A method for preparing functionalized fluorescent protein multicolor coded microspheres

Abstract

The present invention belongs to the field of fluorescent microsphere coding technology, and specifically relates to a method for preparing functionalized fluorescent protein multicolor coded microspheres. Fluorescent protein is used instead of organic fluorescent dyes and nanomaterials, and the fluorescent protein is assembled on the surface of the microsphere by chemical coupling. The protein type, concentration and microsphere particle size are used as coding elements to obtain single-color and multicolor fluorescent microspheres with ultra-large coding capacity; the surface of the fluorescent microsphere is modified by biotin to fix streptavidin to the surface of the microsphere, and combined with biotin-labeled capture probes to achieve high-throughput rapid analysis of multiple target molecules. The preparation process of the present invention is simple, the coding capacity is large, the performance is stable, and it has strong versatility and is environmentally friendly. The microspheres of the present invention can be applied to disease diagnosis, food safety, environmental protection, proteomics, genomics and drug screening.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent microsphere coding, and in particular relates to a method for preparing functionalized fluorescent protein multicolor coding microspheres. Background Art

[0002] In recent years, array technology based on optically encoded microspheres (including liquid biochip technology and microsphere-based planar array chip technology) has incomparable advantages in achieving high-throughput, high-sensitivity, and multi-target simultaneous analysis in basic and applied research. It is particularly suitable for the analysis and determination of complex samples and has become a particularly attractive research field. As a signal carrier, the encoded microspheres should have the following properties: a large number of codes, good coding signal stability, good uniformity, functionalized microsphere surface, convenient decoding, good biocompatibility, and environmental friendliness.

[0003] As shown in Table 1, the existing preparation methods of fluorescent microspheres usually adopt physical methods (such as swelling method and emulsification method, etc.) and chemical methods (such as copolymerization method and self-assembly method, etc.), and the main fluorescent materials used are organic dyes and inorganic nanomaterials. The advantages of using organic fluorescent dyes to prepare coded microspheres are: organic fluorescent dyes have high quantum yields and are easy to couple and adsorb to microspheres. Its disadvantages are: organic fluorescent dyes have poor stability and anti-bleaching ability. Aggregation and fluorescence self-quenching will occur at high concentrations, which seriously limits its coding quantity and coding stability. Among inorganic nanomaterials, quantum dots have excellent optical properties, such as: wide and continuous absorption spectrum, narrow and symmetrical emission spectrum, high quantum efficiency and high stability. However, the reabsorption phenomenon and agglomeration effect caused by the FRET effect between quantum dots are also the biggest challenges of quantum dot coding, resulting in its coding capacity being far lower than its theoretical value, and even lower than the number of coded microspheres obtained by Luminex Co. using organic dyes (500 coding groups). Upconversion nanomaterials rely on atomic transitions to emit light. Compared with quantum dots and organic dyes, they have better fluorescence stability and no FRET effect, but their low quantum yield limits the increase in coding capacity. In addition, the results reported in existing literature show that the following problems still exist in the process of preparing fluorescent coded microspheres using traditional organic fluorescent dyes and inorganic fluorescent nanomaterials: the preparation steps of fluorescent coded microspheres are complicated and time-consuming; a variety of toxic chemical reagents are used in the preparation of fluorescent materials and coded microspheres, and these toxic chemical reagents will cause serious environmental pollution problems; the corresponding fluorescent coded microspheres may cause secondary disasters to the environment and human health during use. Therefore, this field urgently needs to develop a new environmentally friendly fluorescent material and a new method for preparing fluorescent coded microspheres to break through the current technical bottlenecks such as low coding capacity and potential pollution in the preparation and use of existing fluorescent materials, so as to meet the urgent needs of basic research and applied research in the rapidly developing clinical diagnosis, genomics and proteomics.

[0004] Table 1 Comparison of patent literature related to fluorescent coded microspheres

[0005] Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a method for preparing functional fluorescent protein multicolor coded microspheres, which can be applied to different research fields. The method is simple and easy to operate.

[0007] The technical solution of the present invention is as follows:

[0008] First, the functional groups modified on the surface of the microspheres were activated using an activation reagent, and then the fluorescent protein was assembled onto the surface of the microspheres by chemical coupling. The type, concentration and particle size of the fluorescent protein were used as coding elements to obtain a library of single-color, dual-color and tri-color fluorescent coded microspheres with ultra-large coding capacity. At the same time, the biotin-streptavidin system was introduced on the surface of the coded microspheres to make them functional fluorescent coded microspheres.

[0009] Specifically, the technical solution of the present invention is as follows:

[0010] A method for preparing functionalized fluorescent protein multicolor coded microspheres, comprising the following steps:

[0011] (1) obtaining fluorescent protein by microbial expression and purification, and diluting the obtained fluorescent protein into a fluorescent protein solution of 0 to 200 μg / mL;

[0012] The concentration of the fluorescent protein solution of 0 to 200 μg / mL in the above step may also be a trace amount to 200 μg / mL.

[0013] (2) Absorb 1×10 7 ~1×10 11 Place microspheres into an EP tube, add activation reagent (components see below) to fully suspend the microspheres, incubate in a constant temperature shaker at 25°C, 120-140 rpm for 0.5-1 h to activate the functional groups modified on the microsphere surface, and then wash with PBS three times to obtain activated microspheres;

[0014] (3) Loading fluorescent protein onto the microsphere surface: After the microspheres are activated, the microspheres are resuspended in the prepared fluorescent protein solution (1×10 7 microspheres, resuspended in a volume of 200 μL), incubated in a constant temperature shaker at 25°C and 120-140 rpm for 2-5 hours; after the reaction is completed, 200 μL of PBS blocking solution containing 1% BSA (bovine serum albumin) is added to each reaction tube to resuspend the microspheres, incubated in a constant temperature shaker at 25°C and 120-140 rpm for 0.5-1 hour to block the unreacted activated functional groups on the surface of the microspheres, and washed three times with phosphate buffered saline, i.e., PBS, after blocking; using the microsphere particle size, fluorescent protein type and loading amount as coding elements, single-color, dual-color and tri-color fluorescent coded microspheres are obtained;

[0015] (4) Add 450 μL PBS and 50 μL 10 mM biotin-NHS solution to the fluorescent encoded microspheres to resuspend the microspheres, and incubate them in a constant temperature shaker at 25°C and 120-140 rpm for 0.5 h; fully suspend the fluorescent microspheres with PBS and wash them three times;

[0016] (5) Add 500 μL of 50 μg / mL streptavidin solution to the biotin-labeled fluorescent microspheres, incubate at 25°C in a constant temperature shaker at 120-140 rpm for 0.5-2 h, wash three times with PBS, and connect streptavidin to the microsphere surface through biotin to obtain functionalized encoded microspheres.

[0017] in:

[0018] The fluorescent protein is one or two or a combination of two or more of mTagBFP, EYFP, EGFP, DsRed, mCherry, phycoerythrin (PE), allophycocyanin (APC), and a tandem complex of PE-Cy3, PE-Cy5, PE-Cy7, APC-Cy3, APC-Cy5 and APC-Cy7 labeled with a PE or APC fluorescent dye, and the loading concentration gradient can be 0 to 200 μg / mL.

[0019] In the above step, the concentration gradient of the loading concentration is 0 to 200 μg / mL, and the concentration gradient may be a trace amount to 200 μg / mL.

[0020] The microspheres are selected from agarose gel microspheres, dextran gel microspheres, polystyrene microspheres, silica microspheres, ferroferric oxide or ferric oxide magnetic microspheres, and the particle size of the microspheres is 1 to 30 μm.

[0021] The surface of the microsphere is modified with one of amino, carboxyl and hydroxyl functional groups.

[0022] The activation reagent is one or a combination of two of glutaraldehyde, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), sulfonated-NHS, epichlorohydrin, and N'N-carbonyldiimidazole.

[0023] The fluorescent protein is loaded onto the surface of the microsphere by utilizing the covalent condensation reaction between the naturally existing amino, carboxyl and other functional groups of the protein and the functional groups modified on the surface of the microsphere to couple the fluorescent protein to the surface of the microsphere; the type and loading amount of the fluorescent protein are adjusted to produce fluorescent coded microspheres with different fluorescent signals of single color, dual color and tri-color.

[0024] The functionalized fluorescent microspheres are obtained by introducing a biotin-streptavidin system on the surface of the encoded microspheres to form microsphere-fluorescent protein-streptavidin composite functional microspheres, which can efficiently bind to biotin-labeled capture probes (such as nucleic acids, antibodies, antigen proteins, receptor proteins, etc.) to achieve simultaneous qualitative and quantitative detection of multiple target analytes.

[0025] Preferably, the microspheres are 5-10 μm carboxyl-modified Fe3O4-polymer microspheres.

[0026] Preferably, the two-color fluorescent protein-encoded microspheres have a fluorescent protein group selected from a combination of blue fluorescent protein (mTagBFP) and green fluorescent protein (EGFP); a combination of blue fluorescent protein (mTagBFP) and red fluorescent protein (DsRed); a combination of green fluorescent protein (EGFP) and red fluorescent protein (DsRed). The three-color fluorescent protein-encoded microspheres are selected from green fluorescent protein (EGFP); red fluorescent protein (DsRed) and blue fluorescent protein (mTagBFP) three proteins are co-coupled to the same microsphere. However, the fluorescent protein combination selected for the fluorescent protein multi-color encoded microspheres in the implementation of the present invention is not limited thereto.

[0027] Preferably, the step (1) specifically comprises:

[0028] Fluorescent proteins were diluted with PBS, and the concentration gradient of mTagBFP was set to 0.1, 0.9, 2.8, 6.5, 60.0 μg / mL. The concentration gradient of EGFP was set to 0.5, 3.5, 15.0, 50.0, 150.0 μg / mL. The concentration gradient of DsRed was set to 0, 0.3, 0.8, 1.7, 3.2, 6.8, 15.0, 35.0 μg / mL.

[0029] Preferably, the step (2) specifically includes:

[0030] Absorb 1×10 7 ~1×10 11 Microspheres (i.e., 5 μm carboxyl-modified Fe3O4-polymer microspheres) were placed in an EP tube, and 500 μL of 100 mM 2-(N-morpholine) sodium ethanesulfonate (MEST, pH 5.0) solution was added to resuspend the magnetic beads, and magnetic separation and washing were performed twice, and the supernatant was removed. 200 μL of freshly prepared 10 mg / mL 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC / NHS, MEST as solvent) were quickly added, and the magnetic beads were fully suspended and dispersed by vortexing. The reaction was carried out at 25°C and 130 rpm on a shaker for 0.5 h. The supernatant was removed by magnetic separation, and 500 μL of phosphate buffer (PBS, 10 mM, pH 7.4, the same below) was added to fully suspend the magnetic beads, and washed 3 times to obtain activated carboxyl magnetic beads.

[0031] Preferably, the step (3) specifically comprises:

[0032] After the carboxyl magnetic beads were activated, the fluorescent protein solution diluted with PBS was added to resuspend the magnetic beads (the amount of magnetic beads for each coding group was 1×10 7 indivual).

[0033] Single-color coding is to couple a fluorescent protein solution of different concentrations with carboxyl magnetic beads to obtain a group of coded microspheres with different single-color fluorescence intensities. Add 200 μL of the fluorescent protein solution diluted in step (2) to each reaction tube and incubate at 25°C with a shaker at 130 rpm for 5 hours.

[0034] Dual-color coding is to combine three fluorescent proteins in pairs (e.g. mTagBFP and EGFP; mTagBFP and DsRed; EGFP and DsRed) to a magnetic microsphere to obtain a set of coded microspheres with different fluorescence intensities of two colors. The concentration gradient of fluorescent proteins is set the same as single-color coding. Add 200 μL of resuspended magnetic beads to each reaction tube of two fluorescent proteins, and incubate at 25°C, 130 rpm shaker for 5 hours.

[0035] Three-color coding is to couple three fluorescent proteins (mTagBFP, EGFP and DsRed) to one microsphere at the same time to obtain a set of coded microspheres with three different fluorescence intensities. The concentration gradient of fluorescent proteins is set the same as single-color coding. Add 200 μL of resuspended magnetic beads to each reaction tube of three fluorescent proteins, and incubate at 25°C, 130 rpm shaker for 5 hours.

[0036] After the reaction was completed, the magnetic beads were fully suspended with PBS and washed three times to obtain fluorescent microspheres.

[0037] Preferably, the step (4) specifically comprises:

[0038] Prepare a 10mM biotin reagent solution by dissolving 2.0mg biotin-NHS in 590μL dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). Add 450μL PBS and 50μL biotin solution to each reaction tube of the prepared encoded microspheres, and incubate at 25°C with a 130rpm shaker for 0.5h. Suspend the magnetic beads fully with PBS and wash 3 times.

[0039] Preferably, the step (5) specifically includes:

[0040] Add 500 μL of 50 μg / mL streptavidin solution to each reaction tube to resuspend the encoded microspheres, incubate at 25°C, shake on a shaker at 130 rpm for 2 h, fully suspend the magnetic beads with PBS and wash 3 times, connect streptavidin to the microsphere surface through biotin, and prepare a functionalized encoded microsphere.

[0041] The functionalized coded microspheres can be used for the detection of one or more targets, and the detection steps include:

[0042] (1) The biotin-labeled capture probe (nucleic acid, antibody, antigen protein, receptor protein, etc.) is specifically bound to the streptavidin on the surface of the functionalized encoded microspheres;

[0043] (2) capturing the target analyte in the sample to be tested onto the surface of the microspheres through the capture probes coupled to the surface of the microspheres;

[0044] (3) The target analyte captured on the surface of the microsphere specifically binds to the reporter probe labeled with a fluorescent substance;

[0045] (4) Microspheres with different fluorescence characteristics are identified by flow cytometry analysis or fluorescence microscopy imaging, and the fluorescence signal of the reporter probe bound to the surface of each microsphere is detected to obtain qualitative and quantitative analysis results of the target analyte.

[0046] Beneficial effects of the present invention:

[0047] (1) Compared with the preparation of fluorescent microspheres by swelling method or polymerization method, the present invention can directly use microspheres modified with functional groups to covalently couple fluorescent proteins for encoding, which greatly simplifies the encoding procedure and ensures the uniformity of its particle size. The operation is simple, time-saving, cost-effective, and decoding is convenient. The fluorescent encoded microspheres have good monodispersity and high fluorescence stability, and can be prepared in batches.

[0048] (2) Fluorescent proteins replace organic fluorescent dyes and inorganic fluorescent nanomaterials used in traditional methods for preparing fluorescent microspheres. They are cheap, easy to obtain, and environmentally friendly. They avoid the aggregation-induced quenching effect of fluorescent dyes and quantum dots and the pollution to the environment, and obtain a large-capacity fluorescent protein-encoded microsphere library. Taking the carboxyl-modified Fe3O4-polymer microspheres with a diameter of 5 μm as an example, the flow cytometer was used for decoding. In the single-color fluorescent encoded microspheres, MBs@mTagBFP obtained 5 coding groups, MBs@EGFP obtained 5 coding groups, and MBs@DsRed obtained 8 coding groups; in the dual-color fluorescent encoded microspheres, MBs@mTagBFP / EGFP obtained 25 coding groups, MBs@mTagBFP / DsRed obtained 40 coding groups, and MBs@DsRed / EGFP obtained 40 coding groups; in the three-color fluorescent encoded microspheres, MBs@mTagBFP / EGFP / DsRed obtained 160 coding groups. Fe3O4-polymer microspheres modified with carboxyl groups with a diameter of 10 μm achieved the same encoding effect under the same encoding conditions. The actual encoding capacity obtained by this patent has exceeded that of commercially available encoded microspheres.

[0049] (3) The streptavidin was fixed to the surface of the microspheres by self-assembly to form microsphere-fluorescent protein-streptavidin composite functional microspheres, which avoided the complicated steps of modifying the functional groups on the microsphere surface and reduced the cost of microsphere preparation. The functional microspheres can efficiently bind to the biotin-labeled capture probes to achieve qualitative and quantitative detection of multiple target analytes simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : Detection graph of forward scatter angle (FSC) and side scatter angle (SSC) signals of the encoded microspheres with a diameter of 5 μm (red) in the embodiment of the present invention by flow cytometry.

[0051] Figure 2 : Laser confocal microscopy images of single-color, dual-color and multi-color fluorescent protein combination encoded microspheres (diameter 5 μm) in the embodiments of the present invention. Figure 2 Figure a shows mTagBFP single-color coded microspheres; Figure 2 Figure b shows EGFP single-color coded microspheres; Figure 2 Figure c shows DsRed single-color coded microspheres; Figure 2 Figure d is a microscope image of the magnetic microspheres in bright field; Figure 2 Figure e shows mTagBFP and DsRed dual-color coded microspheres; Figure 2 Figure f shows the DsRed and EGFP dual-color coded microspheres; Figure 2 Figure g shows mTagBFP and EGFP dual-color coded microspheres; Figure 2 Figure h shows the three-color coded microspheres of mTagBFP, EGFP and DsRed.

[0052] Figure 3 : Cluster diagram and histogram of monochrome coded microspheres decoded by flow cytometry in the embodiment of the present invention. Explanation of reference numerals: The diameter of the microsphere is 5 μm, wherein Figure 3 Figure a1 in the middle shows the five groups of DsRed single-color coded microspheres; Figure 3 b1 in the middle shows five groups of EGFP single-color-encoded microspheres; Figure 3 Figure c1 in the middle shows 8 groups of mTagBFP single-color coded microspheres; Figure 3 Middle A2 picture, Figure 3 Figure b2, Figure 3 The c2 graphs are Figure 3 Middle A1 picture, Figure 3 Figure b1, Figure 3 The histogram corresponding to the c1 figure.

[0053] Figure 4 : A cluster diagram of dual-color coded microspheres decoded by flow cytometry in an embodiment of the present invention. Explanation of reference numerals: The diameter of the microsphere is 5 μm, Figure 4 Figure a in the middle shows 25 groups of mTagBFP and EGFP dual-color coded microspheres; Figure 4 Figure b in the middle shows 40 groups of DsRed and EGFP dual-color coded microspheres; Figure 4 Figure c shows 40 groups of mTagBFP and DsRed dual-color coded microspheres.

[0054] Figure 5 : A cluster diagram of three-color coded microspheres decoded by flow cytometry in an embodiment of the present invention. Explanation of reference numerals: The diameter of the microspheres is 5 μm, wherein Figure 5 Figure P shows the four groups encoded when the concentrations of mTagBFP were 0.1, 0.9, 2.8, and 6.5 μg / mL; Figure 5 Figures P1 to P4 in the figure are the grouping diagrams of EGFP and DsRed in the detection channels FL1-FL2 when the concentrations of mTagBFP are 0.1, 0.9, 2.8, and 6.5 μg / mL, respectively.

[0055] Figure 6 : A three-segment structure diagram formed when the encoded microspheres in the embodiment of the present invention detect the specific nucleic acid sequence on the uvrC gene of Mycoplasma bovis MB (black is the specific nucleic acid sequence MB-uvrC-T on the uvrC gene of Mycoplasma bovis; blue is the capture probe sequence MB-uvrC-C labeled with biotin; red is the reporter probe sequence MB-uvrC-R labeled with Cy5).

[0056] Figure 7 : Fluorescence signal diagram of the encoding microspheres in the embodiment of the present invention detecting the specific nucleic acid sequence on the uvrC gene of Mycoplasma bovis MB by flow cytometry. Figure 7 Middle A1 picture, Figure 7 Figure a2 in the middle shows the Cy5 detection value of the negative group and its corresponding histogram. Figure 7 Figure b1, Figure 7 Figure b2 shows the Cy5 detection value of the positive group and its corresponding histogram. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0058] It should be understood that the terms such as "and", "or", "having", "including" and "comprising" used herein do not exclude the existence or addition of one or more other elements or their combinations. Unless otherwise specified, the reagents used in the embodiments of the present invention are all commercially available products. The term "encoded microspheres" used herein refers to particles with identifiable characteristics that can be decoded by fluorescence microscopy and flow cytometry.

[0059] Example 1 (Preparation Example)

[0060] This embodiment provides a functional fluorescent protein multicolor coded microsphere, which is prepared by the following method, and the specific steps are as follows:

[0061] (1) Commercial carboxyl magnetic beads (Fe3O4-polymer microspheres, 5 μm in diameter) were used as substrate microspheres for encoding microspheres. Figure 1 , represents the forward scatter angle (FSC) and side scatter angle (SSC) signal detection diagram of magnetic beads with a diameter of 5 μm (red) analyzed by flow cytometry. The beads are activated with an activator to form carboxyl groups on their surface;

[0062] 1-1) After the carboxyl magnetic beads are mixed, 2.83×10 9 Add 100 μL of 100 mM sodium 2-(N-morpholine)ethanesulfonate (MEST, pH 5.0) solution to resuspend the magnetic beads. Place the reaction tube on a magnetic separation rack and let stand for 2 min. Remove the supernatant and wash twice.

[0063] 1-2) Rapidly add 200 μL of freshly prepared 10 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC / NHS, MEST as solvent) to the magnetic beads treated in step 1-1), vortex and mix to fully suspend and disperse the magnetic beads, and shake on a shaker at 130 rpm at 25°C for 0.5 h.

[0064] 1-3) Place the reaction tube on a magnetic separation rack and let it stand for 2 minutes, remove the supernatant, add 500 μL phosphate buffer (PBS, pH 7.4) to fully suspend the magnetic beads, place the reaction tube on a magnetic separation rack and let it stand for 2 minutes, remove the supernatant, and wash with PBS three times to obtain activated carboxyl magnetic beads.

[0065] (2) loading fluorescent protein onto the surface of magnetic beads;

[0066] 2-1) mTagBFP was diluted with PBS to 5 concentrations of 0.1, 0.9, 2.8, 6.5, and 60.0 μg / mL (respectively recorded as B1 to B5); EGFP was diluted to 5 concentrations of 0.5, 3.5, 15.0, 50.0, and 150.0 μg / mL (respectively recorded as G1 to G5); DsRed was diluted to 8 concentrations of 0, 0.3, 0.8, 1.7, 3.2, 6.8, 15.0, and 35.0 μg / mL (respectively recorded as R0 to R7).

[0067] 2-2) Take the carboxyl magnetic beads activated in step 1-3) and covalently couple the fluorescent protein. The amount of magnetic beads used for each coding group is 1×10 7 indivual.

[0068] The concentration gradient of mTagBFP in the single-color fluorescent protein-encoded microspheres was set to B1, B2, B3, B4, B5; the concentration gradient of EGFP was set to G1, G2, G3, G4, G5; the concentration gradient of DsRed was set to R0, R1, R2, R3, R4, R5, R6, R7. 200 μL of different concentrations of mTagBFP, EGFP and DsRed solutions were added to each reaction tube to resuspend the magnetic beads, and the mixture was incubated at 25°C and 130 rpm on a shaker for 5 hours.

[0069] Dual-color fluorescent protein-encoded microspheres are a combination of mTagBFP, EGFP and DsRed, three fluorescent proteins, coupled to a magnetic microsphere in pairs. The combination of mTagBFP and EGFP can be divided into 25 groups (B1G1, B1G2, B1G3, B1G4, B1G5, B2G1, B2G2, B2G3, B2G4, B2G5, B3G1, B3G2, B3G3, B3G4, B3G5, B4G1, B4G2, B4G3, B4G4, B4G5, B5G1, B5G2 The combination of mTagBFP and DsRed can be divided into 40 groups (B1R0, B1R1, B1R2, B1R3, B1R4, B1R5, B1R6, B1R7, B2R0, B2R1, B2R2, B2R3, B2R4, B2R5, B2R6, B2R7, B3R0, B3R1, B3R2, B3R3, B3R4, B3R5, B3R6, B3R7, B4R0, B4R1, B4R2, B4R3, The combination of DsRed and EGFP can be divided into 40 groups (G1R0, G1R1, G1R2, G1R3, G1R4, G1R5, G1R6, G1R7, G2R0, G2R1, G2R2, G2R3, G In each reaction tube, 200 μL of resuspended magnetic beads were added to each of the two fluorescent proteins, and the mixture was incubated at 25°C with a shaker at 130 rpm for 5 h.

[0070] The three-color fluorescent protein-encoded microspheres use mTagBFP, EGFP and DsRed coupled to a magnetic microsphere at the same time. The concentration of mTagBFP is set to B1~B4, the concentration of EGFP is set to G1~G5, and the concentration of DsRed is set to R0~R7, that is, the three-color fluorescent protein-encoded microspheres can be combined 4×5×8, a total of 160 groups. Add 200μL of resuspended magnetic beads to each reaction tube of mTagBFP, EGFP and DsRed, and incubate at 25℃, 130rpm shaker for 5h.

[0071] 2-3) After the magnetic bead-coupled fluorescent protein reaction is completed, the reaction tube is placed on a magnetic separation rack and allowed to stand for 2 minutes. The supernatant is removed and washed three times with PBS to obtain fluorescent protein-encoded magnetic microspheres. Figure 2 . Figure 2 Confocal laser scanning microscopy image showing microspheres encoded with a combination of fluorescent proteins.

[0072] 2-4) Fluorescent protein-encoded microspheres were analyzed and decoded using flow cytometry. In the single-color fluorescent protein-encoded microspheres, MBs@DsRed was detected in the FL2-PE channel of the flow cytometer and can be divided into 8 groups, see Figure 3 a1 in the figure; MBs@EGFP can be divided into five groups when detected in the FL1-FITC channel of a flow cytometer. Figure 3 b1 in the figure; MBs@mTagBFP can be divided into five groups when detected in the FL8-PB450 channel of a flow cytometer. Figure 3 c1 in . Figure 3 a2, b2, and c3 are Figure 3 -a1, a2, a3 corresponding histogram. Dual-color fluorescent protein-encoded microspheres (see Figure 4 ), the MBs@mTagBFP / EGFP combination can achieve 5×5 encodings in the FL8-FL1 detection channel (see Figure 4 -a), MBs@EGFP / DsRed combination can achieve 5×8 encodings in FL8-FL2 detection channels (see Figure 4 -b), MBs@mTagBFP / DsRed combination can achieve 5×8 encodings in FL1-FL2 detection channel (see Figure 4 -c). Three-color fluorescent protein-encoded microspheres (see Figure 5 ), MBs@mTagBFP / EGFP / DsRed can be divided into four groups in the FL8-PB450 channel (see Figure 5 -P), each group can be divided into 40 groups in the FL1-FL2 channel, a total of 4×40 groups (see Figure 5 P1~P4 in it).

[0073] (3) introducing the biotin-streptavidin system into the fluorescent protein-encoded surface to form microsphere-fluorescent protein-streptavidin composite functional microspheres;

[0074] 3-1) Add 200 μL of PBS blocking solution containing 1% BSA to each reaction tube to resuspend the encoded microspheres prepared in step (2), and incubate at 25°C, 130 rpm in a constant temperature shaker for 1 hour to block the unreacted activated functional groups on the surface of the microspheres. Place the reaction tube on a magnetic separation rack and let it stand for 2 minutes, remove the supernatant, wash with PBS three times and resuspend with 450 μL PBS.

[0075] 3-2) Dissolve 2.0 mg of biotin-NHS in 590 μL of dimethyl sulfoxide (DMSO) to prepare a 10 mM biotin reagent solution.

[0076] 3-3) Take 50 μL of the biotin-NHS solution prepared in step 3-2) and add it to the fluorescent encoded microspheres resuspended in step 3-1), and incubate at 25°C with a shaker at 130 rpm for 0.5 h. Place the reaction tube on a magnetic separation rack and let it stand for 2 min, remove the supernatant, and wash with PBS three times.

[0077] 3-4) Resuspend the biotin-labeled encoded microspheres obtained in step 3-3) with 500 μL of 50 μg / mL streptavidin solution, incubate at 25°C, 130 rpm shaker for 2 hours, and connect streptavidin to the surface of the microspheres through biotin. Place the reaction tube on a magnetic separation rack and let it stand for 2 minutes, remove the supernatant, and wash with PBS 3 times to obtain functionalized encoded microspheres.

[0078] Example 2 (Application Example)

[0079] The functionalized fluorescent protein multicolor coded microspheres prepared in Example 1 were used as carriers for single-stranded DNA fragment detection. The specific application steps were as follows:

[0080] (1) Take 7×10 5 Functionalized fluorescent protein-encoded microspheres (monochrome-encoded microspheres MBs@DsRed, diameter 5μm, DsRed encoding concentration 35.0μg / mL), add 500μL PBS-diluted biotin-labeled capture probes (5nM, MB-uvrC-C, sequence see Table 2). Incubate at 37°C, 130rpm shaker for 0.5h, the biotin labeled at the 5' end of the capture probe specifically binds to the streptavidin on the surface of the encoded microspheres to fix the capture probe to the surface of the microspheres. Place the reaction tube on a magnetic separation rack and let it stand for 2min, remove the supernatant, and wash with PBS 3 times to wash away the unbound capture probes.

[0081] (2) The single-stranded DNA fragment of the target Mycoplasma bovis to be detected (2 nM, MB-uvrC-T, gene accession number: AF003959.1, sequence is shown in Table 2) and the Cy5-labeled reporter probe (0.5 nM, MB-uvrC-R, sequence is shown in Table 2) were pre-incubated at 37°C for 0.5 h. The concentration of the target MB-uvrC-T to be detected in the negative control group was 0 nM. The sequence is shown in Table 2.

[0082] Table 2 A specific nucleic acid sequence in the UvrC gene of Mycoplasma bovis and the probe design sequence

[0083]

[0084] (3) Add the pre-hybridized target and reporter probe prepared in step (2) to the encoding microspheres immobilized with capture probes prepared in step (1), and only add the reporter probe to the encoding microspheres immobilized with capture probes in the negative control group. Incubate at 37°C and 130 rpm on a shaker for 0.5 h to form a three-segment complementary structure (see Figure 6 ).

[0085] (4) After the reaction is completed, the fluorescence signal of Cy5 on the microspheres can be directly detected by flow cytometry without washing. The detection channel of the fluorescence signal of Cy5 in the flow cytometer is FL5-APC. The detection results of the single-stranded nucleic acid MB-uvrC-T are as follows Figure 7 As shown, Figure 7 Figure a1 in Figure 7 Figure a2 in the figure is the scatter plot and histogram of the negative group fluorescent microsphere detection of MB-uvrC-T; Figure 7 The b1 graph and Figure 7 Figure b2 is the scatter plot and histogram of the positive group fluorescent microsphere detection of MB-uvrC-T.

[0086] Note: Experimental materials, reagents and instruments used in the examples:

[0087] Fluorescent protein: The fluorescent protein described in the present invention can be purchased commercially or expressed and purified by biosynthesis. In this embodiment, the expression plasmids of the blue fluorescent protein mTagBFP, the green fluorescent protein EGFP and the red fluorescent protein DsRed were all constructed by Shanghai Shenggong Biotechnology Co., Ltd.; Escherichia coli BL21 (DE3) is a publicly available biological material, but the implementation of the present invention is not limited to this biological material.

[0088] Microspheres: The microspheres described in the present invention can be commercially available or prepared using conventional methods. The microspheres in this embodiment are Fe3O4-polymer microspheres with a diameter of 5 μm, purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.

[0089] Reagents: Bovine serum albumin (BSA), sodium 2-(N-morpholino)ethanesulfonate (MES), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and Tween-20 were purchased from SIGMA. NHS-biotin and streptavidin (SA) were purchased from Shanghai Shenggong Biotechnology Co., Ltd. Disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride were purchased from China National Pharmaceutical Group. Biotin-labeled capture probe (MB-uvrC-C, sequence shown in Table 2), target single-stranded DNA (MB-uvrC-T, sequence shown in Table 2), and Cy5-labeled reporter probe (MB-uvrC-R, sequence shown in Table 2) were synthesized and labeled by Shanghai Shenggong Biotechnology Co., Ltd.

[0090] Sodium 2-(N-morpholino)ethanesulfonate (MEST) solution formula: 100 mM MES, pH 5.0, 0.05% Tween 20.

[0091] Phosphate buffered saline (PBS) formula: 10 mM phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride, pH 7.4.

[0092] Instruments: Magnetic Separator Stand 2 / 15 (magnetic field strength: 4000 Gs, purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.). American SCILOGEX MX-S vortex mixer (purchased from Beijing Kaiyuan Guochuang Technology Co., Ltd.). ZWY-240 shaker (purchased from Shanghai Zhicheng Company). Laser confocal N-STORM super-resolution microscope (purchased from Nikon Corporation, Japan). CytoFLEX flow cytometer (purchased from Beckman Group Co., Ltd., USA).

[0093] Description of some settings set to 0 in the experiment of the present invention:

[0094] When red fluorescent protein is coupled to microspheres, 8 concentration gradients are set, and a gradient with a concentration of 0 is designed, because uncoupled proteins can also have a background signal value, and the coding signal is the distinction between strong and weak fluorescence intensity. In addition, green fluorescent protein and blue fluorescent protein are set with 5 concentration gradients respectively, and there is no concentration of 0. When two-color coding is used, there are 8×5=40 groups for red and green, and there is also a group when red is 0, and there are only 5 gradients of green. This situation is equivalent to green monochrome coding. Similarly, the two-color coding of red and blue, and the three-color coding of red, blue and green all have a group when red is 0. Therefore, in the two-color coding, when red is 0, it overlaps with the two other colors of monochrome coded microspheres. In the three-color coded microspheres, when red is 0, it overlaps with the blue-green two-color coding. When calculating the total coding capacity, the overlapping microspheres are only counted once.

[0095] In the embodiment of the present invention, the 0-200 μg / mL fluorescent protein solution may be a trace amount-200 μg / mL fluorescent protein solution.

[0096] The fluorescent protein is one or a combination of two or more of mTagBFP, EYFP, EGFP, DsRed, mCherry, phycoerythrin (PE), allophycocyanin (APC), and a tandem complex of PE-Cy3, PE-Cy5, PE-Cy7, APC-Cy3, APC-Cy5 and APC-Cy7 labeled with a PE or APC fluorescent dye, and the loading concentration gradient is 0 to 200 μg / mL; the loading concentration gradient can also be a trace to 200 μg / mL.

Claims

1. A method for preparing functionalized fluorescent protein multicolor coded microspheres, comprising the following steps: (1) obtaining fluorescent protein by microbial expression and purification, and diluting the obtained fluorescent protein concentration gradient into a trace amount of fluorescent protein solution of 200 μg / mL; (2) Absorb 1×10 7 ~1×10 11 Place microspheres in an EP tube, add activation reagent to fully suspend the microspheres, incubate in a constant temperature shaker at 25°C and 120-140 rpm for 0.5-1 h to activate the functional groups modified on the microsphere surface, and then wash with PBS three times to obtain activated microspheres; (3) Loading fluorescent protein onto the surface of microspheres: After the microspheres are activated, 1×10 7 The microspheres were resuspended in a fluorescent protein solution with a resuspending volume of 200 μL, and incubated in a constant temperature shaker at 25°C and 120-140 rpm for 2-5 hours; after the reaction was completed, 200 μL of phosphate buffered saline (PBS) blocking solution containing 1% bovine serum albumin (BSA) was added to each reaction tube to resuspend the microspheres, and incubated in a constant temperature shaker at 25°C and 120-140 rpm for 0.5-1 hour to block the unreacted activated functional groups on the surface of the microspheres, and washed with PBS for 3 times after blocking; the microsphere particle size, fluorescent protein type and loading amount were used as coding elements to obtain single-color, dual-color and tri-color fluorescent coded microspheres; (4) Add 450 μL of PBS and 50 μL of 10 mM biotin-NHS solution to the fluorescent encoded microspheres to resuspend the microspheres, and incubate them in a constant temperature shaker at 25°C and 120-140 rpm for 0.5 h. After the reaction is complete, fully suspend the fluorescent microspheres with PBS and wash them three times. (5) Add 500 μL of 50 μg / mL streptavidin solution to the biotin-labeled fluorescent microspheres, incubate in a constant temperature shaker at 25°C and 120-140 rpm for 0.5-2 h, and wash three times with PBS to connect streptavidin to the microsphere surface through biotin to obtain functionalized encoded microspheres.

2. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 1, characterized in that: The fluorescent protein is one or a combination of two or more of mTagBFP, EYFP, EGFP, DsRed, mCherry, phycoerythrin (PE), allophycocyanin (APC), and a tandem complex of PE-Cy3, PE-Cy5, PE-Cy7, APC-Cy3, APC-Cy5, APC-Cy7 with a PE or APC labeled fluorescent dye, and the loading concentration gradient is trace to 200 μg / mL.

3. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 1, characterized in that: The microspheres are selected from agarose gel microspheres, dextran gel microspheres, polystyrene microspheres, silica microspheres, ferroferric oxide or ferric oxide magnetic microspheres, and the particle size of the microspheres is 1 to 30 μm.

4. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 1, characterized in that: The surface of the microsphere is modified with one of amino, carboxyl and hydroxyl functional groups.

5. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 1, characterized in that: The activation reagent is one or a combination of two of glutaraldehyde, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), sulfonated-NHS, epichlorohydrin, and N'N-carbonyldiimidazole.

6. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 1, characterized in that: The fluorescent protein is loaded onto the surface of the microsphere by covalent condensation reaction between the fluorescent protein and the functional groups on the surface of the microsphere to couple the fluorescent protein to the surface of the microsphere; the type and loading amount of the fluorescent protein are adjusted to prepare fluorescent coded microspheres with single-color, dual-color and tri-color fluorescent signals.

7. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 1, characterized in that: The biotin-streptavidin system is introduced into the surface of fluorescent microspheres to form microsphere-fluorescent protein-streptavidin composite functional microspheres, which can efficiently bind to biotin-labeled capture probes to achieve simultaneous qualitative and quantitative detection of multiple target analytes.

8. The method for preparing a functionalized fluorescent protein multicolor coded microsphere according to claim 7, characterized in that: The capture probe is selected from nucleic acid, antibody, antigen protein or receptor protein.

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