Multi-channel fluorescent protein-encoded microspheres for liquid suspension arrays and their use under single excitation light conditions

Abstract

The application discloses a kind of multichannel fluorescent protein encoding microspheres for liquid phase suspension array and its application under single excitation light condition.The multichannel fluorescent protein encoding microspheres provided by the application load the combination of two distinguishable emission spectrum recombinant fluorescent proteins with microsphere carrier, so as to construct the encoding system that can be distinguished and read in multiple channels under single excitation light irradiation. By regulating the load ratio and / or intensity of each fluorescent protein, multi-level coding can be formed; and without increasing multi-laser excitation and complex optical path, the coding capacity and engineering availability of the suspension array are improved. At the same time, it also has good coupling controllability and long-term coding stability to adapt to multi-index detection application scenarios, the preparation process is simple, the coding recognition is stable, multiple coding can be realized without increasing multi-laser excitation and complex optical path, and it is suitable for multi-index detection applications such as liquid phase suspension array.

Description

Technical Field

[0001] This invention relates to the field of fluorescent coding materials technology, specifically to a fluorescent protein-coding microsphere that achieves multi-channel readout under a single excitation light condition and its application, suitable for liquid-phase suspension arrays, multi-index biomolecule detection and related fluorescence analysis and identification. Background Technology

[0002] Liquid-phase suspension arrays and microsphere-based multi-index detection technologies can simultaneously capture and quantify multiple biomolecules (such as nucleic acids, proteins, antigens / antibodies, and cytokines) in the same sample. Typical commercial platforms (such as the xMAP system) typically use microspheres encoded with internal fluorescent dyes in a proportional manner to distinguish different "microsphere regions / codes," and use classification lasers and reporter lasers to identify microspheres and read out detection signals, respectively (xMAP classification laser 635 nm, reporter laser 532 nm). These microspheres can also achieve region differentiation through a combination of red / infrared internal dyes, forming a multiplex detection workflow (Sigma / Merck xMAP product information; Dunbar et al., ClinicalChimica Acta, 2006, review).

[0003] Existing microsphere coding materials mainly include: organic fluorescent dye-doped / embedded microspheres, quantum dot-coded microspheres, and rare-earth upconversion or time-resolved emission-coded microspheres. In engineering implementation, these solutions often require more complex optical excitation and spectroscopic detection configurations, such as using multiple lasers, multiple channels, or higher-resolution spectral demixing to support coding dimensionality expansion and improve inter-code separation. Simultaneously, dye systems may face issues such as photobleaching, signal attenuation, batch fluctuations, and dye leakage, thus affecting coding stability and long-term consistency. Therefore, achieving scalable and stable coding without increasing the number of excitation sources and optical path complexity remains an important direction for the continuous optimization of levitation array platforms.

[0004] Existing patents propose methods to simplify the labeling process and improve labeling efficiency by controlling the ratio of EDC, NHS, and carboxyl groups and rapidly adjusting the pH (CN104897904A). Another patent proposes a method for the rapid and efficient coupling of magnetic microspheres containing primary amino ligands and its preparation (CN104402003A). These technologies provide relatively mature pathways for stable microsphere-protein coupling, but they are mostly used for reporter labeling or monochromatic tracing; further development is still needed for achieving multi-channel distinguishable coding systems under single excitation conditions.

[0005] On the other hand, there are still several limitations to the use of near-infrared fluorescent proteins in industrially viable microsphere-encoded materials. Firstly, BphP-derived NIR FPs often depend on biliverdin / biliverdin-type chromophores (typically biliverdin, BV); the brightness and uniformity may fluctuate due to factors such as chromophore supply, binding affinity, and expression and folding stability. Related reviews and databases also emphasize their BV dependence; for example, iRFP682 requires BV (Shcherbakova et al., Curr Opin Chem Biol, 2015; FPbase, iRFP682 entry). Secondly, within the far-infrared / near-infrared detection window of approximately 640–780 nm, the types of proteins that can be stably encoded and spectrally well separated are relatively limited, and multicolor expansion is often constrained by spectral overlap, channel crosstalk, and photostability requirements (Shcherbakova et al., Curr Opin Chem Biol, 2015). Third, after fluorescent proteins are immobilized on or inside the material surface, they must also meet engineering requirements such as tolerance to coupling chemical environments, conformational stability during storage and use, and reduction of non-specific adsorption background; otherwise, the reliability and reproducibility of the encoding may be reduced.

[0006] Therefore, there is an urgent need for a microsphere coding material that can achieve distinguishable reading of multiple emission channels under a single excitation light condition, and can expand the coding capacity through intensity grading and / or combination strategies; to improve the coding capacity and engineering usability of the levitation array without increasing the number of laser excitations and complex optical paths. Simultaneously, it should possess good coupling controllability and long-term coding stability to adapt to multi-index detection application scenarios. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention aims to provide a multi-channel fluorescent protein-encoded microsphere for liquid-phase suspension arrays, through which multi-channel distinguishable code readout is achieved under a single red light excitation band. The single red light excitation band has a center wavelength of approximately 638 nm, with wavelengths in the range of 635–640 nm; typically corresponding to a far-infrared / near-infrared detection window of approximately 640–780 nm.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a multichannel fluorescent protein-encoded microsphere, comprising a microsphere carrier and a combination of recombinant fluorescent proteins immobilized on and / or inside the microsphere carrier; the recombinant fluorescent protein combination serves as the encoding source, wherein the recombinant fluorescent protein combination is a combination of RE-APC protein and BDFP-FRET protein, or a combination of RE-APC protein and miRFP720-FRET protein. The RE-APC protein is used for APC channel encoding and is a multimeric protein with two subunits, apcA and apcB; the BDFP-FRET protein is used for APC-A700 channel encoding; and the miRFP720-FRET protein is used for APC-A750 channel encoding. The microsphere carrier is a carboxylated magnetic microsphere (MB). The recombinant fluorescent protein combination is covalently fixed to the surface of the microsphere carrier via carboxyl-amine coupling. Specifically, after activating the carboxyl groups on the surface of the microspheres using the EDC / NHS system, the carboxyl groups react with the amino groups on the fluorescent protein molecules to form amide bonds, thereby obtaining MBs@FPs encoded microspheres.

[0009] The amino acid sequence of the RE-APC protein is shown in SEQ ID NO:21, the amino acid sequence of the BDFP-FRET protein is shown in SEQ ID NO:10, and the amino acid sequence of the miRFP720-FRET protein is shown in SEQ ID NO:13.

[0010] Secondly, the present invention provides a method for preparing the recombinant fluorescent protein-encoded microspheres, comprising: constructing a corresponding recombinant plasmid, transforming it into a host cell for recombinant expression, obtaining the expression product, and purifying the protein.

[0011] 1) Encoded microspheres and their preparation (covalent fixation and strength grading) The carboxyl activation step of the microspheres includes: washing the microspheres, adding freshly prepared EDC solution and NHS solution to fully suspend the microspheres and activate them for a predetermined time; then performing a protein coupling reaction. The coupling step includes: mixing the activated microspheres with fluorescent protein solutions of different concentration gradients and incubating with shaking for a predetermined time; washing to remove unbound proteins to obtain encoded microspheres.

[0012] By adjusting the loading amount of fluorescent protein on microspheres to form different coding intensity levels, a distinguishable group of coding microspheres can be constructed.

[0013] 2) Encoding capacity expansion (dual-grained strategy) To further improve coding capacity, this invention provides a dual-grain size expansion strategy. A second grain size (preferably about 5 μm) is introduced into the 10 μm single-grain size system to form a dual-grain size coding system, thereby expanding the number of monochrome codes within the same channel. Preferably, under the dual-grain size system, the number of monochrome codes for the APC, APC-A700, and APC-A750 channels is expanded to 19, 18, and 18 coding groups, respectively.

[0014] 3) Two-color combination coding system (strength combination expansion) Based on single-color coding, this invention further provides a dual-color coding system, which couples two different fluorescent proteins to the same microsphere, so that the same microsphere carries two coding information at the same time, and constructs a coding library by combining the two fluorescence intensities, thereby improving the coding capacity.

[0015] By combining RE-APC with BDFP-FRET, 72-fold coding can be achieved in the APC and APC-A700 channels; by combining RE-APC with miRFP720-FRET, 75-fold coding can be achieved in the APC and APC-A750 channels.

[0016] Thirdly, this invention provides the application of the recombinant fluorescent protein-encoded microspheres under a single excitation light condition, performing flow cytometry detection on the aforementioned encoded microspheres, acquiring fluorescence signals in the corresponding detection channels, and discriminating the microsphere encoding based on the fluorescence signals. The discrimination methods include one or more of intensity threshold segmentation, clustering, and / or comparison with a preset encoding library.

[0017] The single excitation light is a red light band excitation light, suitable for a far-infrared / near-infrared detection window of approximately 640–780 nm, with a wavelength range of 635–640 nm, preferably a center wavelength of approximately 638 nm. Under this single excitation condition, multi-channel encoding and signal readout are achieved, thereby reducing the dependence on multiple laser configurations and complex optical paths.

[0018] Compared with the prior art, the present invention has at least the following beneficial effects: 1. Multi-channel encoding readout achieved by single red light band excitation This invention uses recombinant fluorescent protein as the encoding source, enabling the encoding microspheres to achieve encoding and reading of the APC, APC-A700, and APC-A750 channels under a single red light excitation condition. This reduces the dependence on multiple laser excitations and complex optical path configurations, thereby improving the versatility and engineering feasibility of the method.

[0019] 2. Encoding capacity is scalable and clustering is clear. This invention establishes intensity-level coding by regulating the loading amount of fluorescent proteins on microspheres, and expands the coding capacity by combining a dual-particle-size strategy and a dual-color combination coding strategy. Specifically, the dual-particle-size system is used to increase the number of single-color codes; the dual-color combination is used to further construct higher-multiplicity codes. Therefore, the coding capacity and the clustering effect of the coding recognition can be improved without significantly increasing the complexity of the detection system.

[0020] 3. Covalent fixation improves coding stability and batch consistency. The present invention preferably uses an EDC / NHS system to achieve covalent coupling and immobilization of carboxyl microspheres and fluorescent proteins, forming a stable amide bond connection. This helps to reduce the risk of desorption or leakage of the coding source, improve the long-term stability and batch-to-batch consistency of the coding signal, and thus is suitable for large-scale preparation and application.

[0021] 4. Storage stability and resistance to degradation The fluorescent protein-encoded microspheres of this invention can be stored for a long time at low temperatures with minimal fluorescence signal attenuation; they also maintain a high fluorescence retention rate under accelerated aging conditions, indicating their storage stability and resistance to quenching, thus meeting the storage, transportation, and use requirements in practical detection. The fluorescent protein-encoded microspheres can be stably stored at 4 °C for at least 180 days with fluorescence attenuation of less than 10%; under accelerated aging conditions, the fluorescence retention rate of the encoded microspheres is higher than 90%, indicating their stability and resistance to quenching.

[0022] 5. Tolerance to organic solvent treatment The encoded microspheres of this invention can maintain a high average fluorescence intensity even under treatment conditions containing ethanol or dimethyl sulfoxide (DMSO), indicating that they have chemical environmental tolerance and process adaptability, which is beneficial for compatibility with subsequent bioconjugation, washing and sample processing procedures.

[0023] In summary, this invention has advantages in achieving multi-channel encoding, expanding encoding capacity, and improving stability and process compatibility under a single excitation condition, and is suitable for application scenarios such as multi-index detection of liquid phase suspension arrays. Attached Figure Description

[0024] Figure 1 The plasmid maps of the two plasmids of RE-APC in Example 1: the first recombinant plasmid pCDF-apcA-HoI-pcyA and the second recombinant plasmid pRSF-apcB-cpcS-cpcU; Figure 2 Plasmid maps of the fusion fluorescent proteins BDFP3.5–FRET and miRFP720-FRET in Examples 2 and 3. In these maps, A represents BDFP–FRET; B represents miRFP720-FRET. Figure 3 Example 4 shows the appearance of the three target proteins and the electrophoresis results. A is a photograph of the appearance of the three protein solutions; a, b, and c represent RE-APC, BDFP–FRET, and miRFP720-FRET, respectively. B is the electrophoresis result of RE-APC; C is the electrophoresis result of BDFP–FRET; and D is the electrophoresis result of miRFP720-FRET. Lane M is the marker; lanes 1 and 2 are the lysed bacterial cultures of E. coli Rosetta (DE3) expression strain before and after IPTG induction; lane 3 is the precipitate of the lysed bacterial culture of E. coli Rosetta (DE3) expression strain after induction; lane 4 is the supernatant of the lysed bacterial culture of E. coli Rosetta (DE3) expression strain after induction; lane 5 is the sample effluent; and lanes 6-9 are elution buffers of 50, 100, 300, and 500 mmol·L⁻¹ imidazole buffer, respectively. Figure 4 Example 5 shows the absorption and fluorescence emission spectra of the three target proteins. A, B, and C represent RE-APC, BDFP–FRET, and miRFP720-FRET, respectively. Figure 5 Example 7 shows the flow cytometry results of fluorescent protein-encoded microspheres in different near-infrared fluorescence detection channels. A, C, and E are two-dimensional scatter plots of SSC-A / fluorescence intensity of the encoded microspheres in the APC, APC-A700, and APC-A750 detection channels, respectively; B, D, and F are histograms of fluorescence intensity in the corresponding detection channels. Different colors represent microsphere populations encoded with different fluorescence intensities, showing that the encoded microspheres can form distinguishable fluorescence signal distributions in the corresponding detection channels. Figure 6 Example 7: Flow cytometry scatter plots and histograms of dual-size encoded microspheres. A is the FSC-A / SSC-A scatter plot; B is the FSC-A histogram; C–E are scatter plots of the SSC-A and FL5-A (APC), FL6-A (APC-A700), and FL7-A (APC-A750) channel signals, respectively; different colors are used to distinguish microsphere populations of different sizes. Figure 7 Example 7: Schematic diagram of the streaming scatter plot of the coded microspheres. A and C represent the scatter distribution of the microspheres under FSC-A / SSC-A, respectively; B is the scatter plot of the FL5-A (APC) and FL6-A (APC-A700) channel signals; D is the scatter plot of the FL5-A (APC) and FL7-A (APC-A750) channel signals; different colors are used to distinguish different groups of coded microspheres. Figure 8 Example 8: Schematic diagram of the storage stability of fluorescent protein-encoded microspheres. Wherein, A represents MBs@RE-APC; B represents MBs@BDFP-FRET; C represents MBs@miRFP720-FRET; Figure 9 Example 9: Schematic diagram of fluorescence stability analysis of fluorescent protein-encoded microspheres in organic phase. A1 and A2 represent MBs@RE-APC; B1 and B2 represent MBs@BDFP-FRET; C1 and C2 represent MBs@miRFP720-FRET. Figure 10 Example 10: Flow cytometry results of long-term storage stability of the two-color coded microspheres. A and B are two-dimensional scatter plots of the APC / APC-A700 dual-channel array after storage at 4℃ in the dark for days 0 and 180, respectively; C and D are two-dimensional scatter plots of the APC / APC-A750 dual-channel array after storage at 4℃ in the dark for days 0 and 180, respectively. Detailed Implementation

[0025] To facilitate a clearer and more complete understanding of the present invention, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. In this embodiment, the operation methods without specific experimental conditions are all performed using conventional experimental methods and standard conditions in the art, or with reference to the operating conditions recommended by reagent and instrument manufacturers; all kinds of chemical reagents used in the embodiments can be purchased through conventional commercial channels, and the required amplification primers are all synthesized by biotechnology companies. The materials, reagents, main components, and preparation methods used in the examples are as follows:

[0026] Homologous recombination and PCR mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; carboxyl MBs (10.0 µm in diameter) were purchased from Suzhou Nanomicro Technology Co., Ltd.; sodium 2-(N-morpholine) ethanesulfonate (MES), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and Tween-20 were purchased from SIGMA; disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride were purchased from China National Pharmaceutical Group Corporation.

[0027] Instruments: Magnetic Separator Stand 2 / 15 (magnetic field strength: 4000Gs, purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.); SCILOGEX MX-S vortex mixer (purchased from Beijing Kaiyuan Guochuang Technology Co., Ltd.); ZWY-240 shaker (purchased from Shanghai Zhicheng Co., Ltd.); CytoFLEX flow cytometer (purchased from Beckman Coulter, Inc., USA); MEST (100 mmol / L MES, pH 5.0, 0.05% Tween-20): Weigh 2.13 g of MES and dissolve it in 80 mL of deionized water. Adjust the pH to 5.0, add 50 μL of Tween-20, and bring the volume to 100 mL. Phosphate-Buffered Saline (PBS) (10 mmol / L, pH = 7.4): Weigh 8.0 g of sodium chloride, 2.93 g of Na2HPO4·12H2O, 0.2 g of KH2PO4 and 0.2 g of KCl, dissolve in 800 mL of deionized water, adjust the pH to 7.4, and bring the volume to 1 L. Example 1: Construction and Validation of RE-APC Recombinant Plasmid

[0028] (1) Sequence and vector design: The specific process is as follows: Based on the coding sequence of the RE-APC protein and the gene sequences related to pigment synthesis and pigment linkage required for its in vivo reconstruction, a dual-plasmid recombinant expression system for prokaryotic expression is designed. The dual-plasmid recombinant expression system includes the first recombinant plasmid pCDF-apcA-HoI-pcyA and the second recombinant plasmid pRSF-apcB-cpcS-cpcU.

[0029] The first recombinant plasmid, pCDF-apcA-HoI-pcyA, uses the pCDF series expression vectors as a backbone, sequentially introducing the apcA, HoI, and pcyA genes into the same vector. The apcA gene encodes the α subunit of the RE-APC protein; the HoI gene encodes heme oxygenase, which catalyzes the conversion of heme to biliverdin; and the pcyA gene encodes phycocyanin oxidoreductase, which further catalyzes the conversion of biliverdin to phycocyanin chromophores. By constructing apcA, HoI, and pcyA in the same expression vector, the RE-APC α subunit and its chromophore synthesis-related enzymes can be simultaneously expressed in host cells, providing a foundation for subsequent chromophore generation and assembly.

[0030] The second recombinant plasmid, pRSF-apcB-cpcS-cpcU, uses the pRSF series expression vectors as a backbone, sequentially introducing the apcB, cpcS, and cpcU genes into the same vector. The apcB gene encodes the β subunit of the RE-APC protein; the cpcS and cpcU genes encode chromophore-related enzymes, which can promote the binding of phycobilichromes to specific sites on the RE-APC protein subunits, thereby facilitating the formation of fluorescent recombinant RE-APC proteins.

[0031] The two recombinant plasmids contain promoters, ribosome binding sites, origins of replication, and resistance selection markers suitable for prokaryotic expression in *E. coli*. By co-transforming the first recombinant plasmid pCDF-apcA-HoI-pcyA and the second recombinant plasmid pRSF-apcB-cpcS-cpcU into *E. coli* expression hosts, the co-expression of the RE-APC protein α subunit, β subunit, chromophore synthase, and chromophore ligase can be achieved. This allows for the completion of recombinant expression, chromophore synthesis, and chromophore assembly of the RE-APC protein within the host cell, resulting in a recombinant RE-APC protein with fluorescent encoding function. The plasmid map is shown in Figure 1.

[0032] (2) Cloning and Assembly: In this embodiment, the restriction endonuclease ligation method is used to insert the target fragment into the vector to obtain the RE-APC recombinant plasmid. The specific process is as follows: In this embodiment, the homologous recombination ligation method is used to insert the target fragment into the vector to obtain the RE-APC recombinant plasmid. The specific process is as follows: Using Synechocystis PCC 6803 genomic DNA as a template, apcA, apcB, HoI, pcyA, cpcS, and cpcU gene fragments were amplified, with nucleotide sequences shown in SEQ ID NO:15-SEQ ID NO:20, respectively. Homologous recombination sequences matching the corresponding linearized vector or adjacent insert fragments were introduced at both ends of the amplification primers. After PCR amplification, the amplification products were detected by agarose gel electrophoresis, and the target bands were excised or recovered. The PCR products were then purified using a DNA purification kit to obtain the target gene fragments for recombination ligation.

[0033] The pCDF and pRSF expression vectors were linearized, and the linearized vector fragments were recovered. The linearized vector, target insert, recombination reaction buffer, and recombinase were mixed according to the molar ratio of vector to insert fragment for homologous recombination. Specifically, the apcA, HoI, and pcyA gene fragments were sequentially inserted into the pCDF expression vectors to construct the first recombinant plasmid pCDF-apcA-HoI-pcyA; and the apcB, cpcS, and cpcU gene fragments were sequentially inserted into the pRSF expression vectors to construct the second recombinant plasmid pRSF-apcB-cpcS-cpcU.

[0034] After the homologous recombination reaction, the recombinant product was transformed into *E. coli* DH5α competent cells, incubated on ice, then subjected to heat shock, and subsequently revived in antibiotic-free LB broth. The revived bacterial culture was plated on LB agar plates containing the appropriate antibiotics and incubated upside down to obtain candidate recombinant clones. Single clones were then selected for expansion culture for subsequent positive clone screening, plasmid extraction, and sequence verification.

[0035] (3) Identification and Verification: Single colonies were randomly selected from the transformation plates. Using bacterial suspension or colony lysate as templates, PCR amplification was performed using the corresponding identification primers, and the PCR products were detected by agarose gel electrophoresis. The results showed that all candidate positive clones amplified specific bands consistent with the expected size of the target insert fragment, preliminarily indicating that the apcA, HoI, pcyA, apcB, cpcS, and cpcU gene fragments had been inserted into their respective expression vectors.

[0036] The PCR-positive recombinant plasmids were further extracted and verified by nucleotide sequencing. Sequencing results showed that the gene sequences of apcA, HoI, and pcyA in the first recombinant plasmid pCDF-apcA-HoI-pcyA were consistent with the designed sequences, with correct insertion orientation and each coding sequence within the correct reading frame. Similarly, the gene sequences of apcB, cpcS, and cpcU in the second recombinant plasmid pRSF-apcB-cpcS-cpcU were consistent with the designed sequences, with correct insertion orientation and each coding sequence within the correct reading frame. These results demonstrate that the dual-plasmid system required for RE-APC recombinant expression was successfully constructed and can be used for subsequent co-transformation expression and verification of fluorescent protein recombinant expression. The amino acid sequences of the corresponding apcA, HoI, pcyA, apcB, cpcS, and cpcU protein fragments are shown in SEQ ID NO:1-SEQ ID NO:6, respectively. Example 2: Construction and Validation of BDFP-FRET Recombinant Plasmid

[0037] (1) Configuration Determination: The expression sequence of the BDFP-FRET protein encoding the APC-A700 channel was constructed, and the corresponding recombinant plasmid was constructed. Specifically, based on the amino acid sequence of the BDFP3.5 donor fluorescent protein (SEQ ID NO:7) and the amino acid sequence of the BDFP3.6 receptor fluorescent protein (SEQ ID NO:8) and their far-infrared / near-infrared fluorescence characteristics, the structural sequence of the fusion protein was designed as BDFP3.5-linker-BDFP3.6. The amino acid sequence of this fusion protein is shown in SEQ ID NO:10, where BDFP3.5 is located at the N-terminus and BDFP3.6 is located at the C-terminus, connected by a rigid linker peptide. This linker peptide provides a spatial interval between the two fluorescent protein domains, reducing interference between the domains and keeping them within the same open reading frame. The linker peptide used in this embodiment is AEAAAAKEAAAKA. Based on the above amino acid sequence, the corresponding nucleic acid coding sequence was designed, and codon optimization was performed according to the E. coli expression system to obtain the BDFP3.5-linker-BDFP3.6 fusion coding sequence, the nucleotide sequence of which is shown in SEQ ID NO:11; plasmid map is shown below. Figure 2 Figure A in the middle.

[0038] (2) Assembly and Identification: The BDFP3.5-linker-BDFP3.6 fusion coding sequence was inserted into a prokaryotic expression vector using homologous recombination, restriction endonuclease ligation, or cloning of commercially synthesized genes to obtain the BDFP-FRET recombinant plasmid. The correctness of the recombinant plasmid was verified by colony PCR, restriction enzyme digestion, and sequencing. The results showed that cloning primers were designed based on the fusion coding sequences of BDFP3.5, the linker peptide, and BDFP3.6, so that the BDFP3.5 coding sequence was located at the N-terminus of the fusion protein, the BDFP3.6 coding sequence was located at the C-terminus of the fusion protein, and the two were linked by the linker peptide coding sequence and were located in the same open reading frame. The codon-optimized BDFP3.5-linker-BDFP3.6 fusion coding fragment was inserted into the multiple cloning site or the corresponding expression frame of the prokaryotic expression vector to obtain the candidate BDFP-FRET recombinant expression plasmid.

[0039] The ligation or recombinant product was transformed into *E. coli* clone host cells. After screening with antibiotic plates, single clones were selected for colony PCR identification. Colony PCR results showed that candidate positive clones amplified specific bands consistent with the theoretical size of the BDFP3.5-linker-BDFP3.6 fusion fragment. Further extraction of the plasmid from positive clones and restriction enzyme digestion identification showed that the size of the digested bands was consistent with the expected plasmid conformation. Subsequent nucleotide sequencing of the positive recombinant plasmid showed that the inserted fragment sequence was consistent with the designed sequence, the BDFP3.5, linker peptide, and BDFP3.6 coding sequences were correctly ligated, and all were within the same open reading frame, without frameshift mutations or premature stop codons. These results indicate that the BDFP-FRET recombinant expression plasmid for encoding the APC-A700 channel was successfully constructed. Example 3: Construction and Validation of miRFP720-FRET Recombinant Plasmid

[0040] (1) Configuration determination: The miRFP720-FRET protein expression sequence for encoding the APC-A750 channel was constructed, and the corresponding recombinant plasmid was constructed. The specific process was as follows: Based on the coding sequences of BDFP3.5 and miRFP720, the two were designed for fusion using sequence analysis software. During the design, the stop codon at the end of the BDFP3.5 coding sequence was removed, so that the BDFP3.5 coding sequence, the linker peptide coding sequence, and the miRFP720 coding sequence were in the same continuous open reading frame, and the stop codon at the end of the miRFP720 coding sequence was retained. The linker peptide coding sequence was reverse translated according to the selected linker peptide amino acid sequence and codon adaptation was performed to ensure that the fusion protein could be translated normally in the E. coli expression system. After reading frame inspection, stop codon inspection, and restriction endonuclease recognition site analysis, the BDFP3.5-linker-miRFP720 fusion coding sequence was determined, and its nucleotide sequence is shown in SEQ ID NO:14; wherein, the amino acid sequence of the receptor fluorescent protein miRFP720 is shown in SEQ ID NO:12.

[0041] The linker peptide used in this embodiment is AEAAAAKEAAAKA (SEQ ID NO:9), and its plasmid map can be found in [link to plasmid map]. Figure 2 -B.

[0042] (2) Assembly and evaluation: The BDFP3.5-linker-miRFP720 fusion coding sequence was inserted into a prokaryotic expression vector using homologous recombination and restriction endonuclease ligation to obtain the miRFP720-FRET recombinant plasmid. The correct insertion fragment and expression cassette were verified by colony PCR, restriction enzyme digestion, and sequencing. The results are as follows: Cloning primers were designed based on the fusion coding sequences of BDFP3.5, the linker peptide, and miRFP720, positioning the BDFP3.5 coding sequence at the N-terminus of the fusion protein and the miRFP720 coding sequence at the C-terminus, linked by the linker peptide coding sequence and both within the same open reading frame. The stop codon at the end of the BDFP3.5 coding sequence was removed, while the stop codon at the end of the miRFP720 coding sequence was retained, allowing for the sequential translation of BDFP3.5, the linker peptide, and miRFP720 to form the fusion protein. The codon-optimized BDFP3.5-linker-miRFP720 fusion coding fragment was inserted into the multiple cloning site of a prokaryotic expression vector or the corresponding expression frame to obtain the candidate miRFP720-FRET recombinant expression plasmid.

[0043] The ligation or recombinant product was transformed into *E. coli* clone host cells. After screening with antibiotic plates, single clones were selected for colony PCR identification. Colony PCR results showed that candidate positive clones amplified specific bands consistent with the theoretical size of the BDFP3.5-linker-miRFP720 fusion fragment. Further extraction of the plasmid from positive clones and restriction enzyme digestion identification showed that the size of the digested bands was consistent with the expected plasmid conformation. Subsequent nucleotide sequencing of the positive recombinant plasmid showed that the inserted fragment sequence was consistent with the designed sequence, the BDFP3.5, linker peptide coding sequences, and miRFP720 coding sequences were correctly ligated, and all were within the same open reading frame, without frameshift mutations or premature stop codons. These results indicate that the miRFP720-FRET recombinant expression plasmid for encoding the APC-A750 channel was successfully constructed. Example 4: Expression and purification of recombinant fluorescent protein

[0044] (1) Expression: The recombinant plasmids obtained in Examples 1-3 were transformed into host cells and expressed to obtain RE-APC protein (RE-APC protein is a multimeric protein with two subunits, apcA and apcB; the amino acid sequence of RE-APC protein is shown in SEQ ID NO:21), BDFP-FRET protein (i.e., the fusion fluorescent protein BDFP3.5-linker-BDFP3.6; its amino acid sequence is shown in SEQ ID NO:10) and miRFP720-FRET protein (i.e., the fusion fluorescent protein BDFP3.5-linker-miRFP720; its amino acid sequence is shown in SEQ ID NO:13) expression products. The specific process is as follows: pCDF-apcA-HoI-pcyA and pRSF-apcB-cpcS-cpcU obtained in Example 1 were co-transformed into Escherichia coli Rosetta expression host cells; the BDFP-FRET recombinant plasmid obtained in Example 2 was transformed into Escherichia coli Rosetta. Expression in host cells: The miRFP720-FRET recombinant plasmid constructed in Example 3 was transformed into Escherichia coli Rosetta expression host cells. After screening with appropriate antibiotic plates, positive single clones were picked and inoculated into LB liquid medium containing the appropriate antibiotic to obtain seed culture.

[0045] Transfer the seed culture at an inoculation rate of 1% to new LB liquid medium containing the appropriate antibiotic, and incubate until the bacterial culture reaches OD500. 600 The concentration was approximately 0.6. IPTG was then added to the bacterial culture to a final concentration of 0.5 mmol / L, and the culture was induced at 28℃ and 180 r / min for 16–24 h to express the recombinant fluorescent protein. After induction, the bacterial cells were collected by centrifugation, and the resulting cell pellet was used for subsequent cell disruption and protein purification. The expression product is shown in Figure 3-A.

[0046] (2) Purification: The protein expression product was purified by resuspending the induced bacterial cell precipitate in an appropriate amount of PBS buffer and performing high-pressure disruption at low temperature and a disruption pressure of 1264 bar. The disruption was repeated 3-5 times until the bacterial solution was clear. The disruption solution was then centrifuged at high speed, and the supernatant was collected to obtain a crude extract of soluble protein containing recombinant fluorescent protein.

[0047] Since the RE-APC, BDFP-FRET, and miRFP720-FRET recombinant proteins all carry His tags, Ni-NTA affinity chromatography was used for purification. First, the Ni-NTA affinity chromatography column was equilibrated with 5 column volumes of PBS buffer. Then, the crude protein extract was added to the column to bind the His-tagged target protein to the Ni-NTA packing material. Next, non-specifically bound proteins were eluted sequentially with wash buffers containing 50 mmol / L and 100 mmol / L imidazole. Finally, the target protein was eluted with elution buffer containing 300 mmol / L imidazole, and the eluent was collected.

[0048] The obtained target protein eluent was placed in a dialysis bag with a molecular weight cutoff of 10 kDa and dialyzed with PBS buffer to remove imidazole and excess salt ions. The dialyzed protein solution was concentrated and aliquoted for storage to obtain purified RE-APC, BDFP-FRET, and miRFP720-FRET protein samples for subsequent coupling. Gel images of RE-APC, BDFP-FRET, and miRFP720-FRET proteins are shown in Figures 3-B, 3-C, and 3-D.

[0049] (3) Quality control: The purity and integrity of the purified product were verified. The specific process was as follows: SDS-PAGE was used to analyze the expression product, the lysed supernatant, the purified flow-through buffer, the washing buffer, and the final elution buffer. The sample to be tested was mixed with the SDS-PAGE loading buffer and heated to denature, followed by polyacrylamide gel electrophoresis. After electrophoresis, the gel was stained and destained, and the target protein bands were observed. The results showed that the purified RE-APC, BDFP-FRET, and miRFP720-FRET samples showed major protein bands near their corresponding theoretical molecular weights, and the bands of other proteins were significantly reduced, indicating that the above purification method can obtain recombinant fluorescent proteins with high purity. These were then used for subsequent spectral characterization. Example 5: Spectral properties of recombinant fluorescent protein

[0050] Spectroscopic verification: Absorption and / or fluorescence emission spectra of RE-APC, BDFP-FRET, and miRFP720-FRET proteins were measured respectively. See the attached results for details. Figure 4 This was to verify that it possesses distinguishable emission characteristics across the detection channels of APC, APC-A700, and APC-A750, thereby meeting the spectral requirements for multi-channel encoding and recognition, such as... Figure 4It can be seen that RE-APC, BDFP-FRET, and miRFP720-FRET proteins all exhibit corresponding absorption signals in the visible to near-infrared region, indicating that the recombinant fluorescent proteins can form optically active chromophore structures. Further fluorescence emission spectroscopy results show that the three recombinant fluorescent proteins exhibit different emission distribution characteristics in the red to near-infrared detection region. Specifically, RE-APC mainly produces a strong fluorescence emission signal within the APC detection channel (660 nm); BDFP-FRET has a detectable fluorescence emission signal within the APC-A700 detection channel (700 nm); and miRFP720-FRET has a relatively longer wavelength emission distribution within the APC-A750 detection channel (720 nm). The above results indicate that RE-APC, BDFP-FRET, and miRFP720-FRET can generate differential fluorescence signals in the APC, APC-A700, and APC-A750 detection channels of flow cytometers, providing a spectral basis for use as multichannel fluorescently encoded proteins. Example 6: Preparation of fluorescent protein-encoded microspheres—Activation of carboxyl groups on the surface of magnetic beads

[0051] (1) Preparation and washing of magnetic beads: Mix the carboxylated magnetic beads (Magrose-COOH magnetic beads are used in this example), take 100 μL into a 2 mL centrifuge tube, and remove the supernatant by magnetic separation; add 200 μL of MEST solution to wash twice, and remove the supernatant by magnetic separation after each wash.

[0052] (2) EDC / NHS activation: Quickly add 200 μL of freshly prepared EDC solution (10 mg / mL, with MEST as solvent) and 200 μL of NHS solution (10 mg / mL, with MEST as solvent), vortex to mix and fully suspend the magnetic beads; activate at 25 ℃ and 120 r / min for 30 min; use the activated magnetic beads for protein coupling reaction as soon as possible.

[0053] (3) The activated carboxyl magnetic beads were washed three times with MEST (pH 5.0) and grouped for subsequent coupling reactions; (4) Protein gradient and coupling: Fluorescent proteins were prepared in different concentrations using MEST. 200 μL of protein solutions of different concentrations were taken to resuspend the magnetic beads and coupled at 25 °C and 120 r / min for 2 h. Example 7: Preparation and coding construction of single-color and dual-color fluorescent protein-encoded microspheres

[0054] (1) Preparation of monochromatic encoded microspheres: MBs@RE-APC, MBs@BDFP-FRET, and MBs@miRFP720-FRET were prepared using fluorescent protein type and concentration as encoding elements. Multi-level monochromatic coding groups for APC channels, APC-A700 channels, and APC-A750 channels were constructed in a single particle size system at 10 μm; each channel could be constructed into 10 coding groups. Specific coding results are shown in […]. Figure 5 .

[0055] (2) Dual-particle size expansion: A dual-particle size system is further introduced: 10 μm and 5 μm, to expand the number of monochrome codes within the same channel; the results of this embodiment show that it can be expanded to 19 coding groups for the APC channel, 18 coding groups for the APC-A700 channel, and 18 coding groups for the APC-A750 channel. The larger the number of coding groups obtained, the more detections can be performed subsequently; see the specific coding results. Figure 6 .

[0056] (3) Preparation of dual-color encoded microspheres: Two fluorescent proteins were co-coupled to the same magnetic bead to form two types of dual-color encoded microspheres: MBs@RE-APC / BDFP-FRET and MBs@RE-APC / miRFP720-FRET; encoding libraries were constructed by combining the fluorescence intensities of the two channels. Figure 7 As shown, under single laser excitation, 72-75 recoding can be achieved in two channels (APC and APC-A700) or (APC and APC-A750) respectively.

[0057] (4) Example of coupling conditions: Single color coupling or two color coupling are performed under the same conditions as in Example 6, namely 25 ℃, 120 r / min, 2 h; after the reaction, the mixture is washed 3 times with PBS to remove unbound proteins. Example 8: Evaluation of long-term storage stability and accelerated aging of single fluorescent protein-encoded microspheres

[0058] (1) Accelerated aging experiment: The three groups of single fluorescent protein-encoded microspheres, MBs@RE-APC, MBs@BDFP-FRET and MBs@miRFP720-FRET, were incubated at 37 °C for 7 days, and their MFI changes were monitored every 1 day to calculate the fluorescence retention rate (FR).

[0059] (2) Results: After incubation at 37 ℃ for 7 days, the fluorescence filtration rate (FR) of the three groups of single fluorescent protein-encoded microspheres was higher than 90%, corresponding to a fluorescence decay rate of less than 10%. These results indicate that the encoded material possesses chemical stability and resistance to quenching; and equivalent conversion can be performed based on the Arrhenius kinetic model to support the conclusion that it can be stably stored for more than one year at 4 ℃. The above results are as follows:Figure 8 As shown. Example 9: Stability and tolerance of single fluorescent protein-encoded microspheres in organic solvents

[0060] (1) Solvent treatment: The three groups of single fluorescent protein-encoded microspheres (MBs@RE-APC, MBs@BDFP-FRET, MBs@miRFP720-FRET) were mixed and resuspended in PBS (pH 7.4); the PBS contained 0%, 1%, 10%, 20%, and 30% (v / v) DMSO or ethanol, respectively; and were continuously shaken at room temperature for 2 h, 12 h, and 24 h.

[0061] (2) Detection: After the treatment, the microspheres were washed three times with PBS, resuspended, and then flow cytometry was performed and the MFI was recorded. At the same time, the dispersibility of the microspheres could be observed using a fluorescence microscope.

[0062] (3) Results: Under the above conditions, the MFI of the three groups of single fluorescent protein-encoded microspheres remained stable, indicating their tolerance to DMSO or ethanol treatment. The results are as follows: Figure 9 As shown. Example 10: Evaluation of long-term storage stability of dual-color coded microspheres

[0063] (1) Long-term storage experiment: The dual-color coded microspheres prepared in Example 7 were resuspended in PBS buffer (pH 7.4) and stored at 4 °C in the dark. The dual-color coded microspheres included MBs@RE-APC / BDFP-FRET and MBs@RE-APC / miRFP720-FRET. Samples were taken on day 0 and day 180 of storage, and their fluorescence coding distribution was detected by flow cytometry.

[0064] (2) Detection methods: For MBs@RE-APC / BDFP-FRET two-color coded microspheres, the two-dimensional fluorescence distribution in the APC channel and APC-A700 channel was detected; for MBs@RE-APC / miRFP720-FRET two-color coded microspheres, the two-dimensional fluorescence distribution in the APC channel and APC-A750 channel was detected. By comparing the two-dimensional scatter plots on day 0 and day 180, the changes in the coding population distribution and resolvability of the two-color coded microspheres after long-term low-temperature storage were evaluated.

[0065] (3) Results: The results are as follows Figure 10As shown, after 180 days of storage at 4 ℃ in the dark, the two-dimensional scatter distribution of MBs@RE-APC / BDFP-FRET dual-color coded microspheres in both APC and APC-A700 dual channels maintained good population separation characteristics, with no obvious fusion or overall drift among the coding groups. Similarly, after 180 days of storage at 4 ℃ in the dark, the two-dimensional scatter distribution of MBs@RE-APC / miRFP720-FRET dual-color coded microspheres in both APC and APC-A750 dual channels remained stable, and each coding group could still be effectively distinguished. These results indicate that the dual-color coded microspheres prepared in this application can maintain stable fluorescence coding distribution and good population distinguishability under long-term low-temperature storage conditions, and can be used as a stable coding carrier in multiplex detection systems.

[0066] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0067] SEQ ID NO:1 Amino acid sequence of apcA subunit: MSIVTKSIVNADAEARYLSPGELDRIKAFVTGGAARLRIAETLTGSRETIVKQAGDRLFQKRPDIVSPGGNAYGEEMTATCLRDMDYYLRLVTYGVVSGDVTPIEEIGLVGVREMYRSLGTPIEAVAQSVREMKEVASGLMSSDDAAEASAYFDFVIGKMS* SEQ ID NO:2 Amino acid sequence of apcB protein: MQDAITAVINSADVQGKYLDGAAMDKLKSYFASGELRVRAASVISANAATIVKEAVAKSLLYSDVTRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDASILDERVLNGLKETYNSLGVPISSTVQAIQAIKEVTASLVGADAGKEMGVYLDYICSGLS* SEQ ID NO:3 Amino acid sequence of heme oxidase gene HoI: SVNLASQLREGTKKSHSMAENVGFVKCFLKGVVEKNSYRKLVGNLYFVYSAMEEEMAKFKDHPILSHIYFPELNRKQSLEQDLQFYYGSNWRQEVKISAAGQAYVDRVRQVAATAPELLVAHSYTRYLGDLSGGQILKKIAQNAMNLHDGGTAFYEFADIDDEKAFKNTYRQAMNDLPIDQATAERIVDEANDAFAMNMKMFNELEGNLIKAIGIMVFNSLTRRRSQGSTEVGLATSEG* SEQ ID NO:4 Amino acid sequence of biliverdin reductase gene pcyA: MAVTDLSLTNSSLMPTLNPMIQQLALAIAASWQSLPLKPYQLPEDLGYVEGRLEGEKLVIENRCYQTPQFRKMHLELAKVGKGLDILHCVMFPEPLYGLPLFGCDIVAGPGGVSAAIADLSPTQSDRQLPAAYQKSLAELGQPEFEQQRELPPWGEIFSEYCLFIRPSNVTEEERFVQRVVDFLQIHCHQSIVAEPLSEAQTLEHRQGQIHYCQQQQKNDKTRRVLEKAFGEAWAERYMSQVLFDVIQ* SEQ ID NO:5 Amino acid sequence of the lyase gene cpcS: MDAMEFFRNSSGNWRSQRTTHHLAFRRAETGTSEIFVEALGADDQKIVEICEMHDCDPAKAVGGAFVRWESAMAWDKEDENHEGTTVFALIPDEDNPQQGLLLRERGYAEIVPIAGRYHIDEEEALVLVTEYETMTTIERFWFANPDMRLRTSTVQRFGGFNTATYCTEMRVKEENTVSASPAPAYEQFCGW* SEQ ID NO:6 Amino acid sequence of the lyase gene cpcU: MKPVAPRSFFAMDFLEFITAWEGKWLSQRTNYSFEQNEAGNAKSDVTVQRWDSQTELGRSLLERAGLKNQGELILLQLSWDNSVDWGKSKQKGTIYYGFLPDADHGDRGQLWRGTTNSNLAFLRGDYHLGSDECLTLNLGDGETSLTERHWYASPNLRLRTSLIQQGEGYSHSAFYSDIRRLPPAE* The amino acid sequence of the donor fluorescent protein BDFP3.5 (SEQ ID NO:7): MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVQTAVSQLFQEYPELVRPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGFRDTFNSSLGIPLCPAARGIQLMKKIVKEKLAAAGMTNIAFVDEPFDYIARVISETEI The amino acid sequence of the receptor fluorescent protein BDFP3.6 of SEQ ID NO:8: MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVQTAVSQLFQEYPELVRPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGLRDTFNSLGIPLGPVARGIQLMKKIVKEKLAAAGMTNIAFVDEPFDYIARVISETEI* The amino acid sequence of the rigid linker peptide (AEAAAKEAAAKA) of SEQ ID NO:9: AEAAAKEAAAKA The amino acid sequence of SEQ ID NO:10 fusion fluorescent protein BDFP3.5-linker-BDFP3.6: MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVQTAVSQLFQEYPELVRPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGFRDTFNSLGIPLCPAARGIQLMKKIVKEKLAAAGMTNIAFVDEPFDYIARVISETEIAEAAK EAAAKAMANREVVETLKELLADGEKRVQVAGVIGTNAAEVVQTAVSQLFQEYPELVRPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGLRDTFNSLGIPLGPVARGIQLMKKIVKEKLAAAGMTNIAFVDEPFDYARVISETEI* Nucleotide sequence of the fusion fluorescent protein BDFP3.5-linker-BDFP3.6 of SEQ ID NO: 11: ATGGCTAACCGTGAAGTGGTTGAAACTCTGAAAGAACTTCTGGCTGACGGTGAAAAACGTGTGCAGGTTGCTGGCGTTATCGGTACTAACGCTGCGGAAGTTGTTCAGACCGCGGTTTCTCAGCTGTTCCAAGAATATCCAGAACTGGTGCGTCCGGGTGGTTGCGCGTATACCACTCGTCGTTACAACAAGTGCGTTCGTGACATGAACTACTTTCTGCGTATGTGCAGCTACGCGATTGTTGCAGGTGGTGCTTCTGTTCTGGATGGTCGTATGCTGGCTGGTTTCCGTGACACCTTCAACAGCCTGGGTATTCCGCTGTGTCCGGCTGCACGTGGTATCCAGCTGATGAAGAAGATCGTTAAAGAGAAGCTGGCTGCTGCTGGTATGACCAACATCGCATTCGTTGATGAACCGTTCGATTACATCGCGCGTGTTATCAGCGAAACCGAGATCGCGGAAGCGGCGGCGAAAGAAGCGGCGGCGAAAGCGATGGCGAACCGTGAAGTTGTTGAAACGCTGAAAGAACTGCTGGCTGATGGTGAGAAACGTGTTCAGGTTGCGGGTGTTATCGGCACCAACGCGGCTGAAGTTGTGCAGACCGCAGTTTCTCAGCTCTTCCAGGAATACCCGGAACTGGTTCGTCCGGGTGGTTGTGCGTACACCACTCGTCGCTACAACAAATGCGTTCGTGATATGAACTACTTCTTGCGTATGTGTAGCTACGCTATCGTTGCGGGTGGTGCGTCTGTGCTGGACGGTCGTATGTTGGCGGGTCTGCGTGACACTTTCAACTCTCTGGGTATCCCGCTGGGTCCGGTAGCTCGTGGTATCCAGCTCATGAAGAAGATCGTGAAAGAGAAACTGGCTGCGGCAGGTATGACTAACATCGCGTTCGTTGACGAACCGTTCGACTACATCGCACGTGTTATCTCTGAAACCGAAATCTAA Amino acid sequence of the receptor fluorescent protein miRFP720 set forth in SEQ ID NO:12: MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE* Amino acid sequence of the fusion fluorescent protein BDFP3.5-linker-miRFP720 set forth in SEQ ID NO:13: MANREVVETLKELLADGEKRVQVAGVIGTNAAEVVQTAVSQLFQEYPELVRPGGCAYTTRRYNKCVRDMNYFLRMCSYAIVAGGASVLDGRMLAGFRDTFNSLGIPLCPAARGIQLMKKIVKEKLAAAGMTNIAFVDEPFDYIARVISETEIAEAAAKEAAAKAMAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGSDNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFIGSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKITGFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDINYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGLIVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE* SEQ ID NO: 15 Nucleotide sequence of apcA subunit: ATGAGTATCGTCACGAAATCAATCGTGAATGCTGATGCAGAAGCCCGCTACCTGAGCCCCGGCGAACTGGATCGTATTAAAGCTTTTGTTACCGGCGGTGCCGCCCGTCTTCGCATTGCTGAAACCCTAACTGGTTCTCGGGAAACCATCGTTAAACAAGCTGGCGATCGCCTTTTCCAAAAACGTCCTGACATCGTTTCCCCCGGCGGCAATGCCTACGGTGAAGAAATGACCGCCACCTGCTTGCGCGACATGGACTACTACCTCCGTCTCGTCACCTACGGTGTGGTTTCCGGTGACGTTACCCCCATCGAAGAAATCGGTCTAGTGGGCGTACGGGAAATGTATCGTTCCCTCGGCACCCCCATCGAAGCCGTTGCTCAAAGTGTCCGGGAAATGAAAGAAGTTGCTTCCGGTTTGATGTCCTCCGATGATGCCGCCGAAGCCTCTGCCTACTTTGACTTCGTTATCGGAAAAATGAGCTAG Nucleotide sequence of the apcB subunit of SEQ ID NO:16: ATGCAAGACGCAATTACGGCTGTAATCAACTCTGCTGACGTTCAAGGTAAGTACCTCGACGGTGCTGCCATGGACAAACTGAAAAGCTACTTCGCCAGCGGTGAACTGCGGGTGCGGGCTGCCAGTGTAATCAGCGCCAACGCCGCCACCATCGTTAAAGAAGCTGTGGCCAAATCCCTGTTGTACTCCGACGTCACCCGTCCCGGCGGCAACATGTACACCACCCGTCGCTATGCGGCTTGTATCCGTGACTTGGACTACTACCTCCGCTATGCCACCTATGCCATGTTGGCCGGTGATGCTTCCATTCTCGATGAGCGCGTACTCAACGGTTTGAAAGAAACCTACAACTCCCTCGGTGTACCCATCTCTTCCACCGTTCAAGCTATCCAAGCCATCAAAGAAGTTACCGCTAGCTTGGTTGGTGCTGATGCCGGCAAAGAAATGGGCGTTTACCTCGACTACATCTGCTCTGGCTTGAGCTAG SEQ ID NO: 17 Nucleotide sequence of the heme oxygenase gene HoI: AGTGTCAACTTAGCTTCCCAGTTGCGGGAAGGGACGAAAAAATCCCACTCCATGGCGGAGAACGTCGGCTTTGTCAAATGCTTCCTCAAGGGCGTTGTCGAGAAAAATTCCTACCGTAAGCTGGTTGGCAATCTCTACTTTGTCTACAGTGCCATGGAAGAGGAAATGGCAAAATTTAAGGACCATCCCATCCTCAGCCACATTTACTTCCCCGAACTCAACCGCAAACAAAGCCTAGAGCAAGACCTGCAATTCTATTACGGCTCCAACTGGCGGCAAGAAGTGAAAATTTCTGCCGCTGGCCAAGCCTATGTGGACCGAGTCCGGCAAGTGGCCGCTACGGCCCCTGAATTGTTGGTGGCCCATTCCTACACCCGTTACCTGGGGGATCTTTCCGGCGGTCAAATTCTCAAGAAAATTGCCCAAAATGCCATGAATCTCCACGATGGTGGCACAGCTTTCTATGAATTTGCCGACATTGATGACGAAAAGGCTTTTAAAAATACCTACCGTCAAGCTATGAATGATCTGCCCATTGACCAAGCCACCGCCGAACGGATTGTGGATGAAGCCAATGACGCCTTTGCCATGAACATGAAAATGTTCAACGAACTTGAAGGCAACCTGATCAAGGCGATCGGCATTATGGTGTTCAACAGCCTCACCCGTCGCCGCAGTCAAGGCAGCACCGAAGTTGGCCTCGCCACCTCCGAAGGCTAG SEQ ID NO: 18 Nucleotide sequence of the biliverdin reductase gene pcyA: ATGGCCGTCACTGATTTAAGTTTGACCAATTCTTCCCTGATGCCTACGTTGAACCCGATGATTCAACAGTTGGCCCTGGCGATCGCCGCTAGTTGGCAAAGTTTACCCCTCAAGCCCTATCAATTGCCGGAGGATTTGGGCTACGTAGAAGGCCGCCTGGAAGGGGAAAAGTTAGTGATTGAAAATCGGTGCTACCAAACGCCCCAGTTTCGCAAAATGCATTTGGAGTTGGCCAAGGTGGGCAAAGGGTTGGATATTCTCCACTGTGTAATGTTTCCTGAGCCTTTATACGGTCTACCTTTGTTTGGCTGTGACATTGTGGCCGGCCCCGGTGGAGTAAGTGCGGCTATTGCGGATCTATCCCCCACCCAAAGCGATCGCCAATTGCCCGCAGCGTACCAAAAATCATTGGCAGAGCTAGGCCAGCCAGAATTTGAGCAACAACGGGAATTGCCCCCCTGGGGAGAAATATTTTCTGAATATTGTTTATTCATCCGTCCCAGCAATGTCACTGAAGAAGAAAGATTTGTACAAAGGGTAGTGGACTTTTTGCAAATTCATTGTCACCAATCCATCGTTGCCGAACCCTTGTCTGAAGCTCAAACTTTGGAGCACCGTCAGGGGCAAATTCATTACTGCCAACAACAACAGAAAAATGATAAAACCCGTCGGGTACTGGAAAAAGCTTTTGGGGAAGCTTGGGCGGAACGGTATATGAGCCAAGTCTTATTTGATGTTATCCAATAA Nucleotide sequence of the lyase gene cpcS of SEQ ID NO:19: ATGGATGCAATGGAATTCTTTCGCAATAGTTCCGGCAACTGGCGATCGCAACGTACCACCCATCACCTGGCCTTCCGTCGGGCAGAAACCGGCACCTCGGAAATTTTTGTTGAAGCTCTGGGTGCTGATGATCAAAAAATTGTCGAGATTTGTGAAATGCACGACTGCGACCCAGCCAAAGCCGTGGGGGGAGCCTTTGTCCGCTGGGAAAGCGCCATGGCCTGGGATAAAGAGGATGAAAATCATGAAGGAACAACTGTATTTGCCCTCATTCCCGATGAAGACAATCCCCAACAGGGACTACTACTGCGGGAGAGGGGCTATGCGGAAATTGTCCCCATTGCCGGTCGCTATCACATCGACGAAGAGGAAGCCCTGGTGCTAGTGACGGAGTATGAGACCATGACCACCATTGAACGGTTCTGGTTTGCCAACCCGGATATGCGCCTTAGAACCAGTACTGTGCAAAGGTTTGGGGGATTCAATACCGCCACCTACTGTACGGAAATGCGGGTCAAAGAGGAAAACACCGTGTCTGCGTCCCCTGCCCCTGCCTACGAACAATTTTGTGGCTGGTAGG Nucleotide sequence of the lyase gene cpcU of SEQ ID NO:20: ATGAAGCCTGTTGCCCCAAGAAGCTTTTTCGCTATGGATTTTTTGGAATTTATCACCGCCTGGGAAGGAAAATGGCTTTCCCAACGTACTAATTATAGTTTTGAGCAAAATGAAGCGGGCAATGCCAAATCCGATGTGACGGTGCAAAGGTGGGATAGTCAAACTGAGCTTGGGCGATCGCTCCTGGAGCGGGCCGGGTTAAAAAATCAAGGGGAGTTAATCCTGCTACAACTGAGTTGGGATAATTCCGTAGACTGGGGCAAGTCCAAACAAAAGGGCACCATCTATTATGGCTTTTTACCCGATGCTGACCATGGAGATCGGGGTCAACTATGGCGGGGCACAACGAATTCCAACTTGGCTTTCCTAAGGGGTGATTATCATTTAGGTTCTGATGAATGTTTGACCCTAAATCTAGGGGATGGGGAGACTAGTTTAACGGAAAGGCATTGGTATGCTAGTCCTAATCTCCGACTCCGCACCAGTTTGATCCAGCAGGGAGAAGGTTATAGTCATAGTGCCTTCTATTCTGATATTCGTCGTTTGCCTCCGGCAGAGTAA Amino acid sequence of the RE-APC protein of SEQ ID NO:21: MSIVTKSIVNADAEARYLSPGELDRIKAFVTGGAARLRIAETLTGSRETIVKQAGDRLFQKRPDIVSPGGNAYGEEMTATCLRDMDYYLRLVTYGVVSGDVTPIEEIGLVGVREMYRSLGTPIEAVAQSVREMKEVASGLMSSDDAAEASAYFDFVIGKMSMQDAITAVINSADVQGKYLDGAAMDKLKSYFASGELRVRAASVISANAATIVKEAVAKSLLYSDVTRPGGNMYTTRRYAACIRDLDYYLRYATYAMLAGDASILDERVLNGLKETYNSLGVPISSTVQAIQAIKEVTASLVGADAGKEMGVYLDYICSGLS

Claims

1. A multichannel fluorescent protein-encoded microsphere under a single excitation light condition, characterized in that, The multichannel fluorescent protein-encoded microspheres include a microsphere carrier and a fluorescent protein-encoded layer immobilized on the microsphere carrier; the fluorescent protein-encoded layer contains at least two recombinant fluorescent proteins, which are excited by a single excitation light; the single excitation light is visible red light, and the center wavelength of the single excitation light is in the range of 635–640 nm; the microsphere carrier is one of polymer microspheres, inorganic microspheres, or magnetic microspheres.

2. The coded microsphere according to claim 1, characterized in that, The recombinant fluorescent protein combination is a combination of RE-APC protein and BDFP-FRET protein, or a combination of RE-APC protein and miRFP720-FRET protein; the fluorescent protein coding layer is immobilized on the surface or inside the microsphere carrier by one of the following methods: covalent coupling, affinity binding, electrostatic adsorption, embedding, or cross-linking; the amino acid sequence of the RE-APC protein is shown in SEQ ID NO:21, the amino acid sequence of the BDFP-FRET protein is shown in SEQ ID NO:10, and the amino acid sequence of the miRFP720-FRET protein is shown in SEQ ID NO:13; the microsphere carrier is a single-size system or a multi-size system.

3. The method for preparing the coded microspheres according to claim 2, characterized in that, The process includes the following steps: (1) providing a microsphere carrier and having reactive sites on its surface for protein immobilization; (2) immobilizing at least two recombinant fluorescent proteins with the microsphere carrier to fix the recombinant fluorescent proteins on the surface and / or inside the microsphere carrier, forming a fluorescent protein coding layer; (3) by adjusting the concentration of each recombinant fluorescent protein, the resulting coded microspheres exhibit a preset intensity level in at least two emission detection channels under a single excitation light irradiation, thereby obtaining coded microspheres with multi-level coding and / or combined coding.

4. The preparation method according to claim 3, characterized in that, In step (1), when the surface of the microsphere carrier contains carboxyl groups, it is activated by a carbodiimide coupling system to form an intermediate that can react with amino groups, using an EDC / NHS activation system.

5. The preparation method according to claim 3, characterized in that, The immobilization reaction in step (2) is a covalent coupling, which includes amide bond coupling, thiol-maleimide coupling, click chemical coupling, or a combination thereof.

6. The preparation method according to claim 3, characterized in that, In step (2), different recombinant fluorescent proteins are immobilized simultaneously or in stages. When immobilization is used in stages, washing is performed after each immobilization to remove unbound fluorescent proteins.

7. The preparation method according to claim 3, characterized in that, The method further includes a blocking treatment to reduce nonspecific adsorption, wherein the blocking treatment employs a protein blocking agent, a polymer blocking agent, or a combination thereof.

8. The application of the coded microspheres according to claim 1 or 2 in liquid phase suspension array detection, characterized in that, It is used for the differentiation and encoding of multi-index samples and signal readout, and only a single excitation light source is used to complete the multi-channel encoding and recognition during detection; the emission detection channel is a spectroscopic detection channel, and at least two spectroscopic detection channels are provided; the emission detection channels are respectively located in the emission band range of 660 nm, 700 nm, or 750 nm.

9. The application of the coded microspheres according to claim 1 in multiplex biomolecular detection, characterized in that, Used for multiplex detection of one or more substances among nucleic acids, proteins, antigens and antibodies, cytokines, or metabolites.

Images