Method for detecting microRNA based on core-shell structure up-conversion nanoparticles

Abstract

The invention discloses a method for detecting microRNA based on core-shell structure up-conversion nanoparticles, and belongs to the technical field of biomarker detection. The detection method comprises the following steps: chemically bonding a capture probe H1 to a sodium citrate modified core-shell structure up-conversion nanoparticle through oscillation incubation, preparing a capture probe H1 modified core-shell structure up-conversion nanoparticle buffer solution, preparing a detection solution from the buffer solution, microRNA and a catalytic probe H2 through oscillation incubation, and finally, detecting the microRNA in the detection solution. The method comprises the following steps: respectively testing the fluorescence intensity of a detection solution at 566 nm and 654 nm under the action of 514 nm laser and 980 nm laser, drawing a standard working curve of fluorescence quenching efficiency-microRNA concentration and a standard working curve of fluorescence recovery efficiency-microRNA concentration, and establishing a linear working equation; the detection method provided by the invention can realize rapid detection of the biomarker microRNA-222 in a serum sample, has the advantages of low detection limit, high sensitivity and the like, and has a wide application prospect in the field of low-abundance biomarker detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarker detection, and in particular relates to a method for detecting microRNA based on core-shell structure upconversion nanoparticles. Background Art

[0002] MicroRNA is an important class of endogenous non-coding RNA that plays an important role in controlling gene expression, cell differentiation and apoptosis. Recent studies have shown that abnormal expression levels of microRNA are associated with a variety of diseases, including cancer. Therefore, microRNA has been used as a prospective biomarker for early disease diagnosis and prognosis. Sensitive and specific determination of microRNA not only helps to understand the pathogenesis of these diseases, but also improves the accuracy of clinical diagnosis and prognosis. Due to the small size of most microRNAs, low abundance in biological samples and sequence homology between family members, traditional microRNA detection methods have the disadvantages of low sensitivity, long time consumption, susceptibility to environmental influences and high equipment requirements. Therefore, it is increasingly urgent to explore a new method for detecting microRNAs in a simple, rapid, sensitive, accurate and specific manner. Summary of the invention

[0003] The present invention aims at the disadvantages of low sensitivity, long time consumption, susceptibility to environmental influence and high equipment requirements of the current microRNA detection method, and provides a method for detecting microRNA based on core-shell structure upconversion nanoparticles. The detection method is to first chemically bond the capture probe H1 to the sodium citrate modified core-shell structure upconversion nanoparticles by oscillating incubation, prepare the capture probe H1 modified core-shell structure upconversion nanoparticle buffer, and then prepare the detection solution by oscillating incubation of the above buffer with microRNA and catalytic probe H2, and finally, test the fluorescence intensity of the detection solution at 566nm and 654nm under the action of 514nm and 980nm lasers, draw the standard working curves of "fluorescence quenching efficiency-microRNA concentration" and "fluorescence recovery efficiency-microRNA concentration", and establish a linear working equation. The detection method of the present invention can realize the rapid detection of the biomarker microRNA-222 in serum samples, and has the advantages of low detection limit and high sensitivity, and has a wide range of application prospects in the field of low-abundance biomarker detection.

[0004] To achieve the above object, the present invention adopts the following technical solution:

[0005] A method for detecting microRNA based on core-shell structure upconversion nanoparticles is to first chemically bond the capture probe H1 to the sodium citrate modified core-shell structure upconversion nanoparticles by oscillating incubation, prepare the capture probe H1 modified core-shell structure upconversion nanoparticle buffer, then oscillate and incubate the buffer with microRNA and catalytic probe H2 to prepare a detection solution, finally, test the fluorescence intensity of the detection solution at 566nm and 654nm under the action of 514nm and 980nm lasers, draw the standard working curves of "fluorescence quenching efficiency-microRNA concentration" and "fluorescence recovery efficiency-microRNA concentration", and establish a linear working equation; specifically, the following steps are included:

[0006] (1) First, 150-250 μL of a 0.1 mg / mL sodium citrate-modified core-shell structure upconversion nanoparticle deionized water dispersion was added to 1.5-3 mL of MES buffer, and magnetically stirred at room temperature for 30-60 min. Then, 10-30 mg of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a weight ratio of 1:2 was added, and magnetic stirring was continued at room temperature for 30-60 min. Finally, 10-60 μL of a 15 mg / L capture probe H1 TE buffer was added, and the mixture was shaken and incubated at 37° C. for 2-4 h. After centrifugation and washing with deionized water, the mixture was dispersed in 500 μL of PBS buffer to prepare a capture probe H1-modified core-shell structure upconversion nanoparticle buffer;

[0007] (2) adding 10 μL of TE buffer containing microRNA-222 of different concentrations, 20 μL of TE buffer containing 7 mg / L catalytic probe H2, and 20 μL of capture probe H1 modified core-shell structure upconversion nanoparticle buffer prepared in step (1) to 150 μL of PBS buffer, and incubating at room temperature for 30 min to prepare a detection solution;

[0008] (3) First, the fluorescence intensity of the detection solution at 566 nm and 654 nm was tested under the action of 514 nm and 980 nm lasers, respectively. Then, the concentration of microRNA-222 was used as the horizontal axis, and the fluorescence quenching efficiency at 566 nm and the fluorescence recovery efficiency at 654 nm were used as the vertical axes to draw the standard working curves of "fluorescence quenching efficiency-microRNA-222 concentration" and "fluorescence recovery efficiency-microRNA concentration-222", and a linear working equation was established.

[0009] Furthermore, the sodium citrate modified core-shell structure upconversion nanoparticles are prepared according to the literature (Li Baoming, Lin Yingying, Xing Yifeng, Lv Haixia; Method for detecting tert-butylhydroquinone based on core-shell structure upconversion nanoparticles, Chinese invention patent, application number: 202111395473.2, 2023-03-17); the core of the nanoparticle is lithium thulium co-doped sodium erbium tetrafluoride, wherein the molar ratio of erbium and lithium elements is 85-95:5-15, and the molar ratio of erbium and thulium elements is 98-99:1-2; the shell of the nanoparticle is ytterbium-doped sodium tetrafluoride, wherein the molar ratio of gadolinium and ytterbium elements is 70-90:10-30.

[0010] Furthermore, the MES buffer is a 10 mmol / L 2-(N-morpholine)ethanesulfonic acid aqueous solution with a pH value of 6.

[0011] Furthermore, the TE buffer solution is a mixed aqueous solution of tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid with a pH value of 8, wherein the concentration of tris(hydroxymethyl)aminomethane is 10 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1 mmol / L.

[0012] Furthermore, the concentrations of the microRNA-222 buffer in step (2) are 0 nmol / L, 0.5 nmol / L, 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L, respectively.

[0013] Compared with the prior art, the present invention has the following significant advantages:

[0014] (1) The method for detecting microRNA of the present invention uses a fluorescence spectrometer as the main detection device, which has good repeatability, fast response speed, simple instrumentation, and easy operation. Deionized water is used as the solvent, which is green and safe, and the sample does not require complicated pretreatment.

[0015] (2) The fluorescence detection method of microRNA described in the present invention uses core-shell structure upconversion nanoparticles as energy donors and near-infrared light as an excitation light source to induce the upconversion nanoparticles to emit fluorescence, which can significantly reduce the fluorescence background of the detection and improve the sensitivity of the detection. The black hole quencher-2 is used as an energy acceptor, which has the advantages of low background fluorescence, high signal-to-noise ratio, high sensitivity, and excellent quenching effect.

[0016] (3) The target detection object microRNA of the present invention can be regarded as a catalyst for the assembly of multiple H1 / H2 double chains. Each cycle of amplification reaction will restore the red light at 654nm and quench the green light at 566nm, thereby enhancing the intensity of the microRNA detection signal. In addition, the dual-color detection signal can avoid false positive signals and improve the measurement accuracy.

[0017] (4) The detection method of the present invention can detect microRNA-222 in serum samples, and has the advantages of rapidity, low detection limit, and high sensitivity. At 566nm, the fluorescence quenching efficiency is linearly related to the concentration range of microRNA-222 from 0.5 to 4.5nmol / L, the linear correlation coefficient is 0.9847 to 0.9941, and the detection limit is 6.3 to 8.7pmol / L. At 654nm, the fluorescence recovery efficiency is linearly related to the concentration range of microRNA-222 from 0.5 to 4.5nmol / L, the linear correlation coefficient is 0.9754 to 0.9886, and the detection limit is 4.6 to 6.1pmol / L; it has broad application prospects in the field of low-abundance biomarker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the UV-visible absorption spectra of the sodium citrate modified core-shell structure upconversion nanoparticles and the capture probe H1 modified core-shell structure upconversion nanoparticles prepared in Example 1.

[0019] Figure 2 The fluorescence emission spectra of the detection solution prepared in Example 1 at different microRNA-222 concentrations under the action of 514nm (a) and 980nm (b) lasers.

[0020] Figure 3 The standard working curve diagram (a) of “fluorescence quenching efficiency-microRNA-222 concentration” at 566 nm and the standard working curve diagram (b) of “fluorescence recovery efficiency-microRNA-222 concentration” at 654 nm obtained in Example 1 are shown.

[0021] Figure 4 This is the principle of detecting microRNA-222 in Example 1. DETAILED DESCRIPTION

[0022] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0023] Example 1

[0024] A method for detecting microRNA based on core-shell structure upconversion nanoparticles, specifically comprising the following steps:

[0025] (1) First, 200 μL of 0.1 mg / mL sodium citrate modified core-shell structure upconversion nanoparticle deionized water dispersion was added to 2.5 mL MES buffer, and magnetically stirred at room temperature for 45 min. Then, 20 mg of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a weight ratio of 1:2 was added, and magnetic stirring was continued at room temperature for 45 min. Finally, 35 μL of 15 mg / L capture probe H1 in TE buffer was added, and the mixture was shaken and incubated at 37°C for 3 h. After centrifugation and washing with deionized water, the mixture was dispersed in 500 μL PBS buffer to prepare capture probe H1 modified core-shell structure upconversion nanoparticle buffer;

[0026] (2) adding 10 μL of TE buffer containing microRNA-222 of different concentrations, 20 μL of TE buffer containing 7 mg / L catalytic probe H2, and 20 μL of capture probe H1 modified core-shell structure upconversion nanoparticle buffer prepared in step (1) to 150 μL of PBS buffer, and incubating at room temperature for 30 min to prepare a detection solution;

[0027] (3) First, the fluorescence intensity of the detection solution at 566 nm and 654 nm was tested under the action of 514 nm and 980 nm lasers, respectively. Then, the concentration of microRNA-222 was used as the horizontal axis, and the fluorescence quenching efficiency at 566 nm and the fluorescence recovery efficiency at 654 nm were used as the vertical axes to draw the standard working curves of "fluorescence quenching efficiency-microRNA-222 concentration" and "fluorescence recovery efficiency-microRNA concentration-222", and a linear working equation was established.

[0028] Furthermore, the core of the sodium citrate modified core-shell structure upconversion nanoparticles is lithium thulium co-doped sodium erbium tetrafluoride, wherein the molar ratio of erbium to lithium elements is 90:10, and the molar ratio of erbium to thulium elements is 98.5:1.5; the shell of the nanoparticles is ytterbium doped sodium tetrafluoride, wherein the molar ratio of gadolinium to ytterbium elements is 80:20.

[0029] Furthermore, the MES buffer is a 10 mmol / L 2-(N-morpholine)ethanesulfonic acid aqueous solution with a pH value of 6.

[0030] Furthermore, the TE buffer solution is a mixed aqueous solution of tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid with a pH value of 8, wherein the concentration of tris(hydroxymethyl)aminomethane is 10 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1 mmol / L.

[0031] Furthermore, the concentrations of the microRNA-222 buffer in step (2) are 0 nmol / L, 0.5 nmol / L, 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L, respectively.

[0032] Figure 1 The UV-visible absorption spectra of the sodium citrate modified core-shell structure upconversion nanoparticles and the capture probe H1 modified core-shell structure upconversion nanoparticles prepared in this example. As can be seen from the figure, the sodium citrate modified core-shell structure upconversion nanoparticles have no obvious absorption peak within the test wavelength range, and the capture probe H1 modified core-shell structure upconversion nanoparticles have an obvious characteristic absorption peak of the capture probe H1 at a wavelength of 260nm, which indicates that the capture probe H1 is successfully modified on the surface of the core-shell structure upconversion nanoparticles.

[0033] Figure 2 Fluorescence emission spectra of the detection solution prepared for the present embodiment under the action of 514nm (a) and 980nm (b) lasers when the concentration of microRNA-222 is 0nmol / L, 0.5nmol / L, 1.5nmol / L, 2.5nmol / L, 3.5nmol / L and 4.5nmol / L, respectively. As can be seen from the figure, the fluorescence emission peak of the detection solution under the action of 514nm laser is located at 566nm, and the fluorescence emission peak under the action of 980nm laser is located at 654nm, and as the concentration of microRNA-222 increases, the fluorescence emission peak intensity at 566nm gradually decreases, and the fluorescence emission peak intensity at 654nm gradually increases, and when the concentration of microRNA-222 is 4.5nmol / L, the fluorescence emission peak intensity at 566nm reaches the minimum value, and the fluorescence emission peak intensity at 654nm reaches the maximum value, which shows that the detection solution prepared by the present invention can be used for dual signal and high sensitivity detection of microRNA.

[0034] Figure 3 The standard working curves of "fluorescence quenching efficiency-microRNA-222 concentration" (a) and "fluorescence recovery efficiency-microRNA-222 concentration" (b) for the detection solution prepared in this example at emission peaks at 566nm and 654nm, respectively. In the figure, the fluorescence quenching efficiency is defined as (F 0 -F) / F 0 , the fluorescence recovery efficiency is defined as (FF 0 ) / F 0 , where F 0is the fluorescence intensity of the detection solution when the concentration of microRNA-222 is 0 nmol / L, and F is the fluorescence intensity of the detection solution when the concentration of microRNA-222 is 0.5 nmol / L, 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L, respectively. It can be seen from the figure that the fluorescence quenching efficiency at the emission peak of 566nm and the fluorescence recovery efficiency at 654nm show a good linear relationship with the microRNA-222 concentration in the range of 0.5-4.5nmol / L. The linear working equations are Y=4.69X+0.415 and Y=4.415X+0.2125, respectively. The linear correlation coefficients are 0.9941 and 0.9886, respectively. According to the literature (Qi Honglan, Li Jiani, Zhang Chengxiao; Discussion on the relationship between detection limit and sensitivity and influencing factors, University Chemistry, 2021, (9): 219-225), the detection limits are calculated to be 6.3pmol / L and 4.6pmol / L, respectively.

[0035] Figure 4 The principle of detecting microRNA in this embodiment. As shown in the figure, the detection solution is composed of a core-shell structure upconversion nanoparticle with a surface-modified hairpin-shaped capture probe H1, a hairpin-shaped catalytic probe H2 and microRNA-222, wherein the end of the capture probe H1 carries a black hole quencher-2, the end of the catalytic probe H2 carries a fluorescent dye Cy3, the fluorescence emission peak of the fluorescent dye Cy3 is located at 566nm, the fluorescence emission peak of the core-shell structure upconversion nanoparticle is located at 654nm, and the molecular chains of the capture probe H1, the catalytic probe H2 and the microRNA-222 all contain specific domains and complementary sequences.

[0036] When the detection solution does not contain microRNA-222, the hairpin structures of the capture probe H1 and the catalytic probe H2 are stable. Since the black hole quencher-2 is close to the nanoparticles, the black hole quencher-2 can quench the fluorescence of the nanoparticles through fluorescence resonance energy transfer. Since the black hole quencher-2 is far from the fluorescent dye Cy3, the fluorescence of the fluorescent dye Cy3 of the catalytic probe H2 is not affected. Therefore, when the concentration of microRNA-222 in the detection solution is 0 nmol / L, the emission peak at 566 nm has the highest intensity, and the emission peak at 654 nm has the lowest intensity.

[0037] When microRNA-222 is added to the detection solution, the domain of microRNA-222 first combines with the complementary domain on the capture probe H1, and the hairpin-shaped molecular chain of the capture probe H1 is transformed into a straight chain. Then, the exposed straight chain domain of the capture probe H1 combines with the complementary domain on the catalytic probe H2, and the hairpin-shaped molecular chain of the catalytic probe H2 is transformed into a straight chain. Finally, the domain of the capture probe H1 completely combines with the complementary domain of the catalytic probe H2, causing the microRNA-222 molecular chain to fall off the capture probe H1 molecular chain. The detached microRNA-222 then triggers the next cycle. At this time, the distance between the black hole quencher-2 and the nanoparticles becomes farther, and the quenching ability of the black hole quencher-2 on the fluorescence of the nanoparticles weakens, while the distance between the black hole quencher-2 and the fluorescent dye Cy3 becomes closer. Through the fluorescence resonance energy transfer, the quenching ability of the black hole quencher-2 on the fluorescence of the fluorescent dye Cy3 is enhanced. Therefore, as the concentration of microRNA-222 in the detection solution increases, the emission peak at 566nm gradually weakens, and the emission peak at 654nm gradually increases.

[0038] In summary, through the above-mentioned circulation effect, trace amounts of microRNA-222 can cause significant changes in the fluorescence emission peak intensity of core-shell structure upconversion nanoparticles and fluorescent dye Cy3. Therefore, the present invention can achieve high-precision detection of microRNA-222 through changes in dual-color fluorescence signals.

[0039] Example 2

[0040] A method for detecting microRNA based on core-shell structure upconversion nanoparticles, specifically comprising the following steps:

[0041] (1) First, 150 μL of 0.1 mg / mL sodium citrate modified core-shell structure upconversion nanoparticle deionized water dispersion was added to 1.5 mL MES buffer, and magnetically stirred at room temperature for 30 min. Then, 10 mg of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a weight ratio of 1:2 was added, and magnetic stirring was continued at room temperature for 30 min. Finally, 10 μL of TE buffer containing capture probe H1 at a concentration of 15 mg / L was added, and the mixture was incubated at 37°C for 2 h with shaking. After centrifugation and washing with deionized water, the mixture was dispersed in 500 μL PBS buffer to prepare capture probe H1 modified core-shell structure upconversion nanoparticle buffer;

[0042] (2) adding 10 μL of TE buffer containing microRNA-222 of different concentrations, 20 μL of TE buffer containing 7 mg / L catalytic probe H2, and 20 μL of capture probe H1 modified core-shell structure upconversion nanoparticle buffer prepared in step (1) to 150 μL of PBS buffer, and incubating at room temperature for 30 min to prepare a detection solution;

[0043] (3) First, the fluorescence intensity of the detection solution at 566 nm and 654 nm was tested under the action of 514 nm and 980 nm lasers, respectively. Then, the concentration of microRNA-222 was used as the horizontal axis, and the fluorescence quenching efficiency at 566 nm and the fluorescence recovery efficiency at 654 nm were used as the vertical axes to draw the standard working curves of "fluorescence quenching efficiency-microRNA-222 concentration" and "fluorescence recovery efficiency-microRNA concentration-222", and a linear working equation was established.

[0044] Furthermore, the core of the sodium citrate modified core-shell structure upconversion nanoparticles is lithium thulium co-doped sodium erbium tetrafluoride, wherein the molar ratio of erbium to lithium elements is 85:15, and the molar ratio of erbium to thulium elements is 98:2; the shell of the nanoparticles is ytterbium doped sodium tetrafluoride, wherein the molar ratio of gadolinium to ytterbium elements is 70:30.

[0045] Furthermore, the MES buffer is a 10 mmol / L 2-(N-morpholine)ethanesulfonic acid aqueous solution with a pH value of 6.

[0046] Furthermore, the TE buffer solution is a mixed aqueous solution of tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid with a pH value of 8, wherein the concentration of tris(hydroxymethyl)aminomethane is 10 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1 mmol / L.

[0047] Furthermore, the concentrations of the microRNA-222 buffer in step (2) are 0 nmol / L, 0.5 nmol / L, 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L, respectively.

[0048] The fluorescence quenching efficiency of the detection solution prepared in this example at the emission peak of 566 nm and the fluorescence recovery efficiency at 654 nm showed a good linear relationship with the microRNA-222 concentration in the range of 0.5 to 4.5 nmol / L. The linear working equations were Y=4.471X+0.589 and Y=4.025X+0.2462, respectively. The linear correlation coefficients were 0.9853 and 0.9814, respectively. The calculated detection limits were 8.7 pmol / L and 6.1 pmol / L, respectively.

[0049] Example 3

[0050] (1) First, 250 μL of 0.1 mg / mL sodium citrate modified core-shell structure upconversion nanoparticle deionized water dispersion was added to 3 mL of MES buffer, and magnetically stirred at room temperature for 60 min. Then, 30 mg of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a weight ratio of 1:2 was added, and magnetic stirring was continued at room temperature for 60 min. Finally, 60 μL of 15 mg / L capture probe H1 in TE buffer was added, and the mixture was shaken and incubated at 37°C for 4 h. After centrifugation and washing with deionized water, the mixture was dispersed in 500 μL of PBS buffer to prepare capture probe H1 modified core-shell structure upconversion nanoparticle buffer;

[0051] (2) adding 10 μL of TE buffer containing microRNA-222 of different concentrations, 20 μL of TE buffer containing 7 mg / L catalytic probe H2, and 20 μL of capture probe H1 modified core-shell structure upconversion nanoparticle buffer prepared in step (1) to 150 μL of PBS buffer, and incubating at room temperature for 30 min to prepare a detection solution;

[0052] (3) First, the fluorescence intensity of the detection solution at 566 nm and 654 nm was tested under the action of 514 nm and 980 nm lasers, respectively. Then, the concentration of microRNA-222 was used as the horizontal axis, and the fluorescence quenching efficiency at 566 nm and the fluorescence recovery efficiency at 654 nm were used as the vertical axes to draw the standard working curves of "fluorescence quenching efficiency-microRNA-222 concentration" and "fluorescence recovery efficiency-microRNA concentration-222", and a linear working equation was established.

[0053] Furthermore, the core of the sodium citrate modified core-shell structure upconversion nanoparticles is lithium thulium co-doped sodium erbium tetrafluoride, wherein the molar ratio of erbium to lithium elements is 95:5, and the molar ratio of erbium to thulium elements is 99:1; the shell of the nanoparticles is ytterbium doped sodium tetrafluoride, wherein the molar ratio of gadolinium to ytterbium elements is 90:10.

[0054] Furthermore, the MES buffer is a 10 mmol / L 2-(N-morpholine)ethanesulfonic acid aqueous solution with a pH value of 6.

[0055] Furthermore, the TE buffer solution is a mixed aqueous solution of tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid with a pH value of 8, wherein the concentration of tris(hydroxymethyl)aminomethane is 10 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1 mmol / L.

[0056] Furthermore, the concentrations of the microRNA-222 buffer in step (2) are 0 nmol / L, 0.5 nmol / L, 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L, respectively.

[0057] The fluorescence quenching efficiency of the detection solution prepared in this example at the emission peak of 566 nm and the fluorescence recovery efficiency at 654 nm showed a good linear relationship with the microRNA-222 concentration in the range of 0.5 to 4.5 nmol / L. The linear working equations were Y=4.535X+0.8362 and Y=4.36X+0.521, respectively. The linear correlation coefficients were 0.9847 and 0.9754, respectively. The calculated detection limits were 7.4 pmol / L and 5.3 pmol / L, respectively.

[0058] Table 1 Comparison of the method for detecting microRNA-222 of the present invention with the existing detection method

[0059]

[0060] As can be seen from Table 1, compared with existing detection methods, the method for detecting microRNA of the present invention has the advantages of rapidity, low detection limit, high sensitivity, etc.

[0061] The above description is only an illustrative embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be pointed out that, for ordinary technicians in this technical field, a number of improvements and supplements made without departing from the method of the present invention should also be regarded as the protection scope of the present invention; any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the protection scope of the present invention.

Claims

1. A method for detecting microRNA based on core-shell structure upconversion nanoparticles, characterized in that: The detection method comprises the following steps: firstly chemically bonding the capture probe H1 to the sodium citrate modified core-shell structure upconversion nanoparticles by oscillating incubation, preparing a capture probe H1 modified core-shell structure upconversion nanoparticle buffer, then oscillating incubating the buffer with microRNA and catalytic probe H2 to prepare a detection solution, and finally testing the fluorescence intensity of the detection solution at 566nm and 654nm under the action of 514nm and 980nm lasers, respectively, drawing the standard working curves of "fluorescence quenching efficiency-microRNA concentration" and "fluorescence recovery efficiency-microRNA concentration", and establishing a linear working equation; specifically comprising the following steps: (1) First, 150-250 μL of a 0.1 mg / mL sodium citrate-modified core-shell structure upconversion nanoparticle deionized water dispersion was added to 1.5-3 mL of MES buffer, and magnetically stirred at room temperature for 30-60 min. Then, 10-30 mg of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a weight ratio of 1:2 was added, and magnetic stirring was continued at room temperature for 30-60 min. Finally, 10-60 μL of a 15 mg / L capture probe H1 TE buffer was added, and the mixture was shaken and incubated at 37° C. for 2-4 h. After centrifugation and washing with deionized water, the mixture was dispersed in 500 μL of PBS buffer to prepare a capture probe H1-modified core-shell structure upconversion nanoparticle buffer; (2) adding 10 μL of TE buffer containing microRNA-222 of different concentrations, 20 μL of TE buffer containing 7 mg / L catalytic probe H2, and 20 μL of capture probe H1 modified core-shell structure upconversion nanoparticle buffer prepared in step (1) to 150 μL of PBS buffer, and incubating at room temperature for 30 min to prepare a detection solution; (3) First, the fluorescence intensity of the detection solution at 566 nm and 654 nm was tested under the action of 514 nm and 980 nm lasers, respectively. Then, the concentration of microRNA-222 was used as the horizontal axis, and the fluorescence quenching efficiency at 566 nm and the fluorescence recovery efficiency at 654 nm were used as the vertical axes to draw the standard working curves of "fluorescence quenching efficiency-microRNA-222 concentration" and "fluorescence recovery efficiency-microRNA concentration-222", and a linear working equation was established.

2. The method according to claim 1, characterized in that: The core of the sodium citrate modified core-shell structure upconversion nanoparticles is lithium thulium co-doped sodium erbium tetrafluoride, wherein the molar ratio of erbium to lithium is 85-95:5-15, and the molar ratio of erbium to thulium is 98-99:1-2; the shell of the nanoparticles is ytterbium doped sodium tetrafluoride, wherein the molar ratio of gadolinium to ytterbium is 70-90:10-30.

3. The method according to claim 1, characterized in that: The MES buffer solution is a 10 mmol / L 2-(N-morpholino)ethanesulfonic acid aqueous solution with a pH value of 6.

4. The method according to claim 1, characterized in that: The TE buffer solution is a mixed aqueous solution of tris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid with a pH value of 8, wherein the concentration of tris(hydroxymethyl)aminomethane is 10 mmol / L and the concentration of ethylenediaminetetraacetic acid is 1 mmol / L.

5. The method according to claim 1, characterized in that: The concentrations of the microRNA-222 buffer in step (2) are 0 nmol / L, 0.5 nmol / L, 1.5 nmol / L, 2.5 nmol / L, 3.5 nmol / L, and 4.5 nmol / L, respectively.

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