A rare earth complex fluorescent microsphere and its preparation method and application

Abstract

The invention provides a rare earth complex fluorescent microsphere and a preparation method and application thereof. The preparation method comprises: (1) preparing a rare earth complex from thenoyltrifluoroacetone, europium ions, other lanthanide metal ions, sodium hydroxide, phenanthroline and an ethanol solution; (2) dissolving the obtained rare earth complex and polystyrene microspheres in dichloromethane to further prepare the rare earth complex fluorescent microspheres. The rare earth complex fluorescent microspheres synthesized by the preparation method can encapsulate tens of thousands of rare earth complex fluorescent molecules in the microspheres, and can achieve a fluorescence signal amplification effect without dissociation enhancement. The obtained microspheres have a controllable particle size range, good uniformity, monodispersity, stability and repeatability, and have important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of rare earth nano material synthesis, and specifically relates to a rare earth complex fluorescent microsphere and a preparation method and application thereof. Background Art

[0002] Medical testing is an important measure to protect people's lives and health. Among them, instant diagnosis technology is the top priority and one of the important directions for the development of in vitro diagnosis in the future. The key to the development of instant diagnosis technology lies in the continuous improvement of detection materials and diagnostic methods. Compared with traditional fluorescent immunoassay technology and colloidal gold technology, rare earth nanoprobe detection technology has obvious advantages, and compared with chemiluminescence technology, rare earth nanoprobe detection technology has lower instrument costs and has greater advantages in market competition. These advantages mainly lie in the optical properties of rare earth nanoprobes such as long fluorescence lifetime, narrow emission peak half-width and large Stokes shift, which enable the detection technology based on rare earth nanoprobes to effectively reduce the interference of background fluorescence in detection, improve detection sensitivity and signal-to-noise ratio; not only that, compared with traditional molecular probes, rare earth nanoprobes have higher optical stability, can maintain the stability of optical signals during the detection process, improve the accuracy of detection results, and be applied to medical molecular diagnosis. It can increase the sensitivity of traditional molecular probe diagnosis by 2 to 3 orders of magnitude, greatly improve the accuracy of molecular diagnosis, and make in vitro detection faster and more accurate.

[0003] In recent years, scientists at home and abroad have made gratifying progress in the controlled synthesis of rare earth nanoluminescent materials and fluorescent immunoassays of various disease markers. CN112180083A discloses an IgM antibody detection kit and its detection card and a method for preparing the detection card, wherein the IgM antibody detection card comprises: a substrate, a coating film, a sample pad and absorbent paper; the sample pad is sprayed with a microsphere line at one end close to the coating film, and the microsphere line is an anti-human IgM antibody labeled with a rare earth nanoprobe and a rabbit IgG antibody labeled with a rare earth nanoprobe; the coating film is provided with a detection line and a quality control line in sequence, and the detection line is close to the sample pad; the detection line is coated with a recombinant antigen of a new coronavirus, a recombinant antigen of influenza A virus and a recombinant antigen of influenza B virus, and the quality control line is coated with a goat anti-rabbit antibody. The nanoprobe of the invention is a rare earth fluoride nanomaterial with the advantages of low background, long luminescence lifetime, strong fluorescence signal, high signal-to-noise ratio, etc. The labeled product is stable, with high sensitivity, high accuracy, fast and easy detection, etc. It can be used for screening and detection of early suspected COVID-19 patients, and can quickly and specifically help clinical diagnosis. CN111234819A discloses a rare earth doped nanoprobe, which is a core-shell structure of praseodymium-doped sodium lutetium fluoride coated with sodium yttrium fluoride, and its composition is: NaLu 1-x F4:Pr x @NaYF4, among them, NaLu 1-xF4 is the matrix, and the doping ion is Pr; the colon : indicates praseodymium doping; x is the rare earth ion doping molar ratio, and the range of x is 0.0005-0.05; NaYF4 is the shell, and @ indicates that NaYF4 is coated on NaLu 1-x F4:Pr x Surface; it provides a rare earth doped nanoprobe with low background interference and high sensitivity and its preparation and new coronavirus detection probe. CN112730832A discloses a COVID-19 antigen detection card, its preparation method and application, wherein the rare earth nanoprobe used is a core-shell structure sodium erbium fluoride coated with sodium yttrium fluoride, with a particle size of 20 to 30 nm. The invention uses rare earth fluoride as a matrix and synthesizes high-performance sodium erbium fluoride coated with sodium yttrium fluoride nanoprobes by doping with different rare earth ions. The fully erbium-doped nanomaterial is coated with a layer of sodium yttrium fluoride shell on the surface to improve the efficiency of energy conversion, and has the characteristics of high sensitivity, which facilitates its detection, so a rare earth nanoprobe with stable photochemical properties and long luminescence life is prepared.

[0004] However, the application and industrialization of rare earth nanoprobes are still in their infancy, and the rare earth nanoprobes provided by the above patents still have much room for improvement in terms of fluorescence and detection sensitivity.

[0005] Therefore, developing a rare earth complex fluorescent microsphere with controllable particle size and good uniformity and repeatability is a technical problem that urgently needs to be solved in this field. Summary of the invention

[0006] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a rare earth complex fluorescent microsphere and a preparation method and application thereof. The preparation method uses polystyrene microspheres to encapsulate thousands of rare earth complexes in the polystyrene microspheres, thereby achieving a fluorescence signal amplification effect without dissociation enhancement. The obtained rare earth complex fluorescent microspheres have a controllable particle size range, good uniformity, monodispersity, stability and repeatability, and can achieve high-sensitivity in vitro detection after antibodies are connected to them.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing rare earth complex fluorescent microspheres, the preparation method comprising the following steps:

[0009] (1) mixing thenoyltrifluoroacetone, europium ions, other lanthanide metal ions, sodium hydroxide, phenanthroline and ethanol solution, and filtering to obtain a rare earth complex;

[0010] (2) The rare earth complex obtained in step (1), dichloromethane and polystyrene microspheres are mixed, subjected to ultrasound, introduced with nitrogen, dichloromethane is removed, and centrifuged to obtain the rare earth complex fluorescent microspheres.

[0011] The invention provides a method for preparing rare earth complex fluorescent microspheres. Firstly, thenoyl trifluoroacetone (TTA), europium ions, other lanthanide metal ions and phenanthroline (Phen) are mixed, and a rare earth complex (rare earth probe) is synthesized by using a multi-ligand. The luminescence efficiency of the prepared rare earth complex can be improved through the synergistic enhancement effect between the ligands. Meanwhile, lanthanide metal ions are added in the synthesis process for doping, so as to sensitize the luminescence of europium ions, increase the fluorescence intensity, and further improve the optical properties of the rare earth complex. Then, the obtained rare earth complex, polystyrene microspheres and dichloromethane are mixed, ultrasonicated, the dichloromethane is removed, and centrifuged to obtain the rare earth complex fluorescent microspheres (Eu x Ln 1-x (TTA)3Phen).

[0012] The present invention adopts monomer copolymerization method to synthesize rare earth complex fluorescent microspheres. Thousands of rare earth complex fluorescent molecules can be encapsulated in the rare earth complex fluorescent microspheres, thereby achieving an exponential improvement in the intensity of fluorescent signals. When applied to in vitro detection, the detection sensitivity can be greatly increased, which has important research significance.

[0013] In the present invention, "other lanthanide metal ions" refer to lanthanide metal ions other than europium ions.

[0014] Preferably, the molar ratio of the europium ion to thenoyltrifluoroacetone in step (1) is 1:(3-6), for example, 1:3.3, 1:3.6, 1:3.9, 1:4, 1:4.5, 1:5, 1:5.3.7, 1:5.8 or 1:5.9, etc.

[0015] Preferably, the molar ratio of the europium ions to the other lanthanide metal ions in step (1) is 1:(0.2-1), for example, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9 or 1:1, etc.

[0016] Preferably, the other lanthanide metal ions in step (1) include Gd 3+ 、Lu 3+ , Y 3+ or Nd 3+ Any one or a combination of at least two of the following.

[0017] Preferably, the hydroxide in step (1) comprises sodium hydroxide and / or potassium hydroxide.

[0018] Preferably, the molar ratio of the europium ion to phenanthroline in step (1) is 1:(1-2), for example, 1:1.1, 1:1:2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2, etc.

[0019] Preferably, tri-n-octylphosphine oxide (TOPO) is also added to the mixing in step (1).

[0020] As a preferred technical solution of the present invention, TOPO is also added in step (1), and the synthesized rare earth complex fluorescent microspheres are Eu x Ln 1-x (TTA)3Phen(TOPO)2.

[0021] Preferably, the molar ratio of the europium ion to tri-n-octylphosphine oxide in step (1) is 1:(2-4), for example 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.1, 1:3.2, 1:3.3, 1:3.4, 1:3.5, 1:3.6, 1:3.7 or 1:3.8, etc.

[0022] Preferably, the mixing temperature in step (1) is 50-65°C, for example, 52°C, 54°C, 56°C, 58°C, 60°C, 62°C or 64°C.

[0023] Preferably, the mixing in step (1) is carried out in a water bath.

[0024] Preferably, the mixing in step (1) is carried out under stirring conditions, and more preferably, is carried out under stirring conditions at a rotation speed of 600 to 800 rpm (e.g., 620 rpm, 640 rpm, 660 rpm, 680 rpm, 700 rpm, 720 rpm, 740 rpm, 760 rpm or 780 rpm, etc.).

[0025] Preferably, step (1) further includes a condensation reflux step before the suction filtration.

[0026] Preferably, the condensation reflux temperature is 50-65°C, for example, 52°C, 54°C, 56°C, 58°C, 60°C, 62°C or 64°C.

[0027] Preferably, the condensation reflux time is 0.5 to 3 h, for example, 0.7 h, 0.9 h, 1.3 h, 1.6 h, 1.9 h, 2.3 h, 2.6 h or 2.9 h.

[0028] Preferably, the filtration in step (1) further includes washing and vacuum drying steps.

[0029] Preferably, the washing includes washing with ultrapure water and washing with ethanol.

[0030] Preferably, the ultrapure water washing and ethanol washing are performed 2 to 3 times independently.

[0031] Preferably, the polystyrene microspheres in step (2) are prepared by the following method, which comprises: dispersing an initiator, a styrene monomer and an acrylic acid monomer in ultrapure water, reacting, and centrifuging to obtain the polystyrene microspheres.

[0032] Preferably, the initiator comprises a persulfate.

[0033] Preferably, the persulfate comprises potassium persulfate or ammonium persulfate.

[0034] Preferably, the reaction temperature is 60-70°C, for example, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C or 69°C.

[0035] Preferably, the reaction is carried out under stirring conditions, and more preferably, under stirring conditions at a rotation speed of 600 to 800 rpm (e.g., 620 rpm, 640 rpm, 660 rpm, 680 rpm, 700 rpm, 720 rpm, 740 rpm, 760 rpm or 780 rpm, etc.).

[0036] Preferably, the reaction time is 6 to 10 h, for example, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h or 9.5 h.

[0037] Preferably, a washing step is further included after the centrifugation.

[0038] Preferably, the mass ratio of the rare earth complex and the polystyrene microspheres in step (2) is (1-3):1, for example, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.6:1 or 2.8:1, etc.

[0039] Preferably, the ultrasonication time in step (2) is 10 to 20 h, for example, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h or 19 h.

[0040] Preferably, the step (2) further comprises a magnetic stirring step after the ultrasonication.

[0041] Preferably, the magnetic stirring time is 12 to 16 h, for example, 12.5 h, 13 h, 13.5 h, 14 h, 14.5 h, 15 h or 15.5 h.

[0042] Preferably, the removal of dichloromethane in step (2) is accomplished by magnetic stirring at 45-55°C (e.g., 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C or 54°C, etc.).

[0043] Preferably, step (2) further includes a washing step after the centrifugation.

[0044] Preferably, the washing includes washing with ultrapure water and washing with ethanol.

[0045] Preferably, the ultrapure water washing and ethanol washing are performed 2 to 3 times independently.

[0046] As a preferred technical solution, the preparation method comprises the following steps:

[0047] (1) dissolving europium ions, lanthanide metal ions, phenanthroline, thenoyl trifluoroacetone and tri-n-octylphosphine oxide in an ethanol solution, adding hydroxide, stirring and mixing in a water bath at 50-65° C. and a rotation speed of 600-800 rpm, condensing and refluxing at 50-65° C. for 0.5-3 h, filtering, washing with ultrapure water and ethanol for 2-3 times respectively, and vacuum drying to obtain a rare earth complex;

[0048] (2) The rare earth complex obtained in step (1) is dissolved in dichloromethane, mixed with polystyrene microspheres, ultrasonicated for 10 to 20 hours, magnetically stirred for 12 to 16 hours, introduced with nitrogen, removed the dichloromethane under magnetic stirring at 45 to 55° C., centrifuged, and washed to obtain the rare earth complex fluorescent microspheres.

[0049] In a second aspect, the present invention provides a rare earth complex fluorescent microsphere, wherein the rare earth complex fluorescent microsphere is prepared by the preparation method described in the first aspect.

[0050] Preferably, the particle size of the rare earth complex fluorescent microspheres is 170-280 nm, for example, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm or 270 nm.

[0051] The rare earth complex fluorescent microsphere provided by the invention has a strong fluorescent signal under 365nm excitation.

[0052] In a third aspect, the present invention provides a detection microsphere, wherein the detection microsphere comprises the rare earth complex fluorescent microsphere surface as described in the second aspect and an antibody coupled to the rare earth complex fluorescent microsphere surface.

[0053] The detection microspheres provided by the present invention can be obtained by coupling antibodies on the surface of rare earth complex fluorescent microspheres; different antibodies can be coupled according to actual needs to achieve different biological detections; the detection microspheres provided by the present invention are used for in vitro detection.

[0054] In a fourth aspect, the present invention provides a method for preparing the detection microspheres as described in the third aspect, the preparation method comprising: using EDC / NHS to activate the surface of the rare earth complex fluorescent microspheres as described in the second aspect, grafting antibodies, and obtaining the detection microspheres.

[0055] The preparation method of the detection microspheres provided by the present invention successfully carries out antibody labeling on the surface of the rare earth complex fluorescent microspheres through a two-step method. Before the microsphere surface is activated by EDC / NHS, polyacrylic acid (PAA) needs to be modified on the microsphere surface so that the microsphere surface has carboxyl groups. The two-step method of coupling antibodies not only improves the material stability of the finally obtained detection microspheres, but also increases the distance between the antibody and the microspheres, effectively reduces the steric hindrance effect, and thus significantly improves the detection sensitivity of the prepared detection microspheres, which can be used for high-sensitivity in vitro detection.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The preparation method of the rare earth complex fluorescent microspheres provided by the present invention adopts a multi-ligand to synthesize the rare earth complex, which can improve the luminescence efficiency of the rare earth complex through the synergistic enhancement effect between the ligands; at the same time, doping with other lanthanide metal ions can have a sensitization effect on the luminescence of europium ions, increase the fluorescence intensity, and further improve the optical properties of the rare earth complex; specifically, the fluorescence lifetime of the rare earth complex fluorescent microspheres obtained by the preparation method provided by the present invention is 0.634-0.702ms, and the fluorescence intensity value is 5.02×10 5 ~9.28×10 5 .

[0058] (2) The preparation method provided by the present invention utilizes monomer copolymerization to synthesize rare earth complex fluorescent microspheres, which can encapsulate thousands of rare earth complex fluorescent molecules, thereby achieving a multiple increase in the intensity of the fluorescent signal and increasing the detection sensitivity.

[0059] (3) The preparation method provided by the present invention has simple process and strong operability, which can further meet the needs of production and application.

[0060] (4) The rare earth complex fluorescent microspheres obtained by the preparation method provided by the present invention have a controllable particle size range and have the advantages of good uniformity, monodispersity, stability and repeatability, which lays a good foundation for further biological detection applications; and the rare earth complex fluorescent microspheres have a strong fluorescence signal under 365nm excitation and a long fluorescence lifetime, which can be used to develop the field of time-resolved detection and further improve the detection sensitivity of various materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a scanning electron microscope image of the rare earth complex fluorescent microspheres provided in Example 1;

[0062] Figure 2 This is a scanning electron microscope image of the rare earth complex fluorescent microspheres provided in Example 2;

[0063] Figure 3 This is a scanning electron microscope image of the rare earth complex fluorescent microspheres provided in Example 3;

[0064] Figure 4 The excitation spectrum and emission spectrum of the rare earth complex fluorescent microspheres provided in Example 1, wherein 1-laser spectrum, 2-emission spectrum;

[0065] Figure 5 This is a fluorescence attenuation curve of the rare earth complex fluorescent microspheres provided in Example 1. DETAILED DESCRIPTION

[0066] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0067] Preparation Example 1

[0068] A polystyrene microsphere, the preparation method of which comprises: dispersing 1 mL of 30 mg / mL potassium persulfate, 1 mL of styrene monomer and 55 μL of acrylic acid monomer in ultrapure water (50 mL of ultrapure water is placed in a 100 mL round-bottom flask, and a water pump is used to evacuate for 30 minutes to remove oxygen in the water to obtain ultrapure water), stirring at a speed of 700 rpm and reacting at 70° C. for 10 hours, centrifuging at 4000 rpm for 5 minutes, centrifuging at 12000 rpm for 10 minutes, and washing with water 3 times to obtain the polystyrene microsphere;

[0069] 0.9 g of the polystyrene microspheres obtained in Preparation Example 1 were dispersed in 30 mL of 0.25% SDS solution for later use.

[0070] Preparation Example 2

[0071] A polystyrene microsphere, the preparation method of which comprises: dispersing 1 mL of 30 mg / mL potassium persulfate, 845 μL of styrene monomer and 55 μL of acrylic acid monomer in ultrapure water (50 mL of ultrapure water is placed in a 100 mL round-bottom flask, and vacuumed for 30 minutes with a water pump to remove oxygen in the water to obtain ultrapure water), stirring at a speed of 700 rpm and reacting at 70° C. for 8 hours, centrifuging at 4000 rpm for 5 minutes, centrifuging at 12000 rpm for 10 minutes, and washing with water 3 times to obtain the polystyrene microsphere;

[0072] 0.8 g of the polystyrene microspheres obtained in Preparation Example 2 were dispersed in 30 mL of 0.25% SDS solution for later use.

[0073] Preparation Example 3

[0074] A polystyrene microsphere, the preparation method of which comprises: dispersing 1 mL of 30 mg / mL potassium persulfate, 745 μL of styrene monomer and 55 μL of acrylic acid monomer in ultrapure water (50 mL of ultrapure water is placed in a 100 mL round-bottom flask, and vacuumed for 30 minutes with a water pump to remove oxygen in the water to obtain ultrapure water), stirring at a speed of 700 rpm and reacting at 70° C. for 6 hours, centrifuging at 4000 rpm for 5 minutes, centrifuging at 12000 rpm for 10 minutes, and washing with water 3 times to obtain the polystyrene microsphere;

[0075] 0.7 g of the polystyrene microspheres obtained in Preparation Example 3 were dispersed in 30 mL of 0.25% SDS solution for later use.

[0076] Example 1

[0077] A method for preparing rare earth complex fluorescent microspheres comprises the following steps:

[0078] (1) 3 mmol TTA, 5 mL 0.1 mmol / mL europium chloride hexahydrate, 5 mL 0.1 mmol / mL gadolinium chloride, 1 mmol Phen and 2 mmol TOPO were dissolved in 95% ethanol solution, 2 mL 2M NaOH solution was added, and the mixture was stirred and mixed in a 60°C water bath at a speed of 700 rpm, condensed and refluxed at 60°C for 1 h, filtered, washed with ultrapure water and ethanol for 3 times respectively, and vacuum dried for 7 h to obtain a rare earth complex;

[0079] (2) The rare earth complex obtained in step (1) was dissolved in 0.5 mL of dichloromethane, mixed with 5 mL of SDS solution of polystyrene microspheres (Preparation Example 1), ultrasonicated for 15 h, magnetically stirred for 15 h, introduced nitrogen, magnetically stirred at 50 ° C for 2 h to remove dichloromethane, centrifuged at 12000 rpm for 10 min, washed twice with ethanol, centrifuged at 4000 rpm for 5 min, and removed the supernatant to obtain the rare earth complex fluorescent microspheres.

[0080] Example 2

[0081] A method for preparing rare earth complex fluorescent microspheres comprises the following steps:

[0082] (1) 3 mmol TTA, 5 mL 0.1 mmol / mL europium chloride hexahydrate, 5 mL 0.1 mmol / mL gadolinium chloride, 1 mmol Phen and 2 mmol TOPO were dissolved in 95% ethanol solution, 2 mL 2M NaOH solution was added, and the mixture was stirred in a 50°C water bath at a speed of 700 rpm, condensed and refluxed at 50°C for 3 h, filtered, washed twice with ultrapure water and ethanol, and dried in vacuum for 7 h to obtain a rare earth complex;

[0083] (2) The rare earth complex obtained in step (1) was dissolved in 0.5 mL of dichloromethane and mixed with 5 mL of SDS solution of polystyrene microspheres (Preparation Example 2), ultrasonicated for 20 h, magnetically stirred for 12 h, nitrogen was introduced, magnetically stirred at 45 ° C for 2 h to remove dichloromethane, centrifuged at 12000 rpm for 10 min, washed twice with ethanol, centrifuged at 4000 rpm for 5 min, and the supernatant was removed to obtain the rare earth complex fluorescent microspheres.

[0084] Example 3

[0085] A method for preparing rare earth complex fluorescent microspheres comprises the following steps:

[0086] (1) 3 mmol TTA, 8 mL 0.1 mmol / mL europium chloride hexahydrate, 2 mL 0.1 mmol / mL gadolinium chloride, 1 mmol Phen and 2 mmol TOPO were dissolved in 95% ethanol solution, 2 mL 2M NaOH solution was added, and the mixture was stirred and mixed in a 65° C. water bath at a speed of (600-800) rpm, condensed and refluxed at 65° C. for 0.5 h, filtered, washed with ultrapure water and ethanol for 3 times respectively, and vacuum dried for 7 h to obtain a rare earth complex;

[0087] (2) The rare earth complex obtained in step (1) was dissolved in 0.5 mL of dichloromethane, mixed with 5 mL of SDS solution of polystyrene microspheres (Preparation Example 3), ultrasonicated for 10 h, magnetically stirred for 16 h, introduced nitrogen, magnetically stirred at 55 ° C for 2 h to remove dichloromethane, centrifuged at 12000 rpm for 10 min, washed with ethanol three times, centrifuged at 4000 rpm for 5 min, and removed the supernatant to obtain the rare earth complex fluorescent microspheres.

[0088] Example 4

[0089] A method for preparing rare earth complex fluorescent microspheres, which differs from Example 1 only in that gadolinium chloride is replaced by lutetium chloride in step (1), and other substances, parameters and steps are the same as those in Example 1.

[0090] Example 5

[0091] A method for preparing rare earth complex fluorescent microspheres, which differs from Example 1 only in that gadolinium chloride is replaced by neodymium chloride in step (1), and other substances, parameters and steps are the same as those in Example 1.

[0092] Comparative Example 1

[0093] A method for preparing rare earth complex fluorescent microspheres, which differs from Example 1 only in that gadolinium chloride is not added in step (1), and other substances, parameters and steps are the same as those in Example 1.

[0094] Application Example 1

[0095] A detection microsphere, the preparation method of which comprises the following steps:

[0096] (1) 0.1 g of the rare earth complex fluorescent microspheres obtained in Example 1 were dispersed in 10 mL of ultrapure water, 10 mL of 20 mg / mL polyacrylic acid (PAA) aqueous solution was added, ultrasonicated for 4 h, stirred at room temperature for 4 h, centrifuged and washed with water 3 times, and redispersed in 10 mL of ultrapure water to obtain PAA-modified rare earth complex fluorescent microspheres;

[0097] (2) sonicating and shaking the PAA-modified rare earth complex fluorescent microspheres obtained in step (1), taking 200 μL into a round-bottom centrifuge tube, centrifuging at 14,000 rpm and 4° C. for 15 min, and slowly aspirating the supernatant with a pipette to obtain a supernatant;

[0098] (3) adding 1 mL of primary wash buffer (50 mM MES, pH = 5-6) to the supernatant obtained in step (2), sonicating for 2 min to resuspend the precipitate, centrifuging at 14000 rpm and 4°C for 15 min, slowly aspirating the supernatant with a pipette, adding 1 mL of primary wash buffer, and sonicating for 2 min to obtain a mixed solution;

[0099] (4) Weigh 5 mg of EDC and add it to a round-bottom centrifuge tube. Add 50 μL of the initial wash buffer to dissolve it evenly. Take 50 μL and add it to the mixed solution obtained in step (3). Mix and react for 5 minutes. Ultrasonicate for 1 minute to obtain EDC activated microspheres.

[0100] (5) Weigh 15 mg of sulfo-NHS, add it to a round-bottom centrifuge tube, add 150 μL of initial wash buffer to dissolve and mix, pipette 150 μL and add it to the EDC activated microsphere solution obtained in step (4), mix and react for 15 minutes, and sonicate for 1 minute to obtain EDC / NHS activated microspheres;

[0101] (6) Add 1 mL of buffer (50 mM MES, pH = 5.0-6.0) to the EDC / NHS activated microspheres obtained in step (5), resuspend by sonication for 2 min, add 500 μg of antibody (c-reactive protein antibody), couple for 2 h, and sonicate for 2 min; add 1 mL of blocking buffer (50 mM Tris-HCl, 0.5% BSA and 0.1% ProClin), mix and react for 1 h, and sonicate for 2 min. Centrifuge at 14000 rpm and 4°C for 15 min, and slowly aspirate the supernatant with a pipette to obtain the supernatant;

[0102] (7) Add 1 mL of final wash buffer ((pH = 8.0) 1% NaCl, 0.5% BSA, 0.1% Tween 20, 0.1% ProClin in 50 mM Tris-HCl, pH = 8.0) to the supernatant obtained in step (6), sonicate for 2 min to resuspend the precipitate, centrifuge at 14000 rpm and 4°C for 15 min, slowly aspirate the supernatant using a pipette, and repeat the wash twice; add 200 μL of final wash buffer to the washed precipitate, resuspend the precipitate, and obtain the detection microspheres.

[0103] Application Example 2

[0104] A detection microsphere, which differs from Application Example 1 only in that procalcitonin antibody is used to replace the C-reactive protein antibody in step (6) of Application Example 1, and other substances, parameters and preparation methods are the same as those in Application Example 1.

[0105] Application Example 3

[0106] A detection microsphere, which differs from Application Example 1 only in that D-dimer antibody is used to replace the C-reactive protein antibody in step (6) of Application Example 1, and other substances, parameters and preparation methods are the same as those in Application Example 1.

[0107] Application Example 4

[0108] A detection microsphere, which differs from Application Example 1 only in that urine microalbumin antibody is used to replace the C-reactive protein antibody in step (6) of Application Example 1, and other substances, parameters and preparation methods are the same as those in Application Example 1.

[0109] Performance Test:

[0110] (1) Morphology observation: The rare earth complex fluorescent microspheres obtained in Examples 1 to 3 were observed using an Apreo S LoVac field emission scanning electron microscope. The scanning electron microscope image of the rare earth complex fluorescent microspheres obtained in Example 1 is shown in FIG. Figure 1 The scanning electron microscope image of the rare earth complex fluorescent microspheres obtained in Example 2 is shown as follows: Figure 2 The scanning electron microscope image of the rare earth complex fluorescent microspheres obtained in Example 3 is shown as follows: Figure 3 shown; from Figures 1 to 3 It can be seen that the particle sizes of the rare earth complex fluorescent microspheres prepared by the preparation methods provided in Examples 1 to 3 are very uniform; the average particle size of the rare earth complex fluorescent microspheres obtained in Example 1 is about 280nm; the average particle size of the rare earth complex fluorescent microspheres obtained in Example 2 is about 210m; the average particle size of the rare earth complex fluorescent microspheres obtained in Example 3 is about 170nm.

[0111] (2) The rare earth complex fluorescent microspheres obtained in Example 1 were tested using an FLS980 fluorescence spectrometer to obtain the excitation spectrum and emission spectrum of the rare earth complex fluorescent microspheres obtained in Example 1. Figure 4 As shown, 1 represents the laser spectrum and 2 represents the emission spectrum; Figure 4 It can be seen that the rare earth complex fluorescent microspheres obtained in Example 1 exhibit fluorescence emission at 615 nm under the excitation of a 365 nm light source.

[0112] (3) The rare earth complex fluorescent microspheres obtained in Example 1 were tested using an FLS980 fluorescence spectrometer to obtain a fluorescence attenuation curve of the rare earth complex fluorescent microspheres obtained in Example 1. Figure 5 shown; from Figure 5 It can be seen that the fluorescence lifetime of the rare earth complex fluorescent microspheres obtained in Example 1 is about 0.702 ms; the same method is used to detect the rare earth complex fluorescent microspheres obtained in Comparative Examples 2 to 5 and Comparative Example 1, and the test results are shown in Table 1.

[0113] (4) Fluorescence intensity: The rare earth complex fluorescent microspheres obtained in Examples 1 to 5 and Comparative Example 1 were tested using an FLS980 fluorescence spectrometer at the same Eu concentration (350 μg / mL). The test results are shown in Table 1.

[0114] Table 1

[0115] Fluorescence lifetime (ms) Fluorescence intensity value Example 1 0.702 <![CDATA[9.28×10 5 ]]> Example 2 0.681 <![CDATA[8.67×10 5 ]]> Example 3 0.656 <![CDATA[6.08×10 5 ]]> Example 4 0.677 <![CDATA[5.02×10 5 ]]> Example 5 0.634 <![CDATA[8.97×10 4 ]]> Comparative Example 1 0.587 <![CDATA[3.96×10 4 ]]>

[0116] It can be seen from the data in Table 1 that the rare earth complex fluorescent microspheres obtained by the preparation method provided by the present invention have a longer fluorescence lifetime and a higher fluorescence intensity.

[0117] Specifically, the fluorescence lifetime of the rare earth complex fluorescent microspheres obtained in Examples 1 to 5 is 0.634 to 0.702 ms, and the fluorescence intensity value is 5.02×10 5 ~9.28×10 5 However, the fluorescence lifetime and fluorescence intensity of the rare earth complex fluorescent microspheres obtained in Comparative Example 1 without doping with other lanthanide metals are shorter.

[0118] (5) Detection sensitivity: A portable rare earth fluorescence immunoassay analyzer (provided by Xiamen Aode Biotechnology Co., Ltd.) was used as the test instrument. The zero-value sample was used for repeated measurement 20 times. The mean M and standard deviation SD of the 20 results were calculated. The detection limit of the method was reported as the blank mean plus twice the standard deviation (M+2SD).

[0119] (6) Linear range: Eight concentrations of C-reactive protein between 0 and 200 mg / L, procalcitonin between 0 and 80 μg / L, D-dimer between 0 and 10 mg / L, and urine microalbumin between 0 and 300 mg / L were taken respectively. A portable rare earth fluorescence immunoassay analyzer (provided by Xiamen Aode Biotechnology Co., Ltd.) was used as the test instrument. Each concentration was measured three times. The average measured concentration was linearly analyzed with the theoretical concentration to obtain a linear equation for calculation.

[0120] The detection microspheres obtained in Examples 1 to 4 were tested according to test methods (5) and (6). The test results are shown in Table 2:

[0121] Table 2

[0122] Detection object Detection sensitivity Linear range Application Example 1 C-reactive protein 0.5mg / L 0.5~200mg / L Application Example 2 Procalcitonin 0.1 μg / L 0.1~80μg / L Application Example 3 D-Dimer 0.1mg / L 0.1~10mg / L Application Example 4 Urine microalbumin 2mg / L 2~300mg / L

[0123] According to the data in Table 2, it can be seen that the use of the rare earth complex fluorescent microspheres synthesized by the present invention as detection probes can achieve high sensitivity for various disease markers and can achieve detection in a wide linear range.

[0124] The applicant declares that the present invention illustrates a rare earth complex fluorescent microsphere and its preparation method and application through the above-mentioned embodiment, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing detection microspheres, characterized in that: The preparation method comprises: using EDC / NHS to perform surface activation on rare earth complex fluorescent microspheres and grafting antibodies to obtain the detection microspheres; The method for preparing rare earth complex fluorescent microspheres comprises the following steps: (1) mixing thenoyltrifluoroacetone, europium ions, other lanthanide metal ions, hydroxide, phenanthroline, tri-n-octylphosphine oxide and ethanol solution, and filtering to obtain a rare earth complex; (2) mixing the rare earth complex obtained in step (1), polystyrene microspheres and dichloromethane, ultrasonicating, introducing nitrogen, removing dichloromethane, and centrifuging to obtain the rare earth complex fluorescent microspheres; The molar ratio of the europium ions to the other lanthanide metal ions in step (1) is 1:1; The molar ratio of the europium ion to the thenoyltrifluoroacetone in step (1) is 1:(3-6); The molar ratio of the europium ion to the phenanthroline in step (1) is 1:(1-2); The molar ratio of the europium ion to tri-n-octylphosphine oxide in step (1) is 1:(2-4).

2. The preparation method according to claim 1, characterized in that: The other lanthanide metal ions in step (1) include Gd 3+ 、Lu 3+ , Y 3+ or Nd 3+ Any one or a combination of at least two of the following.

3. The preparation method according to claim 1, characterized in that: The hydroxide in step (1) includes sodium hydroxide and / or potassium hydroxide.

4. The preparation method according to claim 1, characterized in that: The mixing temperature in step (1) is 50-65°C.

5. The preparation method according to claim 1, characterized in that: The mixing in step (1) is carried out in a water bath.

6. The preparation method according to claim 1, characterized in that: The mixing in step (1) is carried out under stirring conditions.

7. The preparation method according to claim 6, characterized in that: The mixing in step (1) is carried out under stirring conditions at a rotation speed of 600 to 800 rpm.

8. The preparation method according to claim 1, characterized in that: The step (1) also includes a condensation reflux step before the suction filtration.

9. The preparation method according to claim 8, characterized in that: The condensation reflux temperature is 50-65°C.

10. The preparation method according to claim 8, characterized in that: The condensation reflux time is 0.5 to 3 hours.

11. The preparation method according to claim 1, characterized in that: The method further comprises washing and vacuum drying steps after the suction filtration in step (1).

12. The preparation method according to claim 11, characterized in that: The washing includes washing with ultrapure water and washing with ethanol.

13. The preparation method according to claim 12, characterized in that: The number of ultrapure water washing and ethanol washing is independently 2 to 3 times.

14. The preparation method according to claim 1, characterized in that: The polystyrene microspheres in step (2) are prepared by the following method, which comprises: dispersing an initiator, a styrene monomer and an acrylic acid monomer in ultrapure water, reacting, and centrifuging to obtain the polystyrene microspheres.

15. The preparation method according to claim 14, characterized in that: The initiator includes a persulfate.

16. The preparation method according to claim 15, characterized in that: The persulfate includes potassium persulfate or ammonium persulfate.

17. The preparation method according to claim 14, characterized in that: The reaction temperature is 60-70°C.

18. The preparation method according to claim 14, characterized in that: The reaction is carried out under stirring conditions.

19. The preparation method according to claim 18, characterized in that: The reaction is carried out under stirring conditions at a rotation speed of 600 to 800 rpm.

20. The preparation method according to claim 14, characterized in that: The reaction time is 6 to 10 hours.

21. The preparation method according to claim 14, characterized in that: The centrifugation step also includes a washing step.

22. The preparation method according to claim 1, characterized in that: The mass ratio of the rare earth complex and the polystyrene microspheres in step (2) is (1-3):

1.

23. The preparation method according to claim 1, characterized in that: The ultrasonic treatment time in step (2) is 10 to 20 hours.

24. The preparation method according to claim 1, characterized in that: The step (2) further comprises a magnetic stirring step after the ultrasound.

25. The preparation method according to claim 24, characterized in that: The magnetic stirring time is 12 to 16 hours.

26. The preparation method according to claim 1, characterized in that: The removal of dichloromethane in step (2) is accomplished by magnetic stirring at 45-55°C.

27. The preparation method according to claim 1, characterized in that: Step (2) also includes a washing step after the centrifugation.

28. The preparation method according to claim 27, characterized in that: The washing includes washing with ultrapure water and washing with ethanol.

29. The preparation method according to claim 28, characterized in that: The number of ultrapure water washing and ethanol washing is independently 2 to 3 times.

30. The preparation method according to claim 1, characterized in that: The preparation method comprises the following steps: (1) dissolving europium ions, other lanthanide metal ions, phenanthroline, thenoyl trifluoroacetone and tri-n-octylphosphine oxide in an ethanol solution, adding hydroxide, stirring and mixing in a water bath at 50-65° C. and a rotation speed of 600-800 rpm, condensing and refluxing at 50-65° C. for 0.5-3 h, filtering, washing with ultrapure water and ethanol for 2-3 times respectively, and vacuum drying to obtain a rare earth complex; (2) The rare earth complex obtained in step (1) is dissolved in dichloromethane, mixed with polystyrene microspheres, ultrasonicated for 10 to 20 hours, magnetically stirred for 12 to 16 hours, introduced with nitrogen, removed the dichloromethane under magnetic stirring at 45 to 55° C., centrifuged, and washed to obtain the rare earth complex fluorescent microspheres.

31. The preparation method according to claim 1, characterized in that: The particle size of the rare earth complex fluorescent microsphere is 170-280nm.

32. A detection microsphere, characterized in that: The detection microspheres are prepared by the preparation method according to any one of claims 1 to 31.

Images