A multi-target miRNA bioassay kit based on upconversion fluorescence encoded magnetic beads and a construction method and application thereof

Abstract

The application discloses a multi-target miRNA biological detection kit based on up-conversion fluorescent coded magnetic beads and a construction method and application thereof. The kit comprises three kinds of up-conversion fluorescent coded magnetic beads with different emitting colors (yellow-green, red and blue-violet), capture probes for different target miRNAs connected respectively, and corresponding fluorescent labeled detection probes. The capture probes are specifically hybridized with the target miRNAs, and then sandwich complexes are formed with the detection probes, the characteristics of the up-conversion fluorescent coded signal and the detection signal without spectral interference under different excitation wavelengths are utilized, and the magnetic separation technology is combined, so that high-throughput, high-sensitivity and high-specificity simultaneous detection of multiple miRNAs is realized. The application effectively solves the problems of insufficient miRNA detection specificity, low throughput and spectral overlap in the prior art, and provides a new detection tool for early multi-index diagnosis of tumors and other diseases.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a multi-target miRNA biological detection kit based on upconversion fluorescent encoded magnetic beads, its construction method, and its application. Background Technology

[0002] Among numerous diseases, tumors are highly malignant, characterized by a high rate of recurrence and metastasis, resulting in generally shorter patient survival. Precision medicine enables early diagnosis and personalized treatment plans by detecting changes in the concentration of tumor markers; therefore, tumor marker detection has significant clinical value. MicroRNAs (miRNAs), as non-coding small RNA molecules, are closely related to the dysregulation of various diseases and cancer progression. Research on miRNAs not only helps to elucidate biological mechanisms but also serves as diagnostic, prognostic biomarkers and therapeutic targets, making it a research hotspot in the biomedical field.

[0003] However, miRNAs are present in extremely low concentrations in body fluids, have short and unstable sequences, and require highly sensitive and stable detection environments. Furthermore, the high similarity among miRNA molecules within the same family poses a significant challenge to detection specificity. Traditional single-target detection methods, such as chemiluminescence immunoassays, have low detection rates, while parallel multi-target detection can increase the sensitivity and specificity of tumor detection, significantly improving the detection rate. Therefore, there is an urgent need to develop a versatile, highly sensitive, and high-throughput biological detection method for multiple target miRNAs.

[0004] The current mainstream miRNA detection technology is real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This method first reverse transcribes miRNA into complementary DNA (cDNA), and then achieves quantitative analysis through amplification and real-time monitoring of fluorescence signals. However, this technology has two significant drawbacks: first, due to the high sequence homology, the kit is prone to cross-reactivity, making it difficult to distinguish specific miRNAs and resulting in insufficient specificity; second, each well can only detect one target, limiting throughput and making it unsuitable for simultaneous detection of multiple targets. These limitations restrict its application in clinical multi-indicator diagnosis.

[0005] Upconversion luminescent materials possess unique advantages such as low background signal, high signal-to-noise ratio, narrow emission peak, and good stability. Fluorescently encoded magnetic beads constructed from these materials hold promise for overcoming the shortcomings of existing technologies. However, miRNA bioassay kits based on upconversion fluorescently encoded magnetic beads are currently unavailable both domestically and internationally. Therefore, designing novel miRNA capture and detection probes, and developing multi-target miRNA detection kits based on upconversion fluorescently encoded magnetic beads, holds significant research value and market potential for achieving high-throughput, multi-component, and accurate miRNA detection. Summary of the Invention

[0006] This invention discloses a multi-target miRNA biodetection kit based on upconversion fluorescently encoded magnetic beads, its construction method, and its application. The method involves preparing three types of upconversion fluorescently encoded magnetic beads (yellow-green, red, and blue-violet), connecting them to corresponding miRNA capture probes, and designing fluorescently labeled detection probes. These probes form a complex through sandwich hybridization. Combined with magnetic separation and dual-wavelength excitation, this achieves high-throughput, high-sensitivity simultaneous detection of multiple target miRNAs without spectral interference.

[0007] On the one hand, the present invention provides a multi-target miRNA biodetection kit based on upconversion fluorescently encoded magnetic beads, which adopts the following technical solution: A multi-target miRNA bioassay kit based on upconversion fluorescently encoded magnetic beads, comprising: Yellow-green upconversion fluorescently encoded magnetic beads, red upconversion fluorescently encoded magnetic beads, and blue-violet upconversion fluorescently encoded magnetic beads; Biotinylated capture probes were attached to the surfaces of three types of coding magnetic beads. The capture probes were single-stranded DNA sequences complementary to half of the target miRNA. A detection probe that is complementary to the other half of the target miRNA sequence and labeled with a fluorescent reporter molecule; Positive control, negative control and buffer solution.

[0008] Preferably, the yellow-green upconversion fluorescently encoded magnetic beads are Fe3O4@SiO2-NH-CO-[NaYF4:Yb / Er / Gd]; the red upconversion fluorescently encoded magnetic beads are Fe3O4@SiO2-NH-CO-[NaErF4:Tm@NaYF4]; and the blue-violet upconversion fluorescently encoded magnetic beads are Fe3O4@SiO2-NH-CO-[NaLuF4:Yb / Gd / Tm].

[0009] Preferably, the fluorescent reporter molecule is Rhodamine B.

[0010] Preferably, the target miRNAs are miRNA-10b, miRNA-145, and miRNA-155; The sequences of the capture probes are SEQ ID NO:2, SEQ ID NO:5, and SEQ ID NO:8, respectively; The sequences of the detection probes are SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9, respectively.

[0011] Preferably, the buffer solution includes TE buffer, washing buffer, and blocking buffer; the TE buffer is composed of 10 mM Tris-HCl, 1 mM EDTA, and 2 M NaCl, with a pH of 8.0; the washing buffer is TE buffer with 0.05% Tween 20 added; and the blocking buffer is PBS buffer with 1% BSA added.

[0012] On the other hand, the present invention also provides a method for constructing a multi-target miRNA biological detection kit based on upconversion fluorescently encoded magnetic beads, using the following technical solution: A method for constructing a multi-target miRNA bioassay kit based on upconversion fluorescently encoded magnetic beads includes the following steps: Step 1: Prepare three types of upconversion fluorescent encoded magnetic beads for yellow-green, red, and blue-violet light respectively; Step 2: Attach streptavidin to the surface of the coded magnetic beads to obtain streptavidin-modified coded magnetic beads; Step 3: Incubate the biotinylated capture probe with streptavidin-modified encoded magnetic beads to couple the capture probe to the surface of the magnetic beads, thus obtaining the encoded magnetic bead-capture probe complex. Step 4: Synthesize detection probes labeled with fluorescent reporter molecules; Step 5: Prepare positive control, negative control, and buffer solution; Step 6: Assemble the above components into a kit.

[0013] Preferably, in step one, the method for preparing the encoded magnetic beads is as follows: aminated magnetic beads and carboxylated upconversion nanoparticles are subjected to an amide condensation reaction in the presence of EDC and NHS, the reaction temperature is room temperature, and the reaction time is 4 hours.

[0014] Preferably, in step two, the binding conditions for streptavidin are: rotational reaction at room temperature for 6 hours; in step three, the binding conditions for the biotinylated capture probe are: rotational reaction at room temperature for 2 hours.

[0015] This invention also provides a detection method for a multi-target miRNA biological detection kit based on upconversion fluorescently encoded magnetic beads, employing the following technical solution: A method for simultaneous detection of multiple target miRNAs using a multi-target miRNA bioassay kit based on upconversion fluorescently encoded magnetic beads, comprising the following steps: (1) The test sample, positive control and negative control are mixed with the magnetic bead-capture probe complex and incubated to allow the capture probe to hybridize with the target miRNA to form the magnetic bead-capture probe-miRNA complex. (2) Magnetic separation washing to remove unbound impurities; (3) Add a detection probe labeled with a fluorescent reporter molecule and incubate to form a sandwich complex encoding a magnetic bead-capture probe-miRNA-detection probe; (4) Magnetic separation and washing to remove unbound detection probes; (5) 980 nm laser excitation was used to detect the fluorescence signal of the encoded magnetic beads to distinguish different types of target miRNAs; 488 nm laser excitation was used to detect the fluorescence intensity of the fluorescent reporter molecule for quantitative analysis. (6) Calculate the content of target miRNA in the sample to be tested based on the standard curve established by the positive control.

[0016] Preferably, the incubation conditions are: 37°C with shaking reaction for 30 minutes; magnetic separation washing is performed using TE buffer containing 0.05% Tween 20; the fluorescent reporter molecule is Rhodamine B, with an emission wavelength of 580 nm.

[0017] In summary, the beneficial effects of the present invention are as follows: The beneficial effects of this invention are mainly reflected in two aspects: detection performance and ease of operation. Firstly, this invention uses upconversion fluorescent encoded magnetic beads as the encoding carrier, which has low background signal, high signal-to-noise ratio, and narrow emission peak, effectively avoiding spectral interference and overlap between the encoded signal and the detection signal, and is expected to improve the accuracy and sensitivity of simultaneous detection of multiple target miRNAs. The upconversion material emits light under 980 nm excitation, while the detection probe (Rhodamine B) emits light under 488 nm excitation. This dual-wavelength excitation enables crosstalk-free independent signal acquisition, overcoming the technical defect of overlapping encoded and reporter signals in traditional organic fluorescent dyes.

[0018] Secondly, this invention combines the rapid separation and purification capabilities of magnetic microspheres, enabling the enrichment of encoded magnetic beads within 30 seconds. This simplifies the operation process, improves detection efficiency and accuracy, and enhances the controllability and automation of the detection process. By designing highly specific capture and detection probes and combining them with multivariate detection technology, it is possible to simultaneously integrate and analyze multiple miRNA molecular variables, potentially solving the problem of miRNAs being difficult to detect due to their short sequences and high homology. This kit has broad application prospects in the early multi-indicator diagnosis of diseases such as tumors. Attached Figure Description

[0019] Figure 1 This is based on the principle of multi-target miRNA detection; Figure 2 Image 'a' in the image is a SEM image of a yellow-green light-coded magnetic bead. Figure 2 Image b is a near-infrared laser confocal scanning microscope (CLSM) image of a yellow-green light-coded magnetic bead. Figure 2 Image c in the middle is a near-infrared laser confocal scanning microscope (CLSM) image of a yellow-green light-coded magnetic bead. Figure 2 Image d shows the dispersion of yellow-green optically encoded magnetic beads under a magnetic field irradiation with a 980nm laser. Figure 2 In the image, 'e' represents the SEM image of the red-light encoded magnetic bead. Figure 2 f is a near-infrared laser confocal scanning microscope (CLSM) image of a red light-coded magnetic bead. Figure 2 In the middle, g is a near-infrared laser confocal scanning microscope (CLSM) image of a red light-encoded magnetic bead. Figure 2 The image in the middle (h) shows the dispersion of red-encoded magnetic beads under a magnetic field irradiation by a 980nm laser. Figure 2 In the image, i represents the SEM image of the blue-violet light-coded magnetic bead; Figure 2 The image in middle j is a near-infrared laser confocal scanning microscope (CLSM) image of a blue-violet light-coded magnetic bead. Figure 2 In the middle, k is a near-infrared laser confocal scanning microscope (CLSM) image of a blue-violet light-coded magnetic bead. Figure 2 The image in the middle is a photograph of the dispersion of blue-violet light-coded magnetic beads under a magnetic field irradiation by a 980nm laser. Figure 3 In Figure 'a', the fluorescence spectrum of a mixture of magnetic beads encoded by three commercial organic fluorescent dyes is shown. Figure 3 Figure b shows the mixed fluorescence spectrum of three types of upconversion fluorescently encoded magnetic beads: yellow-green, red, and blue-violet. Figure 4 In Figure 'a', the fluorescence spectrum of a mixture of magnetic beads encoded by three commercial organic fluorescent dyes is shown. Figure 4 b shows the fluorescence spectrum of a mixture of three commercially available organic fluorescent dye-encoded magnetic beads and Rhodamine B. Figure 4 In the middle, c represents the fluorescence spectrum of a mixture of three commercially available organic fluorescent dye-encoded magnetic beads and Cy5. Figure 5 In Figure a, the mixed fluorescence spectrum of upconversion fluorescent encoded magnetic beads in yellow-green, red, and blue-violet colors is shown. Figure 5 Figure b shows the mixed fluorescence spectrum of yellow-green, red, and blue-violet upconversion fluorescently encoded magnetic beads with Rhodamine B. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments.

[0021] Example Example 1 The specific steps for preparing a yellow-green optically encoded magnetic bead are as follows: Take 1 mg of aminated magnetic beads (Fe3O4@SiO2-NH2), wash twice with MES buffer (0.1 M, pH 5.5), and magnetically separate, discarding the supernatant. Add 2 mg of carboxylated yellow-green upconversion nanoparticles (NaYF4:Yb / Er / Gd) to the magnetic beads, add EDC (final concentration 2 mM) and NHS (final concentration 5 mM), and rotate the reaction at room temperature for 4 h. Magnetically separate, wash three times with deionized water to obtain yellow-green encoded magnetic beads (Fe3O4@SiO2-NH-CO-[NaYF4:Yb / Er / Gd]). Disperse the encoded magnetic beads in deionized water, store at 4℃, and adjust the final concentration to 1 mg / mL.

[0022] Example 2 The specific steps for preparing a red light-encoded magnetic bead are as follows: Take 1 mg of aminated magnetic beads (Fe3O4@SiO2-NH2), wash twice with MES buffer (0.1 M, pH 5.5), and magnetically separate, discarding the supernatant. Add 2 mg of carboxylated red upconversion nanoparticles (NaErF4:Tm@NaYF4) to the magnetic beads, add EDC (final concentration 2 mM) and NHS (final concentration 5 mM), and rotate the reaction at room temperature for 4 h. Magnetically separate, wash three times with deionized water to obtain red-encoded magnetic beads (Fe3O4@SiO2-NH-CO-[NaErF4:Tm@NaYF4]). Disperse the coded magnetic beads in deionized water, store at 4℃, and adjust the final concentration to 1 mg / mL.

[0023] Example 3 The specific steps for preparing a blue-violet light-coded magnetic bead are as follows: Take 1 mg of aminated magnetic beads (Fe3O4@SiO2-NH2), wash twice with MES buffer (0.1 M, pH 5.5), and magnetically separate, discarding the supernatant. Add 2 mg of carboxylated blue-violet upconversion nanoparticles (NaLuF4:Yb / Gd / Tm) to the magnetic beads, add EDC (final concentration 2 mM) and NHS (final concentration 5 mM), and rotate the reaction at room temperature for 4 h. Magnetically separate, wash three times with deionized water to obtain blue-violet encoded magnetic beads (Fe3O4@SiO2-NH-CO-[NaLuF4:Yb / Gd / Tm]). Disperse the encoded magnetic beads in deionized water, store at 4℃, and adjust the final concentration to 1 mg / mL.

[0024] Example 4 The coupling of the capture probe (encoded magnetic bead - probe1) is carried out through the following steps: S1. Take 30 µL of each of the three fluorescently encoded magnetic bead suspensions (yellow-green, red, and blue-violet) prepared in Examples 1-3 (1 mg / mL) and place them in centrifuge tubes. Magnetic separation is performed, and the supernatant is discarded. Add 10 µL of 1 mg / mL streptavidin to each tube, and then add PBS buffer to a total volume of 200 µL. Vortex the mixture for 6 h at room temperature. Magnetic separation is performed, and the beads are washed three times with deionized water to obtain streptavidin-modified fluorescently encoded magnetic beads.

[0025] S2. Magnetic separation of the streptavidin-modified magnetic beads and aspiration of the supernatant yields 100 µL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl). Add 21 µL of 100 µM biotinylated DNA probe (probe1, corresponding to the target miRNA-10b, -145, or -155 sequence, see Table 1). Yellow-green light-encoded magnetic beads correspond to miRNA-10b-probe1; red light-encoded magnetic beads correspond to miRNA-145-probe1; and blue-violet light-encoded magnetic beads correspond to miRNA-155-probe1. Vortex the mixture at room temperature for 2 h. Magnetic separation and washing three times with TE buffer yields the "magnetic bead-probe1 encoded" complex. Disperse the three complexes separately in TE buffer and store at 4°C for later use, adjusting the final concentration to 1 mg / mL.

[0026] Table 1. List of probes and miRNA sequences designed in the kit of this invention.

[0027] Example 5 The design and synthesis of the detection probe (probe2) follow these steps: Based on the sequences of three target miRNAs, a complementary DNA single strand (probe2) was designed. The probe design followed these principles: DNA fragments completely complementary to the target miRNA sequence were selected as probe sequences; probe length was controlled to 10-12 nucleotides; hairpin structures or dimers were avoided within the sequence; the melting temperatures (Tm) of probe-target hybridization were ensured to be similar to guarantee effective capture of all three targets under the same reaction conditions; and non-specific hybridization with non-target sequences was eliminated through BLAST alignment. Based on these principles, three sets of capture probes (probe1) and detection probes (probe2) were designed for miRNA-10b, miRNA-145, and miRNA-155, respectively. Specific sequences are shown in Table 1. The three oligonucleotide chains were synthesized by Shanghai Sangon Biotech Co., Ltd., and Rhodamine B fluorescent molecules were linked to the 3' end via a spacer arm ((CH2)6). After synthesis, the molecules were purified by HPLC and lyophilized. Before use, they were dissolved in TE buffer to 100 µM, aliquoted, and stored at -20°C protected from light.

[0028] Example 6 The specific steps for preparing the reference standard and buffer solution are as follows: S1 positive control Three miRNA mimics targeting the target RNA were synthesized (sequences shown in Table 1), dissolved and mixed in TE buffer to a final concentration of 1 µM for each miRNA. Then, 10-fold serial dilutions were performed (10... -1 10 -2 10 -3 10 -4 10 -5 µM), as a series of standards for establishing standard curves.

[0029] S2, Negative Control Whole blood was collected from healthy individuals, centrifuged at 1000 rpm for 10 min, and the supernatant serum was collected. After testing, it was confirmed that the serum did not contain the three target miRNAs. The serum was then aliquoted and stored at -80℃.

[0030] S3, buffer solution TE buffer: 10 mM Tris-HCl, 1 mM EDTA, 2 M NaCl, pH 8.0.

[0031] Washing solution: Add 0.05% Tween 20 to TE buffer.

[0032] Blocking solution: Add 1% (w / v) BSA to PBS buffer (pH 7.4).

[0033] Example 7 The specific steps for assembling and storing the reagent kit are as follows: S1. Take 10 µL (1 mg / mL) of each of the three coded magnetic bead-probe1 mixtures prepared in Example 4 and mix them in one tube, for a total volume of 30 µL; take 5 µL (100 µM) of each of the three probe2 mixtures prepared in Example 5 and mix them in one tube, for a total volume of 15 µL; take 10 µL of each of the six concentration gradients of the positive control series prepared in Example 7; take 10 µL of the negative control in one tube, for a total of two tubes; take 5 mL of TE buffer in one bottle, 10 mL of washing buffer (TE + Tween 20) in one bottle, and 5 mL of blocking buffer (PBS-BSA) in one bottle.

[0034] S2. Please attach the instruction manual, which includes: reagent kit composition and storage conditions; a schematic diagram of the detection principle (e.g., Figure 1 (As shown in the figure); detailed operating steps are shown in Example 8; result interpretation and quantitative analysis methods.

[0035] Example 8 The experimental procedure for simultaneous detection of multiple target miRNAs based on this kit is as follows: S1, Sample Pretreatment Take the whole blood sample to be tested and centrifuge at 1000 rpm for 10 min at 4°C. Carefully aspirate the upper serum layer (avoiding the leukocyte layer) and transfer it to a new centrifuge tube as the test sample. Prepare six concentrations of positive control and prepare TE buffer as a blank control (concentration 0). Prepare the negative control, which is a serum sample free of the three target miRNAs; use it directly without dilution.

[0036] S2, Hybridization capture reaction (formation of a complex encoding magnetic beads-probe1-miRNA) Three parallel groups were set up, with 5 wells in each group, as shown in Table 2. 30 µL of a mixture of fluorescently encoded magnetic beads-probe1 (10 µL each of yellow-green, red, and blue-violet, pre-mixed) was added to each well. The mixture was gently blown into the wells with a pipette. The 96-well plate was placed in a 37°C constant-temperature shaker and reacted at 200 rpm for 30 min.

[0037] Table 2 Detection Sample System

[0038] S3. Magnetic separation washing (removal of unbound impurities) Place the 96-well plate on a magnetic separator and let it stand for 1 min to allow the magnetic beads to adhere to the bottom and sidewalls of the wells. Carefully aspirate the supernatant using a pipette (avoid touching the magnetic beads). Add 100 µL of washing buffer (TE + 0.05% Tween 20) to each well, remove the magnetic separator, and gently pipette or agitate to redisperse the magnetic beads. Place the plate back on the magnetic separator and let it stand for 1 min, then aspirate the supernatant. Repeat the washing steps three times. After the final wash, add 100 µL of blocking buffer (PBS + 1% BSA) to each well, remove the magnetic separator, and resuspend the magnetic beads. Block at room temperature for 30 min to block non-specific binding sites on the surface of the magnetic beads.

[0039] S4. Sandwich complex formation (with addition of detection probe 2) Place the sealed 96-well plate on a magnetic separator and let it stand for 1 min, then discard the blocking solution. Add 5 µL of a mixture of three probe2 solutions (the final concentration of each probe2 is approximately 5 µM, which can be optimized as needed) and 95 µL of TE buffer to each well, making a total volume of 100 µL. Mix thoroughly by pipetting to ensure complete dispersion of the magnetic beads. Place the plate in a 37°C constant-temperature shaker and react at 200 rpm for 30 min.

[0040] S5. Secondary magnetic separation washing (removal of free probe2) After the reaction, place the plate on a magnetic separator and let it stand for 1 min, then discard the supernatant. Add 100 µL of washing buffer to each well, remove the magnetic separator, and resuspend the magnetic beads. Perform magnetic separation and discard the supernatant. Repeat the washing process three times to thoroughly remove unbound probe2 and other background substances. After the final wash, add 100 µL of TE buffer to each well, resuspend the magnetic beads, and prepare for signal detection.

[0041] S6. Dual-wavelength excitation and fluorescence signal detection The resuspended sample was transferred to a small test tube. First, the fluorescence spectra of the three types of coded magnetic beads were recorded using a 980 nm laser (peak intensities at 540 nm, 660 nm, and 475 nm) to confirm the normality of the coded signals in each well and to distinguish the target types. Then, the fluorescence intensity (Rhodamine B signal) at 580 nm was recorded using a 488 nm laser. The detection conditions are shown in Table 3.

[0042] Table 3 Detection Signal and Excitation Wavelength

[0043] S7. Quantitative Analysis and Result Calculation For each concentration in the positive standard group (S1~S6 and blank control), a standard curve was plotted with the Rhodamine B fluorescence intensity at 580 nm as the ordinate (Y-axis) and the logarithm of the target miRNA concentration as the abscissa (X-axis). Standard curves were established for each of the three miRNAs (miRNA-10b, -145, and -155). Since the three targets were detected simultaneously in the same reaction well, they could be distinguished by the peak positions of the encoding magnetic beads: yellow-green beads corresponded to miRNA-10b, red beads to miRNA-145, and blue-violet beads to miRNA-155. Therefore, at each standard concentration, three fluorescence intensity values ​​were actually obtained simultaneously, corresponding to three standard curves. The Rhodamine B fluorescence intensity at 580 nm was read from each well in the test sample group, and the three corresponding miRNAs were distinguished by the peak positions of the magnetic beads (these were removed). The fluorescence intensity of the test sample was substituted into the standard curve equation for the corresponding miRNA to calculate the concentration of that miRNA in the sample. The fluorescence intensity at 580 nm in the negative control group should be close to that of the blank control, with no obvious background signal. The correlation coefficient R of the standard curve... 2 The coefficient of variation (CV) should be ≥0.99; otherwise, retesting is required. The coefficient of variation (CV) between parallel wells should be ≤15%.

[0044] Test Example 1 A small number of samples were taken for SEM morphology characterization, CLSM fluorescence imaging, and luminescence observation under 980 nm laser excitation. The specific steps are as follows: CLSM fluorescence imaging and luminescence observation under 980 nm laser excitation were performed using a laser scanning confocal microscope (Olympus FV3000); SEM morphology characterization was performed using a scanning electron microscope (MIRA3 LMU).

[0045] Figure 2 These are scanning electron microscope (SEM) images, near-infrared laser confocal scanning microscope (CLSM) images, and a photograph of the dispersion of the fluorescently encoded magnetic beads under a magnetic field irradiated by a 980 nm laser. SEM images of the yellow-green encoded magnetic beads show that the prepared encoded magnetic beads have good monodispersity, an average particle size of 5 μm, a rough surface, and are coated with a large amount of particulate material. Figure 2 a). Furthermore, the CLSM images show a distinct yellow-green coating on the surface of the encoded magnetic beads, demonstrating that we have successfully fabricated high-intensity yellow-green encoded magnetic beads. The upconversion luminescent material is mainly concentrated on the surface of the magnetic beads, and the fluorescence distribution of the encoded magnetic beads is uniform. Figure 2 b, 2c). Furthermore, from photographs of the dispersion of yellow-green light-coded magnetic beads in an aqueous solution under a magnetic field, it can be concluded that ( Figure 2d) Magnets placed on the side of the test tube can enrich yellow-green light-encoded magnetic beads within 30 seconds, which plays a significant role in the subsequent rapid bioseparation process. Furthermore, the beads emit yellow-green light when excited by a 980 nm laser. Similarly, the morphology of the red light-encoded magnetic beads was characterized by SEM, such as... Figure 2 As shown in Figure e, the upconversion material attached to the surface of the magnetic beads is clearly visible, and the prepared coded magnetic beads have good monodispersity with an average particle size of 5 μm. Figure 2 The results of f and 2g CLSM images show that the surface of the encoded magnetic beads has a significant red light coating, proving that we have successfully prepared red-light encoded magnetic beads with high luminescence intensity. The upconversion luminescent material is mainly concentrated on the surface of the magnetic beads, and the fluorescence distribution of the encoded magnetic beads is uniform. Furthermore, the dispersion images of the red-light encoded magnetic beads in aqueous solution under a magnetic field show that… Figure 2 (h) A magnet on the side of the test tube can enrich red-encoded magnetic beads within 30 seconds. When excited by a 980 nm laser, the beads emit red light. Blue-violet coded magnetic beads were also characterized by SEM, CLSM, and bright-dark field photographs of aqueous solution dispersion under a magnetic field. The characterization results were consistent with those of the yellow-green and red coded magnetic beads described above. Figure 2 i-2l).

[0046] Test Example 2 The comparison of the signal resolution capabilities of upconversion encoded magnetic beads and organic dye encoded magnetic beads is conducted through the following steps: S1, a mixed sample of upconversion fluorescently encoded magnetic beads Take 30 µL (1 mg / mL) each of the yellow-green, red, and blue-violet upconversion fluorescently encoded magnetic beads prepared in Examples 1-3 and add them to the same centrifuge tube. Add TE buffer to a total volume of 1 mL, vortex for 30 seconds to fully disperse the magnetic beads. Magnetic separation for 1 min, discard the supernatant, and wash once with TE buffer. Finally, resuspend in 1 mL of TE buffer as the "upconversion mixed sample".

[0047] S2, Mixed sample of organic dye-coded magnetic beads Take 30 µL (1 mg / mL) each of three commercially available organic fluorescent dye-coded magnetic beads (green, yellow / orange, and red), purchased from Shenzhen Weigong Technology Co., Ltd., and add them to the same centrifuge tube. Add TE buffer to a total volume of 1 mL, vortex for 30 seconds to fully disperse the magnetic beads. Magnetic separation for 1 min, discard the supernatant, and wash once with TE buffer. Finally, resuspend in 1 mL TE buffer to obtain the "organic dye mixed sample".

[0048] S3, Fluorescence Spectroscopy Detection Transfer the "upconversion mixed sample" to a quartz cuvette. Set the fluorescence spectrometer parameters: excitation wavelength: 980 nm (laser); emission scan range: 400 nm ~ 800 nm; slit width: 5 nm (excitation and emission); integration time: 0.5 s; scan speed: 600 nm / min. Acquire the emission spectrum and record the peak intensity and full width at half maximum (FWHM) at 475 nm (blue-violet), 540 nm (yellow-green), and 660 nm (red).

[0049] Transfer the "organic dye mixture sample" into a quartz cuvette. Set the fluorescence spectrometer parameters as follows: excitation wavelength: 488 nm; emission scan range: 500 nm ~ 750 nm; slit width: 5 nm; integration time: 0.5 s; scan speed: 600 nm / min; acquire the emission spectrum and observe the separation degree and full width at half maximum (FWHM) of the three emission peaks.

[0050] S4. Data Processing: Normalize the spectra of the two samples and then overlay them. Compare the following indicators: Full width at half maximum (FWHM) of each emission peak: the upconversion sample should be significantly narrower than the organic dye sample; Peak separation: there should be obvious valleys between the three peaks of the upconversion sample; the three peaks of the organic dye sample should show "peak envelopment" or severe overlap; record typical spectra.

[0051] like Figure 3 As shown in Figure a, when three commercially available organic fluorescent dye-encoded magnetic beads are mixed, the measured fluorescence spectral peaks exhibit a peak-envelope pattern, which is not conducive to subsequent differentiation from the detection signal. However, when the three upconversion fluorescent-encoded magnetic beads are mixed, the measured emission bands are sharp, and the peak shapes are easily distinguishable. Figure 3 b).

[0052] Test Example 3 The spectral crosstalk experiment for the control group (organic dye-encoded magnetic beads) is conducted as follows: S1, Mixed sample of organic dye-coded magnetic beads Take 30 µL (1 mg / mL) of each of the three commercially available yellow-green, red, and blue-violet organic dye-coded magnetic beads and add them to the same centrifuge tube. Add TE buffer to a total volume of 1 mL, vortex for 30 seconds to fully disperse the magnetic beads. Magnetic separation for 1 min, discard the supernatant, and wash once with TE buffer. Finally, resuspend in 1 mL of TE buffer. This is the "organic dye mixed sample".

[0053] S2, Organic dye-coded magnetic beads + Rhodamine B sample Take 1 mL of the above "organic dye mixture sample". Add Rhodamine B stock solution (1 mM, dissolved in water) to a final concentration of 10 µM. Vortex mix for 30 seconds and incubate at room temperature in the dark for 10 min.

[0054] S3, Organic Dye-Encoded Magnetic Beads + Cy5 Sample Take another 1 mL of the "organic dye mixture sample" and add Cy5 stock solution (1 mM, dissolved in DMSO or water) to make a final concentration of 10 µM. Vortex mix for 30 seconds and incubate at room temperature in the dark for 10 min.

[0055] S4, organic dye-coded magnetic bead mixed sample Transfer the sample to a quartz cuvette. Set the following conditions: excitation wavelength: 488 nm; emission scan range: 500 nm–750 nm; slit width: 5 nm; integration time: 0.5 s; scan speed: 600 nm / min. Record the spectrum as a reference.

[0056] S5, Organic dye-coded magnetic beads + Rhodamine B sample Transfer the sample to a quartz cuvette. Set the parameters: excitation wavelength: 488 nm (can simultaneously excite the organic dye and Rhodamine B); emission scan range: 500 nm ~ 750 nm. Record the spectrum and observe: whether the encoded signal peaks of the organic dye itself (~520 nm, ~580 nm, ~660 nm) are still distinguishable; whether the emission peak of Rhodamine B (~580 nm) overlaps with the encoded signal.

[0057] S6, Organic Dye-Encoded Magnetic Beads + Cy5 Sample Transfer the sample to a quartz cuvette. Excitation wavelength: 488 nm (for the organic dye); emission scan range: 550 nm ~ 750 nm. Record the spectrum and observe the degree of overlap between the Cy5 emission peak (~670 nm) and the signal from the red-coded magnetic beads (~660 nm).

[0058] In the control group, such as Figure 4 As shown, the reporter signal obtained under 488 nm excitation overlaps with the encoded signal of the organic fluorescent dye-encoded magnetic bead, regardless of whether the emission peak is at 580 nm for Rhodamine B ( Figure 4 b) or Cy5 with an emission peak at 660 nm ( Figure 4 c) The spectral peaks overlapped, and there was no obvious reportable detection signal.

[0059] Test Example 4 The crosstalk-free verification experiment of upconversion encoded magnetic beads is carried out in the following steps: S1, Upconversion Encoded Magnetic Bead Mixed Sample Take 30 µL each of the yellow-green, red, and blue-violet upconversion fluorescently encoded magnetic beads and mix them in a centrifuge tube. Add TE buffer to 1 mL, vortex to mix, and wash once with magnetic separation. Resuspend in 1 mL TE buffer to obtain the "UCNP mixed sample".

[0060] S2, Upconversion Encoded Magnetite Beads + Rhodamine B Sample Take 1 mL of the above "UCNP mixed sample" and add Rhodamine B stock solution (1 mM) to make a final concentration of 10 µM. Vortex mix for 30 seconds and incubate at room temperature in the dark for 10 min to obtain the "UCNP + RhB sample".

[0061] S3. Fluorescence spectroscopy detection (dual-wavelength excitation) The coded signal was excited at 980 nm. The "UCNP + RhB" sample was transferred to a quartz cuvette. Spectrometer parameters were set as follows: excitation wavelength: 980 nm (laser); emission scan range: 400 nm ~ 800 nm; slit width: 5 nm; emission spectrum was acquired and recorded: ~475 nm (blue-violet light, code 1); ~540 nm (yellow-green light, code 2); ~660 nm (red light, code 3). The excitation signal was detected at 488 nm. This was done for the same sample, but with a different excitation source. Spectrometer parameters were set as follows: excitation wavelength: 488 nm (xenon lamp or laser); emission scan range: 500 nm ~ 750 nm; emission spectrum was acquired and recorded: ~580 nm (Rhodamine B emission peak). S4, dual-spectral superposition The two spectral curves obtained in (1) and (2) were normalized using Origin software and then superimposed on the same coordinate system. The relative positions of the two spectral curves on the wavelength axis were observed to confirm that the encoded signal region (~475, 540, 660 nm) and the reporting signal region (~580 nm) were completely separated and had no overlap.

[0062] like Figure 5 As shown, the fluorescence spectrum of the encoded signal was obtained under 980 nm excitation, while the fluorescence spectrum of the detected signal was obtained under 488 nm excitation, and no encoded signal was detected. Figure 5 a). A clear distinction was also observed when the peaks excited by two different excitation lights were superimposed. Figure 5 b).

[0063] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A multi-target miRNA bioassay kit based on upconversion fluorescently encoded magnetic beads, characterized in that, include: Yellow-green upconversion fluorescently encoded magnetic beads, red upconversion fluorescently encoded magnetic beads, and blue-violet upconversion fluorescently encoded magnetic beads; Biotinylated capture probes are respectively attached to the surface of the three types of encoded magnetic beads, wherein the capture probe is a single-stranded DNA complementary to half of the target miRNA sequence; A detection probe that is complementary to the other half of the target miRNA sequence and labeled with a fluorescent reporter molecule; Positive control, negative control and buffer solution.

2. The reagent kit according to claim 1, characterized in that, The yellow-green upconversion fluorescently encoded magnetic beads are Fe3O4@SiO2-NH-CO-[NaYF4:Yb / Er / Gd]; the red upconversion fluorescently encoded magnetic beads are Fe3O4@SiO2-NH-CO-[NaErF4:Tm@NaYF4]; and the blue-violet upconversion fluorescently encoded magnetic beads are Fe3O4@SiO2-NH-CO-[NaLuF4:Yb / Gd / Tm].

3. The reagent kit according to claim 1, characterized in that, The fluorescent reporter molecule is Rhodamine B.

4. The reagent kit according to claim 1, characterized in that, The target miRNAs are miRNA-10b, miRNA-145, and miRNA-155; The sequences of the capture probes are SEQ ID NO:2, SEQ ID NO:5 and SEQ ID NO:8, respectively; The sequences of the detection probes are SEQ ID NO:3, SEQ ID NO:6 and SEQ ID NO:9, respectively.

5. The reagent kit according to claim 1, characterized in that, The buffer solution includes TE buffer, washing buffer, and blocking buffer; the TE buffer is composed of 10 mM Tris-HCl, 1 mM EDTA, and 2 M NaCl, with a pH of 8.0; the washing buffer is TE buffer with 0.05% Tween 20 added; and the blocking buffer is PBS buffer with 1% BSA added.

6. A method for constructing a reagent kit according to any one of claims 1 to 5, characterized in that, Includes the following steps: Step 1: Prepare three types of upconversion fluorescent encoded magnetic beads for yellow-green, red, and blue-violet light respectively; Step 2: Attach streptavidin to the surface of the coded magnetic beads to obtain streptavidin-modified coded magnetic beads; Step 3: Incubate the biotinylated capture probe with streptavidin-modified encoded magnetic beads to couple the capture probe to the surface of the magnetic beads, thus obtaining the encoded magnetic bead-capture probe complex. Step 4: Synthesize detection probes labeled with fluorescent reporter molecules; Step 5: Prepare positive control, negative control, and buffer solution; Step 6: Assemble the above components into a kit.

7. The construction method according to claim 6, characterized in that, In step one, the method for preparing the encoded magnetic beads is as follows: aminated magnetic beads and carboxylated upconversion nanoparticles are subjected to an amide condensation reaction in the presence of EDC and NHS. The reaction temperature is room temperature and the reaction time is 4 hours.

8. The construction method according to claim 6, characterized in that, In step two, the streptavidin ligation conditions are: room temperature rotation reaction for 6 hours; in step three, the biotinylated capture probe ligation conditions are: room temperature rotation reaction for 2 hours.

9. A method for simultaneous detection of multiple target miRNAs using the kit according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) The test sample, positive control, and negative control are respectively mixed with the encoded magnetic bead-capture probe complex and incubated to allow the capture probe to hybridize with the target miRNA, forming a encoded magnetic bead-capture probe-miRNA complex; (2) Magnetic separation washing to remove unbound impurities; (3) Add a detection probe labeled with a fluorescent reporter molecule and incubate to form a sandwich complex encoding a magnetic bead-capture probe-miRNA-detection probe; (4) Magnetic separation and washing to remove unbound detection probes; (5) 980 nm laser excitation was used to detect the fluorescence signal of the encoded magnetic beads to distinguish different types of target miRNAs; 488 nm laser excitation was used to detect the fluorescence intensity of the fluorescent reporter molecule for quantitative analysis. (6) Calculate the content of target miRNA in the sample to be tested based on the standard curve established by the positive control.

10. The detection method according to claim 9, characterized in that, The incubation conditions were 37°C with shaking for 30 minutes; the magnetic separation and washing used TE buffer containing 0.05% Tween 20; the fluorescent reporter molecule was Rhodamine B, with an emission wavelength of 580 nm.

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