Fluorescent reagent for detecting tumor markers and preparation method thereof, method for constructing fluorescent sensor array sensing unit and application thereof

Abstract

The present invention provides a fluorescent reagent for detecting tumor markers, a preparation method thereof, a method for constructing a fluorescent sensor array sensing unit, and an application thereof. The fluorescent reagent includes a probe composed of one or more carbon dots including at least three sites of amino, carboxyl, hydroxyl or sulfhydryl groups, or a probe constructed by combining carbon dots with IRMOF‑3. Its preparation method includes a hydrothermal method, a microwave method or a method of combining carbon dots with IRMOF‑3. The fluorescent reagent is used to construct a fluorescent sensor array sensing unit and is used to quantitatively detect the concentration of tumor markers in the blood of unknown samples, and to verify whether it exceeds a threshold value to infer whether the patient is a tumor patient. The actual blood sample consumption is low, the operation is simple, and the detection efficiency is greatly improved. The present invention can simultaneously distinguish multiple tumor markers, such as carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 15‑3 (CA15‑3), alpha-fetoprotein (AFP), etc., and the required sample amount is small and the detection time is short.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid screening of tumor markers, and in particular relates to a fluorescent reagent for detecting tumor markers, a preparation method thereof, and a method for constructing a fluorescent sensor array sensing unit and its application. Background Art

[0002] With the rapid development of the modern economy, people's concern for health is increasing year by year. Cancer, the leading cause of death in today's society, has been a hot topic of research for scientists worldwide. Tumor markers are substances that are secreted and synthesized by tumor cells during their growth and proliferation through their own endogenous gene expression, or that are secreted or increased in response to abnormal expression by the body. Therefore, people can monitor and differentiate tumor markers to conduct relevant biomedical research.

[0003] Traditional methods for analyzing and detecting proteins mainly include two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Among them, as the most commonly used method, in ELISA, proteins are measured by reacting the corresponding antigen-antibody on the surface of a carrier. The classic protein sensing scheme using ELISA is to immobilize high-affinity and specific receptors on the surface of a matrix (such as polystyrene), bind to the analyzed protein, and then use a secondary receptor to bind to a colorimetric or fluorescent substrate to generate a detectable signal. Since small molecular weight proteins are not easy to coat on the surface of the matrix, they are usually coupled to a carrier protein. However, the preparation and purification process of protein conjugates is complicated and requires high technical skills from the experimenter.

[0004] Fluorescent carbon dots have been extensively studied in the field of sensing due to their excellent optical properties, including strong photostability and high quantum yield, as well as their inherent low toxicity, good biocompatibility, and environmental friendliness. Their ease of preparation and ease of preparation have led to their widespread application in the field of sensing. However, developing a rapid and simple fluorescent sensor array approach for the simultaneous detection and differentiation of multiple tumor markers remains a pressing technical challenge.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a fluorescent reagent for detecting tumor markers and a preparation method thereof, a method for constructing a fluorescent sensor array sensing unit and its application, so as to solve the above problems.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a fluorescent reagent for detecting tumor markers, comprising at least three of the following probes:

[0009] a. A probe comprising one or more carbon dots containing one or more amino, carboxyl, hydroxyl or sulfhydryl groups;

[0010] b. Probe constructed by combining carbon dots with IRMOF-3.

[0011] Furthermore, the tumor markers include one or more of carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 15-3 (CA15-3) or alpha-fetoprotein (AFP).

[0012] In a second aspect, the present invention further provides a method for preparing the fluorescent reagent for detecting tumor markers, comprising preparing the following carbon dots or probes and selecting at least three of the carbon dots and / or probes for combination:

[0013] c. Preparation of carbon dots by hydrothermal or microwave method;

[0014] d. Probe constructed by combining synthetic carbon dots with IRMOF-3.

[0015] In a third aspect, the present invention further provides a method for constructing a fluorescent sensor array sensing unit using the fluorescent reagent: adding different concentrations of any one or more tumor markers to the three probes of the fluorescent reagent and mixing them evenly;

[0016] Preferably, several proteins and any one or more tumor markers are added to the three probes of the fluorescent reagent and mixed evenly;

[0017] Preferably, the several proteins include one or more of horseradish peroxidase, ovalbumin, lysozyme, trypsin, pepsin, myoglobin, papain, cytochrome C, bovine serum albumin, human hemoglobin or bovine hemoglobin.

[0018] In a fourth aspect, the present invention further provides an application of a fluorescent sensor array sensing unit constructed by the method as described above: adding any one or more tumor markers of different concentrations to PBS buffer and mixing evenly, or adding several proteins and any one or more tumor markers to PBS buffer and mixing evenly; using normalized data F1 / F0, where F1 is the fluorescence value after adding the tumor marker and F0 is the fluorescence value of the carbon dots themselves; using the obtained fingerprint data to draw a two-dimensional linear discriminant analysis LDA graph; linearizing Factor (1) in the LDA graph with the tumor marker concentration to calculate the tumor marker content in the unknown actual blood sample;

[0019] Preferably, the concentration of the tumor marker in the blood of an unknown sample is quantitatively detected by comparing the linearity with the corresponding Factor (1) in the LDA graph of the actual blood, and verifying whether it exceeds the threshold value to infer whether the patient is a tumor patient.

[0020] Compared to existing technologies, the present invention uses simple, homemade fluorescent sensing reagents as sensing units to construct a fluorescent sensor array. This allows for simultaneous differentiation of multiple tumor markers or cancer patient blood, quantitatively detecting the concentration of the tumor marker in an unknown blood sample, verifying whether it exceeds a threshold, and inferring whether the patient has a cancer. This approach uses a low amount of actual blood sample and is simple to operate, significantly improving detection efficiency for tumor marker differentiation and cancer patient detection. The present invention can simultaneously differentiate multiple tumor markers, such as carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 15-3 (CA15-3), and alpha-fetoprotein (AFP), requiring minimal sample volume and shortening detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 This is a transmission electron microscopy photograph of carbon dots with glutathione (GSH) as a precursor obtained in Example 1;

[0023] Figure 2 This is a linear discriminant analysis (LDA) graph drawn in Example 1 when 11 proteins and the tumor marker CEA were added;

[0024] Figure 3 This is a linear discriminant analysis (LDA) graph drawn in Example 2 when 11 proteins and two tumor markers, CEA and CA125, were added;

[0025] Figure 4 middle Figure 4 A is a linear discriminant analysis (LDA) graph drawn in Example 1 when different concentrations of the tumor marker CEA were added; Figure 4 B is a linear graph of Factor (1) in the LDA graph and the tumor marker concentration.

[0026] Figure 5 This is the linear discriminant analysis (LDA) graph of Example 1 with blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) and healthy people (C1, C2) added.

[0027] Figure 6 This is the linear discriminant analysis (LDA) diagram for distinguishing different tumor markers in Example 11. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] It should be noted that the proportions, values ​​and units in the embodiments are exemplary, especially the values ​​and units, which are experimental data and are not intended to limit the scope of protection. Those skilled in the art are aware that they can be converted into values ​​and units for industrial production.

[0030] The present invention provides a fluorescent reagent for detecting tumor markers, comprising at least three of the following probes:

[0031] a. A probe comprising one or more carbon dots containing one or more amino, carboxyl, hydroxyl or sulfhydryl groups;

[0032] b. Probe constructed by combining carbon dots with IRMOF-3.

[0033] Optionally, the tumor markers include one or more of carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 15-3 (CA15-3), and alpha-fetoprotein (AFP).

[0034] The present application also provides a method for preparing a fluorescent reagent for detecting tumor markers, comprising preparing the following carbon dots or probes and selecting at least three of the carbon dots and / or probes for combination:

[0035] c. Preparation of carbon dots by hydrothermal or microwave method;

[0036] d. Probe constructed by combining synthetic carbon dots with IRMOF-3.

[0037] Furthermore, with exemplary numerical values ​​and units, the preparation of carbon dots by the hydrothermal method may be the following representative method:

[0038] For example, one method involves adding 0.4-0.6g of glutathione (GSH) to 5-20mg of Congo red (CR) and 0.1-0.3g of ammonium persulfate (APS) and dissolving them in 10-50mL of deionized water. The solution is sonicated for 5-20 minutes until completely dissolved, then transferred to an autoclave, heated in an oven to 160-220°C, reacted for 4-8 hours, and cooled to room temperature. Impurities are removed by filtration using a 0.22μm microporous membrane, and the solution is dialyzed using a 1000Da dialysis bag for 1-3 days. The dialyzed carbon dot solution is then placed in a refrigerator at 4°C until ready for use.

[0039] For example, another method involves adding 5-20 mg of Congo red (CR) and 0.1-0.3 g of ammonium persulfate (APS) to 0.2-0.4 g of o-phenylenediamine (o-PD) and dissolving it in 10-50 mL of deionized water. The solution is sonicated for 5-20 minutes until completely dissolved, then transferred to an autoclave, heated in an oven to 160-220°C, reacted for 4-8 hours, and cooled to room temperature. Impurities are removed by filtration through a 0.22 μm microporous membrane, and the solution is dialyzed using a 1000 Da dialysis bag for 1-3 days. The dialyzed carbon dot solution is then placed in a refrigerator at 4°C until ready for use.

[0040] For example, another method involves adding 5-20 mg of Congo red (CR) and 0.1-0.3 g of ammonium persulfate (APS) to 0.8-1.2 g of malic acid (MA) and dissolving it in 10-50 mL of deionized water. The solution is sonicated for 5-20 minutes until completely dissolved, then transferred to an autoclave, heated in an oven to 160-220°C, reacted for 4-8 hours, and cooled to room temperature. Impurities are removed by filtration through a 0.22 μm microporous membrane, and the solution is dialyzed using a 1000 Da dialysis bag for 1-3 days. The dialyzed carbon dot solution is then placed in a refrigerator at 4°C until ready for use.

[0041] Furthermore, the preparation of carbon dots by microwave method comprises:

[0042] For example, 0.4-0.6g of glutathione (GSH) is dissolved in 10-50mL of deionized water by adding 5-20mg of Congo red (CR) and 0.1-0.3g of ammonium persulfate (APS). This solution is placed in a 100mL beaker and placed in a microwave oven with the power set to medium-high for 5-10 minutes. After the reaction is complete, the solution is cooled to room temperature, 5-10mL of deionized water with a pH of 6 is added, and sonication is performed for 5-20 minutes to dissolve the resulting colloid. Impurities are removed by filtration using a 0.22μm filter membrane. The filtered solution is pale yellow. The solution is then dialyzed for 1-3 days using a 1000Da dialysis bag. The dialyzed carbon dot solution is then placed in a refrigerator at 4°C until ready for use.

[0043] For example, another method involves adding 5-20 mg of Congo red (CR) and 0.1-0.3 g of ammonium persulfate (APS) to 0.2-0.4 g of o-phenylenediamine (o-PD) and dissolving them in 10-50 mL of deionized water. The solution is placed in a 100 mL beaker and placed in a microwave oven with the power set to medium-high for 5-10 minutes. After the reaction is complete, the solution is cooled to room temperature, 5-10 mL of deionized water with a pH of 6 is added, and ultrasonication is performed for 5-20 minutes to dissolve the resulting colloid. Impurities are removed by filtration using a 0.22 μm filter membrane, and the filtered solution is light yellow. The solution is then dialyzed for 1-3 days using a 1000 Da dialysis bag. The dialyzed carbon dot solution is then placed in a refrigerator at 4°C until ready for use.

[0044] For example, another method is to add 5-20mg of Congo red (CR) and 0.1-0.3g of ammonium persulfate (APS) to 0.8-1.2g of malic acid (MA) and dissolve it in 10-50mL of deionized water. The above solution is placed in a 100mL beaker and placed in a microwave oven with the power set to medium-high and the microwave time set to 5-10min. After the reaction is completed, cool to room temperature, add 5-10mL of deionized water with a pH of 6, and sonicate for 5-20min to dissolve the colloid formed after the reaction. Filter with a 0.22μm filter membrane to remove impurities. The filtered solution is light yellow. Then dialyze for 1-3 days using a 1000Da dialysis bag. The dialyzed carbon dot solution is placed in a refrigerator at 4°C for use.

[0045] It should be noted that the method for preparing carbon dots is not limited to the above examples. Glutathione (GSH), o-phenylenediamine (o-PD), and malic acid (MA) carbon dots can all be prepared by hydrothermal or microwave methods.

[0046] Furthermore, as a parallel technical solution, the probe constructed by combining the synthesized carbon dots with IRMOF-3 can be: 1.5-2.5g of o-PD is thoroughly dissolved in 50-80mL of DMF by ultrasonication, transferred to a 25mL reactor, and heated in a 160-220°C oven for 4-8 hours. After the reaction is complete, the reaction is allowed to stand until it cools to room temperature. The reactor is opened to obtain a dark brown solution, which is filtered through a 0.22μm filter to remove impurities. The solution is then dialyzed using a 1000Da dialysis bag for 1-3 days. The dialyzed carbon dot solution is placed in a refrigerator at 4°C until ready for use. Alternatively, the solution is placed in a 100mL beaker and microwaved at medium-high power for 5-10 minutes. After the reaction is complete, cool to room temperature, add 5-10mL of deionized water with a pH of 6, and ultrasonicate for 5-20 minutes to dissolve the resulting colloid. Filter through a 0.22μm filter to remove impurities, and the filtered solution is light yellow. The solution was then dialyzed using a 1000Da dialysis bag for 1-3 days. The dialyzed carbon dot solution was then placed in a refrigerator at 4°C until ready for use. 400-600mg of Zn(NO3)2·6H2O and 100-300mg of 2-aminoterephthalic acid were dissolved in 5-20mL of o-PD-CDs, followed by the addition of 1-3mL of triethylamine and stirring at room temperature for 1-3 days. The product was washed 1-3 times with DMF and then transferred to a 70°C vacuum oven for drying and activation.

[0047] As a parallel technical solution, the synthetic carbon dots combined with IRMOF-3 can also be constructed by combining the following: 1.0-3.0g of Zn(NO3)2·6H2O and 0.8-1.2g of 2-aminoterephthalic acid are mixed and dissolved in 80-120mL of DMF using magnetic stirring to obtain a clear mixed solution. 3-8mL of triethylamine is then slowly added dropwise to the mixed solution, instantly producing a white precipitate. The mixture is then stirred at room temperature for 2-4 hours to obtain the product IRMOF-3. The product is washed three times with DMF and then transferred to a 70°C vacuum drying oven for drying and activation. 80-120 mg of IRMOF-3 was dissolved in 10-30 mL of DMF. After complete dissolution, 0.4-0.8 g of o-PD was added and stirred at room temperature for 1-3 days. The mixture was transferred to a 25 mL reactor and placed in an oven at 160-220°C for 4-8 hours. After the reaction was completed, the mixture was allowed to stand until it cooled to room temperature. The reactor was opened to obtain a dark brown solution. Impurities were removed by filtering with a 0.22 μm disposable filter membrane and the solution was transferred to a dialysis bag (MW = 1000 Da) for further purification for 1-3 days. The solution was then placed in a refrigerator at 4°C for later use.

[0048] As a parallel technical solution, the probe constructed by combining the synthetic carbon dots with IRMOF-3 can also be: 100-300 mg o-PD, 400-600 mg Zn(NO3)2·6H2O and 100-300 mg 2-aminoterephthalic acid are completely dissolved in 20-50 mL DMF, stirred at room temperature for 1-3 days, transferred to a 100 mL reactor, placed in a 160-220 ° C oven to react for 4-8 hours, and after the reaction is completed, allowed to stand until cooled to room temperature, the reactor is opened to obtain a dark brown solution, which is filtered using a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 1-3 days, and placed in a 4 ° C refrigerator for use.

[0049] The present application also provides a method for constructing a fluorescent sensor array sensing unit using the fluorescent reagent: adding 50-80 μL of any one or more tumor markers at different concentrations (0.025-1 ng / mL) to 50-80 μL of the three probes and mixing them evenly (incubating at 37°C), and measuring the fluorescence emission spectrum of each sample (five parallel samples are required for each group of samples).

[0050] Preferably, 50-80 μL of several different proteins and any one or more tumor markers are added to 50-80 μL of the three probes and mixed evenly (incubated at 37° C.), and the fluorescence emission spectrum of each sample is measured (five parallel samples are required for each group of samples).

[0051] Optionally, the several proteins include one or more of horseradish peroxidase (HRP), ovalbumin (Alb), lysozyme (Lys), trypsin (Try), pepsin (Pep), myoglobin (Myo), papain (Pap), cytochrome C (Cyt-C), bovine serum albumin (BSA), human hemoglobin (Hb) and bovine hemoglobin (Hem).

[0052] The present application also provides an application of a fluorescent sensor array sensing unit constructed by the method described above: adding 50-80 μL of any one or more tumor markers at different concentrations (0.025-1 ng / mL) to 50-80 μL PBS buffer (pH = 7.4, 25 mM) and mixing uniformly (incubating at 37°C), and measuring the fluorescence emission spectrum of each sample (five parallel samples are required for each group of samples); or adding 50-80 μL of several different proteins and any one or more tumor markers to 50-80 μL PBS buffer (pH = 7.4, 25 mM) and mixing uniformly (incubating at 37°C), and measuring the fluorescence emission spectrum of each sample (five parallel samples are required for each group of samples). Normalized data F1 / F0 is used, where F1 is the fluorescence value after adding the tumor marker and F0 is the fluorescence value of the carbon dots themselves; an LDA graph is drawn using the obtained fingerprint data; and a linear relationship is drawn between Factor (1) in the LDA graph and the tumor marker concentration to calculate the tumor marker content in the unknown actual blood sample.

[0053] Furthermore, the linearity is compared with the corresponding Factor (1) of the actual blood in the LDA graph to quantitatively detect the concentration of the tumor marker in the unknown sample blood, and to verify whether it exceeds the threshold, to infer whether the patient is a tumor patient.

[0054] Example 1

[0055] 5 mg Congo red (CR) and 0.1 g ammonium persulfate (APS) were added to 0.4 g glutathione (GSH) and 0.4 g o-phenylenediamine (o-PD) and dissolved in 10 mL deionized water. The solution was ultrasonicated for 5 minutes until completely dissolved and then transferred to a high-pressure reactor, which was placed in an oven and heated to 160°C. The reaction was performed for 8 hours and cooled to room temperature. The impurities were removed by filtering with a 0.22 μm microporous filter membrane, and the solution was dialyzed with a 1000 Da dialysis bag for 1 day. The dialyzed carbon dot solution was placed in a refrigerator at 4°C for use, and two types of carbon dots containing different functional groups were obtained. Among them, the transmission electron microscope photo of GSH-CR-CDs carbon dots is shown in FIG. Figure 1 shown.

[0056] 0.8g malic acid (MA) was dissolved in 10mL of deionized water, along with 5mg of Congo red (CR) and 0.1g of ammonium persulfate (APS). The solution was placed in a 100mL beaker and microwaved at a medium power setting (450W) for 5 minutes. After the reaction, the mixture was cooled to room temperature and 10mL of deionized water (pH 6) was added. Ultrasonication was performed for 5 minutes to dissolve the colloid formed after the reaction. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was light yellow. The solution was then dialyzed for one day using a 1000Da dialysis bag. The dialyzed carbon dot solution was then placed in a refrigerator at 4°C until ready for use.

[0057] To 50 μL of the three aforementioned probes and 50 μL of PBS buffer (pH = 7.4, 25 mM), 50 μL of 11 different proteins (horseradish peroxidase (HRP), ovalbumin (Alb), lysozyme (Lys), trypsin (Try), pepsin (Pep), myoglobin (Myo), papain (Pap), cytochrome C (Cyt-C), bovine serum albumin (BSA), human hemoglobin (Hb), and bovine hemoglobin (Hem)) and CEA were added and mixed (incubation was performed at 37°C). The fluorescence emission spectrum of each sample was measured (five replicates were measured for each sample group). The normalized data F1 / F0 was used, where F1 is the fluorescence value after the addition of the tumor marker CEA and F0 is the fluorescence value of the carbon dots themselves. The obtained fingerprint data was input into SYSTAT 12.5 software to draw the LDA graph, as shown in Figure 2. Figure 2 shown.

[0058] To 50 μL of each of the three probes and 50 μL of PBS buffer (pH = 7.4, 25 mM), 50 μL of CEA at different concentrations (0.025-1 ng / mL) was added and mixed (incubated at 37°C). The fluorescence emission spectrum of each sample was measured (five parallel samples were measured for each group of samples). The normalized data F1 / F0 was used, where F1 is the fluorescence value after adding the tumor marker CEA and F0 is the fluorescence value of the carbon dots themselves. The obtained fingerprint data was input into SYSTAT 12.5 software to draw the LDA graph, as shown in Figure 2. Figure 4 As shown in A.

[0059] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient. Figure 4 As shown in B.

[0060] Add 50 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 50 μL of PBS buffer to each of the three probes (50 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0061] Normalized data F1 / F0 is used, where F1 is the fluorescence value after adding tumor markers and F0 is the fluorescence value of carbon dots themselves. The obtained fingerprint data is input into SYSTAT 12.5 software to draw the LDA graph, as shown in the following figure: Figure 5 As shown in Figure 2, the presence or absence of excessive tumor markers in the blood is determined based on the area of ​​the sample in the LDA graph that is subjected to linear discriminant analysis.

[0062] Example 2

[0063] 0.5g of glutathione (GSH) and 0.2g of o-phenylenediamine (o-PD) were added with 10mg of Congo red (CR) and 0.3g of ammonium persulfate (APS) and dissolved in 30mL of deionized water. The solution was ultrasonicated for 10 minutes until completely dissolved and then transferred to an autoclave. The autoclave was heated to 220°C in an oven, reacted for 4 hours, and cooled to room temperature. Impurities were removed by filtration using a 0.22μm microporous filter membrane and dialyzed using a 1000Da dialysis bag for 2 days. The dialyzed carbon dot solution was placed in a refrigerator at 4°C for use. Two types of carbon dots containing different functional groups were obtained.

[0064] 1.0 g of Zn(NO₃)₂·6H₂O and 1.2 g of 2-aminoterephthalic acid were mixed and dissolved in 80 mL of DMF using magnetic stirring to obtain a clear mixed solution. 8 mL of triethylamine was slowly added dropwise to the mixed solution, instantly producing a white precipitate. Stirring was continued at room temperature for 2 h to obtain the product, IRMOF-3. The product was washed three times with DMF and then transferred to a 70°C vacuum drying oven for drying and activation. 80 mg of IRMOF-3 was dissolved in 10 mL of DMF. Once completely dissolved, 0.4 g of o-PD was added and stirred at room temperature for 1 day. The product was then transferred to an autoclave and placed in a 160°C oven for 8 h. After the reaction was complete, the product was allowed to cool to room temperature. The autoclave was opened to obtain a dark brown solution. It was filtered through a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 1 day. The solution was then refrigerated at 4°C until ready for use.

[0065] 60 μL of 11 different proteins (horseradish peroxidase (HRP), ovalbumin (Alb), lysozyme (Lys), trypsin (Try), pepsin (Pep), myoglobin (Myo), papain (Pap), cytochrome C (Cyt-C), bovine serum albumin (BSA), human hemoglobin (Hb), and bovine hemoglobin (Hem)), CEA, and CA125 were added to 60 μL of three probes and 60 μL of PBS buffer (pH = 7.4, 25 mM) and mixed evenly (incubation was performed at 37°C). The fluorescence emission spectrum of each sample was measured (5 replicates were measured for each sample group). The normalized data F1 / F0 was used, where F1 is the fluorescence value after adding the tumor marker CEA or CA125, and F0 is the fluorescence value of the carbon dots themselves. The obtained fingerprint data was input into SYSTAT 12.5 software to draw the LDA graph, as shown in Figure 2. Figure 3 shown.

[0066] Example 3

[0067] 1.2g of malic acid (MA) was added with 20mg of Congo red (CR) and 0.3g of ammonium persulfate (APS) and dissolved in 20mL of deionized water. The solution was placed in a 100mL beaker and placed in a microwave oven with the power set to high (750W) for 10 minutes. After the reaction, the mixture was cooled to room temperature and 20mL of deionized water (pH 6) was added. Ultrasonication was performed for 10 minutes to dissolve the colloid formed after the reaction. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was light yellow. The solution was then dialyzed for 3 days using a 1000Da dialysis bag. The dialyzed carbon dot solution was placed in a refrigerator at 4°C until ready for use.

[0068] 1.5 g of o-PD was thoroughly dissolved in 50 mL of DMF by ultrasonication, transferred to an autoclave, and reacted in a 160°C oven for 8 h. After the reaction was complete, the mixture was allowed to cool to room temperature. The autoclave was opened to yield a dark brown solution, which was filtered through a 0.22 μm disposable filter to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 1 day, yielding a brownish-yellow CDs solution. The solution was then refrigerated at 4°C until further use. 400 mg of Zn(NO₃)₂·6H₂O and 100 mg of 2-aminoterephthalic acid were each dissolved in 5 mL of o-PD-CDs, followed by the addition of 1 mL of triethylamine and stirring at room temperature for 1 day. The product was washed once with DMF and then dried and activated in a 70°C vacuum oven.

[0069] 100 mg o-PD, 400 mg Zn(NO3)2·6H2O and 100 mg 2-aminoterephthalic acid were completely dissolved in 20 mL DMF, stirred at room temperature for 1 day, transferred to a high-pressure reactor, and placed in a 160°C oven for reaction for 8 h. After the reaction was completed, the mixture was allowed to stand until it cooled to room temperature. The reactor was opened to obtain a dark brown solution, which was filtered using a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 1 day. The solution was then placed in a 4°C refrigerator for use.

[0070] To each of the three probes and 70 μL of PBS buffer (pH 7.4, 25 mM), 70 μL of 11 different proteins (horseradish peroxidase (HRP), ovalbumin (Alb), lysozyme (Lys), trypsin (Try), pepsin (Pep), myoglobin (Myo), papain (Pap), cytochrome C (Cyt-C), bovine serum albumin (BSA), human hemoglobin (Hb), and bovine hemoglobin (Hem)) and CA15-3 were added and mixed (incubation at 37°C). The fluorescence emission spectra of each sample were measured (five replicates per group). The normalized data (F1 / F0) was used, where F1 represents the fluorescence value after the addition of the tumor marker CA15-3 and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were input into SYSTAT 12.5 software to generate LDA plots.

[0071] To each of the three probes and 70 μL of PBS buffer (pH 7.4, 25 mM) was added 70 μL of CA15-3 at varying concentrations (0.025-1 ng / mL) and mixed thoroughly (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker CA15-3 and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0072] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0073] Add 70 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 70 μL of PBS buffer to each of the three probes (70 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0074] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0075] Example 4

[0076] 3.0 g of Zn(NO₃)₂·6H₂O and 1.0 g of 2-aminoterephthalic acid were mixed and dissolved in 120 mL of DMF using magnetic stirring to obtain a clear mixed solution. 3 mL of triethylamine was slowly added dropwise to the mixed solution, instantly producing a white precipitate. Stirring was continued at room temperature for 3 h to obtain the product, IRMOF-3. The product was washed three times with DMF and then transferred to a 70°C vacuum drying oven for drying and activation. 100 mg of IRMOF-3 was dissolved in 20 mL of DMF. Once completely dissolved, 0.8 g of PD was added and stirred at room temperature for 3 days. The product was then transferred to an autoclave and reacted in a 220°C oven for 4 h. After the reaction was complete, the product was allowed to cool to room temperature. The autoclave was opened to obtain a dark brown solution. It was filtered through a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 3 days. The solution was then refrigerated at 4°C until ready for use.

[0077] 2.0 g of o-PD was thoroughly dissolved in 80 mL of DMF by ultrasonication, transferred to an autoclave, and placed in a 220°C oven for 4 h. After the reaction was complete, the mixture was allowed to cool to room temperature. The autoclave was opened to yield a dark brown solution, which was filtered through a 0.22 μm disposable filter to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 3 days to yield a brownish-yellow CDs solution, which was refrigerated at 4°C until further use. 600 mg of Zn(NO3)2·6H2O and 300 mg of 2-aminoterephthalic acid were separately dissolved in 20 mL of o-PD-CDs, followed by the addition of 2 mL of triethylamine and stirring at room temperature for 2 days. The product was washed twice with DMF and then transferred to a 70°C vacuum drying oven for drying and activation.

[0078] 200 mg o-PD, 500 mg Zn(NO3)2·6H2O and 250 mg 2-aminoterephthalic acid were completely dissolved in 30 mL DMF, stirred at room temperature for 2 days, transferred to a high-pressure reactor, and placed in a 220°C oven for reaction for 4 hours. After the reaction was completed, the solution was allowed to stand until it cooled to room temperature. The reactor was opened to obtain a dark brown solution, which was filtered using a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 3 days. The solution was then placed in a 4°C refrigerator for use.

[0079] To each of the three probes and 80 μL of PBS buffer (pH 7.4, 25 mM) was added 80 μL of AFP at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker AFP and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0080] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0081] Add 80 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 80 μL of PBS buffer to each of the three probes (80 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0082] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0083] Example 5

[0084] 0.6 g of glutathione (GSH) was dissolved in 50 mL of deionized water, along with 20 mg of Congo red (CR) and 0.2 g of ammonium persulfate (APS). The solution was sonicated for 20 minutes until completely dissolved, then transferred to an autoclave, heated to 180°C in an oven, and allowed to react for 6 hours before cooling to room temperature. Impurities were removed by filtration using a 0.22 μm microporous membrane. The solution was then dialyzed using a 1000 Da dialysis bag for 3 days. The dialyzed carbon dot solution was then stored in a refrigerator at 4°C until ready for use.

[0085] To 1.0g of malic acid (MA), add 15mg of Congo red (CR) and 0.2g of ammonium persulfate (APS) and dissolve in 50mL of deionized water. Place the solution in a 100mL beaker and microwave at high power (750W) for 7 minutes. After the reaction, cool to room temperature and add 8mL of deionized water (pH 6). Ultrasonicate for 5 minutes to dissolve the resulting gel. Filter through a 0.22μm filter to remove impurities. The filtered solution is light yellow. Dialysis is then performed for 2 days using a 1000Da dialysis bag. The dialyzed carbon dot solution is then placed in a refrigerator at 4°C until ready for use.

[0086] 2.5 g of Zn(NO₃)₂·6H₂O and 0.8 g of 2-aminoterephthalic acid were mixed and dissolved in 90 mL of DMF using magnetic stirring to obtain a clear mixed solution. 5 mL of triethylamine was slowly added dropwise to the mixed solution, instantly producing a white precipitate. Stirring was continued at room temperature for 4 h to obtain the product, IRMOF-3. The product was washed three times with DMF and then transferred to a 70°C vacuum drying oven for drying and activation. 120 mg of IRMOF-3 was dissolved in 30 mL of DMF. Once completely dissolved, 0.7 g of o-PD was added and stirred at room temperature for 2 days. The mixture was then transferred to an autoclave and reacted in a 200°C oven for 5 h. After completion of the reaction, the mixture was allowed to cool to room temperature. The autoclave was opened to obtain a dark brown solution. Impurities were removed by filtration using a 0.22 μm disposable filter membrane and transferred to a dialysis bag (MW = 1000 Da) for further purification for 2 days. The solution was then refrigerated at 4°C until ready for use.

[0087] To each of the three probes and 75 μL of PBS buffer (pH 7.4, 25 mM) was added 75 μL of CA125 at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker CA125 and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0088] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0089] Add 75 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 75 μL of PBS buffer to each of the three probes (75 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0090] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0091] Example 6

[0092] 0.3 g of o-phenylenediamine (o-PD) was dissolved in 50 mL of deionized water with 20 mg of Congo red (CR) and 0.2 g of ammonium persulfate (APS). The solution was sonicated for 20 minutes until completely dissolved, then transferred to an autoclave, heated to 210°C in an oven, and allowed to react for 7 hours before cooling to room temperature. Impurities were removed by filtration using a 0.22 μm microporous membrane. The solution was then dialyzed using a 1000 Da dialysis bag for 3 days. The dialyzed carbon dot solution was then stored in a refrigerator at 4°C until ready for use.

[0093] 2.5 g of o-PD was thoroughly dissolved in 70 mL of DMF by ultrasonication, transferred to an autoclave, and placed in an oven at 180°C for 7 h. After the reaction was complete, the mixture was allowed to cool to room temperature. The autoclave was opened to yield a dark brown solution, which was filtered through a 0.22 μm disposable filter to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for two days, yielding a brownish-yellow CDs solution. The solution was then refrigerated at 4°C until further use. 500 mg of Zn(NO₃)₂·6H₂O and 200 mg of 2-aminoterephthalic acid were each dissolved in 12 mL of o-PD-CDs, followed by the addition of 3 mL of triethylamine and stirring at room temperature for 3 days. The product was washed three times with DMF and then dried and activated in a vacuum drying oven at 70°C.

[0094] 300 mg o-PD, 600 mg Zn(NO3)2·6H2O and 300 mg 2-aminoterephthalic acid were completely dissolved in 50 mL DMF, stirred at room temperature for 3 days, transferred to a high-pressure reactor, and placed in a 190°C oven for reaction for 6 h. After the reaction was completed, the solution was allowed to stand until it cooled to room temperature. The reactor was opened to obtain a dark brown solution, which was filtered using a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 2 days. The solution was then placed in a 4°C refrigerator for use.

[0095] To each of the three probes and 65 μL of PBS buffer (pH 7.4, 25 mM) was added 65 μL of AFP at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker AFP and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0096] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0097] Add 65 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 65 μL of PBS buffer to each of the three probes (65 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0098] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0099] Example 7

[0100] To 0.8 g of malic acid (MA), 10 mg of Congo red (CR) and 0.2 g of ammonium persulfate (APS) were added and dissolved in 30 mL of deionized water. The solution was sonicated for 15 minutes until completely dissolved, then transferred to an autoclave, heated to 180°C in an oven, reacted for 6 hours, and cooled to room temperature. Impurities were removed by filtration using a 0.22 μm microporous membrane. The solution was dialyzed using a 1000 Da dialysis bag for 2 days. The dialyzed carbon dot solution was stored in a refrigerator at 4°C until ready for use.

[0101] 0.4g glutathione (GSH) and 0.2g o-phenylenediamine (o-PD) were added with 10mg Congo red (CR) and 0.2g ammonium persulfate (APS) and dissolved in 40mL deionized water. The solution was placed in a 100mL beaker and placed in a microwave oven with the power set to medium (450W) and the microwave time set to 7min. After the reaction, the mixture was cooled to room temperature and 8mL deionized water with a pH of 6 was added. The reaction mixture was sonicated for 15min to dissolve the colloid formed after the reaction. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was light yellow. The solution was then dialyzed for 2 days using a 1000Da dialysis bag. The dialyzed carbon dot solution was placed in a refrigerator at 4°C until use.

[0102] To each of the three probes and 70 μL of PBS buffer (pH 7.4, 25 mM) was added 70 μL of AFP at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker AFP and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0103] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0104] 70 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 70 μL of PBS buffer were added to each of the three probes (65 μL) and mixed evenly (incubated at 37°C). The fluorescence emission spectrum of each sample was measured (three parallel samples were required for each group of samples).

[0105] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0106] Example 8

[0107] 1.2 g of malic acid (MA) was dissolved in 10 mL of deionized water, along with 5 mg of Congo red (CR) and 0.1 g of ammonium persulfate (APS). The solution was sonicated for 5 minutes until completely dissolved, then transferred to an autoclave, heated in an oven to 220°C, reacted for 4 hours, and cooled to room temperature. Impurities were removed by filtration using a 0.22 μm microporous membrane. The solution was dialyzed using a 1000 Da dialysis bag for one day. The dialyzed carbon dot solution was then stored in a refrigerator at 4°C until ready for use.

[0108] 0.6g of glutathione (GSH) was dissolved in 10mL of deionized water, along with 5mg of Congo red (CR) and 0.1g of ammonium persulfate (APS). The solution was placed in a 100mL beaker and microwaved at high power (750W) for 5 minutes. After the reaction, the mixture was cooled to room temperature and 5mL of deionized water (pH 6) was added. Ultrasonication was performed for 5 minutes to dissolve the resulting gel. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was pale yellow. The solution was then dialyzed for one day using a 1000Da dialysis bag. The dialyzed carbon dot solution was then placed in a refrigerator at 4°C until ready for use.

[0109] 1.5 g of o-PD was thoroughly dissolved in 70 mL of DMF by ultrasonication. The solution was placed in a 100 mL beaker and microwaved at medium power (450 W) for 9 minutes. After the reaction, the mixture was cooled to room temperature, and 7.5 mL of deionized water (pH 6) was added. The resulting gel was sonicated for 10 minutes to dissolve the resulting gel. The solution was filtered through a 0.22 μm filter to remove impurities, resulting in a pale yellow solution. The solution was then dialyzed for 2 days using a 1000 Da dialysis bag. The dialyzed carbon dot solution was then stored in a refrigerator at 4°C until ready for use. 400 mg of Zn(NO₃)₂·6H₂O and 100 mg of 2-aminoterephthalic acid were each dissolved in 10 mL of o-PD-CDs. 2 mL of triethylamine was then added and stirred at room temperature for 2 days. The product was washed twice with DMF and then transferred to a 70°C vacuum oven for drying and activation.

[0110] To each of the three probes and 60 μL of PBS buffer (pH 7.4, 25 mM) was added 60 μL of CEA at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker CEA and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0111] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0112] Add 60 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 65 μL of PBS buffer to each of the three probes (60 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0113] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0114] Example 9

[0115] Dissolve 1.0g of malic acid (MA) in 50mL of deionized water, adding 20mg of Congo red (CR) and 0.3g of ammonium persulfate (APS). Allow the solution to dissolve completely over 20 minutes, then transfer it to an autoclave, heat it in an oven to 160°C, react for 8 hours, and cool it to room temperature. Filter the solution through a 0.22μm microporous membrane to remove impurities, then dialyze it for 3 days using a 1000Da dialysis bag. Place the dialyzed carbon dot solution in a refrigerator at 4°C until ready for use.

[0116] 0.4g o-phenylenediamine (o-PD) was dissolved in 10mL of deionized water with 5mg of Congo red (CR) and 0.1g of ammonium persulfate (APS). The solution was placed in a 100mL beaker and microwaved at high power (750W) for 5 minutes. After the reaction, the solution was cooled to room temperature and 5mL of deionized water (pH 6) was added. Ultrasonication was performed for 5 minutes to dissolve the colloid formed after the reaction. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was light yellow. The solution was then dialyzed for 1 day using a 1000Da dialysis bag. The dialyzed carbon dot solution was placed in a refrigerator at 4°C until ready for use.

[0117] 2.5 g of o-PD was thoroughly dissolved in 50 mL of DMF by ultrasonication. The solution was placed in a 100 mL beaker and placed in a microwave oven at medium-high power for 5-10 minutes. After the reaction, the mixture was cooled to room temperature, and 5 mL of deionized water (pH 6) was added. The resulting gel was sonicated for 5 minutes to dissolve. The resulting solution was filtered through a 0.22 μm filter to remove impurities. The filtered solution was pale yellow. The solution was then dialyzed for one day using a 1000 Da dialysis bag. The dialyzed carbon dot solution was stored in a refrigerator at 4°C until ready for use. 400 mg of Zn(NO₃)₂·6H₂O and 100 mg of 2-aminoterephthalic acid were each dissolved in 5 mL of o-PD-CDs. 1 mL of triethylamine was then added and stirred at room temperature for one day. The product was washed once with DMF and then transferred to a 70°C vacuum oven for drying and activation.

[0118] To each of the three probes and 75 μL of PBS buffer (pH 7.4, 25 mM) was added 75 μL of CA15-3 at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker CA15-3 and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0119] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0120] Add 75 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 75 μL of PBS buffer to each of the three probes (75 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0121] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0122] Example 10

[0123] 0.5g of glutathione (GSH) was dissolved in 50mL of deionized water with 20mg of Congo red (CR) and 0.3g of ammonium persulfate (APS). The solution was placed in a 100mL beaker and microwaved at high power (750W) for 10 minutes. After the reaction, the mixture was cooled to room temperature and 10mL of deionized water (pH 6) was added. Ultrasonication was performed for 20 minutes to dissolve the colloid formed after the reaction. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was light yellow. The solution was then dialyzed for 3 days using a 1000Da dialysis bag. The dialyzed carbon dot solution was then placed in a refrigerator at 4°C until ready for use.

[0124] 0.3g o-phenylenediamine (o-PD) was dissolved in 50mL of deionized water with 20mg of Congo red (CR) and 0.3g of ammonium persulfate (APS). The solution was placed in a 100mL beaker and microwaved at a medium power setting (450W) for 10 minutes. After the reaction, the solution was cooled to room temperature and 10mL of deionized water (pH 6) was added. Ultrasonication was performed for 20 minutes to dissolve the colloid formed after the reaction. Impurities were removed by filtration using a 0.22μm filter membrane. The filtered solution was light yellow. The solution was then dialyzed for 3 days using a 1000Da dialysis bag. The dialyzed carbon dot solution was placed in a refrigerator at 4°C until ready for use.

[0125] 2.0 g of o-PD was thoroughly dissolved in 80 mL of DMF by ultrasonication. The solution was placed in a 100 mL beaker and placed in a microwave oven at medium-high power for 10 minutes. After the reaction, the mixture was cooled to room temperature, and 10 mL of deionized water (pH 6) was added. The resulting gel was sonicated for 20 minutes to dissolve the resulting gel. The solution was filtered through a 0.22 μm filter to remove impurities, resulting in a pale yellow solution. The solution was then dialyzed for 3 days using a 1000 Da dialysis bag. The dialyzed carbon dot solution was then stored in a refrigerator at 4°C until ready for use. 500 mg of Zn(NO₃)₂·6H₂O and 200 mg of 2-aminoterephthalic acid were each dissolved in 20 mL of o-PD-CDs. 3 mL of triethylamine was then added and stirred at room temperature for 3 days. The product was washed three times with DMF and then transferred to a 70°C vacuum oven for drying and activation.

[0126] To each of the three probes and 80 μL of PBS buffer (pH 7.4, 25 mM) was added 80 μL of AFP at varying concentrations (0.025-1 ng / mL) and mixed (incubation at 37°C). Fluorescence emission spectra were measured for each sample (five replicates per group). The normalized data, F1 / F0, was used, where F1 represents the fluorescence value after addition of the tumor marker AFP and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data was input into SYSTAT 12.5 software to generate LDA plots.

[0127] By making a linear relationship between Factor (1) and tumor marker concentration in the LDA graph, the content of tumor marker in the unknown actual blood sample can be calculated. According to the Factor (1) corresponding to the actual blood in the LDA graph, compared with the previously obtained linear relationship, it is calculated whether the concentration of the tumor marker in the unknown sample blood exceeds the threshold, and it is inferred whether the patient has the tumor, so as to screen the patient.

[0128] Add 80 μL of blood samples from cancer patients (Y1, Y2, Y3, Y4, Y5) or healthy subjects (C1, C2) (original human blood diluted 1000 times) and 80 μL of PBS buffer to each of the three probes (80 μL) and mix well (incubate at 37°C). Measure the fluorescence emission spectrum of each sample (three parallel samples are required for each group of samples).

[0129] Normalized data (F1 / F0) were used, where F1 represents the fluorescence value after adding the tumor marker and F0 represents the fluorescence value of the carbon dots themselves. The resulting fingerprint data were then input into SYSTAT 12.5 software to create an LDA plot. The presence of excessive tumor markers in the blood was determined based on the region of the LDA plot where the sample was detected by linear discriminant analysis.

[0130] Example 11

[0131] 0.3 g of o-phenylenediamine (o-PD) was dissolved in 50 mL of deionized water with 20 mg of Congo red (CR) and 0.2 g of ammonium persulfate (APS). The solution was sonicated for 20 minutes until completely dissolved, then transferred to an autoclave, heated to 210°C in an oven, and allowed to react for 7 hours before cooling to room temperature. Impurities were removed by filtration using a 0.22 μm microporous membrane. The solution was then dialyzed using a 1000 Da dialysis bag for 3 days. The dialyzed carbon dot solution was then stored in a refrigerator at 4°C until ready for use.

[0132] 2.5 g of o-PD was thoroughly dissolved in 70 mL of DMF by ultrasonication, transferred to an autoclave, and placed in an oven at 180°C for 7 h. After the reaction was complete, the mixture was allowed to cool to room temperature. The autoclave was opened to yield a dark brown solution, which was filtered through a 0.22 μm disposable filter to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for two days, yielding a brownish-yellow CDs solution. The solution was then refrigerated at 4°C until further use. 500 mg of Zn(NO₃)₂·6H₂O and 200 mg of 2-aminoterephthalic acid were each dissolved in 12 mL of o-PD-CDs, followed by the addition of 3 mL of triethylamine and stirring at room temperature for 3 days. The product was washed three times with DMF and then dried and activated in a vacuum drying oven at 70°C.

[0133] 300 mg o-PD, 600 mg Zn(NO3)2·6H2O and 300 mg 2-aminoterephthalic acid were completely dissolved in 50 mL DMF, stirred at room temperature for 3 days, transferred to a high-pressure reactor, and placed in a 190°C oven for reaction for 6 h. After the reaction was completed, the solution was allowed to stand until it cooled to room temperature. The reactor was opened to obtain a dark brown solution, which was filtered using a 0.22 μm disposable filter membrane to remove impurities and transferred to a dialysis bag (MW = 1000 Da) for further purification for 2 days. The solution was then placed in a 4°C refrigerator for use.

[0134] 65 μL of three probes and 65 μL of PBS Buffer (pH = 7.4, 25 mM) were added to 65 μL of CEA, CA125, CA15-3, and AFP, and mixed (incubated at 37 ° C), and the fluorescence emission spectrum of each sample was measured (5 parallel samples were measured for each group of samples). The normalized data F1 / F0 was used, where F1 is the fluorescence value after adding tumor markers CEA, CA125, CA15-3, and AFP, and F0 is the fluorescence value of the carbon dots themselves. The obtained fingerprint data was input into SYSTAT 12.5 software to draw the LDA graph. LDA converted the matrix (3 probes × 4 tumor markers × 5 repetitions) into three standardized scores. The 4 different tumor markers were clearly classified without any overlap, such as Figure 6 Schematic diagram of LDA shown.

[0135] The present invention uses simple-to-prepare carbon dots as sensing units to construct a sensor array for detecting and distinguishing tumor markers. It can simultaneously detect and distinguish multiple samples, requires a very low sample amount, has a short detection time, and is easy to operate.

[0136] Those skilled in the art will appreciate that the present invention is not limited to the details of the exemplary embodiments described above and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. For example, it should be understood that any carbon dots with different functional groups (not limited to the three types mentioned herein) can serve as sensing elements in a fluorescence sensor array for detecting and distinguishing tumor markers. Furthermore, it should be understood that the constructed fluorescence sensor array can distinguish different tumor markers because each tumor marker has its own unique characteristics, which are consistent with the differential response of the sensor array signal (not limited to the four types mentioned herein).

[0137] Therefore, no matter from which point of view, the embodiments should be regarded as illustrative rather than restrictive, and the scope of the present invention is limited by the appended claims rather than the above description, and it is intended that all changes that fall within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved. Please note that the technical features of the above embodiments can be combined in any way. In order to make the description concise, not all possible combinations of the various technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above embodiments only express several implementation methods of the present application, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application should be based on the appended claims.

Claims

1. A fluorescent reagent for detecting tumor markers, characterized in that: Include at least three of the following probes: a. A probe comprising one or more carbon dots having one or more amino, carboxyl, hydroxyl, or sulfhydryl groups; wherein the carbon dots are prepared by reacting glutathione, o-phenylenediamine, or malic acid with Congo red and ammonium persulfate; b. Probe constructed by combining carbon dots with IRMOF-3; the carbon dots were prepared using o-phenylenediamine, and IRMOF-3 was prepared by reacting Zn(NO₃)₂·6H₂O with 2-aminoterephthalic acid.

2. The fluorescent reagent for detecting tumor markers according to claim 1, characterized in that The tumor markers include one or more of carcinoembryonic antigen, cancer antigen 125, cancer antigen 15-3 or alpha-fetoprotein.

3. A method for preparing a fluorescent reagent for detecting tumor markers according to claim 1 or 2, characterized in that: The method comprises preparing the following carbon dots or probes and selecting at least three of the carbon dots and / or probes for combination: c. preparing carbon dots by a hydrothermal method or a microwave method; wherein the carbon dots are prepared by reacting glutathione, o-phenylenediamine, or malic acid with Congo red and ammonium persulfate; d. Synthesis of a probe constructed by combining carbon dots with IRMOF-3; the carbon dots were prepared using o-phenylenediamine, and IRMOF-3 was prepared by reacting Zn(NO₃)₂·6H₂O with 2-aminoterephthalic acid.

4. The preparation method according to claim 3, characterized in that The hydrothermal method for preparing carbon dots includes: Dissolve glutathione, Congo red, and ammonium persulfate in deionized water, heat to 160-220°C in an autoclave by hydrothermal method, react for 4-8 hours, cool, filter to remove impurities, and dialyze for 1-3 days; and / or, dissolving o-phenylenediamine, Congo red, and ammonium persulfate in deionized water, heating to 160-220° C. in an autoclave by a hydrothermal method, reacting for 4-8 hours, cooling, filtering to remove impurities, and dialyzing for 1-3 days to obtain; And / or, malic acid, Congo red and ammonium persulfate are dissolved in deionized water, heated to 160-220° C. in an autoclave by a hydrothermal method, reacted for 4-8 hours, cooled, filtered to remove impurities, and dialyzed for 1-3 days to obtain the product.

5. The preparation method according to claim 3, characterized in that The method of preparing carbon dots by microwave method comprises: Dissolve glutathione, Congo red, and ammonium persulfate in deionized water, heat under microwave for 5-10 minutes, cool, add deionized water with a pH of 5.5-6.5, sonicate for 5-20 minutes, filter to remove impurities, and dialyze for 1-3 days to obtain the product. Dissolve o-phenylenediamine, Congo red, and ammonium persulfate in deionized water, heat under microwave for 5-10 minutes, cool, add deionized water with a pH of 5.5-6.5, sonicate for 5-20 minutes, filter to remove impurities, and dialyze for 1-3 days to obtain the product. Dissolve malic acid, Congo red and ammonium persulfate in deionized water, heat under microwave for 5-10 min, cool, add deionized water with a pH of 5.5-6.5, sonicate for 5-20 min, filter to remove impurities, and dialyze for 1-3 days to obtain the product.

6. The preparation method according to claim 3, characterized in that The probe constructed by combining the synthesized carbon dots with IRMOF-3 includes: o-phenylenediamine is dissolved in DMF and heated to 160-220°C in an autoclave by a hydrothermal method for 4-8 hours. After cooling, impurities are removed by filtration and the solution is purified by dialyzing for 1-3 days to obtain a brown-yellow CDs solution. Alternatively, o-phenylenediamine is dissolved in deionized water and microwave-heated for 5-10 minutes. After cooling, deionized water is added and the solution is ultrasonicated for 5-20 minutes. Impurities are removed by filtration and the solution is dialyzed for 1-3 days to obtain a brown-yellow CDs solution. Dissolve Zn(NO3)2·6H2O and 2-aminoterephthalic acid in the CDs solution respectively and mix, then add triethylamine, stir, wash with DMF, dry and activate to obtain the product.

7. The preparation method according to claim 3, characterized in that The probe constructed by combining the synthesized carbon dots with IRMOF-3 includes: dissolving Zn(NO3)2·6H2O and 2-aminoterephthalic acid in DMF, adding triethylamine, and continuing to stir to obtain IRMOF-3, washing, drying and activating; Dissolve IRMOF-3 in DMF, add o-phenylenediamine, stir for 1-3 days, react at 160-220°C for 4-8 hours, cool, filter to remove impurities, and dialysis for purification for 1-3 days to obtain the product.

8. The preparation method according to claim 3, characterized in that The probe constructed by combining the synthesized carbon dots with IRMOF-3 comprises: dissolving o-phenylenediamine, Zn(NO3)2·6H2O and 2-aminoterephthalic acid in DMF, stirring for 1-3 days, reacting at 160-220°C for 4-8 hours, filtering to remove impurities after cooling, and purifying by dialyzing for 1-3 days to obtain the probe.

9. A method for constructing a fluorescent sensor array sensing unit using the fluorescent reagent according to claim 1, characterized in that: Adding any one or more tumor markers at different concentrations to the three probes of the fluorescent reagent and mixing them evenly; Adding several proteins and any one or more tumor markers to the three probes of the fluorescent reagent and mixing them evenly; The several proteins include one or more of horseradish peroxidase, ovalbumin, lysozyme, trypsin, pepsin, myoglobin, papain, cytochrome C, bovine serum albumin, human hemoglobin or bovine hemoglobin; The tumor markers include one or more of carcinoembryonic antigen, cancer antigen 125, cancer antigen 15-3 or alpha-fetoprotein.

10. An application of a fluorescence sensor array sensing unit constructed by the method according to claim 9, characterized in that: Add any one or more tumor markers at different concentrations to PBS buffer and mix them evenly, or add several proteins and any one or more tumor markers to PBS buffer and mix them evenly; use normalized data F1 / F0, where F1 is the fluorescence value after adding the tumor marker and F0 is the fluorescence value of the carbon dots themselves; use the obtained fingerprint data to draw a two-dimensional linear discriminant analysis graph; linearly compare Factor (1) in the two-dimensional linear discriminant analysis graph with the tumor marker concentration to calculate the tumor marker content in the unknown actual blood sample.

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