Ratio-dependent fluorescence sensor for simultaneously detecting various microRNAs of colorectal cancer as well as detection method and application of ratio-dependent fluorescence sensor

Abstract

The invention relates to the technical field of biosensing, in particular to a ratiometric fluorescence sensor for simultaneously detecting microRNAs (Ribonucleic Acid) of various colorectal cancers, which comprises aminated UiO-66, a fluorescent probe, a fluorescent probe and a fluorescent probe, the single-stranded nucleic acid probe group is complementarily paired with the colorectal cancer related microRNA bases; the single-stranded nucleic acid probe group comprises a plurality of single-stranded nucleic acid probes which are complementarily paired with different colorectal cancer related microRNA bases; one end of the single-stranded nucleic acid probe is modified with a fluorophore; and various different single-stranded nucleic acid probes are modified with different fluorophores. According to the invention, simultaneous quantitative detection of various microRNAs related to colorectal cancer in a tube can be realized, and the detection efficiency is improved; compared with a traditional fluorescent probe, the ratio-type fluorescent probe can reduce background interference to the maximum extent and shows better sensitivity and accuracy, so that the analysis performance is improved.

Description

Technical Field

[0001] The present invention relates to the field of biosensor technology, and in particular to a ratiometric fluorescent sensor for simultaneously detecting a plurality of colorectal cancer microRNAs, a detection method and an application thereof. Background Art

[0002] Colorectal cancer is a gastrointestinal malignancy and the third most common cancer in the world. Most colorectal cancers originate from precancerous colorectal adenomas, gradually accumulate mutations and epigenetic changes, and eventually progress to malignant tumors. Although colorectal cancer usually progresses relatively slowly, its onset is often hidden, and most patients have no clinical symptoms in the early stages, leading to delayed treatment. Early detection and precise treatment are the key to improving the survival rate of patients with colorectal cancer. At present, histopathological biopsy remains the gold standard for diagnosing colorectal cancer. However, biopsy is an invasive procedure, and due to the spatiotemporal heterogeneity of tumors, a single histopathological biopsy often fails to capture the comprehensive biological characteristics and dynamic changes of colorectal cancer. Therefore, there is an urgent need for minimally invasive methods to screen high-risk populations, reduce unnecessary tissue biopsies, and identify asymptomatic colorectal cancer patients at an early and curable stage.

[0003] MicroRNA is a widely studied genetic biomarker with great potential in cancer diagnosis. Sensitive detection of abnormal microRNA concentrations is a frontier in the early diagnosis of malignant tumors. However, human physiological processes are usually coordinated by a group of microRNAs rather than regulated by a single microRNA. Therefore, quantitative detection of a single microRNA is insufficient for comprehensive microRNA research. Dysregulation of multiple microRNA expression profiles has been reported in colorectal cancer. Therefore, microRNA has important clinical value for early screening of colorectal cancer patients, monitoring recurrence, evaluating treatment efficacy, and guiding precision treatment, and can be used as an important molecular marker for the diagnosis and treatment of colorectal cancer. Current strategies for detecting colorectal cancer-related microRNAs mainly focus on improving the detection limit, sensitivity or selectivity of a single target, ignoring the complex interactions and combinatorial patterns of microRNA expression. In addition, since abnormal expression of the same microRNA can occur in different cancer types, simultaneous detection of multiple microRNA levels associated with colorectal cancer can provide better data correction, avoid false positives or false negatives, and ultimately improve test accuracy.

[0004] In recent years, fluorescence sensing technology has been widely used in biomarker detection due to its versatility, fast response time and high sensitivity. In order to improve the quantitative sensitivity of multi-targets, nanomaterials have become a key component in fluorescent biosensors. The nanometer size of these materials provides ideal biosensing properties and has significant advantages in amplifying signals and improving the sensitivity of biomarker analysis. UiO-66-NH2 is a typical metal organic framework nanomaterial with bright blue fluorescence and customizable molecular properties. It has many advantages over traditional porous materials such as zeolites and porous carbons, but the application of UiO-66-NH2-based fluorescent probes in tumor-associated nucleic acid analysis is still limited. In addition, compared with fluorescent sensors based on single emission probes, ratiometric fluorescent sensors have self-regulation capabilities, which helps to reduce the interference of variables and further improve the analysis accuracy. Therefore, ratiometric fluorescent sensors can significantly improve the accuracy of tumor-associated nucleic acid analysis, making them a more attractive alternative to single emission strategies. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a ratiometric fluorescent sensor for simultaneously detecting multiple colorectal cancer microRNAs, as well as a detection method and use thereof, so as to solve the problems in the prior art.

[0006] To achieve the above objectives and other related objectives, the present invention first provides a ratiometric fluorescence sensor for simultaneously detecting multiple colorectal cancer microRNAs, the ratiometric fluorescence sensor comprising:

[0007] Amination of UiO-66;

[0008] A single-stranded nucleic acid probe set that is complementary to colorectal cancer-related microRNA bases, wherein the single-stranded nucleic acid probe set includes a plurality of single-stranded nucleic acid probes that are complementary to different colorectal cancer-related microRNA bases, and one end of the single-stranded nucleic acid probe is modified with a fluorescent group;

[0009] Various single-stranded nucleic acid probes are modified with different fluorescent groups.

[0010] The present invention also provides a method for quantitatively detecting a variety of colorectal cancer microRNAs for purposes other than disease diagnosis or treatment, and uses the above-mentioned ratiometric fluorescent sensor to perform quantitative detection on samples to be tested.

[0011] The quantitative detection method comprises the following steps:

[0012] 1) mixing the sample to be tested and a single-stranded nucleic acid probe set that is complementary to the sample and has one end modified with a fluorescent group, and then incubating the mixture to obtain a first mixed system;

[0013] 2) mixing the first mixed system prepared in step 1) with the amino-modified UiO-66 and incubating the mixture to obtain a second mixed system,

[0014] The fluorescence intensity in the second mixed system is measured.

[0015] The present invention also provides the use of the ratiometric fluorescent sensor for simultaneously detecting multiple colorectal cancer microRNAs in the preparation of colorectal cancer diagnostic products.

[0016] The present invention also provides a colorectal cancer diagnostic product, which includes the ratiometric fluorescent sensor for simultaneously detecting multiple colorectal cancer microRNAs.

[0017] As described above, the ratiometric fluorescent sensor, detection method and use of the present invention for simultaneously detecting multiple colorectal cancer microRNAs have the following beneficial effects:

[0018] 1) It can realize the simultaneous quantitative detection of multiple microRNAs related to colorectal cancer in one tube, avoiding the tedious process of one-by-one detection by traditional methods, significantly improving the detection efficiency, shortening the detection time, and helping to quickly obtain comprehensive microRNA expression profile information, providing a more timely basis for the early diagnosis and disease monitoring of colorectal cancer, and improving the detection efficiency and diagnosis rate;

[0019] 2) Compared with traditional fluorescent probes, ratiometric fluorescent probes can minimize background interference. By using the fluorescence intensity ratio of the fluorescent group and the amino-modified UiO-66 as the signal output, it can effectively eliminate the influence of external conditions on the detection results, thereby showing better sensitivity and accuracy, improving the reliability and repeatability of the detection, and helping to reduce the misdiagnosis rate;

[0020] 3) The single-stranded nucleic acid probe is designed based on the sequence of the sample to be tested, and can complementarily pair with specific microRNA bases to achieve specific recognition and binding of the target microRNA. In the reaction system, only the target microRNA that specifically binds to the probe can cause changes in the fluorescent signal, and other non-target microRNAs or impurity molecules have little interference with the detection signal, thus ensuring the high selectivity and specificity of the detection;

[0021] 4) The detection method of the present invention is easy to operate, does not require complex instruments and cumbersome steps, and only requires the target microRNA to be mixed and incubated with a single-stranded nucleic acid probe, and then mixed and incubated with amino-modified UiO-66, and then the fluorescence intensity is measured to complete the detection. The whole process is simple and easy to implement automation and high-throughput detection, and is suitable for promotion and application in clinical diagnosis and large-scale screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shown is a time optimization diagram of the incubation time of UiO-66-NH2 in response to microRNA-21 in Example 2 of the present invention.

[0023] Figure 2 Shown is a concentration optimization diagram of UiO-66-NH2 in Example 3 of the present invention in response to microRNA-21, microRNA-17 and microRNA-92a.

[0024] Figure 3 Shown is a concentration optimization diagram for simultaneous detection of microRNA-21, microRNA-17 and microRNA-92a in Example 4 of the present invention.

[0025] Figure 4 Shown is the selectivity result diagram for microRNA-21 in the reaction system in Example 5 of the present invention.

[0026] Figure 5 Shown is the selectivity result diagram for microRNA-17 in the reaction system in Example 6 of the present invention.

[0027] Figure 6 It shows the selectivity result diagram of microRNA-92a in the reaction system in Example 7 of the present invention.

[0028] Figure 7 The graph shows the result of simultaneous detection of microRNA-21, microRNA-17 and microRNA-92a in colorectal cancer tissue and adjacent cancer tissue in the reaction system in Example 8 of the present invention. DETAILED DESCRIPTION

[0029] The present invention constructs a novel ratiometric fluorescence sensor, which combines UiO-66-NH2 and a nucleic acid probe modified with a fluorescent dye and can be used to simultaneously and quantitatively detect the concentrations of multiple microRNA (for example, microRNA-21, microRNA-17, and microRNA-92a) markers related to colorectal cancer.

[0030] The method first synthesized the metal organic framework UiO-66-NH2 that emits blue fluorescence, and at the same time designed nucleic acid probes modified with different fluorescent groups that specifically capture the target microRNA. When the target microRNA is not present, due to the unique surface chemical properties and pore structure of UiO-66-NH2, the single-stranded nucleic acid probe can be adsorbed on the surface of the regular octahedron UiO-66-NH2 through strong binding, and the fluorescence of the nucleic acid probe is quenched. When the target microRNA is present, they can respectively bind to the corresponding nucleic acid fluorescent probe to form a stable rigid double-stranded structure. The complex is difficult to adsorb on the surface of UiO-66-NH2, and the fluorescence of the probe remains unchanged. At the same time, the blue fluorescence of UiO-66-NH2 itself remains almost unchanged and can be used as an internal standard for ratiometric probes. The ratio of the fluorescence intensity of the fluorescent group to that of UiO-66-NH2 is used as the signal output. According to the fluorescence intensity ratio F 荧光基团 / F UiO-66-NH2 The concentration of microRNA is quantitatively detected respectively. This method has high sensitivity, good selectivity and simple detection, and can be used for early screening of colorectal cancer to improve the diagnosis rate and specificity.

[0031] The present invention first provides a ratiometric fluorescence sensor for simultaneously detecting multiple colorectal cancer microRNAs, wherein the ratiometric fluorescence sensor comprises:

[0032] Amination of UiO-66;

[0033] A single-stranded nucleic acid probe set that is complementary to colorectal cancer-related microRNA bases, wherein the single-stranded nucleic acid probe set includes a plurality of single-stranded nucleic acid probes that are complementary to different colorectal cancer-related microRNA bases, and one end of the single-stranded nucleic acid probe is modified with a fluorescent group;

[0034] Various single-stranded nucleic acid probes are modified with different fluorescent groups.

[0035] In the present invention, a single-stranded nucleic acid probe that is complementary to a specific microRNA base pairing is defined as "a" probe, and the "single-stranded nucleic acid probe group" refers to a set of probes that are complementary to different types of microRNA base pairing; the "different single-stranded nucleic acid probes" refer to single-stranded nucleic acid probes that are complementary to different types of microRNA base pairing, and the fluorescent groups modified on the various single-stranded nucleic acid probes are different.

[0036] In certain embodiments of the present invention, the aminated UiO-66 is UiO-66-NH2.

[0037] In certain embodiments of the present invention, the emission wavelength of the fluorescent group is different from the emission wavelength of the amino-modified UiO-66.

[0038] In a preferred embodiment of the present invention, the fluorescent group is selected from one or more of FAM, TAMRA, Cy5, Cy3, Cy5.5, Cy7, NED or TET.

[0039] In certain embodiments of the present invention, the length of the single-stranded nucleic acid probe is 20 to 30 nt.

[0040] In certain embodiments of the present invention, the microRNA associated with colorectal cancer is selected from multiple ones of microRNA-21, microRNA-17 and / or microRNA-92a.

[0041] In certain embodiments of the present invention, the single-stranded nucleic acid probe complementary to the base pairing of microRNA-21 is denoted as P1, and the nucleotide sequence of P1 is shown in SEQ ID NO.1, specifically: 5'-FAM-TCAACATCAGTCTGATAAGCTA-3'.

[0042] In certain embodiments of the present invention, the single-stranded nucleic acid probe complementary to the base pairing of microRNA-17 is denoted as P2, and the nucleotide sequence of P2 is shown in SEQ ID NO.2, specifically: 5'-TAMRA-CTACCTGCACTGTAAGCACTTTG-3'.

[0043] In certain embodiments of the present invention, the single-stranded nucleic acid probe complementary to the base pairing of microRNA-92a is denoted as P3, and the nucleotide sequence of P3 is shown in SEQ ID NO.3, specifically: 5'-Cy5-ACAGGCCGGGACAAGTGCAATA-3'.

[0044] The present invention also provides a method for quantitatively detecting a variety of colorectal cancer microRNAs for purposes other than disease diagnosis or treatment, and uses the above-mentioned ratiometric fluorescent sensor to perform quantitative detection on samples to be tested.

[0045] The quantitative detection method comprises the following steps:

[0046] 1) mixing the sample to be tested and a single-stranded nucleic acid probe set that is complementary to the sample and has one end modified with a fluorescent group, and then incubating the mixture to obtain a first mixed system;

[0047] 2) mixing the first mixed system prepared in step 1) with the amino-modified UiO-66 and incubating the mixture to obtain a second mixed system,

[0048] The fluorescence intensity of the second mixed system is measured.

[0049] In certain embodiments of the present invention, in step 1), the sample to be tested is selected from microRNA associated with colorectal cancer in tissues, cells or blood; the microRNA is selected from multiple microRNAs selected from microRNA-21, microRNA-17 and / or microRNA-92a.

[0050] In certain embodiments of the present invention, in step 1), based on the volume of the first mixed system, the concentration of the single-stranded nucleic acid probe set complementary to the base of the sample to be tested is 800-1800 nM.

[0051] The concentration of the single-stranded nucleic acid probe complementary to the base of the sample to be tested is selected from any of the following ranges: 800-1000 nM, 1000-1200 nM, 1200-1400 nM, 1400-1600 nM, 1600-1800 nM.

[0052] In the present invention, the single-stranded nucleic acid probe set includes two or more single-stranded nucleic acid probes, and different single-stranded nucleic acid probes are used to specifically bind to different types of colorectal cancer-related microRNAs.

[0053] In certain embodiments of the present invention, the concentration of each single-stranded nucleic acid probe in the single-stranded nucleic acid probe set is 400-600 nM.

[0054] In a preferred embodiment of the present invention, the concentration of each single-stranded nucleic acid probe in the single-stranded nucleic acid probe set is 500 nM.

[0055] In certain embodiments of the present invention, the single-stranded nucleic acid probe that is complementary to the base pairing of microRNA-21 is denoted as P1, and the nucleotide sequence of P1 is shown in SEQ ID NO.1; the single-stranded nucleic acid probe that is complementary to the base pairing of microRNA-17 is denoted as P2, and the nucleotide sequence of P2 is shown in SEQ ID NO.2; and / or the single-stranded nucleic acid probe that is complementary to the base pairing of microRNA-92a is denoted as P3, and the nucleotide sequence of P3 is shown in SEQ ID NO.3.

[0056] In certain embodiments of the present invention, in step 1), the fluorescent group is selected from one or more of FAM, TAMRA, Cy5, Cy3, Cy5.5, Cy7, NED or TET.

[0057] In certain embodiments of the present invention, in step 1), the incubation temperature is 25°C to 37°C.

[0058] In a preferred embodiment of the present invention, in step 1), the incubation temperature is 37°C.

[0059] In certain embodiments of the present invention, in step 1), the incubation time is 20 to 40 minutes.

[0060] In a preferred embodiment of the present invention, in step 1), the incubation time is 30 minutes.

[0061] In certain embodiments of the present invention, in step 1), the sample to be tested and the single-stranded nucleic acid probe set that is complementary to the base pairing thereof are mixed in a buffer.

[0062] In a preferred embodiment of the present invention, in step 1), the buffer is a Tris-HCl buffer having a pH of 8.0.

[0063] In certain embodiments of the present invention, in step 2), the ratio of each single-stranded nucleic acid probe in the single-stranded nucleic acid probe group to the aminated UiO-66 is 500nM: (0.1-2) mg / mL. The ratio of each single-stranded nucleic acid probe to the aminated UiO-66 is selected from any of the following ranges: 500nM: (0.1-0.5) mg / mL, 500nM: (0.5-1) mg / mL, 500nM: (1-1.5) mg / mL, 500nM: (1.5-2) mg / mL.

[0064] In a preferred embodiment of the present invention, in step 2), the ratio of the single-stranded nucleic acid probe to the amino-modified UiO-66 is 500 nM:0.8 mg / mL.

[0065] In certain embodiments of the present invention, in step 2), the incubation time is less than 60 min.

[0066] In a preferred embodiment of the present invention, in step 2), the incubation time is 30 min to 40 min.

[0067] In certain embodiments of the present invention, in step 2), the incubation temperature is 25-45°C.

[0068] In a preferred embodiment of the present invention, in step 2), the incubation temperature is 37°C.

[0069] In certain embodiments of the present invention, in step 2), the aminated UiO-66 is UiO-66-NH2.

[0070] In certain embodiments of the present invention, in step 2), the preparation method of UiO-66-NH2 comprises the following steps:

[0071] a) mixing ZrCl4 and H2BDC-NH2 in a DMF solution and dissolving them to obtain a mixed solution;

[0072] b) subjecting the mixed solution prepared in step a) to a solvothermal reaction and collecting the generated precipitate;

[0073] c) vacuum drying the precipitate obtained in step b) to obtain powdered UiO-66-NH2.

[0074] In certain embodiments of the present invention, in step a), based on the volume of the mixed solution, the molar concentration ratio of ZrCl4 to H2BDC-NH2 is 1:(1-1.5).

[0075] In a preferred embodiment of the present invention, in step a), the molar concentration ratio of ZrCl4 and H2BDC-NH2 is 1:1 based on the volume of the mixed solution.

[0076] In certain embodiments of the present invention, in step b), the solvothermal reaction time is 10 to 16 hours. The solvothermal reaction time is selected from any of the following ranges: 10 to 12 hours, 12 to 14 hours, and 14 to 16 hours.

[0077] In a preferred embodiment of the present invention, in step b), the solvent thermal reaction time is 12 hours.

[0078] In certain embodiments of the present invention, in step b), the temperature of the solvothermal reaction is 110-130°C.

[0079] In a preferred embodiment of the present invention, in step b), the temperature of the solvothermal reaction is 120°C.

[0080] In certain embodiments of the present invention, in step b), the generated precipitate is collected by centrifugation.

[0081] In certain embodiments of the present invention, step b) further comprises washing the generated precipitate with ethanol, the number of washings being 1 to 3 times.

[0082] In certain embodiments of the present invention, in step c), the vacuum drying time is 12 to 18 hours.

[0083] In certain embodiments of the present invention, in step 2), the fluorescence intensity in the second mixed system is determined by setting parameters such as excitation wavelength, emission wavelength, detection range, etc. according to the fluorescent group modified on the single-stranded nucleic acid probe, reading the fluorescence signal, and detecting the sample to be tested.

[0084] The present invention also provides the use of the ratiometric fluorescent sensor for simultaneously detecting multiple colorectal cancer microRNAs in the preparation of colorectal cancer diagnostic products.

[0085] The present invention also provides a colorectal cancer diagnostic product, which includes the ratiometric fluorescent sensor for simultaneously detecting multiple colorectal cancer microRNAs.

[0086] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0087] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0088] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0089] Example 1 Synthesis of UiO-66-NH2

[0090] Reagents: ZrCl4 was purchased from Aladdin Biochemical Technology Co., Ltd., with the catalog number Z109460; H2BDC-NH2 was purchased from Aladdin Biochemical Technology Co., Ltd., with the catalog number A151463; DMF solution was purchased from Aladdin Biochemical Technology Co., Ltd., with the catalog number D112004.

[0091] First, 1.03 mmol of ZrCl4 and 1.02 mmol of H2BDC-NH2 were mixed with 20 mL of DMF solution and sonicated at room temperature until they were completely dissolved;

[0092] Then, the solution was transferred to a reaction vessel for solvothermal reaction and reacted at 120 °C for 12 h;

[0093] Finally, the resulting pale yellow precipitate was collected by centrifugation and washed three times with ethanol; the powder was dried in a vacuum overnight and kept at room temperature until use.

[0094] Example 2 Experiment to investigate the effect of UiO-66-NH2 incubation time on target microRNA response

[0095] 500nM microRNA-21 and 500nM P1 were hybridized in Tris-HCl buffer at 37°C to form the first mixed system. After incubation for 30min, 0.3mg / mL UiO-66-NH2 was added and incubated at 37°C for 0min, 5min, 10min, 15min, 20min, 30min, 45min and 60min, and the fluorescence intensity of the eight mixed solutions at 521nm and 450nm was measured respectively. The results are shown in Figure 1 shown.

[0096] Depend on Figure 1 It can be seen that with the increase of time, when microRNA-21 is present, F 521 / F 450 The fluorescence ratio of UiO-66-NH2 first decreased and then maintained. When UiO-66-NH2 and the first mixed system were incubated for 30 to 40 minutes, the fluorescence ratio of 521 / F 450 The fluorescence ratio of UiO-66-NH2 reached a plateau value. Therefore, the optimal incubation time of UiO-66-NH2 was 30 min.

[0097] Example 3 Experiment on the effect of UiO-66-NH2 concentration on the response of microRNA-21, microRNA-17 and microRNA-92a

[0098] 500nM microRNA-21, 500nM microRNA-17 and 500nM microRNA-92a were hybridized with single-stranded nucleic acid probes 500nM P1, 500nM P2 and 500nM P3 in Tris-HCl buffer at 37°C for 30min, and different concentrations of UiO-66-NH2 (0.1-2mg / mL) were added. After incubation at 37°C for 30min, the fluorescence intensity at 521nm, 581nm, 670nm and 450nm was measured. The results are shown in Figure 2 A and 2B. The reaction system is shown in Table 1 below:

[0099] Table 1 Experimental study on the effect of UiO-66-NH2 concentration on the responses of three microRNAs

[0100]

[0101] Depend on Figure 2 As shown in A, with the increase of UiO-66-NH2 concentration, when there is no target, the fluorescence intensity of FAM, TAMRA and Cy5 channels first decreases and then maintains. When there is target microRNA, the corresponding fluorescence intensity of FAM, TAMRA and Cy5 channels first decreases and then maintains. At the same UiO-66-NH2 concentration, the F / F 450 Both were higher than the F / F of no-target microRNA 450 .Depend on Figure 2 B shows that with the increase of UiO-66-NH2 concentration, the signal-to-noise ratio of FAM, TAMRA and Cy5 channels first increases and then decreases. Therefore, the optimal concentration of UiO-66-NH2 is 0.8 mg / mL.

[0102] Example 4 Simultaneous quantitative detection of colorectal cancer-related markers microRNA-21, microRNA-17 and microRNA-92a

[0103] Different concentrations of microRNA-21, microRNA-17 and microRNA-92a were hybridized with 500nM P1, 500nM P2 and 500nM P3 in Tris-HCl buffer at 37°C for 30min, and then 0.8mg / mL UiO-66-NH2 was added and incubated at 37°C for 30min. The fluorescence intensity at 521nm, 581nm, 670nm and 450nm was measured. The results are shown in Figure 2. Figure 3 The specific reaction system is shown in Table 2:

[0104] Table 2 Simultaneous quantitative detection of three microRNAs related to colorectal cancer

[0105]

[0106]

[0107] Depend on Figure 3 As shown in A, the fluorescence intensity of FAM, TAMRA and Cy5 channels gradually increased with the increase of the concentration of target microRNA-21, microRNA-17 and microRNA-92a, respectively. The fluorescence intensity of UiO-66-NH2 channel remained unchanged. Figure 3 B shows that F 521 / F 450 、F 581 / F 450 and F 670 / F 450 The expression of these proteins gradually increased with the increase of concentrations of microRNA-21, microRNA-17 and microRNA-92a respectively.

[0108] Depend on Figure 3 C, 3D and 3E show that the fluorescence intensity ratio F 521 / F 450 、F 581 / F 450 and F 670 / F 450 The linear relationship of microRNA-21 with the concentration of microRNA-21, microRNA-17 and microRNA-92a in the range of 5-1000nM, 5-800nM and 5-800nM was shown to be F 521 / F 450 =0.001C microRNA-21 +0.0297(R 2 =0.985), LOD was 0.97nM; microRNA-17 linear relationship was F 581 / F 450 =0.0001C microRNA-17 +0.0674(R 2 =0.9742), LOD was 1.66nM; microRNA-92a linear relationship was F 670 / F 450 =0.0007C microRNA-92a +0.305(R 2 =0.9768), LOD was 3.43 nM.

[0109] Example 5 Selective study of colorectal cancer-related microRNA-21 in the reaction system

[0110] 500nM microRNA-21, microRNA-17, microRNA-92a, microRNA-19a, microRNA-31, microRNA-133a, microRNA-200c and microRNA-221 were hybridized with 500nM P1, 500nM P2 and 500nM P3 in Tris-HCl buffer at 37℃ for 30min, then 0.8mg / mL UiO-66-NH2 was added and incubated at 37℃ for 30min, and the fluorescence intensity at 521nm and 450nm was measured. The results are shown in Figure 4 The specific sample volume and reaction system are shown in Table 3:

[0111] Table 3 Selective study of colorectal cancer-related microRNA-21

[0112]

[0113]

[0114] Depend on Figure 4 It can be seen that the reaction system of the present invention has good selectivity for colorectal cancer-related microRNA-21.

[0115] Example 6 Selective study of colorectal cancer-related microRNA-17 in the reaction system

[0116] 500nM microRNA-17, microRNA-21, microRNA-92a, microRNA-19a, microRNA-31, microRNA-133a, microRNA-200c and microRNA-221 were hybridized with 500nM P1, 500nM P2 and 500nM P3 in Tris-HCl buffer at 37°C for 30min, then 0.8mg / mL UiO-66-NH2 was added and incubated at 37°C for 30min, and the fluorescence intensity at 581nm and 450nm was measured. The results are shown in Figure 5 The specific sample volume and reaction system are shown in Table 4:

[0117] Table 4 Selective study of colorectal cancer-related microRNA-17

[0118]

[0119] Depend on Figure 5 It can be seen that the reaction system of the present invention has good selectivity for colorectal cancer-related microRNA-17.

[0120] Example 7 Selective study of colorectal cancer-related microRNA-92a in the reaction system

[0121] 500nM microRNA-92a, microRNA-21, microRNA-17, microRNA-19a, microRNA-31, microRNA-133a, microRNA-200c and microRNA-221) were hybridized with 500nM P1, 500nM P2 and 500nM P3 in Tris-HCl buffer at 37°C for 30min, then 0.8mg / mL UiO-66-NH2 was added and incubated at 37°C for 30min, and the fluorescence intensity at 670nm and 450nm was measured. The results are shown in Figure 6 The specific sample volume and reaction system are shown in Table 5:

[0122] Table 5 Selective study of colorectal cancer-related microRNA-92a

[0123]

[0124] Depend on Figure 6 It can be seen that the reaction system of the present invention has good selectivity for colorectal cancer-related microRNA-92a.

[0125] Example 8 Simultaneous quantitative detection of related markers microRNA-21, microRNA-17 and microRNA-92a in tissue samples of colorectal cancer patients

[0126] Sample 1: 5 μL colorectal cancer tissue sample was hybridized with 500 nM P1, 500 nM P2, and 500 nM P3 in Tris-HCl buffer at 37°C for 30 min, then 0.8 mg / mL UiO-66-NH2 was added and incubated at 37°C for 30 min, and the fluorescence intensity at 521 nm, 581 nm, 670 nm, and 450 nm was measured;

[0127] Sample 2: 5 μL colorectal cancer adjacent tissue sample was hybridized with 500 nM P1, 500 nM P2, and 500 nM P3 in Tris-HCl buffer at 37°C for 30 min, then 0.8 mg / mL UiO-66-NH2 was added and incubated at 37°C for 30 min, and the fluorescence intensity at 521 nm, 581 nm, 670 nm, and 450 nm was measured;

[0128] The relative expression of sample markers was calculated based on the fluorescence intensity and related linear relationships. The results are as follows: Figure 7 shown.

[0129] Depend on Figure 7 It can be seen that the expression levels of related markers microRNA-21, microRNA-17 and microRNA-92a in colorectal cancer tissue samples are higher than those in adjacent cancer tissues, which proves that this method has good practical application capabilities.

[0130] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A ratiometric fluorescence sensor for simultaneously detecting multiple colorectal cancer microRNAs, characterized in that: The ratiometric fluorescence sensor comprises: Amination of UiO-66; A single-stranded nucleic acid probe set that is complementary to colorectal cancer-related microRNA bases, wherein the single-stranded nucleic acid probe set includes a plurality of single-stranded nucleic acid probes that are complementary to different colorectal cancer-related microRNA bases, and one end of the single-stranded nucleic acid probe is modified with a fluorescent group; Various single-stranded nucleic acid probes are modified with different fluorescent groups.

2. The fluorescence sensor according to claim 1, characterized in that The aminated UiO-66 is UiO-66-NH2; and / or the length of the single-stranded nucleic acid probe is 20 to 30 nt; And / or, the emission wavelength of the fluorescent group is different from the emission wavelength of the amino-modified UiO-66; preferably, the fluorescent group is selected from one or more of FAM, TAMRA, Cy5, Cy3, Cy5.5, Cy7, NED or TET.

3. The fluorescence sensor according to claim 1, characterized in that: The microRNA associated with colorectal cancer is selected from multiple ones of microRNA-21, microRNA-17 and / or microRNA-92a.

4. The fluorescence sensor according to claim 3, characterized in that: The nucleotide sequence of the single-stranded nucleic acid probe that is complementary to the microRNA-21 base is shown in SEQ ID NO.1; the nucleotide sequence of the single-stranded nucleic acid probe that is complementary to the microRNA-17 base is shown in SEQ ID NO.2; and / or the nucleotide sequence of the single-stranded nucleic acid probe that is complementary to the microRNA-92a base is shown in SEQ ID NO.

3.

5. A method for quantitatively detecting multiple colorectal cancer microRNAs for purposes other than disease diagnosis or treatment, characterized in that: The ratiometric fluorescence sensor described in any one of claims 1 to 4 is used to perform quantitative detection on a sample to be tested.

6. The quantitative detection method according to claim 5, characterized in that: The quantitative detection method comprises the following steps: 1) mixing the sample to be tested and a single-stranded nucleic acid probe set that is complementary to the sample and has one end modified with a fluorescent group, and then incubating the mixture to obtain a first mixed system; 2) The first mixed system prepared in step 1) is mixed with the amino-modified UiO-66 and then incubated to obtain a second mixed system, and the fluorescence intensity of the second mixed system is measured.

7. The detection method according to claim 6, characterized in that: In step 1), any one or more of the following features are included: 11) The sample to be tested is selected from microRNA associated with colorectal cancer in tissues, cells or blood; preferably, the microRNA associated with colorectal cancer is selected from multiple types of microRNA-21, microRNA-17 and / or microRNA-92a; more preferably, the nucleotide sequence of the single-stranded nucleic acid probe complementary to the base pairing of microRNA-21 is shown in SEQ ID NO.1; the nucleotide sequence of the single-stranded nucleic acid probe complementary to the base pairing of microRNA-17 is shown in SEQ ID NO.2; and / or the nucleotide sequence of the single-stranded nucleic acid probe complementary to the base pairing of microRNA-92a is shown in SEQ ID NO.3; 12) The fluorescent group is selected from one or more of FAM, TAMRA, Cy5, Cy3, Cy5.5, Cy7, NED or TET; 13) The incubation temperature is 25°C to 37°C; 14) The incubation time is 20 to 40 minutes; 15) Based on the volume of the first mixed system, the concentration of the single-stranded nucleic acid probe group complementary to the base of the sample to be tested is 800-1800 nM; preferably, the single-stranded nucleic acid probe group includes more than two single-stranded nucleic acid probes; more preferably, the concentration of each single-stranded nucleic acid probe is 400-600 nM; 16) The sample to be tested and the single-stranded nucleic acid probe set that is complementary to its base are mixed in a buffer; preferably, the buffer is Tris-HCl buffer.

8. The detection method according to claim 6, characterized in that: In step 2), any one or more of the following features are included: 21) Based on the volume of the second mixed system, the ratio of each single-stranded nucleic acid probe in the single-stranded nucleic acid probe set to amino-modified UiO-66 is 500 nM: (0.1-2) mg / mL; 22) The incubation time is less than 60 min; 23) The incubation temperature is 25-45°C; 24) The aminated UiO-66 is UiO-66-NH2; 25) Determining the fluorescence intensity in the second mixed system is to set parameters such as excitation wavelength, emission wavelength, detection range, etc. according to the fluorescent group modified on the single-stranded nucleic acid probe, read the fluorescence signal, and detect the sample to be tested.

9. Use of the ratiometric fluorescence sensor as claimed in any one of claims 1 to 4 in the preparation of a colorectal cancer diagnosis product.

10. A colorectal cancer diagnostic product, characterized in that: The diagnostic product comprises the ratiometric fluorescence sensor according to any one of claims 1 to 4.

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