A single-line-state oxygen self-circulation aggregation-induced emission homogeneous microsphere and a preparation method and a no-washing detection method thereof

Abstract

This invention discloses a homogeneous microsphere based on singlet oxygen self-circulating aggregation-induced emission, its preparation, and a wash-free detection method, which improves the sensitivity and efficiency of wash-free luminescent oxygen channel immunoassay. This invention successfully prepared AIE receptor microspheres by embedding the photosensitizer AIER dye, ultra-bright AIEG dye, and singlet oxygen-responsive dye SO into carboxyl-modified polystyrene nanoparticles. Simultaneously, an antibody-1-labeled AIE receptor microsphere solution, an antigen-containing test solution, and biotinylated antibody-2 were added to a 96-well plate, followed by streptavidin-modified donor microspheres. After wash-free incubation, the fluorescence signal value was measured, and the concentration of antigen in the test solution was obtained according to a standard curve. This platform completes wash-free, highly sensitive detection within 5 minutes, innovatively combining the aggregation-induced emission characteristics of AIEgens and a singlet oxygen cycling strategy, providing an efficient and universal solution for wash-free diagnostics.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection and analysis, and particularly relates to a kind of homogenous microspheres based on singlet oxygen self-circulation aggregation-induced emission (AIE) and a preparation method and a no-wash high-sensitivity detection method. BACKGROUND

[0002] Luminescent oxygen channeling immunoassay (LOCI) based on donor-acceptor microbeads plays an important role in the "no-wash" immunoassay platform, making it widely used in clinical diagnosis, protein profiling, food safety and early diagnosis, etc. ACS Nano 2017, 11, 5238-5292.】. In which the target analyte brings the donor beads (DBs) and the acceptor beads (ABs) close to each other, and connects them through the formation of an immune complex. Under the irradiation of 680 nm laser, the donor microbeads will produce singlet oxygen, which will diffuse to the nearby acceptor microbeads and trigger a series of chemical reactions, finally producing chemiluminescence at 615 nm. On the contrary, in the absence of analyte, the donor beads and the acceptor beads remain spatially separated, which prevents the transfer of singlet oxygen, and thus no chemiluminescence signal will be generated Proceedings of the National Academy of Sciences 1994, 91, 5426-5430. 】. Therefore, the LOCI platform can achieve high sensitivity and high specificity of no-wash quantitative detection without the need for complex reagent handling or washing steps required in traditional immunoassay Advanced Healthcare Materials 2013, 2, 921-921.】. The sensitivity of LOCI detection is highly dependent on the performance of the reporter acceptor microbeads (ABs), especially their signal intensity and the stability of the dye. However, most fluorescent materials, such as traditional LOCI probes of aggregation-induced emission acceptor microspheres europium chelate or organic fluorophores, face major challenges such as low intrinsic quantum yield (QY) and fluorescence quenching (ACQ) effect due to aggregation Coordination Chemistry Reviews 2025, 542,216843.】. Low emission efficiency limits the performance of fluorescent probes, making it difficult to achieve high sensitivity and trace detection of low-abundance target analytes. In the LOCI system, singlet oxygen ( 1 O2) is a key medium in the chemiluminescence process. However, its generation and transfer in aqueous solution usually lasts only about 10 microseconds, and it cannot be maintained for a long time in acceptor beads, thus limiting the improvement of detection sensitivity Coordination Chemistry Reviews 2021, 427, 213575.】. Therefore, it is of great significance to develop acceptor beads that can recycle 1 O2 and maintain high quantum efficiency. However, there are still few reports on such materials.

[0003] AIEgens, first discovered by Tang et al. in 2001, are fluorophores that exhibit weak fluorescence in dilute solutions but strong luminescence in aggregated or solid states. Journal of Materials Chemistry [2001, 11, 2974-2978.]. The AIE phenomenon overcomes the limitations of traditional aggregation-induced quenching (ACQ) fluorophores. Incorporating AIEgens into a solid matrix can enhance luminescence by restricting intramolecular motion. Benefiting from their excellent photostability and biocompatibility, Tang's research group successfully encapsulated AIEgens in polystyrene nanoparticles, thereby developing a highly sensitive diagnostic platform. Aggregate [2021, 2, e51]. Among them, Zhang's research group was the first to use... 1 The use of O2-cycled dye (TTMN, photosensitizer) AIE nanoparticles to improve the sensitivity of lateral flow immunoassay has been confirmed. 1 O2 cycling can enhance the intensity of luminescent signals. Biosens Bioelectron 2022, 212, 114411. Therefore, a design is needed. 1 O2-circulating AIE homogeneous acceptor microspheres are a promising candidate for next-generation wash-free LOCI platforms. Summary of the Invention

[0004] Luminescent oxygen channel immunoassay is a homogeneous chemiluminescence detection platform with high specificity and wash-free advantages. However, the short-lived singlet oxygen and low luminescence quantum yield of traditional fluorescent nanospheres (such as europium chelate microspheres) limit its performance improvement. To address the shortcomings of existing technologies, this invention aims to provide a homogeneous microsphere based on singlet oxygen self-circulation aggregation-induced luminescence and its wash-free detection method. This method is intended to improve the sensitivity and efficiency of wash-free luminescent oxygen channel immunoassay.

[0005] The present invention adopts the following technical solution:

[0006] A wash-free detection method based on singlet oxygen self-circulating aggregation-induced emission homogeneous microspheres includes the following steps: adding antibody-1 labeled aggregation-induced emission receptor microspheres (i.e., AIE fluorescent microspheres) solution and biotinylated antibody-2 to the test solution containing antigen, then adding streptavidin-modified donor microspheres, incubating without washing, measuring the fluorescence signal value, and obtaining the concentration of antigen in the test solution according to the standard curve.

[0007] Preferably, the mass amount of the antibody-1 labeled aggregation-induced emission acceptor microspheres is 0.1% to 50% of the mass amount of the donor spheres; the mass amount of the biotinylated antibody-2 is 0.01% to 10% of the mass amount of the antibody-1 labeled aggregation-induced emission acceptor microspheres. Further preferably, the mass amount of the antibody-1 labeled aggregation-induced emission acceptor microspheres is 0.5% to 20% of the mass amount of the donor spheres; the mass amount of the biotinylated antibody-2 is 0.05% to 2% of the mass amount of the antibody-1 labeled aggregation-induced emission acceptor microspheres.

[0008] Preferably, the detection method is used in the field of in vitro diagnosis, such as inflammation, cytokine interleukin-6 (IL-6), food quality detection and environmental monitoring, etc.

[0009] The present application uses specific immune response to test the LOCI detection effect of the aggregation-induced emission acceptor microspheres. The donor spheres serve as output starting singlet oxygen, and activate the singlet oxygen self-circulation of the acceptor microspheres. The antibody-1 labeled aggregation-induced emission acceptor microspheres serve as a fluorescence output unit; the antibody-1 labeled aggregation-induced emission acceptor microspheres, the antigen sample and the biotin modified antibody-2 are added in a 96-well plate, and then the donor spheres are added, without separation operation, and the fluorescence signal value is directly determined using a photochemical chemiluminescence instrument.

[0010] Preferably, the antibody-1 and the antibody-2 can specifically bind to the antigen; the antibody-1 and the antibody-2 can be the same or different, the antibody-1 used in the embodiments of the present application is IL-6, and the antibody-2 is a biotinylated antibody; the fluorescence signal value is determined using a photochemical chemiluminescence instrument.

[0011] Preferably, the particle size ratio of the antibody-1 labeled aggregation-induced emission acceptor microspheres and the donor spheres is 0.5 to 0.9; the particle size of the antibody-1 labeled aggregation-induced emission acceptor microspheres is 200 nm or less.

[0012] Further preferably, the particle size ratio of the antibody-1 labeled aggregation-induced emission acceptor microspheres and the antibody-2 labeled magnetic microspheres is 0.66; the particle size of the antibody-1 labeled aggregation-induced emission microspheres is 200 nm.

[0013] Preferably, the composition of the antibody-1 labeled aggregation-induced emission microspheres comprises a carboxyl modified nanoscale microsphere matrix, an AIEG molecule, an AIER molecule and a dimethyl thiophene derivative dye; the preparation method comprises the following steps:

[0014] (1) dispersing the carboxyl modified nanoscale microsphere matrix and an emulsifier (water-soluble emulsifier) in water to obtain an aqueous phase solution;

[0015] (2) dissolving the AIEG molecule, the AIER molecule and the dimethylthiophene derivative (SO) dye in an organic solvent to obtain an oil phase solution;

[0016] (3) adding the oil phase solution of step (2) to the water phase solution of step (1), sealing and swelling, and then volatilizing the solvent to obtain an AIE fluorescent microsphere emulsion; centrifuging and dispersing in water to obtain an aggregation-induced emission acceptor microsphere;

[0017] (4) adding the aggregation-induced emission acceptor microsphere prepared in step (3) to an acidic buffer solution, ultrasonically dispersing, adding a carbodiimide condensation reagent, and activating the carboxyl group in a mixer; centrifuging to remove the supernatant to obtain an activated aggregation-induced emission microsphere;

[0018] (5) adding the activated aggregation-induced emission microsphere of step (4) to a basic buffer solution and an antibody-1, incubating in a mixer, centrifuging to remove the supernatant, adding a blocking solution and a preservation solution, and ultrasonically dispersing to obtain an antibody-1-labeled aggregation-induced emission acceptor microsphere.

[0019] Further preferably, in step (1), the mass amount of the carboxyl-modified nanoscale microsphere matrix is 0.01% to 10% of the mass amount of water.

[0020] Further preferably, in step (1), the particle size of the carboxyl-modified nanoscale microsphere matrix is 50 nm to 400 nm. The carboxyl-modified nanoscale microsphere matrix is a nanoscale microsphere matrix known in the art, such as polystyrene, polymethyl methacrylate microspheres. Preferably, the particle size is 100 nm to 200 nm.

[0021] Further preferably, in step (1), the mass amount of the emulsifier is 0.01% to 5% of the mass amount of water. More preferably, the mass amount of the emulsifier is 0.1% to 2% of the mass amount of water.

[0022] Further preferably, in step (1), the emulsifier is selected from at least one of MOA-50, O-50, sodium dodecyl sulfate, polyvinyl alcohol, Tween 20, and sodium dodecyl benzene sulfonate.

[0023] Further preferably, in step (2), the mass amount of the AIEG molecule is 0.01% to 5% of the mass amount of the organic solvent; more preferably, the mass amount of the AIEG molecule is 0.05% to 1% of the mass amount of the organic solvent; the mass amount of the AIER molecule is 0.005% to 1% of the mass amount of the organic solvent; more preferably, the mass amount of the AIEG molecule is 0.01% to 0.1% of the mass amount of the organic solvent.

[0024] Further preferably, in step (2), the mass amount of the organic solvent is 0.1% to 80% of the mass of water; more preferably, the mass amount of the organic solvent is 5% to 10% of the mass of water.

[0025] Further preferably, in step (2), the organic solvent is selected from one or a mixture of two of dichloromethane, tetrahydrofuran, chloroform, benzyl alcohol, anisole, toluene.

[0026] Further preferably, in step (2), the AIEG molecule is selected from at least one of the following AIEG-1 to AIEG-4 molecules:

[0027]

[0028] Further preferably, in step (2), the AIER molecule is selected from at least one of the following AIER-1 to AIER-4 molecules:

[0029]

[0030] Further preferably, in step (2), the dimethylthiophene derivative (SO) dye is selected from at least one of the following SO-1 to SO-3 molecules.

[0031]

[0032] Further preferably, in step (3), the time for closing and swelling is 1 h to 5 h; the time for volatilizing the solvent is 5 h to 24 h; the centrifugal speed is 8000 rpm to 12000 rpm; and the number of centrifugation is more than three times.

[0033] Further preferably, in step (4), the acid buffer is 2-(N-morpholino)ethanesulfonic acid buffer, glycine-hydrochloric acid buffer, or phthalic acid-hydrochloric acid buffer; the pH value of the acid buffer is 5.0 to 6.5; and the mass amount of the acid buffer is 10 to 200 times of the mass amount of the carboxyl-modified nanoscale microsphere matrix.

[0034] Further preferably, in step (4), the carbodiimide condensing agent is selected from one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and the mass amount of the carbodiimide condensing reagent is 0.1% to 10% of the mass amount of the carboxyl-modified nanoscale microsphere matrix.

[0035] In order to maximize the condensation reaction efficiency, the acid buffer is preferably 2-(N-morpholine) ethanesulfonic acid buffer with pH value in the range of 6.0-6.5, and the carbodiimide condensing agent is preferably 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride.

[0036] Further preferably, in step (4), the activation time is 5 min-5 h.

[0037] Further preferably, in step (4), the centrifugation time is 10 min-60 min, the rotation speed is 6000 rpm-14000 rpm, and the centrifugation times are more than three; more preferably, the centrifugation time is 25 min, and the rotation speed is 11000 rpm.

[0038] Further preferably, in step (5), the basic buffer is Tris-HCl buffer, boric acid buffer or phosphate buffered saline (PBS) solution; the pH value of the basic buffer is 7.4-9.0; and the mass amount of the basic buffer is 5-90 times the mass amount of the carboxyl-modified nanoscale microsphere matrix.

[0039] In order to maximize the condensation reaction efficiency, the basic buffer is preferably Tris-HCl buffer with pH value in the range of 7.5-8.5.

[0040] Further preferably, in step (5), the mass amount of the antibody-1 is 0.001%-5% of the mass amount of the carboxyl-modified nanoscale microsphere matrix.

[0041] Further preferably, in step (5), considering that the activated active ester is not completely reacted, the active ester needs to be blocked, and the blocking solution is bovine serum albumin (BSA).

[0042] Further preferably, in step (5), the storage solution is Tris microsphere storage solution.

[0043] Regarding the present application, through in-depth research, the ratio of AIEG molecules and AIER molecules plays an important role in the sensitivity of antigen quantitative detection, and different ratios of dyes can be selected to prepare aggregation-induced emission receptor microspheres with different detection limits; with the decrease of AIEG content, the FRET effect and fluorescence signal value of antigen detection decrease, and the final minimum detection limit of antigen detection increases, which reduces the sensitivity of detection, and the most suitable ratio of AIEG molecules and AIER molecules is between 5-10. Therefore, appropriate dye ratio should be selected according to different product requirements.

[0044] Compared with the prior art, the present application has the following beneficial effects:

[0045] The application provides a novel method for efficiently preparing single oxygen self-circulation aggregation-induced emission acceptor microspheres by combining swelling technology and carbodiimide reaction, and verifies potential application value of the AIE fluorescent microspheres in in-vitro diagnosis by detecting application effects of the aggregation-induced emission acceptor microspheres through specific reaction. AIER molecules, AIEG molecules and SO dyes are embedded in a nanoscale microsphere matrix. Antibody-labeled AIE fluorescent microspheres are efficiently prepared by using carbodiimide activation reaction, and finally, blocking liquid and preservative liquid are added to obtain the antibody-labeled AIE fluorescent microspheres. By combining the single oxygen self-circulation enhanced fluorescence mechanism, the detection sensitivity of IL-6 can be reduced to 0.36 pg·mL -1 The antigen sample can be detected quickly within 5 minutes without washing.

[0046] The single oxygen self-circulation enhanced fluorescence mechanism is explained as follows: when the single oxygen excited by the donor ball is transferred to the acceptor microspheres, the oxygen-receiving dye SO in the acceptor microspheres will trigger chemiluminescence with a wavelength of 300 nm-400 nm; then the AIEG dye and the AIER dye are excited, and the light-emitting wavelength is 525 nm-750 nm, and new single oxygen is generated to circulate and emit light; at the same time, the FRET effect between the AIEG dye and the AIER dye enhances the final fluorescence signal.

[0047] The application provides a no-wash detection method based on single oxygen self-circulation aggregation-induced emission homogeneous microspheres.

[0048] (1) The application first integrates the photosensitizer AIEG molecules, AIER molecules and oxygen-receiving dye SO in a single nanoparticle system, and constructs a novel structure that can realize the light-induced 1 O2 circulation mechanism. Specifically, the SO dye can trigger chemiluminescence after receiving single oxygen, the AIER generates 1 O2 through the intersystem crossing (ISC) process under light excitation, and the introduction of the AIEG optimizes the overlap between spectral absorption and emission, significantly improves the Förster resonance energy transfer (FRET) efficiency. The circulation mechanism not only effectively prolongs the action time of 1 O2 (thus overcoming the limitation of its inherent short lifetime), but also realizes the amplification of the signal output, which is the key to improving the sensitivity of the LOCI detection.

[0049] (2) The aggregation-induced emission characteristics (derived from intramolecular motion restriction) of AIEgens and the polystyrene skeleton produce a synergistic effect, effectively reducing non-radiative energy loss, thereby improving the fluorescence quantum yield. At the same time, since the AIE material has a relatively wide emission spectrum, it can adapt to a 30 nm bandwidth optical filter, which maximizes the capture of real fluorescence signals, further enhancing the overall detection signal strength.

[0050] (3) The strategy provides a general technical paradigm for wash-free diagnosis, has good scalability, and can be applied to the detection of other biomarkers, thereby promoting the development of precision medicine towards high-throughput and rapidity. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Scanning electron microscope image of the AIE fluorescent acceptor microspheres prepared in Example 1.

[0052] Figure 2 Emission spectrum and excitation image of the AIE fluorescent microspheres prepared in Example 1.

[0053] Figure 3 Schematic diagram of the test method of the wash-free LOCI prepared in Example 1.

[0054] Figure 4 Linear relationship curve of the AIE fluorescent microspheres prepared in Example 1 and Comparative Example 1 for detecting different IL-6 antigens. DETAILED DESCRIPTION

[0055] The application will be further described below in conjunction with specific embodiments, but the scope of protection of the application is not limited thereto.

[0056] The nanomicrospheres in the examples are carboxyl-modified polymer magnetic beads of different particle sizes from the Guangdong Greater Bay Area South China University of Technology Aggregation-Induced Emission Research Institute, product number NWKPC-020; the 250 nm streptavidin donor microspheres in the examples are from the Guangzhou Bio-Technology Company;

[0057] The IL-6 antigen in the examples is a protein expressed by prokaryotic recombinant expression. Antibody-1 and biotinylated antibody-2 are from the Feipeng Biological Company, with concentrations of 4.2 mg / mL and 0.2 mg / mL, respectively.

[0058] Example 1

[0059] I. Preparation of antibody-labeled AIE fluorescent acceptor microspheres

[0060] 0.1 g of carboxyl-modified 200 nm polystyrene microsphere matrix and 0.05 g of sodium dodecyl sulfate were weighed and dispersed in 12.5 g of water to serve as an aqueous emulsifier solution for the continuous phase;

[0061] 7.5 mg of AIEG-1 molecules, 1 mg of AIER-1 molecules, and 2.5 mg of SO-1 molecules were dissolved in 1 mL of dichloromethane to obtain an oil phase solution. After mixing and sealing the two solutions for swelling for 1 h, AIE fluorescent microsphere emulsion was prepared; the emulsion was centrifuged and then redispersed in water, and this process was repeated three times to obtain purified AIE fluorescent microspheres;

[0062] Add 5 g of MES buffer (pH = 6.0) to the AIE fluorescent microspheres, ultrasonic dispersion, then add 0.005 g of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, and put it into a mixer to activate the carboxyl group at a speed of 200 rpm for 3 min. The activated AIE fluorescent microspheres are obtained by centrifuging the supernatant, adding 5 g of boric acid buffer (pH = 7.4) and 50 μg of IL-6 antibody, and incubating in a mixer. The supernatant is removed by centrifugation, and the blocking solution BSA and Tris microsphere storage solution are added. After ultrasonic dispersion, the IL-6 antibody-labeled AIE fluorescent microspheres are obtained.

[0063] II. LOCI assay of the wash-free homogeneous aggregation-induced emission microspheres

[0064] Add 5 μL of IL-6 antigen sample, 20 μL of IL-6 antibody-labeled AIE receptor microspheres (10 mg / mL) and 20 μL of biotinylated antibody-2 (5 μg / mL) to a 96-well plate, and then add 100 μL of 250 nm streptavidin-modified donor spheres. After incubation, the fluorescence signal value is directly measured using a photochemical chemiluminescence instrument. The tested fluorescence signal value is 2750 x 10 4 , the minimum detection limit is 0.36 pg / mL, and the wash-free immunoassay is successful.

[0065] Comparative Example 1

[0066] Dissolve 1 mg of AIER-1 molecule and 2.5 mg of SO-1 molecule in 1 mL of dichloromethane, only change the dye type of the oil phase solution, and the preparation method, amount and type of the fluorescent receptor microspheres, the labeled antibody are the same as in Example 1. Finally, the fluorescence signal value is measured using a photochemical chemiluminescence instrument. Because the fluorescent receptor microspheres do not have AIEG dye, they cannot enhance the FRET effect of the dye, and the tested fluorescence signal value is 26 x 10 4 , the minimum detection limit is 38.5 pg / mL, and the wash-free immunoassay is successful.

[0067] Comparative Example 2

[0068] Dissolve 7.5 mg of AIEG-1 molecule and 2.5 mg of SO-1 molecule in 1 mL of dichloromethane, only change the dye type of the oil phase solution, and the preparation method, labeled antibody are the same as in Example 1. Finally, the fluorescence signal value is measured using a photochemical chemiluminescence instrument. Because the AIE receptor microspheres do not have AIER dye, they cannot form singlet oxygen cycle, and the tested fluorescence signal value is 7 x 10 4 , the fluorescence signal value is low, and the minimum detection limit cannot be obtained, and the wash-free immunoassay fails.

[0069] Comparative Example 3

[0070] 7.5 mg AIEG-1 molecules and 1 mg AIER-1 molecules were dissolved in 1 mL of dichloromethane, only the dye type of the oil phase solution was changed, the preparation method of the fluorescent receptor microspheres, the labeled antibody and Example 1 were consistent, and finally the fluorescence signal value was measured using a light-activated chemiluminescence instrument. Because there was no SO dye in the AIE acceptor microspheres, there was no chemiluminescence trigger, and no chemiluminescence could be formed, the fluorescence signal value tested was 0.5 x 10 4 , close to the background signal, and the immunoassay failed.

[0071] Example 2

[0072] I. Preparation of AIE fluorescent receptor microspheres labeled with antibodies

[0073] 0.2 g of carboxyl-modified 200 nm polystyrene microsphere matrix and 0.1 g of sodium dodecyl sulfate were weighed into 25 g of water and used as an aqueous emulsifier solution for the continuous phase;

[0074] 15 mg of AIEG-1 molecules, 2 mg of AIER-1 molecules and 5 mg of SO-1 molecules were dissolved in 2 mL of tetrahydrofuran to obtain an oil phase solution. After mixing and sealing the two solutions and swelling for 1 h, AIE fluorescent microsphere emulsion was prepared; the emulsion was centrifuged and then redispersed in water, and this process was repeated three times to obtain purified AIE fluorescent receptor microspheres;

[0075] The AIE fluorescent microspheres were added to 25 g of MES buffer (pH = 6.0), ultrasonically dispersed, and then 0.01 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to activate the carboxyl groups in a mixing instrument at a speed of 200 rpm for 3 min. The activated AIE fluorescent microspheres were obtained by removing the supernatant using centrifugation, and 10 g of MES buffer (pH = 6.0) and 50 μg of IL-6 antibody were added and incubated in a mixing instrument. The supernatant was removed by centrifugation, and blocking solution BSA and Tris microsphere storage solution were added and ultrasonically dispersed to obtain IL-6 antibody-labeled AIE fluorescent microspheres.

[0076] II. LOCI assay of the self-cleaning homogeneous aggregation-induced emission microspheres

[0077] 5 μL of IL-6 antigen sample, 20 μL of IL-6 antibody-labeled AIE receptor microspheres (10 mg / mL) and 20 μL of biotinylated antibody-2 (5 μg / mL) were added to a 96-well plate, and then 100 μL of 250 nm streptavidin-modified donor spheres were added. After incubation, the fluorescence signal value was directly measured using a light-activated chemiluminescence instrument. The fluorescence signal value tested was 2249 x 10 4The lowest detection limit is 0.48 pg / mL, and the wash-free immunoassay is successful.

[0078] Example 3

[0079] I. Preparation of antibody-labeled AIE fluorescent receptor microspheres

[0080] 0.5 g of carboxyl-modified 200 nm polystyrene microsphere matrix and 0.2 g of sodium dodecyl sulfate were weighed into 50 g of water as an emulsifier aqueous solution for the continuous phase;

[0081] 30 mg of AIEG-1 molecules, 5 mg of AIER-1 molecules, and 12.5 mg of SO-1 molecules were dissolved in 10 mL of tetrahydrofuran to obtain an oil phase solution. After mixing and sealing the two solutions for swelling for 1 h, AIE fluorescent microsphere emulsion was prepared; the emulsion was centrifuged and then redispersed in water, and this process was repeated three times to obtain purified AIE fluorescent receptor microspheres;

[0082] The AIE fluorescent microspheres were added to 50 g of MES buffer (pH=6.0), ultrasonically dispersed, and then 0.025 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added. The carboxyl groups were activated in a mixer at a speed of 200 rpm for 3 min. The activated AIE fluorescent microspheres were obtained by removing the supernatant using centrifugation, and 10 g of MES buffer (pH=6.0) and 50 μg of IL-6 antibody were added. The mixture was incubated in a mixer, and the supernatant was removed by centrifugation. Blocking solution BSA and Tris microsphere storage solution were added, and the mixture was ultrasonically dispersed to obtain IL-6 antibody-labeled AIE fluorescent microspheres.

[0083] II. LOCI assay of wash-free homogeneous aggregation-induced emission microspheres

[0084] In a 96-well plate, 5 μL of IL-6 antigen sample, 20 μL of IL-6 antibody-labeled AIE receptor microspheres (10 mg / mL), and 20 μL of biotinylated antibody-2 (5 μg / mL) were added, followed by the addition of 100 μL of 250 nm streptavidin-modified donor spheres. After incubation, the fluorescence signal value was directly measured using a photochemical luminescence instrument. The tested fluorescence signal value was 2386×10 4 The lowest detection limit is 0.39 pg / mL, and the wash-free immunoassay is successful.

[0085] Example 4

[0086] I. Preparation of antibody-labeled AIE fluorescent receptor microspheres

[0087] Take 1 g of carboxyl-modified 200 nm polystyrene microsphere matrix and 0.3 g of sodium dodecyl sulfate dispersed in 75 g of water as an aqueous emulsifier solution for the continuous phase;

[0088] Dissolve 50 mg of AIEG-1 molecules, 10 mg of AIER-1 molecules and 25 mg of SO-1 molecules in 20 mL of chloroform to obtain an oil phase solution. After mixing and sealing the two solutions for swelling for 2 h, an AIE fluorescent microsphere emulsion is prepared; the emulsion is centrifuged and then redispersed in water, and this process is repeated three times to obtain purified AIE fluorescent receptor microspheres;

[0089] Add the AIE fluorescent microspheres to 100 g of MES buffer (pH=6.5), ultrasonically disperse, then add 0.9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and put it into a mixer to activate the carboxyl group at a speed of 200 rpm for 3 min. Use centrifugation to remove the supernatant to obtain activated AIE fluorescent microspheres, add 25 g of boric acid buffer (pH=6.0) and 50 μg of IL-6 antibody, and put it into a mixer for incubation. Centrifuge to remove the supernatant, add blocking solution BSA and Tris microsphere storage solution, and ultrasonically disperse to obtain IL-6 antibody-labeled AIE fluorescent microspheres.

[0090] II. LOCI assay of the self-cleaning homogeneous phase aggregation-induced emission microspheres

[0091] Add 5 μL of IL-6 antigen sample, 20 μL of IL-6 antibody-labeled AIE receptor microspheres (10 mg / mL) and 20 μL of biotinylated antibody-2 (5 µg / mL) to a 96-well plate, and then add 100 μL of 250 nm streptavidin-modified donor spheres. After incubation, directly use a photochemical chemiluminescence instrument to measure the fluorescence signal value. The tested fluorescence signal value is 2189×10 4 , and the minimum detection limit is 0.42 pg / mL, and the self-cleaning immunoassay is successful.

[0092] Example 5

[0093] I. Preparation of antibody-labeled AIE fluorescent receptor microspheres

[0094] Take 2.5 g of carboxyl-modified 200 nm polystyrene microsphere matrix and 0.5 g of sodium dodecyl sulfate dispersed in 100 g of water as an aqueous emulsifier solution for the continuous phase;

[0095] The 50 mg AIEG-1 molecules, 10 mg AIER-1 molecules and 25 mg SO-1 molecules are dissolved in 20 mL of tetrahydrofuran to obtain an oil phase solution. After the two solutions are mixed and closed for swelling for 2 h, an AIE fluorescent microsphere emulsion is prepared; the emulsion is centrifuged and then re-dispersed in water, and this process is repeated three times to obtain purified AIE fluorescent receptor microspheres;

[0096] The AIE fluorescent microspheres are added to 150 g of MES buffer (pH = 6.5), ultrasonically dispersed, and then 2.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added to activate the carboxyl groups in a mixer at a speed of 200 rpm for 3 min. The activated AIE fluorescent microspheres are obtained by removing the supernatant by centrifugation, and 50 g of boric acid buffer (pH = 8.0) and 50 μg of IL-6 antibody are added and incubated in a mixer. The supernatant is removed by centrifugation, and a blocking solution of BSA and Tris microsphere storage solution is added and ultrasonically dispersed to obtain IL-6 antibody-labeled AIE fluorescent microspheres.

[0097] II. LOCI assay of the self-cleaning homogeneous phase aggregation-induced emission microspheres

[0098] In a 96-well plate, 5 μL of IL-6 antigen sample, 20 μL of IL-6 antibody-labeled AIE receptor microspheres (10 mg / mL) and 20 μL of biotinylated antibody-2 (5 μg / mL) are added, and then 100 μL of donor balls are added. After incubation, the fluorescence signal value is directly measured using a photochemical luminescence instrument. The tested fluorescence signal value is 2319 x 10 4 , the minimum detection limit is 0.45 pg / mL, and the self-cleaning immunoassay is successful.

[0099] Example 6

[0100] I. Preparation of antibody-labeled AIE fluorescent receptor microspheres

[0101] 0.1 g of carboxyl-modified 200 nm polystyrene microsphere matrix and 0.05 g of sodium dodecyl sulfate are weighed and dispersed in 12.5 g of water to serve as an aqueous emulsifier solution for the continuous phase;

[0102] 15 mg of AIEG-1 molecules, 2 mg of AIER-1 molecules and 5 mg of SO-1 molecules are dissolved in 2 mL of tetrahydrofuran to obtain an oil phase solution. After the two solutions are mixed and closed for swelling for 1 h, an AIE fluorescent microsphere emulsion is prepared; the emulsion is centrifuged and then re-dispersed in water, and this process is repeated three times to obtain purified AIE fluorescent receptor microspheres;

[0103] Add AIE fluorescent microspheres to 10 g MES buffer (pH=6.5), ultrasonic dispersion, then add 0.005 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, put into the mixer to activate the carboxyl group, speed 200 rpm, time 3 min. Use centrifugal method to remove the supernatant to obtain activated AIE fluorescent microspheres, add 10 g boric acid buffer (pH=6.0) and 50 μg IL-6 antibody, put into the mixer to incubate, centrifuge to take the supernatant, add blocking liquid BSA and Tris microsphere storage solution, ultrasonic dispersion to obtain IL-6 antibody labeled AIE fluorescent microspheres.

[0104] II. LOCI assay of the self-cleaning homogeneous phase aggregation-induced emission microspheres

[0105] Add 5 μL IL-6 antigen sample, 20 μL IL-6 antibody labeled AIE receptor microspheres (10 mg / mL) and 20 μL biotinylated antibody-2 (5 μg / mL) in a 96-well plate, then add 100 μL 250 nm streptavidin modified donor balls, incubate, and then directly use the photochemical luminescence instrument to determine the fluorescence signal value. The tested fluorescence signal value is 1250×10 4 , the minimum detection limit is 0.89 pg / mL, and the self-cleaning immunological test is successful.

[0106] Example 7

[0107] I. Preparation of antibody labeled AIE fluorescent receptor microspheres

[0108] Weigh 0.1 g of carboxyl modified 200 nm polystyrene microsphere matrix and 0.05 g of sodium dodecyl sulfate into 12.5 g of water to disperse as an aqueous emulsifier solution for the continuous phase;

[0109] Dissolve 15 mg of AIEG-1 molecules, 2 mg of AIER-1 molecules and 5 mg of SO-1 molecules in 2 mL of tetrahydrofuran to obtain an oil phase solution. After mixing and closing the swelling for 1 h, AIE fluorescent microsphere emulsion is prepared; centrifuge the emulsion and re-disperse it in water, and repeat this process three times to obtain purified AIE fluorescent receptor microspheres;

[0110] The AIE fluorescent microspheres were added to 10 g of MES buffer (pH = 6.5), and after ultrasonic dispersion, 0.005 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, and the carboxyl group was activated in a mixer at a speed of 200 rpm for 3 min. The activated AIE fluorescent microspheres were obtained by centrifuging the supernatant, and 10 g of boric acid buffer (pH = 6.0) and 50 μg of IL-6 antibody were added, and incubated in a mixer, and the supernatant was obtained by centrifugation, and blocking liquid BSA and Tris microsphere storage liquid were added, and ultrasonic dispersion was performed to obtain IL-6 antibody-labeled AIE fluorescent microspheres.

[0111] II. LOCI assay of the wash-free homogeneous phase aggregation-induced emission microspheres

[0112] In a 96-well plate, 5 μL of IL-6 antigen sample, 20 μL of IL-6 antibody-labeled AIE receptor microspheres (10 mg / mL), and 20 μL of biotinylated antibody-2 (5 μg / mL) were added, and then 100 μL of 250 nm streptavidin-modified donor spheres were added, and after incubation, the fluorescence signal value was directly determined using a photochemical luminescence instrument. The tested fluorescence signal value was 956×10 4 , the minimum detection limit was 10.46 pg / mL, and the wash-free immunoassay was successful.

[0113] Table 1. Effect of different proportions of AIEG dye, AIER dye, and SO dye on IL-6 detection signal value and detection limit

[0114]

[0115] Figure 1 The scanning electron microscope image of the AIE fluorescent receptor microspheres prepared in Example 1 was spherical, uniform, and the particle size was about 200 nm.

[0116] Figure 2 The emission spectrum and excitation image of the AIE fluorescent microspheres prepared in Example 1 were shown, and the maximum emission wavelength was 590 nm, emitting strong orange-red fluorescence.

[0117] Figure 3 The test method schematic diagram of the wash-free LOCI prepared in Example 1 was shown, and the signal could be detected within 5 min after the test sample was added to the reaction system.

[0118] Figure 4 The linear relationship curve of the AIE fluorescent microspheres prepared in Example 1 and Comparative Example 1 for detecting different IL-6 antigens was shown, and the detection limit LOD of Example 1 could reach 0.36 pg / mL, while Comparative Example 1 only had 38.5 pg / mL.

[0119] In addition to the embodiments provided by the present application, various combinations of the AIEG molecules (such as AIEG-2, AIEG-3, AIEG-4), AIER molecules (AIER-2, AIER-3, AIER-4), and SO molecules (such as SO-2, SO-3) with similar structures mentioned herein can achieve similar effects to the above embodiments.

[0120] The above embodiments are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, and the like made without departing from the spirit and principles of the present application are equivalent replacement manners and are included in the protection scope of the present application.

Claims

1. A homogeneous microsphere based on singlet oxygen autocycling aggregation-induced emission, characterized in that, The microspheres are specifically antibody-1 labeled aggregation-induced emission acceptor microspheres, and are composed of carboxyl modified nanoscale microsphere matrix, AIEG molecules, AIER molecules and dimethyl thiophene derivative dyes, wherein the mass ratio of the AIEG molecules to the AIER molecules is 5-10; The AIEG molecules are selected from at least one of the following AIEG-1 to AIEG-4 molecules: The AIER molecules are selected from at least one of the following AIER-1 to AIER-4 molecules: 。 2. The method for preparing a homogeneous microsphere based on singlet oxygen self-cycling aggregation-induced emission according to claim 1, characterized in that, The method comprises the following steps: (1) dispersing the carboxyl modified nanoscale microsphere matrix and an emulsifier in water to obtain an aqueous solution; (2) dissolving the AIEG molecules, the AIER molecules and the dimethyl thiophene derivative dyes in an organic solvent to obtain an oil phase solution; (3) adding the oil phase solution of step (2) to the aqueous solution of step (1), sealing and swelling, volatilizing the solvent, and preparing an AIE fluorescent microsphere emulsion; centrifuging to obtain a precipitate, dispersing the precipitate in water, and obtaining aggregation-induced emission acceptor microspheres; (4) adding the aggregation-induced emission acceptor microspheres prepared in step (3) to an acidic buffer, ultrasonic dispersing, adding a carbodiimide condensation reagent, and activating the carboxyl group in a mixing instrument; centrifuging to remove the supernatant, and obtaining activated aggregation-induced emission microspheres; (5) adding the activated aggregation-induced emission microspheres of step (4) to an alkaline buffer and antibody-1, incubating in a mixing instrument, centrifuging to obtain the supernatant, adding a blocking solution and a preservation solution, ultrasonic dispersing, and obtaining antibody-1 labeled aggregation-induced emission acceptor microspheres.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass amount of the carboxyl modified nanoscale microsphere matrix is 0.01%-10% of the mass amount of water; and the particle size of the carboxyl modified nanoscale microsphere matrix is 50 nm-400 nm. The mass amount of the emulsifier is 0.01%-5% of the mass amount of water; and the emulsifier is selected from at least one of the following: MOA-50, O-50, sodium dodecyl sulfate, polyvinyl alcohol, Tween 20 and sodium dodecyl benzene sulfonate.

4. The preparation method according to claim 2, characterized in that, In step (2), the mass amount of the AIEG molecules is 0.01%-5% of the mass of the organic solvent; and the mass amount of the AIER molecules is 0.005%-1% of the mass of the organic solvent. The mass amount of the organic solvent is 0.1%-80% of the mass of the water in step (1); and the organic solvent is selected from one or a mixture of two of the following: dichloromethane, tetrahydrofuran, chloroform, benzyl alcohol, benzyl ether and toluene.

5. The preparation method according to claim 2, characterized in that, In step (3), the sealing and swelling time is 1 h-5 h; the volatilization time of the solvent is 5 h-24 h; the centrifugation speed is 8000 rpm-12000 rpm; and the centrifugation times are more than three.

6. The preparation method according to claim 2, characterized in that, In step (4), the acidic buffer is 2-(N-morpholine) ethanesulfonic acid buffer, glycine-hydrochloric acid buffer or phthalic acid-hydrochloric acid buffer; the pH value of the acidic buffer is 5.0-6.5; and the mass amount of the acidic buffer is 10-200 times of the mass amount of the carboxyl modified nanoscale microsphere matrix. The carbodiimide condensing agent is selected from one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the mass amount of the carbodiimide condensing agent is 0.1% to 10% of the mass amount of the carboxyl-modified nanoscale microsphere matrix.

7. The preparation method according to claim 2, characterized in that, In step (4), the activation time is 5 min to 5 h; the centrifugation time is 10 min to 60 min, the rotation speed is 6000 rpm to 14000 rpm, and the centrifugation times are more than three times.

8. The preparation method according to claim 2, characterized in that, In step (5), the alkaline buffer is a tris-hydroxymethyl aminomethane-hydrochloric acid buffer, a boric acid buffer or a phosphate buffered saline (PBS) solution; the pH value of the alkaline buffer is 7.4 to 9.0; the mass amount of the alkaline buffer is 5 to 90 times of the mass amount of the carboxyl-modified nanoscale microsphere matrix. The mass amount of the antibody-1 is 0.001% to 5% of the mass amount of the carboxyl-modified nanoscale microsphere matrix.

9. A method for self-cycling singlet oxygen-based aggregation-induced emission homogenous microsphere detection without washing, characterized in that, The method comprises the following steps: adding the antibody-1 labeled aggregation-induced emission acceptor microsphere solution and the biotinylated antibody-2 into an antigen-containing solution to be tested, then adding the streptavidin-modified donor spheres, incubating without washing, measuring the fluorescence signal value, and obtaining the concentration of the antigen in the solution to be tested according to a standard curve.

10. The no-wash detection method of claim 9, wherein, The antibody-1 and the antibody-2 can be the same or different; the particle size ratio of the antibody-1 labeled aggregation-induced emission acceptor microsphere and the donor sphere is 0.5 to 0.9; and the particle size of the antibody-1 labeled aggregation-induced emission acceptor microsphere is 200 nm or less. The carbodiimide condensing agent is selected from one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the mass amount of the carbodiimide condensing agent is 0.1% to 10% of the mass amount of the carboxyl-modified nanoscale microsphere matrix.

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