CN114660029A - Colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere and application thereof - Google Patents
Colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere and application thereof Download PDFInfo
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Abstract
The invention discloses a colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere and application thereof, and the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere mainly comprises a polystyrene microsphere and a colorimetric-fluorescent double-signal dye; specifically, a polystyrene microsphere is taken as a main body, and a colorimetric-fluorescent dual-signal dye with strong absorption and high luminescence performance is embedded in the polystyrene microsphere through a swelling method to prepare the bifunctional nano microsphere with colorimetric and fluorescent signals; the excitation of the colorimetric-fluorescent dual-signal dye is 365nm, the emission is 625nm, and the strong absorption is at the position with strong absorption at 530 nm. The colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere is used as a beacon probe, is applied to an immunochromatography platform, and simultaneously realizes naked eye qualitative detection and fluorescent quantitative detection of a target analyte. The invention solves the defects of the existing single signal probe and provides the colorimetric-fluorescent double-signal nano microsphere with strong absorption performance, high fluorescence intensity and low biotoxicity.
Description
Technical Field
The invention belongs to the technical field of biological analysis and rapid detection, and particularly relates to a colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere and application thereof.
Background
The immunochromatography technology is widely applied to rapid detection in the fields of clinical diagnosis, food safety, environmental monitoring and the like due to the obvious advantages of rapid reaction, simple operation, economy, low price, visual detection result and the like. The traditional colloidal gold immunochromatographic test strip takes 30-40nm colloidal gold as a color signal probe, and achieves the purpose of naked eye detection by relying on probe enrichment and color development on a detection line. However, the colloidal gold with the particle size has small molar extinction coefficient and weak color generation effect, so that the traditional colloidal gold immunochromatography test strip has low detection sensitivity and poor result accuracy, and can only achieve qualitative coarse screening and hardly meet the purpose of quantitative detection. In order to improve the detection sensitivity, novel signal probes with higher signal intensity are continuously researched and developed, for example, emission spectrum type probes include traditional organic fluorescent microspheres, quantum dot fluorescent microspheres, time-resolved microspheres, up-conversion luminescent microspheres and the like. In addition, within the allowable range of the instrument, the fluorescence intensity and the concentration of the fluorescent material are in a linear relationship, so that the fluorescent signal probe successfully solves the defect that the colloidal gold immunochromatographic test strip cannot quantify. However, the fluorescent probe requires an additional uv excitation light source, which is very inconvenient for poor medical conditions or for home users. And the common fluorescent probe only has single signal output capability and cannot have the multi-functional detection performance of naked eye qualitative detection and fluorescence quantitative detection.
Therefore, in recent years, many researchers develop the colorimetric-fluorescent bifunctional microspheres and apply the colorimetric-fluorescent bifunctional microspheres to an immunochromatography platform to achieve the purposes of naked eye qualitative determination and fluorescent quantitative determination. For example, in patent CN106680496A, the inventor swells quantum dots with fluorescence signals and colloidal gold with absorption signals into polystyrene microspheres by a swelling method to prepare a colorimetric-fluorescent dual-signal probe. However, due to the internal filtering effect, the colloidal gold absorbs the fluorescence of the quantum dots, so that the increase of the absorption signal is accompanied by the loss of the fluorescence signal, and the perfect combination of the two signals is difficult to achieve. Therefore, the research proposes that a bifunctional aggregation-induced emission dye with strong absorption at 530nm, strong emission at 625nm, high molar extinction coefficient and high quantum efficiency is used as a chromogen and a fluorophore, the dye is swelled into a carboxylated polystyrene microsphere through a swelling method to prepare the colorimetric-fluorescent dual-signal nanosphere, and the colorimetric-fluorescent dual-signal nanosphere is applied to an immunochromatography platform to prepare the NT-BNP-pro immunochromatography test strip rapid detection kit.
Disclosure of Invention
Aiming at the defects and problems in the prior art, the invention aims to provide a colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere and application thereof.
The invention is realized by the following technical scheme:
the invention provides a colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere which mainly comprises two parts, namely a polystyrene microsphere and a colorimetric-fluorescent double-signal dye; specifically, a polystyrene microsphere is taken as a main body, and a colorimetric-fluorescent dual-signal dye with strong absorption and high luminescence performance is embedded in the polystyrene microsphere through a swelling method to prepare the bifunctional nano microsphere with colorimetric and fluorescent signals; the excitation of the colorimetric-fluorescent dual-signal dye is 365nm, the emission is 625nm, and the strong absorption is realized at 530 nm.
The specific preparation method of the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere comprises the following steps:
s1, dissolving the carboxyl polystyrene microspheres in an aqueous solution containing a surfactant, performing ultrasonic dispersion, adding an organic solvent to swell for 10-30min, and swelling at room temperature.
S2, dissolving the colorimetric-fluorescent double-signal dye into a good solvent, performing ultrasonic dispersion, and preparing a monodisperse room-temperature double-dye solution, wherein the molecular formula of the colorimetric-fluorescent double-signal dye has typical aggregation-induced emission performance, and the specific structural formula is as follows:
the fluorescent dye is an organic molecule, has a tetraphenylethylene unit, is a typical aggregation-induced emission molecule, and has the advantages of large molar extinction coefficient, high quantum efficiency, no toxicity and no harm. The fluorescent dye has high molecular luminous efficiency, large Stokes displacement, good light stability, photobleaching resistance, strong absorption at 530nm, and colorimetric and fluorescent dual-signal output capability;
and S3, adding the mixed solution of S1 into the dye solution in S2, ultrasonically stirring for 20-40min, carrying out reduced pressure rotary steaming for 3-5 hours, centrifugally washing the reaction mixed solution until the supernatant is free from fluorescence, and carrying out vacuum drying to obtain the colorimetric-fluorescent double-signal aggregation-induced fluorescent microspheres.
Preferably, the surfactant in step S1 may be polyoxyethylene stearyl alcohol, polyoxyethylene cetyl alcohol, sodium lauryl sulfate, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium N-hexadecyl-N-ethyl sulfate, etc., preferably sodium dodecyl sulfate; in step S1, the polystyrene microsphere has a particle size of 350 nm.
The good solvent in S1 and S2 is preferably methanol, ethanol, acetonitrile, acetone, toluene, n-hexane, etc., preferably acetone.
Preferably, in the step S2, the ultrasonic power is 50-100w, and the ultrasonic time is 30S; in step S2, the concentration of aggregation-induced emission dye is 1-10 mg/mL.
Preferably, the volume ratio of the aggregation-inducing luminescent dye solution to the polystyrene microsphere solution in step S3 is (2-5): (4-15); the reaction temperature is 30-37 ℃, and the vacuum degree is 0.05-0.09 mpa; the system of the residual organic solvent in the step S3 is 5-8%; the centrifugation speed was 10000rpm, 4 ℃ and 15 min.
The invention also provides the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere as a beacon probe, which is applied to an immunochromatography platform and can realize naked eye qualitative detection and fluorescent quantitative detection of a target analyte. Preferably, the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere is used for preparing a colorimetric and fluorescent microsphere coupled antibody immunity probe, and the preparation method comprises the following steps:
(a) dissolving the prepared carboxylated colorimetric-fluorescent bifunctional microspheres in 1mL of phosphate buffer solution with the pH value of 6.5 to obtain a final concentration of 1 mg/mL;
(b) adding 0.1mgNT-BNP-pro monoclonal antibody into the microsphere solution, and carrying out electrostatic adsorption for 30min under stirring;
(c) adding 50 μ g of 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (EDC) into the system, stirring for reaction for 30min, repeating the reaction for three times, and adding 10mg of Bovine Serum Albumin (BSA) for blocking for 1 h;
(d)10000rpm, 4 ℃ for 15min, and the pellet was resuspended in probe complex solution (25% sucrose, 1% BSA, 0.1% sodium azide) to a final concentration of 0.2 mg/mL.
Preferably, the colorimetric-fluorescent dual-signal aggregation-induced fluorescent microsphere is used for preparing a colorimetric-fluorescent dual-functional microsphere NT-BNP-pro fluorescent test strip kit, and the preparation method comprises the following steps:
(a) spraying the NT-BNP-pro monoclonal antibody and the IgY antibody marked by the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere on a glass fiber membrane to prepare a binding pad;
(b) the preparation of the nitrocellulose membrane with detection line and quality control line is as follows: respectively spraying an NT-BNP-pro capture antibody with the concentration of 1-2 mg/mL and an anti-IgY secondary antibody solution with the concentration of 1-2 mg/mL on a nitrocellulose membrane to draw two parallel lines, wherein the drawn line of the NT-BNP-pro capture antibody is used as a detection line (T line), and the drawn line of the anti-chicken IgY secondary antibody solution is used as a quality control line (C line);
(c) assembling the colorimetric-fluorescent bifunctional microsphere immunochromatographic test strip: and (3) respectively and sequentially sticking a sample pad, a bonding pad sprayed with the probe, a nitrocellulose membrane with T and C lines and absorbent paper to the plastic lining plate, wherein the length of the adjacent overlapped parts is 2mm, cutting the test paper into test paper strips with the width of 3.9mm by using a test paper strip cutting instrument, sealing and drying, and storing at the temperature.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention takes carboxyl polystyrene microspheres as template spheres, prepares the colorimetric-fluorescent double-signal microspheres by a swelling method, synthesizes the microspheres with colorimetric signals and fluorescent signals, has high molar extinction coefficient and high quantum efficiency, has the surface with the advantages of functional groups which can be directly used for protein coupling, is applied to an immunochromatography platform, and can simultaneously realize the purposes of naked eye qualitative determination and fluorescent quantitative determination. In addition, the microsphere synthesized by the method has the advantages of uniform particle size, high reproducibility, good stability, large-scale synthesis potential and capability of meeting the industrial batch production requirement.
2. The invention uses the red colorimetric-fluorescent double-signal dye as a signal source substance, successfully prepares the colorimetric-fluorescent microsphere, has strong absorption at 530nm, excitation at 365nm and emission at 625nm, has the advantages of large Stokes shift, high molar extinction coefficient, high luminous efficiency and the like, has good fluorescence characteristic of aggregation-induced luminescence, avoids the problem that the common fluorescent microsphere fluorescent dye can generate a fluorescence quenching phenomenon when loaded to a certain concentration, and obviously improves the fluorescence intensity while having strong absorption performance.
Drawings
FIG. 1 shows a schematic diagram of the synthesis of AIE fluorescent microspheres of the present application;
FIG. 2 shows an electron micrograph of the microspheres of example 1;
FIG. 3 shows the UV absorption spectrum and fluorescence emission spectrum of the colorimetric-fluorescent bifunctional microsphere in example 2;
FIG. 4 is a graph showing the comparison of the fluorescence intensity ratio of the control line of the detection line with the actual concentration of NT-BNP-pro when the test strip of the colorimetric-fluorescent dual signal test of example 3 is applied to the detection of NT-BNP-pro;
FIG. 5 shows the application of the aggregation-induced emission test strip of example 3 to the detection of NT-BNP-pro, showing the colorimetric and fluorescent changes with concentration for the T-line (down-going) and C-line (up-going), respectively.
Detailed Description
The invention will be further described with reference to the accompanying drawings. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It should be noted that, in the present application, features in the embodiments may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example 1
The synthesis of the colorimetric-fluorescent double-signal aggregation-induced fluorescent microspheres is shown in the figure 1.
Preparing a dye prefabricated liquid: the colorimetric-fluorescent dual-signal AIE fluorescent dye was weighed and dissolved in tetrahydrofuran to prepare a standard dye stock solution with a final concentration of 200 mg/mL.
Preparing 350nm colorimetric-fluorescent bifunctional microspheres: dissolving 100mg 350nm carboxyl polystyrene microspheres into 10mL aqueous solution containing 1mg/mL surfactant, performing ultrasonic dispersion, and adding 20mL acetone solution for swelling for 10-30 min. Dissolving 0.25mL of standard dye stock in 10mL of acetone solution, and performing ultrasonic dispersion; and (3) dropwise adding the pre-swollen polystyrene microsphere solution into the dye solution, carrying out reduced pressure rotary evaporation at 37 ℃, centrifuging after 3h, washing with ethanol for three times, removing redundant dye, and preparing the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere.
The colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere mainly comprises a polystyrene microsphere and colored and fluorescent double-signal dyes; specifically, the polystyrene microsphere is taken as a main body, and the dye with strong absorption and high luminescence performance is embedded in the polystyrene microsphere through a swelling method. An electron microscope image of the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere is shown in FIG. 2.
Example 2
Microsphere absorption and fluorescence property characterization
Preparing the colorimetric-fluorescent bifunctional aggregation-induced fluorescent microspheres prepared in the example 1 into a dilute solution of 50 μ g/mL, and measuring ultraviolet absorption and fluorescence spectra of the dilute solution respectively, as shown in fig. 3; the microsphere prepared by the invention has good colorimetric and fluorescent properties.
Example 3
The colorimetric-fluorescent bifunctional aggregation-induced fluorescent microspheres are used as a beacon probe to prepare a sandwich immunochromatographic test strip for detecting NT-BNP-PRO. The method comprises the following specific steps:
(1) to a 25mL clean flask was added 10mL of phosphate buffer (pH 6.0, 0.01M), and under stirring, 1mg of the AIE microspheres obtained in example 1, 100. mu.g of EDC, 120. mu.g of mouse anti-NT-BNP-PRO monoclonal antibody was added, and the mixture was stirred at 37 ℃ for 60min, and then 1mL of 1% (w/v) BSA (bovine serum albumin) solution was added and the mixture was blocked at room temperature for 1h to obtain the NT-BNP-PRO fluorescent marker.
(2) The reaction solution was centrifuged at 10000rpm for 15min, and the pellet was redispersed in 2mL of a probe complex solution (PB buffer, pH 7.4, 0.01M, 25% sucrose, 1% BSA, 0.5% polyethylene glycol 2000, 0.5% Tween 20).
(3) And (3) spraying the probe prepared in the step (1) and the probe marked with the IgY antibody on a bonding pad, wherein the spraying amount is 5 mu L/cm, and drying in vacuum for 2h at 37 ℃ for later use.
(4) NT-BNP-PRO polyclonal antibody (1mg/mL) and anti-IgY secondary antibody (0.5mg/mL) with different concentrations are respectively sprayed on a nitrocellulose membrane as a detection line (T line) and a quality control line (C line), and the nitrocellulose membrane is dried for 12h at 65 ℃.
(5) And (3) sequentially sticking the pre-treated nitrocellulose membrane, the bonding pad and the grade absorbent paper on a PVC (polyvinyl chloride) base plate, cutting the nitrocellulose membrane, the bonding pad and the grade absorbent paper into test strips with the width of 3.9mm by using an automatic slitter, and drying and storing the test strips at room temperature for later use.
(6) Adding 70 mu L of sample to be detected into a sample adding hole of a horizontally placed test strip, reacting for 15min, and reading T, C-line colorimetric and fluorescent intensity of the test strip by using a test strip reader. If the test strip T, C shows color, the test strip shows a positive result, and if the test strip T shows no color and the test strip C shows color, the test strip shows a negative result. If the C line does not show a fluorescent strip, the detection is invalid. The results are shown in FIG. 5.
The result pair of the ratio of the fluorescence intensity of the control line of the detection line when the prepared colorimetric-fluorescent dual-signal test strip is applied to the detection of NT-BNP-pro and the actual concentration of NT-BNP-pro is shown in FIG. 4, so that the colorimetric-fluorescent dual-functional aggregation-induced fluorescent microsphere prepared by the invention has very high accuracy when used as a beacon probe for detection.
The foregoing merely represents preferred embodiments of the invention, which are described in some detail and detail, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes, modifications and substitutions can be made without departing from the spirit of the present invention, and these are all within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (5)
1. A colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere is characterized in that: the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere mainly comprises a polystyrene microsphere and a colorimetric-fluorescent double-signal dye;
specifically, a polystyrene microsphere is taken as a main body, and a colorimetric-fluorescent dual-signal dye with strong absorption and high luminescence performance is embedded in the polystyrene microsphere through a swelling method to prepare the bifunctional nano microsphere with colorimetric and fluorescent signals;
the excitation of the colorimetric-fluorescent dual-signal dye is 365nm, the emission is 625nm, and the strong absorption is realized at 530 nm.
2. The colorimetric-fluorescent dual-signal aggregation-induced fluorescence microsphere of claim 1, wherein: the molecular formula of the colorimetric-fluorescent dual-signal dye has typical aggregation-induced emission performance, and the specific structural formula is as follows:
3. the use of a colorimetric-fluorescent dual signal aggregation-induced fluorescence microsphere as claimed in claim 1 or 2, wherein: the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere is used as a beacon probe, is applied to an immunochromatography platform, and realizes naked eye qualitative detection and fluorescent quantitative detection of a target analyte.
4. Use according to claim 3, characterized in that: the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere is used for preparing colorimetric and fluorescent microsphere coupled antibody immune probes, and the preparation method comprises the following steps:
(a) dissolving the prepared carboxylated colorimetric-fluorescent bifunctional microspheres in 1mL of phosphate buffer solution with the pH value of 6.5 to obtain a final concentration of 1 mg/mL;
(b) adding 0.1mgNT-BNP-pro monoclonal antibody into the microsphere solution, stirring, and performing electrostatic adsorption for 30 min;
(c) adding 50 μ g of 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (EDC) into the system, stirring for reaction for 30min, repeating the reaction for three times, and adding 10mg of Bovine Serum Albumin (BSA) for blocking for 1 h;
(d)10000rpm, 4 ℃ for 15min, and the pellet was resuspended in probe complex solution (25% sucrose, 1% BSA, 0.1% sodium azide) to a final concentration of 0.2 mg/mL.
5. Use according to claim 3, characterized in that: the colorimetric-fluorescent dual-signal aggregation induced fluorescent microsphere is used for preparing a colorimetric-fluorescent dual-functional microsphere NT-BNP-pro fluorescent test strip kit, and the preparation method comprises the following steps:
(a) spraying the NT-BNP-pro monoclonal antibody and the IgY antibody marked by the colorimetric-fluorescent double-signal aggregation-induced fluorescent microsphere on a glass fiber membrane to prepare a binding pad;
(b) the preparation of the nitrocellulose membrane with detection line and quality control line is as follows: respectively spraying NT-BNP-pro capture antibody with the concentration of 1-2 mg/mL and anti-IgY secondary antibody solution with the concentration of 1-2 mg/mL on a nitrocellulose membrane to draw two parallel lines, wherein the NT-BNP-pro capture antibody is drawn as a detection line (T line), and the anti-chicken IgY secondary antibody solution is drawn as a quality control line (C line);
(c) assembling the colorimetric-fluorescent bifunctional microsphere immunochromatographic test strip: and (3) respectively and sequentially sticking a sample pad, a bonding pad sprayed with the probe, a nitrocellulose membrane with T and C lines and absorbent paper to the plastic lining plate, wherein the length of the adjacent overlapped parts is 2mm, cutting the test paper into test paper strips with the width of 3.9mm by using a test paper strip cutting instrument, sealing and drying, and storing at the temperature.
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| CN115389760A (en) * | 2022-10-27 | 2022-11-25 | 艾康生物技术(杭州)有限公司 | Detection reagent for immunoassay test strip |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115389760A (en) * | 2022-10-27 | 2022-11-25 | 艾康生物技术(杭州)有限公司 | Detection reagent for immunoassay test strip |
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