversion luminescent coding materials that can be used are greatly increased. Moreover, the Lab color space is widely used in computer vision and has device independence. Therefore, calculating and comparing the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space can effectively avoid errors caused by human judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0072] Example 1: Five-fold detection of nucleic acids using color difference differentiation (the material used for auxiliary separation in this example is a fiber membrane; in practical applications, magnetic particles can also be used as auxiliary separation materials, and the specific choice can be made according to actual needs without specific limitations): In this example, the upconversion luminescent coding material is prepared using radially porous silica nanoparticles as templates. Rare earth-doped calcium fluoride nanocrystals are grown in situ in the mesopores, and magnetic particles are synthesized in situ on the surface. Five rare earth doping methods are used: 1. Yb / Tm co-doping (20 / 1 mol%); 2. Yb / Ho co-doping (20 / 1 mol%); 3. Yb / Tm / Tb triple doping (20 / 1 / 10 mol%); 4. Yb / Tm / Ho triple doping (20 / 1 / 1 mol%); 5. Yb / Tm / Eu triple doping (20 / 1 / 10 mol%). The first photoluminescence spectra of the five upconversion luminescent coding materials were tested under 980nm illumination, and the data of the five first photoluminescence spectra were imported into Origin software to obtain the position distribution of the first photoluminescence spectra of the five upconversion luminescent coding materials in the CIE 1931 chromaticity diagram, as shown below. Figure 4 As shown ( Figure 4 The x-axis represents the X-component of the color coordinates, and the y-axis represents the Y-component of the color coordinates. The colors, RGB values, and corresponding upconversion luminescence images of the five upconversion luminescent coding materials are shown below. Figure 5 As shown.

[0073] Five upconversion luminescent coding materials were surface-modified to amino groups using the silane coupling agent APTES, and then modified to carboxyl groups using PAA. Subsequently, they were coupled with five nucleic acid probes (P1, P2, P3, P4, and P5) labeled with 5' NH2-C12 via an amide reaction. The mixture was dispersed in PBS solution and stored at 4°C for later use. A fiber membrane with a graphene oxide layer was prepared. The five upconversion luminescent coding materials labeled with different nucleic acid probes were bound to the fiber membrane through π-π stacking interactions between single-stranded DNA and graphene. After multiple washings of the fiber membrane, it was mixed with the target nucleic acid T1, heated to 95°C for 5 minutes, and then incubated in a 37°C metal bath or shaker for 1 hour. The target nucleic acid T1 and probe P1 have complementary sequences. The supernatant after the reaction was magnetically separated using a magnet, and the second photoluminescence spectrum of the separated magnetically separated product under 980 nm illumination was measured. The above process was repeated five times to obtain five second photoluminescence spectra. The color difference between the second photoluminescence spectra of the five products and the five first photoluminescence spectra was calculated, and the statistical results are as follows: Figure 6 As shown ( Figure 6 The horizontal axis represents the upconversion luminescent coding material, and the vertical axis represents the color difference data. By comparing the color difference with a preset threshold (in this embodiment, the preset threshold is 20), it can be determined that the product obtained by magnetic separation belongs to the Yb / Tm co-doped coding mode, and the corresponding target nucleic acid to be detected is T1 (that is, the target corresponding to T1 is the corresponding target type).

[0074] Example 2: Multiplex nucleic acid detection achieved by homologous rare earth doping:

[0075] Using the same detection method as in Example 1, the rare earth doping mode of the upconversion luminescent coding material was changed to: 1. Yb / Tm co-doping (20 / 1 mol%); 2. Yb / Tm co-doping (20 / 2 mol%); Yb / Tm co-doping (20 / 4 mol%), and represented by Tm1, Tm2, ​​and Tm4, respectively. The photoluminescence spectra (i.e., the first photoluminescence spectrum) of the three doping modes under 980nm illumination are as follows. Figure 7 As shown ( Figure 7 In the graph, the horizontal axis represents wavelength and the vertical axis represents intensity. The emission peaks are all located at 476 nm, 647 nm, and 700 nm. The photoluminescence colors of Tm1, Tm2, ​​and Tm4 are similar, all exhibiting a bluish-violet hue. The distribution of the three sets of first photoluminescence spectra imported into the CIE 1931 chromaticity diagram is shown below. Figure 8 As shown.

[0076] Using the same surface modification and probe labeling methods as in Example 1, three nucleic acid probes P1 / P2 / P3 were labeled on three upconversion luminescent coding materials (Tm1, Tm2, ​​and Tm4), respectively, and then composited with a graphene oxide-containing fiber membrane. The probes were mixed with three concentrations of target nucleic acid T1 (10 μM, 1 μM, and 1 nM), heated to 95°C and held for 5 minutes to denature the nucleic acid, and then incubated on a shaker at 37°C for 1 hour. The supernatant was then magnetically separated, and the second photoluminescence spectrum of the magnetically separated product was measured. After importing the three sets of second photoluminescence spectra into the CIE 1931 chromaticity diagram, the corresponding RGB values ​​of the colors are (100, 97, 255), (69, 71, 255), and (89, 101, 255), respectively. Specifically, the RGB values ​​for Tm1 are (78, 85, 255), Tm2 are (114, 81, 255), and Tm4 are (142, 87, 255). See details below. Figure 10 As shown, the color difference between the three second photoluminescence spectra and the first photoluminescence spectra corresponding to the three upconversion luminescent coding materials was calculated, and the results are as follows. Figure 9 As shown ( Figure 9 The horizontal axis represents the upconversion luminescent coding material, and the vertical axis represents the color difference data. The specific values ​​of the color difference are as follows: Figure 10As shown, in a 10 μM concentration of target nucleic acid T1, the second photoluminescence spectrum of the magnetically separated product was measured, and the color differences calculated between the second photoluminescence spectrum and the first photoluminescence spectra corresponding to Tm1, Tm2, ​​and Tm4 were 6.95, 10.93, and 12.18, respectively. The color difference less than or equal to a preset threshold (set to 10 in this embodiment) is Tm1. Therefore, the target nucleic acid T1 paired with the nucleic acid probe P1 labeled on Tm1 is the target type of the sample to be tested. In a 1 μM concentration of target nucleic acid T1, the second photoluminescence spectrum of the magnetically separated product was measured, and the color differences calculated between the second photoluminescence spectrum and the first photoluminescence spectra corresponding to Tm1, Tm2, ​​and Tm4 were 8.88, 10.20, and 15.88, respectively. The color difference less than or equal to a preset threshold (set to 10 in this embodiment) is Tm1. Therefore, the target nucleic acid T1 paired with the nucleic acid probe P1 labeled on Tm1 is the target type of the sample to be tested. The target nucleic acid T1 is the target type of the sample to be tested. In the target nucleic acid T1 at a concentration of 1 nM, the second photoluminescence spectrum of the magnetically separated product was measured, and the color differences were calculated to be 8.54, 14.71, and 16.10 respectively compared with the first photoluminescence spectra corresponding to Tm1, Tm2, ​​and Tm4. The color difference less than or equal to a preset threshold (set to 10 in this embodiment) is Tm1. Therefore, the target nucleic acid T1 paired with the nucleic acid probe P1 labeled on Tm1 is the target type of the sample to be tested. From the above experimental data, it can be concluded that regardless of the concentration of the target nucleic acid, whether the photoluminescence colors of the upconversion luminescent coding materials are similar, or whether the photoluminescence spectral peak positions are the same, different indicators can still be distinguished. That is, the in vitro multiplex detection method of this application is not limited by the photoluminescence color and photoluminescence spectral peak positions of the upconversion luminescent coding materials. Figure 10 It is evident from the three test results that the color difference between the target nucleic acid T1 and the upconversion luminescent coding material Tm1 is less than the preset threshold (the preset threshold is 10), and there is a significant statistical difference compared with Tm2 and Tm4 (i.e., p≤0.01). Therefore, the in vitro multiplex detection method of this application can distinguish different indicators in multiplex detection when the photoluminescence spectral peak positions of the upconversion luminescent coding materials are the same and the emission colors are similar.

[0077] In some embodiments, the magnetically separated product is extracted using a double-antibody sandwich method, the specific steps of which include:

[0078] Different primary antibodies were labeled on various upconversion luminescent coding materials;

[0079] Different secondary antibodies were labeled on each magnetic particle;

[0080] All upconversion luminescent coding materials labeled with different primary antibodies, all magnetic particles labeled with different secondary antibodies, and the sample to be tested were mixed to prepare a first detection solution. After operating according to the antigen-antibody binding procedure, the first detection solution was magnetically separated to obtain a magnetically separated product containing magnetic particles.

[0081] Specifically, the magnetic separation product containing magnetic particles is obtained by the above-mentioned double antibody sandwich method.

[0082] In some embodiments, the magnetic separation product is extracted using a sandwich method, the specific steps of which include:

[0083] Different first nucleic acid probes were labeled on various upconversion luminescent coding materials;

[0084] Different second nucleic acid probes were labeled on each magnetic particle;

[0085] A second detection solution was prepared by mixing all upconversion luminescent coding materials labeled with different first nucleic acid probes, all magnetic particles labeled with different second nucleic acid probes, and the sample to be tested. After following the nucleic acid hybridization procedure, the second detection solution was magnetically separated to obtain a magnetically separated product containing magnetic particles.

[0086] Specifically, the magnetic separation product containing magnetic particles is obtained by the sandwich method described above.

[0087] In some implementations, step A3 includes:

[0088] A301. Convert various first and second photoluminescence spectra into points on the CIE chromaticity diagram to obtain the corresponding first RGB and second RGB values;

[0089] A302. Convert each of the first RGB values ​​and the second RGB values ​​into Lab values ​​in the Lab color space, and obtain the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra based on the Lab values.

[0090] Specifically, various first and second photoluminescence spectra are first converted (using existing technology, by importing various first and second photoluminescence spectra into Origin software and plotting CIE chromaticity diagrams to obtain their corresponding points on the CIE chromaticity diagram) to points on the CIE chromaticity diagram, thereby obtaining the corresponding first RGB and second RGB values. Then, each first RGB and second RGB value is converted into Lab values ​​in the Lab color space, and the color difference between the second photoluminescence spectrum and various first photoluminescence spectra is obtained based on the Lab values. Since the Lab color space is device-independent, it does not require the different emission peaks of various first photoluminescence spectra at different wavelengths, nor does it require the generation of obvious differences in photoluminescence colors. This effectively avoids errors caused by human eye judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0091] In some implementations, step A302 includes:

[0092] Convert each of the first RGB and second RGB values ​​to XYZ values ​​in the XYZ color space using the following formula:

[0093] ;

[0094] In the formula, R is the R value in the first RGB value or the second RGB value, G is the G value in the first RGB value or the second RGB value, B is the B value in the first RGB value or the second RGB value, X is the X value in the XYZ value, Y is the Y value in the XYZ value, and Z is the Z value in the XYZ value.

[0095] In practical applications, when converting each first RGB value to an XYZ value in the XYZ color space, R, G, and B in the above calculation formula are the R, G, and B values ​​of the first RGB value, respectively. When converting the second RGB value to an XYZ value in the XYZ color space, R, G, and B in the above calculation formula are the R, G, and B values ​​of the second RGB value, respectively.

[0096] According to the following formula, convert the XYZ values ​​of each first RGB value and second RGB value to Lab values ​​in the Lab color space to obtain the corresponding values. value, Value and value:

[0097] ;

[0098] ;

[0099] , , ;

[0100] In the formula, L is the L value in the Lab numerical value. For Lab values value, For Lab values value, It is the reference value for the X value in the XYZ numerical values. It is the reference value for the Y value in the XYZ numerical values. t is the reference value of Z in the XYZ numerical values, f is the transformation function, and t is the independent variable of the transformation function;

[0101] Based on the L value, Value and The color difference between the second photoluminescence spectrum and various first photoluminescence spectra was calculated.

[0102] Specifically, using the above calculation formula, the XYZ values ​​of each first RGB value are converted into Lab values ​​in the Lab color space to obtain the corresponding L values. Value and The value is denoted as the L1 value. Value and Value; convert the XYZ values ​​of the second RGB value to Lab values ​​in the Lab color space to obtain the corresponding L value. Value and The value is denoted as the L2 value. Value and Value, and thus based on L2 value, Value and Values ​​and individual L1 values, Value and The color difference between the second photoluminescence spectrum and various first photoluminescence spectra can be calculated separately, without relying on human eyes or equipment for judgment, which helps to improve the accuracy of in vitro multiplex detection.

[0103] In some implementations, based on the L value, Value and The steps for calculating the color difference between the second photoluminescence spectrum and various first photoluminescence spectra include:

[0104] Calculate the absolute value deviation of L values ​​between the second photoluminescence spectrum and each of the first photoluminescence spectra. absolute value deviation and Absolute value deviation;

[0105] Based on the absolute value deviation of L, absolute value deviation and The absolute value deviation of the color difference is calculated using the following formula:

[0106] ;

[0107] In the formula, The color difference between the second photoluminescence spectrum and the first photoluminescence spectrum. The absolute value deviation of L. for absolute value deviation for Absolute value deviation.

[0108] Specifically, the absolute value deviation of L is calculated using the following formulas. absolute value deviation and absolute value deviation , , This allows for the calculation of the color difference between the second photoluminescence spectrum and various first photoluminescence spectra. It eliminates the need for different wavelengths of emission peaks in the various first photoluminescence spectra, and also eliminates the need for obvious differences in photoluminescence color. This effectively avoids errors caused by human judgment and equipment limitations, and helps improve the accuracy of in vitro multiplex detection.

[0109] In some implementations, step A1 includes:

[0110] Various upconversion luminescent coding materials were irradiated with light in the wavelength range of 380nm-780nm, and the corresponding first photoluminescence spectra were obtained.

[0111] In some implementations, step A2 includes:

[0112] The magnetically separated products were irradiated with light in the wavelength range of 380nm-780nm to obtain a second photoluminescence spectrum.

[0113] In some implementations, step A4 includes:

[0114] By comparing the various color differences with the preset threshold, if the color difference is less than or equal to the preset threshold, the first photoluminescence spectrum with a color difference less than or equal to the preset threshold is taken as the target photoluminescence spectrum. The antibody or probe labeled on the upconversion luminescence coding material corresponding to the target photoluminescence spectrum is taken as the target antibody or target probe. The detection index corresponding to the target antibody or target probe is taken as the target type of the sample to be tested. The detection index refers to the target nucleic acid that can form a base complementary pair with the probe, or the target antigen that can bind to the antibody.

[0115] As can be seen from the above, the in vitro multiplex detection method provided in this application obtains the first photoluminescence spectra corresponding to various upconversion luminescent coding materials and the second photoluminescence spectra corresponding to magnetic separation products, and obtains the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. By comparing each color difference with a preset threshold, the target type of the sample to be tested can be determined. This application quantifies the differences between different first photoluminescence spectra by obtaining the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. It does not require the difference of different wavelength emission peaks of various first photoluminescence spectra, nor does it require the generation of obvious differences in photoluminescence colors. In multiplex detection applications, the types of upconversion luminescent coding materials that can be used are greatly increased. Moreover, the Lab color space is widely used in computer vision and has device independence. Therefore, calculating and comparing the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space can effectively avoid errors caused by human judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0116] Secondly, this application provides an in vitro multiplex detection device, comprising:

[0117] The first acquisition module 1 is used to acquire the photoluminescence spectra of various upconversion luminescent coding materials. The photoluminescence spectra of various upconversion luminescent coding materials are different. The photoluminescence spectrum of the upconversion luminescent coding material is denoted as the first photoluminescence spectrum.

[0118] The second acquisition module 2 is used to acquire the photoluminescence spectrum of the magnetic separation product, denoted as the second photoluminescence spectrum. The magnetic separation product is the product extracted by the double antibody sandwich method or the sandwich method based on the upconversion luminescence coding material, magnetic particles and the sample to be tested.

[0119] The third acquisition module 3 is used to acquire the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space;

[0120] The judgment module 4 is used to compare the various color differences with the preset threshold to determine the target type of the sample to be tested.

[0121] The preset threshold can be set according to actual needs.

[0122] Specifically, by acquiring the first photoluminescence spectra corresponding to various upconversion luminescent coding materials and the second photoluminescence spectra corresponding to magnetic separation products, and by acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space, and comparing each color difference with a preset threshold, the target type of the sample to be tested can be determined. This application quantifies the differences between different first photoluminescence spectra by acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. It does not require the difference of different wavelength emission peaks of various first photoluminescence spectra, nor does it require the generation of obvious differences in photoluminescence colors. In multiplex detection applications, the types of upconversion luminescent coding materials that can be used are greatly increased. Moreover, the Lab color space is widely used in computer vision and has device independence. Therefore, calculating and comparing the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space can effectively avoid errors caused by human judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0123] Example 1: Five-fold detection of nucleic acids using color difference differentiation (the material used for auxiliary separation in this example is a fiber membrane; in practical applications, magnetic particles can also be used as auxiliary separation materials, and the specific choice can be made according to actual needs without specific limitations): In this example, the upconversion luminescent coding material is prepared using radially porous silica nanoparticles as templates. Rare earth-doped calcium fluoride nanocrystals are grown in situ in the mesopores, and magnetic particles are synthesized in situ on the surface. Five rare earth doping methods are used: 1. Yb / Tm co-doping (20 / 1 mol%); 2. Yb / Ho co-doping (20 / 1 mol%); 3. Yb / Tm / Tb triple doping (20 / 1 / 10 mol%); 4. Yb / Tm / Ho triple doping (20 / 1 / 1 mol%); 5. Yb / Tm / Eu triple doping (20 / 1 / 10 mol%). The first photoluminescence spectra of the five upconversion luminescent coding materials were tested under 980nm illumination, and the data of the five first photoluminescence spectra were imported into Origin software to obtain the position distribution of the first photoluminescence spectra of the five upconversion luminescent coding materials in the CIE 1931 chromaticity diagram, as shown below. Figure 4 As shown ( Figure 4 The x-axis represents the X-component of the color coordinates, and the y-axis represents the Y-component of the color coordinates. The colors, RGB values, and corresponding upconversion luminescence images of the five upconversion luminescent coding materials are shown below. Figure 5 As shown.

[0124] Five upconversion luminescent coding materials were surface-modified to amino groups using the silane coupling agent APTES, and then modified to carboxyl groups using PAA. Subsequently, they were coupled with five nucleic acid probes (P1, P2, P3, P4, and P5) labeled with 5' NH2-C12 via an amide reaction. The mixture was dispersed in PBS solution and stored at 4°C for later use. A fiber membrane with a graphene oxide layer was prepared. The five upconversion luminescent coding materials labeled with different nucleic acid probes were bound to the fiber membrane through π-π stacking interactions between single-stranded DNA and graphene. After multiple washings of the fiber membrane, it was mixed with the target nucleic acid T1, heated to 95°C for 5 minutes, and then incubated in a 37°C metal bath or shaker for 1 hour. The target nucleic acid T1 and probe P1 have complementary sequences. The supernatant after the reaction was magnetically separated using a magnet, and the second photoluminescence spectrum of the separated magnetically separated product under 980 nm illumination was measured. The above process was repeated five times to obtain five second photoluminescence spectra. The color difference between the second photoluminescence spectra of the five products and the five first photoluminescence spectra was calculated, and the statistical results are as follows: Figure 6 As shown ( Figure 6 The horizontal axis represents the upconversion luminescent coding material, and the vertical axis represents the color difference data. By comparing the color difference with a preset threshold (in this embodiment, the preset threshold is 20), it can be determined that the product obtained by magnetic separation belongs to the Yb / Tm co-doped coding mode, and the corresponding target nucleic acid to be detected is T1 (that is, the target corresponding to T1 is the corresponding target type).

[0125] Example 2: Multiplex nucleic acid detection achieved by homologous rare earth doping:

[0126] Using the same detection method as in Example 1, the rare earth doping mode of the upconversion luminescent coding material was changed to: 1. Yb / Tm co-doping (20 / 1 mol%); 2. Yb / Tm co-doping (20 / 2 mol%); Yb / Tm co-doping (20 / 4 mol%), and represented by Tm1, Tm2, ​​and Tm4, respectively. The photoluminescence spectra (i.e., the first photoluminescence spectrum) of the three doping modes under 980nm illumination are as follows. Figure 7 As shown ( Figure 7 In the graph, the horizontal axis represents wavelength and the vertical axis represents intensity. The emission peaks are all located at 476 nm, 647 nm, and 700 nm. The photoluminescence colors of Tm1, Tm2, ​​and Tm4 are similar, all exhibiting a bluish-violet hue. The distribution of the three sets of first photoluminescence spectra imported into the CIE 1931 chromaticity diagram is shown below. Figure 8 As shown.

[0127] Using the same surface modification and probe labeling methods as in Example 1, three nucleic acid probes P1 / P2 / P3 were labeled on three upconversion luminescent coding materials (Tm1, Tm2, ​​and Tm4), respectively, and then composited with a graphene oxide-containing fiber membrane. The probes were mixed with three concentrations of target nucleic acid T1 (10 μM, 1 μM, and 1 nM), heated to 95°C and held for 5 minutes to denature the nucleic acids, and then incubated at 37°C in a shaker for 1 hour. The supernatant was then magnetically separated, and the second photoluminescence spectrum of the magnetically separated product was measured. After importing the three sets of second photoluminescence spectra into the CIE 1931 chromaticity diagram, the corresponding RGB values ​​of the colors are (100, 97, 255), (69, 71, 255), and (89, 101, 255), respectively. Specifically, the RGB values ​​for Tm1 are (78, 85, 255), Tm2 are (114, 81, 255), and Tm4 are (142, 87, 255). See details below. Figure 10 As shown, the color difference between the three second photoluminescence spectra and the first photoluminescence spectra corresponding to the three upconversion luminescent coding materials is calculated as follows: Figure 9 As shown ( Figure 9 The horizontal axis represents the upconversion luminescent coding material, and the vertical axis represents the color difference data. The specific values ​​of the color difference are as follows: Figure 10As shown, in a 10 μM concentration of target nucleic acid T1, the second photoluminescence spectrum of the magnetically separated product was measured, and the color differences calculated between the second photoluminescence spectrum and the first photoluminescence spectra corresponding to Tm1, Tm2, ​​and Tm4 were 6.95, 10.93, and 12.18, respectively. The color difference less than or equal to a preset threshold (set to 10 in this embodiment) is Tm1. Therefore, the target nucleic acid T1 paired with the nucleic acid probe P1 labeled on Tm1 is the target type of the sample to be tested. In a 1 μM concentration of target nucleic acid T1, the second photoluminescence spectrum of the magnetically separated product was measured, and the color differences calculated between the second photoluminescence spectrum and the first photoluminescence spectra corresponding to Tm1, Tm2, ​​and Tm4 were 8.88, 10.20, and 15.88, respectively. The color difference less than or equal to a preset threshold (set to 10 in this embodiment) is Tm1. Therefore, the target nucleic acid T1 paired with the nucleic acid probe P1 labeled on Tm1 is the target type of the sample to be tested. The target nucleic acid T1 is the target type of the sample to be tested. In the target nucleic acid T1 at a concentration of 1 nM, the second photoluminescence spectrum of the magnetically separated product was measured, and the color differences were calculated to be 8.54, 14.71, and 16.10 respectively compared with the first photoluminescence spectra corresponding to Tm1, Tm2, ​​and Tm4. The color difference less than or equal to a preset threshold (set to 10 in this embodiment) is Tm1. Therefore, the target nucleic acid T1 paired with the nucleic acid probe P1 labeled on Tm1 is the target type of the sample to be tested. From the above experimental data, it can be concluded that regardless of the concentration of the target nucleic acid, whether the photoluminescence colors of the upconversion luminescent coding materials are similar, or whether the photoluminescence spectral peak positions are the same, different indicators can still be distinguished. That is, the in vitro multiplex detection method of this application is not limited by the photoluminescence color and photoluminescence spectral peak positions of the upconversion luminescent coding materials. Figure 10 It is evident from the three test results that the color difference between the target nucleic acid T1 and the upconversion luminescent coding material Tm1 is less than the preset threshold (the preset threshold is 10), and there is a significant statistical difference compared with Tm2 and Tm4 (i.e., p≤0.01). Therefore, the in vitro multiplex detection device of this application can distinguish different indicators in multiplex detection when the photoluminescence spectral peak positions of the upconversion luminescent coding materials are the same and the emission colors are similar.

[0128] In some embodiments, the magnetically separated product is extracted using a double-antibody sandwich method, the specific steps of which include:

[0129] Different primary antibodies were labeled on various upconversion luminescent coding materials;

[0130] Different secondary antibodies were labeled on each magnetic particle;

[0131] All upconversion luminescent coding materials labeled with different primary antibodies, all magnetic particles labeled with different secondary antibodies, and the sample to be tested were mixed to prepare a first detection solution. After operating according to the antigen-antibody binding procedure, the first detection solution was magnetically separated to obtain a magnetically separated product containing magnetic particles.

[0132] Specifically, the magnetic separation product containing magnetic particles is obtained by the above-mentioned double antibody sandwich method.

[0133] In some embodiments, the magnetic separation product is extracted using a sandwich method, the specific steps of which include:

[0134] Different first nucleic acid probes were labeled on various upconversion luminescent coding materials;

[0135] Different second nucleic acid probes were labeled on each magnetic particle;

[0136] A second detection solution was prepared by mixing all upconversion luminescent coding materials labeled with different first nucleic acid probes, all magnetic particles labeled with different second nucleic acid probes, and the sample to be tested. After following the nucleic acid hybridization procedure, the second detection solution was magnetically separated to obtain a magnetically separated product containing magnetic particles.

[0137] Specifically, the magnetic separation product containing magnetic particles is obtained by the sandwich method described above.

[0138] In some implementations, when the third acquisition module 3 acquires the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space, it specifically performs the following:

[0139] A301. Convert various first and second photoluminescence spectra into points on the CIE chromaticity diagram to obtain the corresponding first RGB and second RGB values;

[0140] A302. Convert each of the first RGB values ​​and the second RGB values ​​into Lab values ​​in the Lab color space, and obtain the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra based on the Lab values.

[0141] Specifically, various first and second photoluminescence spectra are first converted (using existing technology, by importing various first and second photoluminescence spectra into Origin software and plotting CIE chromaticity diagrams to obtain their corresponding points on the CIE chromaticity diagram) to points on the CIE chromaticity diagram, thereby obtaining the corresponding first RGB and second RGB values. Then, each first RGB and second RGB value is converted into Lab values ​​in the Lab color space, and the color difference between the second photoluminescence spectrum and various first photoluminescence spectra is obtained based on the Lab values. Since the Lab color space is device-independent, it does not require the different emission peaks of various first photoluminescence spectra at different wavelengths, nor does it require the generation of obvious differences in photoluminescence colors. This effectively avoids errors caused by human eye judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0142] In some implementations, the third acquisition module 3 includes:

[0143] The first conversion module is used to convert each of the first RGB values ​​and the second RGB values ​​into XYZ values ​​in the XYZ color space according to the following formula:

[0144] ;

[0145] In the formula, R is the R value in the first RGB value or the second RGB value, G is the G value in the first RGB value or the second RGB value, B is the B value in the first RGB value or the second RGB value, X is the X value in the XYZ value, Y is the Y value in the XYZ value, and Z is the Z value in the XYZ value.

[0146] In practical applications, when converting each first RGB value to an XYZ value in the XYZ color space, R, G, and B in the above calculation formula are the R, G, and B values ​​of the first RGB value, respectively. When converting the second RGB value to an XYZ value in the XYZ color space, R, G, and B in the above calculation formula are the R, G, and B values ​​of the second RGB value, respectively.

[0147] The second conversion module is used to convert the XYZ values ​​of each first RGB value and second RGB value into Lab values ​​in the Lab color space according to the following formula, so as to obtain the corresponding values. value, Value and value:

[0148] ;

[0149] ;

[0150] , , ;

[0151] In the formula, L is the L value in the Lab numerical value. For Lab values value, For Lab values value, It is the reference value for the X value in the XYZ numerical values. It is the reference value for the Y value in the XYZ numerical values. t is the reference value of Z in the XYZ numerical values, f is the transformation function, and t is the independent variable of the transformation function;

[0152] The calculation module is used to calculate based on the L value, Value and The color difference between the second photoluminescence spectrum and various first photoluminescence spectra was calculated.

[0153] Specifically, using the above calculation formula, the XYZ values ​​of each first RGB value are converted into Lab values ​​in the Lab color space to obtain the corresponding L values. Value and The value is denoted as the L1 value. 1 value and 1. Convert the XYZ values ​​of the second RGB value to Lab values ​​in the Lab color space to obtain the corresponding L value. Value and The value is denoted as the L2 value. Value and Value, and thus based on L2 value, Value and Values ​​and individual L1 values, Value and The color difference between the second photoluminescence spectrum and various first photoluminescence spectra can be calculated separately, without relying on human eyes or equipment for judgment, which helps to improve the accuracy of in vitro multiplex detection.

[0154] In some implementations, the calculation module calculates based on the L value, Value and When calculating the color difference between the second photoluminescence spectrum and various first photoluminescence spectra, the specific steps are as follows:

[0155] Calculate the absolute value deviation of L values ​​between the second photoluminescence spectrum and each of the first photoluminescence spectra. absolute value deviation and Absolute value deviation;

[0156] Based on the absolute value deviation of L, absolute value deviation and The absolute value deviation of the color difference is calculated using the following formula:

[0157] ;

[0158] In the formula, The color difference between the second photoluminescence spectrum and the first photoluminescence spectrum. The absolute value deviation of L. for absolute value deviation for Absolute value deviation.

[0159] Specifically, the absolute value deviation of L is calculated using the following formulas. absolute value deviation and absolute value deviation , , This allows for the calculation of the color difference between the second photoluminescence spectrum and various first photoluminescence spectra. It eliminates the need for different wavelengths of emission peaks in the various first photoluminescence spectra, and also eliminates the need for obvious differences in photoluminescence color. This effectively avoids errors caused by human judgment and equipment limitations, and helps improve the accuracy of in vitro multiplex detection.

[0160] In some embodiments, when the first acquisition module 1 acquires the photoluminescence spectra of various upconversion luminescent coding materials, and the photoluminescence spectra of each upconversion luminescent coding material are different, and the photoluminescence spectrum of the upconversion luminescent coding material is recorded as the first photoluminescence spectrum, the following specific actions are performed:

[0161] Various upconversion luminescent coding materials were irradiated with light in the wavelength range of 380nm-780nm, and the corresponding first photoluminescence spectra were obtained.

[0162] In some embodiments, when the second acquisition module 2 acquires the photoluminescence spectrum of the magnetic separation product, denoted as the second photoluminescence spectrum, and the magnetic separation product is the product extracted from the upconversion luminescent coding material, magnetic particles, and the sample to be tested using a double antibody sandwich method, the specific steps are as follows:

[0163] The magnetically separated products were irradiated with light in the wavelength range of 380nm-780nm to obtain a second photoluminescence spectrum.

[0164] In some implementations, when determining the target type of the sample to be tested by comparing various color differences with preset thresholds, the judgment module 4 specifically performs the following:

[0165] By comparing the various color differences with the preset threshold, if the color difference is less than or equal to the preset threshold, the first photoluminescence spectrum with a color difference less than or equal to the preset threshold is taken as the target photoluminescence spectrum. The antibody or probe labeled on the upconversion luminescence coding material corresponding to the target photoluminescence spectrum is taken as the target antibody or target probe. The detection index corresponding to the target antibody or target probe is taken as the target type of the sample to be tested. The detection index refers to the target nucleic acid that can form a base complementary pair with the probe, or the target antigen that can bind to the antibody.

[0166] As can be seen from the above, the in vitro multiplex detection device provided in this application can determine the target type of the sample by acquiring the first photoluminescence spectrum corresponding to various upconversion luminescent coding materials and the second photoluminescence spectrum corresponding to the magnetic separation product, and acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. By comparing each color difference with a preset threshold, the target type of the sample can be determined. This application quantifies the differences between different first photoluminescence spectra by acquiring the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space. It does not require the difference of different wavelength emission peaks of various first photoluminescence spectra, nor does it require the generation of obvious differences in photoluminescence color. In multiplex detection applications, the types of upconversion luminescent coding materials that can be used are greatly increased. Moreover, the Lab color space is widely used in computer vision and has device independence. Therefore, calculating and comparing the color difference between the second photoluminescence spectrum and each first photoluminescence spectrum in the Lab color space can effectively avoid errors caused by human judgment and equipment limitations, thereby improving the accuracy of in vitro multiplex detection.

[0167] Please refer to Figure 3 , Figure 3This is a schematic diagram of the structure of an electronic device provided in an embodiment of this application. The electronic device includes a processor 301 and a memory 302. The processor 301 and the memory 302 are interconnected and communicate with each other via a communication bus 303 and / or other connection mechanisms (not shown). The memory 302 stores a computer program executable by the processor 301. When the electronic device is running, the processor 301 executes the computer program to perform the in vitro multiplex detection method in any optional implementation of the above embodiments, to achieve the following functions: acquiring the photoluminescence spectra of various upconversion luminescent coding materials, wherein the photoluminescence spectra of each upconversion luminescent coding material are different, and the photoluminescence spectrum of the upconversion luminescent coding material is denoted as the first photoluminescence spectrum; acquiring the photoluminescence spectrum of a magnetic separation product, denoted as the second photoluminescence spectrum, wherein the magnetic separation product is a product extracted from the upconversion luminescent coding material, magnetic particles, and the sample to be tested using a double antibody sandwich method or a sandwich method; acquiring the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space; comparing each color difference with a preset threshold to determine the target type of the sample to be tested.

[0168] This application provides a storage medium storing a computer program. When the computer program is executed by a processor, it performs an in vitro multiplex detection method in any optional implementation of the above embodiments to achieve the following functions: acquiring the photoluminescence spectra of various upconversion luminescent coding materials, wherein the photoluminescence spectra of each upconversion luminescent coding material are different, and the photoluminescence spectrum of the upconversion luminescent coding material is denoted as the first photoluminescence spectrum; acquiring the photoluminescence spectrum of a magnetically separated product, denoted as the second photoluminescence spectrum, wherein the magnetically separated product is a product extracted from the upconversion luminescent coding material, magnetic particles, and the sample to be tested using a double antibody sandwich method or a sandwich method; acquiring the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra in the Lab color space; comparing each color difference with a preset threshold to determine the target type of the sample to be tested. The storage medium can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as Static Random Access Memory (SRAM), Electrically Erasable Programmable Read-Only Memory (EEPROM), Erasable Programmable Read Only Memory (EPROM), Programmable Red-Only Memory (PROM), Read-Only Memory (ROM), magnetic storage, flash memory, magnetic disk, or optical disk.

[0169] In the embodiments provided in this application, it should be understood that the disclosed apparatus and methods can be implemented in other ways. The apparatus embodiments described above are merely illustrative. For example, the division of units is only a logical functional division, and there may be other division methods in actual implementation. Furthermore, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Additionally, the coupling or direct coupling or communication connection shown or discussed may be through some communication interface; the indirect coupling or communication connection between apparatuses or units may be electrical, mechanical, or other forms.

[0170] Furthermore, the units described as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.

[0171] Furthermore, the functional modules in the various embodiments of this application can be integrated together to form an independent part, or each module can exist independently, or two or more modules can be integrated to form an independent part.

[0172] In this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, without necessarily requiring or implying any such actual relationship or order between these entities or operations.

[0173] The above are merely embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. An in vitro multiplexed detection method, characterized in that, The method comprises the steps of: A1: obtaining photoluminescence spectra of a plurality of upconversion luminescence encoding materials, the photoluminescence spectra of the upconversion luminescence encoding materials being different from each other, and the photoluminescence spectra of the upconversion luminescence encoding materials being recorded as first photoluminescence spectra; A2: obtaining a photoluminescence spectrum of a magnetic separation product, recorded as a second photoluminescence spectrum, the magnetic separation product being extracted from the upconversion luminescence encoding materials, magnetic particles and a sample to be detected by a double antibody sandwich method or a sandwich method; A3: obtaining color differences between the second photoluminescence spectrum and each of the first photoluminescence spectra in a Lab color space; A4: comparing each of the color differences with a preset threshold to determine a target species of the sample to be detected.

2. The in vitro multiplexed detection method of claim 1, wherein, The magnetic separation product is extracted by the double antibody sandwich method, and the specific steps comprise: different first antibodies are labeled on each of the upconversion luminescence encoding materials; different second antibodies are labeled on each of the magnetic particles; all the upconversion luminescence encoding materials labeled with different first antibodies, all the magnetic particles labeled with different second antibodies and the sample to be detected are mixed to prepare a first detection solution, and after operation according to the steps of antigen-antibody combination, the first detection solution is subjected to magnetic separation to obtain the magnetic separation product with the magnetic particles.

3. The in vitro multiplexed detection method of claim 1, wherein, The magnetic separation product is extracted by the sandwich method, and the specific steps comprise: different first nucleic acid probes are labeled on each of the upconversion luminescence encoding materials; different second nucleic acid probes are labeled on each of the magnetic particles; all the upconversion luminescence encoding materials labeled with different first nucleic acid probes, all the magnetic particles labeled with different second nucleic acid probes and the sample to be detected are mixed to prepare a second detection solution, and after operation according to the steps of nucleic acid hybridization, the second detection solution is subjected to magnetic separation to obtain the magnetic separation product with the magnetic particles.

4. The in vitro multiplexed detection method of claim 1, wherein, Step A3 comprises: A301. converting each of the first photoluminescence spectra and the second photoluminescence spectrum into a point on a CIE chromaticity diagram to obtain corresponding first RGB values and second RGB values; A302. converting each of the first RGB values and the second RGB values into Lab values in a Lab color space, respectively, and obtaining color differences between the second photoluminescence spectrum and each of the first photoluminescence spectra according to the Lab values.

5. The in vitro multiplexed detection method of claim 4, wherein, Step A302 comprises: each of the first RGB values and the second RGB values is converted into XYZ values in an XYZ color space according to the following formula: ； wherein R is an R value in the first RGB value or the second RGB value, G is a G value in the first RGB value or the second RGB value, B is a B value in the first RGB value or the second RGB value, X is an X value in the XYZ value, Y is a Y value in the XYZ value, and Z is a Z value in the XYZ value; The XYZ values of each first RGB value and the second RGB value are converted into Lab values of a Lab color space, respectively, to obtain corresponding values, values and values according to the following formulas: ； ； , , ； wherein L is the L value in the Lab values, is the L value in the Lab values, value, is the L value in the Lab values, value, is the reference value for the X value in the XYZ values, is the reference value for the Y value in the XYZ values, is the reference value for the Z value in the XYZ values, f is a conversion function, and t is an argument of the conversion function. The color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra is calculated based on the L value, the a value and the b value. The color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra is calculated based on the L value, the a value and the b value. The color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra is calculated based 6. The in vitro multiplexed assay method according to claim 5, wherein, said step of calculating the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra comprises: said step of calculating the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra comprises: said step of calculating the color difference between the second photoluminescence spectrum and each of the first photoluminescence spectra comprises: calculating an L-value absolute value deviation between the second photoluminescence spectrum and each of the first photoluminescence spectra, value absolute value deviation and value absolute value deviation; According to the L value absolute value deviation, the value absolute value deviation and the value absolute value deviation, the color difference is calculated by the following formula: ； wherein is the color difference between the second photoluminescence spectrum and the first photoluminescence spectrum, is the absolute value deviation of the L value, is the absolute value deviation of the value absolute value deviation, is the absolute value deviation of the value absolute value deviation.

7. The in vitro multiplexed detection method of claim 1, wherein, Step A1 comprises: The upconversion luminescence encoding materials are irradiated by irradiation light with a wavelength range of 380-780 nm to obtain corresponding first photoluminescence spectra.

8. A device for in vitro multiplex detection, characterized in that The application further provides a computer program product, which comprises a computer program and a computer readable storage medium. The first obtaining module is configured to obtain photoluminescence spectra of a plurality of upconversion luminescence encoding materials, wherein the photoluminescence spectra of the upconversion luminescence encoding materials are different from each other, and the photoluminescence spectra of the upconversion luminescence encoding materials are recorded as first photoluminescence spectra. The second obtaining module is configured to obtain a photoluminescence spectrum of a magnetic separation product, which is recorded as a second photoluminescence spectrum, and the magnetic separation product is extracted by a double antibody sandwich method or a sandwich method according to the upconversion luminescence encoding materials, magnetic particles and a sample to be detected. The third obtaining module is configured to obtain color differences between the second photoluminescence spectrum and each of the first photoluminescence spectra in a Lab color space. The judging module is configured to compare each of the color differences with a preset threshold value to determine a target type of the sample to be detected.

9. An electronic device, comprising: The computer program is executed by the processor to run the steps in the in vitro multiplex detection method according to any one of claims 1-7.

10. A computer storage medium having stored thereon a computer program, characterized in that The computer program is executed by the processor to run the steps in the in vitro multiplex detection method according to any one of claims 1-7.

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