Fluorescent nanoparticles, protein complexes and immunodetection test strips

Abstract

The application discloses fluorescent nanoparticles, protein complexes and immunological detection test strips. In a first aspect, the application provides fluorescent nanoparticles, which comprise AIE ligands and coordinated metal ions. The fluorescent nanoparticles according to the embodiments of the application have at least the following beneficial effects: for the fluorescent nanoparticles formed by the AIE ligands and the coordinated metal ions, when labeling proteins, the proteins can be directly adsorbed to the fluorescent nanoparticles under the action of electrostatic interaction and van der Waals force to be combined to the surface of the fluorescent nanoparticles, thus avoiding the cumbersome labeling steps in the prior art, effectively improving the detection sensitivity, and having good stability and not being easy to be inactivated.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to fluorescent nanoparticles, protein complexes, and immunoassay test strips. Background Technology

[0002] Most existing fluorescent immunoassay test strips are based on quantum dots, fluorescent dye-labeled PS microspheres, micelles, or SiO2 spheres. The key challenge lies in the complex overall preparation process, particularly the cumbersome, inefficient, and costly antibody labeling steps, which limits further improvements in sensitivity. They also suffer from poor stability (e.g., quantum dots are easily inactivated, fluorescent dyes are easily bleached, and microspheres and micelles are prone to aggregation). Antibody labeling often requires pre-modification with carboxyl or amino groups for subsequent covalent conjugation of antibody proteins. The technical route typically involves coating labeled particles with carboxyl or amino-functionalized polymers, followed by covalent conjugation of antibody proteins via an amide reaction. This process is complex and cumbersome, and the low or excessively high antibody-protein conjugation efficiency can lead to inactivation. Summary of the Invention

[0003] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a fluorescent nanoparticle, which can be used to obtain a protein complex that is simple to process, does not easily become inactive after coupling, protein labeling, and an immunoassay strip constructed using this protein complex.

[0004] A first aspect of this application provides a fluorescent nanoparticle comprising an AIE ligand and a coordinating metal ion.

[0005] The fluorescent nanoparticles according to the embodiments of this application have at least the following beneficial effects:

[0006] For fluorescent nanoparticles formed by AIE ligands and coordinating metal ions, when labeling proteins, the proteins can be directly adsorbed onto the surface of the fluorescent nanoparticles under the action of electrostatic interaction and van der Waals forces, thus avoiding the cumbersome labeling steps in the prior art and effectively improving the detection sensitivity.

[0007] In this context, AIE ligands refer to molecules possessing aggregation-induced emission (AIE) properties. Possible emission mechanisms include restricted intramolecular motion (RIM), intramolecular rotation (RIR), and vibrational restriction (RIV). AIE molecules can currently be broadly categorized into compounds containing only carbon and hydrogen atoms, substances containing heteroatoms, and metal complexes. In the embodiments of this application, AIE molecules, acting as ligands, chelate with coordinating metal ions to form fluorescent nanoparticles, which then serve as indicators for labeling proteins adsorbed onto them.

[0008] In some embodiments of this application, the AIE ligand has carboxyl, pyridyl, and imidazole groups, and the AIE ligand forms fluorescent nanoparticles with the coordinated metal ions through these groups.

[0009] In some embodiments of this application, the AIE ligand is at least one of the molecules having the following structures:

[0010]

[0011] Wherein, X has at least one of C = C and CN;

[0012] Y can be either N or P;

[0013] R1 to R4 are each independently selected from any one of hydrogen atom, carboxyl group, 4-carboxyphenyl, pyridyl group, and imidazole group, and at least two of them are not hydrogen atoms;

[0014] R5 to R7 are each independently selected from carboxyl, 4-carboxyphenyl, pyridyl, and imidazolyl.

[0015] In some embodiments of this application, R1 to R4 are each independently selected from any one of hydrogen atom, carboxyl group, 4-carboxyphenyl, 4-pyridyl group, and imidazole group, and at least two of them are not hydrogen atoms; R5 to R7 are each independently selected from any one of carboxyl group, 4-carboxyphenyl, 4-pyridyl group, and imidazole group.

[0016] In some embodiments of this application, R1 to R4 are each independently selected from carboxyl, 4-carboxyphenyl, 4-pyridyl, and imidazolyl.

[0017] In some embodiments of this application, the AIE ligand is any one of the following molecules having the following structure:

[0018]

[0019] In some embodiments of this application, the AIE ligand is any one of the following molecules having the following structure:

[0020]

[0021] Among them, R1 to R4 are each independently selected from any one of hydrogen atom, carboxyl group, 4-carboxyphenyl, pyridyl group, and imidazole group, and at least two of them are not hydrogen atoms;

[0022] R5 to R7 are each independently selected from carboxyl, 4-carboxyphenyl, pyridyl, and imidazolyl.

[0023] In some embodiments of this application, R1 to R4 are simultaneously selected from carboxyl, 4-carboxyphenyl, pyridyl, and imidazolyl groups;

[0024] R5 to R7 are selected from any one of carboxyl, 4-carboxyphenyl, pyridyl, and imidazolyl.

[0025] In some embodiments of this application, the AIE ligand is selected from at least one of the following:

[0026]

[0027]

[0028] The fluorescence wavelength can be changed by adjusting the structure of AIE. Taking the compound in formula (I) above as an example, the fluorescent nanoparticles obtained by this AIE ligand have blue fluorescence, while the fluorescent nanoparticles obtained by the compound in formula (II) formed by introducing additional phenyl groups into R1 to R4 have yellow fluorescence.

[0029] In some embodiments of this application, the metal ions are ions of second subgroup elements and other metal elements with stable d orbitals.

[0030] In some embodiments of this application, the metal ion is selected from at least one of zinc ion, zirconium ion, hafnium ion, gallium ion, indium ion, tin ion and bismuth ion.

[0031] In some embodiments of this application, the particle size of the fluorescent nanoparticles is 50–1000 nm. Preferably, the particle size of the fluorescent nanoparticles is 50–500 nm, 50–200 nm, or 150 nm. By adjusting the particle size of the fluorescent nanoparticles, the amount of protein adsorbed on the fluorescent nanoparticles is increased, thereby further improving the detection sensitivity.

[0032] A second aspect of this application provides a protein complex comprising the aforementioned fluorescent nanoparticles and a protein adsorbed on the fluorescent nanoparticles.

[0033] When using the aforementioned fluorescent nanoparticles to label proteins, the labeling can be directly adsorbed onto the surface of the fluorescent nanoparticles under the influence of electrostatic interactions and van der Waals forces, thus avoiding the cumbersome labeling steps in existing technologies and effectively improving detection sensitivity.

[0034] On the other hand, the protein complexes obtained in this way can be stored at room temperature for more than a year without significant changes in fluorescence intensity, demonstrating good stability and resistance to inactivation.

[0035] A third aspect of this application provides a protein labeling method, comprising the following steps: mixing fluorescent nanoparticles with a protein to be labeled and incubating for 1 to 5 hours to obtain a protein complex; the fluorescent nanoparticles include an AIE ligand and coordinating metal ions.

[0036] This labeling method involves directly incubating fluorescent nanoparticles with the protein to be labeled, thereby adsorbing the protein onto the fluorescent nanoparticles through electrostatic interactions and van der Waals forces. The entire process is simple and highly efficient.

[0037] In some embodiments of this application, after the fluorescent nanoparticles and the protein to be labeled are mixed and incubated, a blocking solution is added and incubation continues. After centrifugation to remove the supernatant, the protein complex is obtained. The incubation time after adding the blocking solution can be selected from 0.1 to 2 hours, preferably 1 hour.

[0038] In some embodiments of this application, the fluorescent nanoparticles are prepared as follows: AIE ligands and metal ions are mixed and reacted at 70–120°C to obtain fluorescent nanoparticles.

[0039] In some embodiments of this application, the mass ratio of AIE ligand to metal ion is (1-10):1.

[0040] In some embodiments of this application, the raw material for the metal ions is a metal salt, which further participates in the reaction in solution form. The metal salts include, but are not limited to, halogen salts, carbonates, nitrates, acetates, and sulfates.

[0041] A fourth aspect of this application provides an immunoassay test strip comprising a primary antibody comprising the aforementioned protein complex, the primary antibody being used to specifically bind to an analyte.

[0042] In some embodiments of this application, a secondary antibody and an anti-antibody are also included, wherein the secondary antibody is used to specifically bind to the analyte, and the anti-antibody is used to specifically bind to the primary antibody. It is understood that the binding sites of the secondary antibody and the primary antibody are different in order to form the secondary antibody-analyte-primary antibody complex.

[0043] In some embodiments of this application, the immunoassay test strip defines a sample area, a binding area, a detection area, and an adsorption area that are sequentially connected. The sample area is used to add the analyte, the binding area is coated with a primary antibody, the detection area contains a detection line (T line) and a control line (C line), a secondary antibody is coated on the detection line, and an anti-antibody is coated on the control line.

[0044] In some embodiments of this application, the immunoassay test strip includes a base plate, on which a sample pad, a labeling pad, a chromatography membrane, and an absorbent pad are connected in sequence.

[0045] In some embodiments of this application, the substrate is a polymer film, such as a polyvinyl chloride membrane. The sample pad is glass fiber treated with a processing solution, which may be a phosphate buffer solution containing Triton X-100 and a preservative. The labeling pad is glass fiber. The chromatography membrane is a nitrocellulose membrane (NC membrane) with a pore size of 3–15 μm, preferably an NC membrane with a pore size of 8 μm. The absorbent pad is filter paper.

[0046] In some embodiments of this application, the size of the immunoassay test strip is (40-100) × (1-10) mm.

[0047] In some embodiments of this application, the chromatography membrane is marked with detection lines and control lines at intervals of 5 to 10 mm.

[0048] In some embodiments of this application, the method of using the immunoassay test strip is as follows: the sample to be tested is applied to the sample area of ​​the immunoassay test strip, the fluorescence signal of the immunoassay test strip is determined, and the content of the analyte in the sample is determined based on the fluorescence signal.

[0049] In some embodiments of this application, the method of using the immunoassay test strip is as follows: the sample to be tested is applied to the sample area of ​​the immunoassay test strip, then working buffer is applied to the sample area, chromatography is performed, the fluorescence signal of the immunoassay test strip is determined after chromatography, and the content of the analyte in the sample is determined based on the fluorescence signal.

[0050] In some embodiments of this application, the method for determining the content of the analyte in the sample based on the fluorescence signal is as follows: determine the fluorescence signals of the detection line and the control line, and determine the content of the analyte in the sample based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the control line.

[0051] A fifth aspect of this application provides a kit comprising the aforementioned protein complex.

[0052] A sixth aspect of this application provides a detection method for non-diagnostic purposes, which uses the aforementioned protein complex, or the aforementioned immunoassay strip, or the aforementioned kit for detection.

[0053] This application also includes the use of the aforementioned protein complex, or the aforementioned immunoassay strip, or the aforementioned kit in the process of performing detection for non-diagnostic purposes.

[0054] This application simplifies the overall preparation process by designing and synthesizing fluorescent nanoparticles with high fluorescence quantum yield and strong protein adsorption. During labeling, no complex additional functionalization modifications are required between the protein and the fluorescent nanoparticles; after uniform mixing, direct incubation on a shaker allows the protein to be adsorbed onto the surface of the fluorescent nanoparticles via electrostatic and van der Waals forces. This method increases the amount of protein adsorbed on the fluorescent nanoparticles, resulting in a significant improvement in the final detection sensitivity. The detection limit is one to two orders of magnitude lower than that of conventional fluorescent test strips.

[0055] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0056] Figure 1 These are electron microscope images of the fluorescent nanoparticles prepared in Example 1 of this application at different scales. The scale bars from left to right are 1 μm, 200 nm and 200 nm, respectively. Detailed Implementation

[0057] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.

[0058] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0059] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0060] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0061] Example 1

[0062] This embodiment provides a fluorescent nanoparticle, and the preparation method of the fluorescent nanoparticle is as follows:

[0063] A ligand and zirconium chloride were mixed at a mass ratio of 1:1, reacted in a water bath at 80°C for 5 hours, centrifuged, washed twice with anhydrous ethanol, and resuspended in deionized water to obtain a dispersion of fluorescent nanoparticles.

[0064] Among them, the AIE ligand selected by formula (Ⅰ) is 4,4',4”,4”'-(ethylene-1,1,2,2-tetramethyl)tetrabenzoic acid, with the following structural formula:

[0065]

[0066] These fluorescent nanoparticles exhibit strong blue fluorescence (470 nm) under ultraviolet light irradiation. (Reference) Figure 1 The image shows an electron microscope image of the fluorescent nanoparticles prepared by this method. The particle size ranges from 50 nm to 1 μm. These fluorescent nanoparticles can be directly incubated with proteins to complete labeling, avoiding the cumbersome labeling steps in existing technologies.

[0067] Example 2

[0068] This embodiment provides an immunoassay test strip, specifically an alpha-fetoprotein (AFP) test strip. The raw materials used in its preparation, by volume, are shown in Table 1 below.

[0069] Table 1. Raw materials for preparing immunoassay test strips

[0070]

[0071]

[0072] The fluorescent nanoparticles were those prepared in Example 1. The blocking and resuspension solutions were 0.02M phosphate buffer (PBS, pH 7.0) containing 1 w / v% BSA (bovine serum albumin), and the working buffer was 0.02M phosphate buffer containing 0.1 w / v% Triton X-100.

[0073] The specific preparation process of this immunoassay test strip is as follows:

[0074] (1) The primary antibody protein AFP-Ab1 was directly mixed with fluorescent nanoparticles and incubated on a shaker for 3 hours. Then, blocking solution was added and incubated for another 1 hour. After centrifugation at 8000 rpm for 10 minutes, the supernatant was removed and the mixture was resuspended in resuspending solution to obtain a primary antibody solution of primary antibody protein AFP-Ab1 adsorbed on fluorescent nanoparticles. Thus, the primary antibody protein was labeled and a solution of the protein complex corresponding to the primary antibody protein was obtained.

[0075] (2) Spray the primary antibody solution onto the labeling pad and dry it for later use; take the secondary antibody protein Anti-AFP-Ab1 and anti-antibody AFP-Ab2 and run them on the chromatography membrane to form T lines and C lines, respectively, and dry them for later use; after the sample pad is pretreated with 0.02M PBS solution (2w / v% Triton X-100, 0.03wt% NaN3, pH=7.0), it is assembled with the labeling pad, chromatography membrane, absorbent pad and polyvinyl chloride base plate to form an immunoassay chromatography plate, and then cut into 60×3mm immunoassay test strips and placed in a dry and sealed aluminum foil bag for later use.

[0076] The method for using this immunoassay test strip is as follows:

[0077] (1) Plotting the standard curve:

[0078] Alpha-fetoprotein (AFP) standard solutions of 0 pg / mL, 0.2 pg / mL, 0.5 pg / mL, 1 pg / mL, 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 500 pg / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL were added to the sample pad, and the corresponding working buffer was added to the sample pad. The chromatography was observed under UV light until the C-line / T-line stabilized. The fluorescence signals of the T-line and C-line were collected by the fluorescence analyzer, and the ratio of the T-line to C-line fluorescence signals was obtained. Based on the relationship between the fluorescence signals and concentration values ​​of different standards, a curve fitting was performed to obtain the standard curve of AFP.

[0079] (2) Sample testing

[0080] Take the sample to be tested and obtain the ratio of the corresponding T-line / C-line fluorescence signal according to the method in step (1). The concentration of alpha-fetoprotein in the sample to be tested is obtained according to its standard curve.

[0081] In this embodiment, in all three replicates, a positive result with obvious blue fluorescence of the T / C lines was observed after adding the standard at a concentration as low as 0.5 pg / mL. Therefore, the detection limit of this immunoassay test strip for alpha-fetoprotein is <1 pg / mL.

[0082] Comparative Example 1

[0083] This comparative example provides an alpha-fetoprotein (AFP) test strip, which differs from Example 2 in that the labeled pad is coated with a protein complex formed by the primary antibody protein AFP-Ab1, multi-walled carbon nanotubes, and water-soluble quantum dots CdTe. The specific preparation method of this protein complex is as follows:

[0084] 0.02 g of aminated carbon nanotubes were dispersed in PBS (pH = 7.4), activated with 0.1 mL EDC and 0.15 mL NHS for 10 min, then 100 mg of primary antibody protein AFP-Ab1 was added, and the mixture was incubated at 37 °C for 1 h. After centrifugation to remove the supernatant, the mixture was reconstituted in PBS, activated again with 0.1 mL LEDC and 0.15 mL NHS for 10 min, then 5 mL of carboxyl-coated CdTe quantum dots was added, and the mixture was incubated at 37 °C for 1 h. After centrifugation to remove the supernatant, the mixture was reconstituted in PBS to obtain a dispersion of the protein complex.

[0085] The standard samples of different concentrations were tested according to the method in Example 2, and the test was repeated 3 times. The results showed that the limit of detection of the alpha-fetoprotein immunoassay test strip was 20 pg / mL.

[0086] Compared with Example 2, it can be seen that the labeling method of the primary antibody provided in this application is simpler. It can be directly incubated without additional functionalization modification, which greatly saves manpower and material costs. It does not use highly toxic Cd cadmium ions, and the sensitivity of the immunoassay test strip is greatly improved.

[0087] Example 3

[0088] This embodiment provides an immunoassay test strip, which differs from Embodiment 2 in that the fluorescent nanoparticles labeled with the primary antibody are obtained by reacting the AIE ligand shown in Formula (II) with zinc sulfate. During detection, the fluorescence signal in the yellow light band is analyzed, and it has a similar detection limit as Embodiment 2.

[0089] Example 4

[0090] This embodiment provides an immunoassay test strip, which differs from Example 2 in that the fluorescent nanoparticles labeled with the primary antibody are obtained by reacting the AIE ligand shown in Formula (III) with gallium nitrate, and has a similar detection limit as Example 2.

[0091] Example 5

[0092] This embodiment provides an immunoassay test strip, which differs from Example 2 in that the fluorescent nanoparticles labeled with the primary antibody are obtained by reacting the AIE ligand shown in Formula (Ⅳ) with bismuth sulfate, and has a similar detection limit as Example 2.

[0093] Example 6

[0094] This embodiment provides an immunoassay test strip, which differs from Example 2 in that the fluorescent nanoparticles labeled with the primary antibody are obtained by reacting the AIE ligand shown in Formula (V) with tin acetate (IV), and has a similar detection limit as Example 2.

[0095] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. Furthermore, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

Claims

1. A protein labeling method, characterized in that, Includes the following steps: Fluorescent nanoparticles are directly mixed with the protein to be labeled and incubated for 1-5 hours to obtain a protein complex; the fluorescent nanoparticles include AIE ligands and coordinating metal ions; The AIE ligand is R1 to R4 are carboxyl groups, respectively; The metal ion is a zirconium ion.

2. The protein labeling method according to claim 1, characterized in that, The fluorescent nanoparticles are prepared as follows: the AIE ligand and the coordinating metal ions are mixed and reacted at 70~120℃ to obtain the fluorescent nanoparticles.

3. The protein labeling method according to claim 1, characterized in that, The fluorescent nanoparticles have a particle size of 50~1000nm.

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