CN112014560A - Near-infrared fluorescence immunochromatographic test strip for detecting human anti-SARS-CoV-2 antibody - Google Patents
Near-infrared fluorescence immunochromatographic test strip for detecting human anti-SARS-CoV-2 antibody Download PDFInfo
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Abstract
The invention belongs to the technical field of nano medical biotechnology detection, and particularly relates to a near-infrared fluorescent dye-doped fluorescent microsphere which comprises a high molecular polymer microsphere and a near-infrared fluorescent dye embedded in the high molecular polymer microsphere; the near-infrared fluorescent dye is loaded into the high molecular polymer microspheres, the obtained near-infrared fluorescent microspheres can be coupled with recombinant SARS-CoV-2 spinous process protein in a covalent coupling mode to prepare the immunofluorescence probe with high fluorescence intensity, good stability and uniform fluorescence signals, the biomarker process is simple, and the problem that the emission peak value of the fluorescence label of the current immunochromatographic test strip is in a visible light region and is interfered by the autofluorescence of an NC membrane and a biological sample is solved.
Description
Technical Field
The invention belongs to the technical field of nano medical biotechnology detection, and particularly relates to a fluorescent microsphere doped with near-infrared fluorescent dye, a preparation method and application thereof in immunochromatography detection of new coronavirus SARS-CoV-2.
Background
The 2019 coronavirus disease (COVID-19) epidemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) rapidly spreads, and causes global pandemics. By 24 days 7 and 24 months in 2020, 15508992 total cases of infection and 633703 total cases of death (4.1% of the total number of infection cases reported worldwide) are reported. Symptomatic or asymptomatic infection has been reported to result in interpersonal transmission. Therefore, there is an urgent need for early screening for SARS-CoV-2 infection to facilitate early intervention and reduce the risk of large-scale infection and spread.
Currently, reverse transcription-polymerase chain reaction (RT-PCR) for the detection of SARS-CoV-2RNA genome is the standard method for clinical diagnosis of COVID-19. However, it relies on expensive equipment and highly skilled analysts, with false negative results, not suitable for point-of-care testing (POCT). In addition, molecular diagnostic methods such as RT-PCR cannot monitor the infection process of SARS-CoV-2 in humans and cannot predict the time of infection. Therefore, a simple, rapid, sensitive detection method is crucial for detecting and monitoring COVID-19.
The serological immunoassay methods such as immunochromatography test strip technology (LFIA) and the like have the advantages of convenience, rapidness and the like. These methods allow prediction of SARS-CoV-2 exposure time by monitoring human anti-SARS-CoV-2 IgM (early immunoglobulin after viral invasion) and human anti-SARS-CoV-2 IgG (neutralizing middle and late viral activity) antibodies. However, the ability of conventional colloidal gold immunochromatographic strip technology (Au-LFIA) to detect low concentrations of IgM and IgG antibodies in SARS-CoV-2 infection early specimens is not ideal. Early infection with low concentrations of human anti-SARS-CoV-2 IgM and human anti-SARS-CoV-2 IgG could not be achieved. Therefore, there is an urgent need to develop a simple, rapid and sensitive detection method for monitoring patients with early SARS-CoV-2 infection.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a near-infrared fluorescent dye-doped fluorescent microsphere, including a high molecular polymer microsphere and a near-infrared fluorescent dye embedded inside the high molecular polymer microsphere; wherein the structure of the near-infrared fluorescent dye is as follows:
as a preferred technical scheme, the high molecular polymer microspheres are carboxylated polystyrene spheres.
As a preferable technical scheme, the particle size of the carboxylated polystyrene sphere is 150-350nm, and the coefficient of variation is less than 5%.
The second aspect of the present invention provides a method for preparing the fluorescent microsphere doped with the near-infrared fluorescent dye, which at least comprises the following steps:
(1) dispersing the near-infrared fluorescent dye in an organic solvent to form an organic phase;
(2) dispersing the high molecular polymer microspheres in an aqueous solution, and performing ultrasonic treatment to form a high molecular polymer microsphere aqueous solution;
(3) adding a surfactant into a high-molecular polymer microsphere aqueous solution to obtain a solution A;
(4) mixing the organic phase and the solution A, heating and continuously stirring for reaction, and carrying out aftertreatment to obtain the catalyst.
As a preferable technical scheme, the concentration of the near-infrared fluorescent dye in the organic phase in the step (1) in the invention is 10-25 mg/mL.
As a preferable technical scheme, in the step (3) of the invention, the concentration of the polymer microspheres in the solution A is 30-50mg/mL, and the concentration of the surfactant is 2-8 mg/mL.
As a preferable technical solution, the volume ratio of the organic phase to the solution a in the step (4) of the present invention is 1: (5-10).
The third aspect of the invention provides a near-infrared fluorescence immunochromatographic test strip for detecting a human anti-SARS-CoV-2 antibody, which comprises a base plate; the bottom plate is sequentially provided with a sample area, a detection area and an adsorption area which are connected;
the sample area contains a fluorescent probe; the fluorescent probe comprises the fluorescent microspheres doped with the near-infrared fluorescent dye in the preparation process; one part of the fluorescent probe is coupled with recombinant SARS-CoV-2 spinous process protein, the other part of the fluorescent probe is coupled with anti-chicken IgY, and the number of microspheres coupled with the recombinant SARS-CoV-2 spinous process protein and the number of microspheres coupled with the anti-chicken IgY are preferably 1: 1-5: 1;
and a detection line and a quality control line are arranged in the detection area.
As a preferred technical scheme, the detection line is sprayed with a mouse anti-human IgM polyclonal antibody and a mouse anti-human IgG polyclonal antibody.
As a preferable technical scheme, the quality control line is sprayed with chicken IgY.
Compared with the prior art, the invention has the following excellent beneficial effects:
the invention provides a near-infrared fluorescent dye-doped fluorescent microsphere, wherein the near-infrared fluorescent dye is loaded into a high-molecular polymer microsphere, the obtained near-infrared fluorescent microsphere can be coupled with recombinant SARS-CoV-2 spinous process protein in a covalent coupling mode to prepare an immunofluorescence probe with high fluorescence intensity, good stability and uniform fluorescence signals, the biomarker process is simple, and the problem that the emission peak value of the fluorescence label of the current immunochromatographic test strip is in a visible light region and is interfered by the autofluorescence of an NC membrane and a biological sample is solved. The near-infrared fluorescent microspheres prepared based on the fluorescent dye with the emission peak value of 800nm can obviously reduce the interference of autofluorescence, and further improve the detection sensitivity.
The following detailed description is provided to facilitate easier and easier understanding of the technical problems, solutions and advantages of the present invention as described above.
Drawings
FIG. 1 shows the absorption and emission spectra of fluorescent microspheres prepared with near-infrared dyes.
FIG. 2 is a scanning electron microscope image of fluorescent microspheres prepared from near-infrared dye.
FIG. 3 is a graph showing the luminescence intensity spectra of fluorescent microspheres prepared from different near-infrared dyes, wherein the structures of the dyes used from top to bottom are formula 1, comparative formula II and comparative formula I.
FIG. 4 is a schematic diagram showing the structure of an immunochromatographic test strip for detecting human anti-SARS-CoV-2 IgM and IgG antibodies.
FIG. 5 is a graph showing the comparison of the results of the method described in example 3 and the conventional colloidal gold assay for human anti-SARS-CoV-2 IgM and IgG antibodies in mixed positive plasma.
Wherein, the labels in fig. 3, fig. 4, fig. 5 are sequentially explained as:
when the 1 is near-infrared fluorescent dye as formula 1, the fluorescent intensity test result of the fluorescent microsphere doped with the near-infrared fluorescent dye is obtained;
2 is the fluorescence intensity test result of the fluorescent microsphere doped with the near-infrared fluorescent dye when the near-infrared fluorescent dye is a comparison formula I;
and 3 is the fluorescence intensity test result of the fluorescent microsphere doped with the near-infrared fluorescent dye when the near-infrared fluorescent dye is in the comparison formula II.
4 is a sample pad; 5 is a bonding pad; 6 is a T1 line; 7 is a T2 line; 8 is a C line; 9 is absorbent paper; 10 is a fluorescent probe.
5-1 is the result of the human anti-SARS-CoV-2 IgM and IgG antibodies after the positive mixed sample is diluted 1000 times by the immunochromatographic test strip of the present invention, wherein 10 times, 100 times, 1000 times, 2500 times and negative control are sequentially performed from left to right.
5-2, the conventional colloidal gold is used as a control, and the colloidal gold immunochromatographic test strip can detect the human anti-SARS-CoV-2 IgM and IgG antibodies after the positive mixed sample is diluted by 10 times.
Detailed Description
For purposes of the following detailed description, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. Moreover, other than in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
The invention provides a near-infrared fluorescent dye-doped fluorescent microsphere, which comprises a high molecular polymer microsphere and a near-infrared fluorescent dye embedded in the high molecular polymer microsphere; wherein the structure of the near-infrared fluorescent dye is as follows:
the synthesis process of the near-infrared fluorescent dye structure is as follows:
i: compound 1(1.11g) and Cs2CO3(2.43g) was dissolved in anhydrous N, N-dimethylformamide (DMF, 15mL), and 1-bromooctane (1.25g) was added under argon and stirred at room temperature for 12 h; after the reaction was completed, the mixture was poured into water, and extracted with dichloromethane 3 times in order over MgSO4Dried and filtered off. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/dichloromethane ═ 9/1) to obtain compound 2.
II: the compound2(667mg) in anhydrous THF (10mL), n-BuLi (0.93mL, 1.5mmol) was added slowly at-78 deg.C under an argon atmosphere, then stirred at-78 deg.C for 3 h; tributyltin chloride (Bu)3SnCl, 0.58mL, 2mmol) was added to the mixture, which was then slowly warmed to room temperature and stirred overnight. The mixture was poured into water and extracted with dichloromethane (30 ml. times.3). The combined organic layers were washed successively with water (100 mL. times.3) over MgSO4Dried and filtered. After removal of the solvent, the residue was a colorless liquid and was used directly in the next step without further purification.
III: compound 3(351.2mg), compound 4(76mg), Pd (PPh)3)4(11.5mg) and toluene (30ml) were charged into a Schlenk tube. The mixture was stirred at 100 ℃ under an argon atmosphere for 20 hours. After cooling to room temperature, the mixture was diluted successively with dichloromethane (100mL), washed with aqueous potassium fluoride (10%, 100 mL. times.3), water (100 mL. times.3), and over MgSO4And drying. The crude product was purified by silica gel column chromatography (petroleum ether/dichloromethane ═ 4/1) to give compound 5.
In some preferred embodiments, the polymeric microspheres are carboxylated polystyrene spheres. In some preferred embodiments, the particle size of the carboxylated polystyrene spheres is 150-350nm, and the coefficient of variation is less than 5%; preferably, the particle size of the carboxylated polystyrene spheres is 300nm, and the coefficient of variation is less than 5%.
The carboxylated polystyrene spheres of the present invention are purchased from national biotechnology limited.
The second aspect of the present invention provides a preparation method of the near-infrared fluorescent dye-doped fluorescent microsphere, which at least comprises the following steps:
(1) dispersing the near-infrared fluorescent dye in an organic solvent to form an organic phase;
(2) dispersing the high molecular polymer microspheres in an aqueous solution, and performing ultrasonic treatment to form a high molecular polymer microsphere aqueous solution;
(3) adding a surfactant into a high-molecular polymer microsphere aqueous solution to obtain a solution A;
(4) mixing the organic phase and the solution A, continuously stirring and reacting for 3-10 hours at the temperature of 25-45 ℃, and carrying out post-treatment to obtain the organic phase.
In some preferred embodiments, in step (4), the post-treatment is centrifugation, washing and storage in ultrapure water.
In some preferred embodiments, in step (4), the organic phase and solution a are mixed, immediately shaken, and stirred.
In some preferred embodiments, in step (4), the organic phase and solution a are added to an aqueous SDS solution for mixing; the concentration of the SDS aqueous solution is 1-6mg mL-1(ii) a Preferably, the concentration of the SDS aqueous solution is 4mg mL-1。
In some preferred embodiments, the volume ratio between the aqueous SDS solution and the organic phase is (5-15): 1; preferably, the volume ratio of the SDS aqueous solution to the organic phase is 10: 1.
and (4) mixing the organic phase with the solution A in the step (4), immediately oscillating, stirring, and gradually volatilizing the organic solvent in the process to enable the fluorescent dye in the near infrared region to successfully enter the interior of the high-molecular polymer microsphere so as to obtain the high-brightness fluorescent microsphere.
In some preferred embodiments, in the step (4), the organic phase and the solution A are mixed, and the mixture is continuously stirred and reacted for 7 hours at the temperature of 35 ℃ and then is subjected to post-treatment.
The fluorescent microspheres doped with the near-infrared fluorescent dye are applied to an immunochromatography test strip for detecting human anti-SARS-CoV-2 IgM and IgG antibodies.
In some embodiments, in step (1), the concentration of the near-infrared fluorescent dye in the organic phase is from 10 to 25 mg/mL; preferably, the concentration of the near-infrared fluorescent dye in the organic phase is 20 mg/mL.
In some embodiments, in step (3), the concentration of the polymeric microspheres in the solution A is 30-50mg/mL, and the concentration of the surfactant is 2-8 mg/mL.
In some preferred embodiments, in step (3), the concentration of the polymeric microspheres in the solution A is 40mg/mL, and the concentration of the surfactant is 4 mg/mL.
In some embodiments, the volume ratio between the organic phase and the solution a in step (4) is 1: (5-10); preferably, the volume ratio of the organic phase to the solution A in the step (4) is 1: 5.
in some embodiments, the organic solvent in step (1) includes, but is not limited to, at least one of tetrahydrofuran, acetone, benzyl alcohol, phenethyl alcohol, toluene, chlorobenzene, and xylene.
In some embodiments, the surfactant includes, but is not limited to, at least one of sodium lauryl sulfate, tween, Thesit.
The Thesit is short for polyethylene glycol monolaurate.
The third aspect of the invention provides a near-infrared fluorescence immunochromatographic test strip for detecting a human anti-SARS-CoV-2 antibody, which comprises a base plate; the bottom plate is sequentially provided with a sample area, a detection area and an adsorption area which are connected; the sample area contains a fluorescent probe; the fluorescent probe comprises the fluorescent microspheres doped with the near-infrared fluorescent dye in the preparation process; one part of the fluorescent probe is coupled with recombinant SARS-CoV-2 spinous process protein, the other part of the fluorescent probe is coupled with anti-chicken IgY, and the number of microspheres coupled with the recombinant SARS-CoV-2 spinous process protein and the number of microspheres coupled with the anti-chicken IgY are preferably 1: 1-5: 1; and a detection line and a quality control line are arranged in the detection area.
In some preferred embodiments, the near-infrared fluorescence immunochromatographic test strip for detecting a human anti-SARS-CoV-2 antibody comprises a substrate; the bottom plate is sequentially provided with a sample area, a detection area and an adsorption area which are connected;
the sample area contains a fluorescent probe; the fluorescent probe comprises the fluorescent microspheres doped with the near-infrared fluorescent dye in the preparation process; one part of the fluorescent probe is coupled with recombinant SARS-CoV-2 spinous process protein, the other part is coupled with anti-chicken IgY, and the number of the microspheres coupled with the recombinant SARS-CoV-2 spinous process protein and the microspheres coupled with the anti-chicken IgY is preferably 2.5: 1; and a detection line and a quality control line are arranged in the detection area.
The fluorescent probe comprises near-infrared fluorescent dye-doped fluorescent microspheres coupled with recombinant SARS-CoV-2 spinous process protein in the preparation process, the first part of the probe is used for detecting luminescence indication on a line T, a sandwich structure is formed through immunoreaction, the probe microspheres are finally combined to the position of the line T and the near-infrared fluorescent dye-doped fluorescent microspheres coupled with goat anti-chicken IgY, the second part of the probe is used for quality control line luminescence indication on a line C, the probe microspheres are finally combined to the position of the line C through immunoreaction, and the number ratio of the two kinds of probe microspheres of the first part to the second part is preferably 1:1 to 5: 1.
Two detection lines (T1 and T2) and a quality control line (C line) are arranged in the detection area; the detection line is respectively sprayed with a mouse anti-human IgM polyclonal antibody and a mouse anti-human IgG polyclonal antibody; the quality control line is sprayed with chicken IgY.
In some embodiments, the near-infrared fluorescence immunochromatographic test strip for detecting a human anti-SARS-CoV-2 antibody is prepared by the following method:
a: preparing a fluorescent probe;
b: preparing an NC membrane of the immunochromatographic test strip;
c: preparing an immunochromatography test strip sample pad;
d: and (5) assembling the immunochromatographic test strip.
In some preferred embodiments, the fluorescent probe in step a is prepared as follows:
s1: centrifuging the fluorescent microspheres doped with the near-infrared fluorescent dye, redissolving the fluorescent microspheres into a first BBS buffer solution, and dispersing the fluorescent microspheres by ultrasonic waves to obtain a dispersion system;
s2: adding EDC and NHSS into the dispersion system respectively, and reacting for 10-60 minutes at room temperature;
s3: after the reaction is finished, the system obtained by S2 is centrifugally washed, redissolved in a second BBS buffer solution, added with recombinant SARS-CoV-2 spinous process protein and reacted for 1 to 5 hours at room temperature;
s4: after the reaction of S3, BSA aqueous solution was added and the reaction was carried out at room temperature for 0.5 to 3 hours.
S5: after the reaction of S4, the reaction solution was washed by centrifugation, redissolved in a third BBS buffer solution, and stored at 4 ℃ for further use.
S6: the preparation process of the near-infrared fluorescent dye-doped fluorescent microsphere coupled anti-chicken sheep IgY is the same as S1-S5, and the difference is that recombinant SARS-CoV-2 spinous process protein in S3 is replaced by anti-chicken sheep IgY.
In some preferred embodiments, the fluorescent probe in step a is prepared as follows:
s1: centrifuging 2mg of the near-infrared fluorescent dye-doped fluorescent microspheres, redissolving the microspheres into 500 mu L of a first BBS buffer solution with the pH value of 7.4, and dispersing the microspheres by ultrasonic waves to obtain a dispersion system;
s2: 0.2mgEDC and 0.112mgNHSS were added to the dispersion system, respectively, and reacted at room temperature for 30 minutes;
s3: after the reaction is finished, the system obtained by S2 is centrifugally washed, redissolved into 500 mu L of second BBS buffer solution with pH7.4, 0.25mg of recombinant SARS-CoV-2 spinous process protein is added into the solution, and the reaction is carried out for 1 to 5 hours at room temperature;
s4: after completion of the reaction of S3, 50. mu.L of 100mg mL was added-1BSA aqueous solution was reacted at room temperature for 1 hour.
S5: after the completion of the S4 reaction, the reaction mixture was washed by centrifugation, redissolved in 500. mu.L of a third BBS buffer solution (pH7.4), and stored at 4 ℃ until use.
S6: the preparation process of the near-infrared fluorescent dye-doped fluorescent microsphere coupled anti-chicken sheep IgY is the same as S1-S5, and the difference is that recombinant SARS-CoV-2 spinous process protein in S3 is replaced by anti-chicken sheep IgY.
Wherein EDC is the abbreviation of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride.
The NHSS is the abbreviation of N-hydroxysuccinimide sulfonic acid sodium salt.
In some embodiments, in step B, the preparation process of the NC membrane of the immunochromatographic test strip is as follows: respectively scratching a mouse anti-human IgM polyclonal antibody, a mouse anti-human IgG polyclonal antibody and chicken IgY on a nitrocellulose membrane by a film scratching instrument at the concentrations of 0.6mg/mL, 0.6mg/mL and 1mg/mL by using a PBS buffer solution (pH6.8), and drying at 37 ℃ overnight to obtain the antibody.
In some embodiments, in step C, the immunochromatographic test strip sample pad is prepared as follows: taking the prepared fluorescent probe, centrifuging, re-dissolving the recombinant SARS-CoV-2 spinous process protein coupled with the fluorescent microsphere doped with the external fluorescent dye and the goat anti-chicken IgY coupled with the fluorescent microsphere doped with the near-infrared fluorescent dye into 20.8mg/mL and 2mg/mL respectively by using a film spraying buffer solution (1% BSA, 1% sucrose), spraying the fluorescent probe on glass fiber at the speed of 1.2 muL/cm by using a film spraying instrument, and baking at 37 ℃ for overnight to obtain the fluorescent probe.
In some embodiments, in step D, the assembly process of the immunochromatographic test strip is as follows: sequentially attaching a sample pad and a combination pad (glass fiber marked with recombinant SARS-CoV-2 spinous process protein and goat anti-chicken IgY coupled fluorescent probe) on a white PVC bottom plate in a staggered manner by 3mm, scratching a mouse anti-human IgM polyclonal antibody, a mouse anti-human IgG polyclonal antibody and a chicken IgY NC membrane, and finally attaching absorbent paper, wherein the structure is shown in figure 4; and then cutting the assembled chromatography plate into test strips with the width of 3.8mm by a high-speed cutting machine, and fixing the test strips by using an upper plastic card shell and a lower plastic card shell which are matched to obtain the immunochromatographic test strips.
The present invention also provides a method of determining the amount of a target analyte in a plasma sample, comprising the steps of:
1) taking 5 mu L of a plasma sample, and diluting the plasma sample with BBS buffer solution (borate buffer solution added with 2% BSA, 0.25% Tween20 and 0.1% Thesit) to obtain a loading solution; 2) adding a sample loading solution into a sample area of the immunochromatographic test strip; 3) measuring the fluorescence intensity of the negative sample at detection areas T1, T2 and C, and calculating T1/C, T2/C to obtain A1 and A2 respectively; 4) measuring the fluorescence intensity of the actual sample detection areas T1, T2 and C, and calculating to obtain T1/C, T2/C to obtain B1 and B2 respectively; 5) if B1> A1 or B2> A2, the determination is positive, that is, human anti-SARS-CoV-2 IgM and IgG antibodies are present, otherwise, the determination is negative.
In some embodiments, in step 1), the BBS buffer is a buffer comprising 2 wt% BSA, 0.25 wt% Tween20, and 0.1 wt% Thesit.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
Example 1
A near-infrared fluorescent dye-doped fluorescent microsphere comprises a high molecular polymer microsphere and a near-infrared fluorescent dye embedded in the high molecular polymer microsphere; wherein the structure of the near-infrared fluorescent dye is as follows:
the high molecular polymer microspheres are carboxylated polystyrene spheres. The carboxylated polystyrene spheres are purchased from PS07C of Biotechnology Limited, have a particle size of 300nm and a coefficient of variation of less than 5%.
The preparation method of the near-infrared fluorescent dye-doped fluorescent microsphere comprises the following steps:
(1) dispersing the near-infrared fluorescent dye in tetrahydrofuran to form an organic phase;
(2) dispersing the high molecular polymer microspheres in an aqueous solution, and performing ultrasonic treatment to form a high molecular polymer microsphere aqueous solution;
(3) adding a surfactant into a high-molecular polymer microsphere aqueous solution to obtain a solution A;
(4) 50 μ L of the organic phase and 250 μ L of solution A were mixed and added to 500 μ L of 4mg mL-1The reaction was heated and stirred continuously in aqueous SDS solution, and the product was centrifuged at 12000rpm for 10 minutes and resuspended in 1mL of Milli-Q water.
In the step (1), the concentration of the near-infrared fluorescent dye in the organic phase is 20 mg/mL.
The concentration of the polymer microspheres in the solution A is 40 mg/mL.
The absorption and emission spectra of the near-infrared fluorochrome-doped fluorescent microspheres obtained in this example were measured, as shown in fig. 1. The transmission electron microscope image of the fluorescent microsphere doped with the near-infrared fluorescent dye is shown in fig. 2, and it can be seen that the shape and size of the fluorescent microsphere doped with the near-infrared fluorescent dye obtained after the fluorescent dye is embedded are uniform.
Comparative example 1
The experiment of example 1 was repeated except that the near-infrared fluorescent dye of formula 1 was replaced with the near-infrared fluorescent dye of comparative formula I.
Comparative example 2
The experiment of example 1 was repeated except that the near-infrared fluorescent dye represented by formula 1 was replaced with the near-infrared fluorescent dye represented by comparative formula II.
The near-infrared fluorescent dye-doped fluorescent microspheres prepared in example 1, comparative example 1 and comparative example 2 were subjected to a luminescence property test, and as shown in fig. 3, the fluorescent microsphere prepared by using formula 1 has the highest luminescence intensity, which is the most preferable fluorescent microsphere for immunochromatography. The near-infrared fluorescent dye-doped fluorescent microspheres prepared by using the comparative formula I and the comparative formula II are lower in luminous intensity than the near-infrared fluorescent dye-doped fluorescent microspheres prepared by using the formula 1. The change of the dye molecule substituent has certain influence on the luminescence property of the prepared fluorescent microsphere, and the formula 1 used in the invention is optimized in detail in structure and is more suitable for immunochromatography application in performance.
In the invention, the test method for the luminous performance comprises the following steps: diluting the prepared near-infrared fluorescent dye-doped fluorescent microspheres with water to the concentration of 100 mu g/mL, testing by using an Edinburgh FL920 fluorometer, and using a 650nm xenon lamp as an excitation light source.
Example 2
An immunochromatographic test strip comprises a bottom plate; the bottom plate is sequentially provided with a sample area, a detection area and an adsorption area which are connected; two detection lines (T1 and T2) and a quality control line (C line) are arranged in the detection area; the detection line is respectively sprayed with a mouse anti-human IgM polyclonal antibody and a mouse anti-human IgG polyclonal antibody; the quality control line is sprayed with chicken IgY. The sample area contains a fluorescent probe; the fluorescent probe comprises the fluorescent microspheres doped with the near-infrared fluorescent dye in the preparation process; the fluorescent probe comprises a near-infrared fluorescent dye-doped fluorescent microsphere coupled recombinant SARS-CoV-2 spinous process protein, the probe is named as P-1 and used for generating a luminescent signal on a T line and forming a sandwich structure through immunoreaction, the probe microsphere is finally combined to the position of the T line, the near-infrared fluorescent dye-doped fluorescent microsphere is coupled with the goat anti-chicken IgY, the probe is named as P-2 and used for generating a luminescent signal on a C line, the probe microsphere is finally combined to the position of the C line through immunoreaction, and the number ratio of the two probe microspheres P-1 to P-2 is preferably 2.5: 1.
The preparation method of the immunochromatographic test strip is as follows:
a: preparing a fluorescent probe;
b: preparing an NC membrane of the immunochromatographic test strip;
c: preparing an immunochromatography test strip sample pad;
d: and (5) assembling the immunochromatographic test strip.
The preparation process of the fluorescent probe in the step A is as follows:
s1: centrifuging 2mg of the near-infrared fluorescent dye-doped fluorescent microspheres, redissolving the microspheres into 500 mu L of a first BBS buffer solution with the pH value of 7.4, and dispersing the microspheres by ultrasonic waves to obtain a dispersion system;
s2: 0.2mgEDC and 0.112mgNHSS were added to the dispersion system, respectively, and reacted at room temperature for 30 minutes;
s3: after the reaction is finished, the system obtained by S2 is centrifugally washed, redissolved into 500 mu L of second BBS buffer solution with pH7.4, 0.25mg of recombinant SARS-CoV-2 spinous process protein is added into the solution, and the reaction is carried out for 1 to 5 hours at room temperature;
s4: after completion of the reaction of S3, 50. mu.L of 100mg mL was added-1BSA aqueous solution was reacted at room temperature for 1 hour.
S5: after the completion of the S4 reaction, the reaction mixture was washed by centrifugation, redissolved in 500. mu.L of a third BBS buffer solution (pH7.4), and stored at 4 ℃ until use.
S6: the preparation process of the near-infrared fluorescent dye-doped fluorescent microsphere coupled anti-chicken sheep IgY is the same as S1-S5, and the difference is that recombinant SARS-CoV-2 spinous process protein in S3 is replaced by anti-chicken sheep IgY.
In the step B, the preparation process of the NC membrane of the immunochromatographic test strip is as follows:
respectively scratching a mouse anti-human IgM polyclonal antibody, a mouse anti-human IgG polyclonal antibody and chicken IgY on a nitrocellulose membrane by a film scratching instrument at the concentrations of 0.6mg/mL, 0.6mg/mL and 1mg/mL by using a PBS buffer solution (pH6.8), and drying at 37 ℃ overnight to obtain the antibody.
In the step C, the preparation process of the immunochromatographic test strip sample pad is as follows:
taking the prepared fluorescent probe, centrifuging, re-dissolving the recombinant SARS-CoV-2 spinous process protein coupled with the fluorescent microsphere doped with the external fluorescent dye and the goat anti-chicken IgY coupled with the fluorescent microsphere doped with the near-infrared fluorescent dye into 20.8mg/mL and 2mg/mL respectively by using a film spraying buffer solution (1% BSA, 1% sucrose), spraying the fluorescent probe on glass fiber at the speed of 1.2 muL/cm by using a film spraying instrument, and baking at 37 ℃ for overnight to obtain the fluorescent probe.
In the step D, the assembly process of the immunochromatographic test strip is as follows:
sequentially attaching a sample pad and a combination pad (glass fiber marked with recombinant SARS-CoV-2 spinous process protein and goat anti-chicken IgY coupled fluorescent probe) on a white PVC bottom plate in a staggered manner by 3mm, scratching a mouse anti-human IgM polyclonal antibody, a mouse anti-human IgG polyclonal antibody and a chicken IgY NC membrane, and finally attaching absorbent paper, wherein the structure is shown in figure 4; and then cutting the assembled chromatography plate into test strips with the width of 3.8mm by a high-speed cutting machine, and fixing the test strips by using an upper plastic card shell and a lower plastic card shell which are matched to obtain the immunochromatographic test strips.
Examples3
The method for determining and detecting the human anti-SARS-CoV-2 IgM and IgG antibodies in the mixed positive plasma comprises the following steps:
1) the positive mixed plasma of human anti-SARS-CoV-2 IgM and IgG antibodies was diluted to different times by 10, 100, 1000 and 2500 times with the negative plasma, and the negative plasma was used for blank control.
2) mu.L of plasma samples and blanks at different dilution times were added to 95. mu.L LBBS buffer (containing 2% BSA, 0.25% Tween20, and 0.1% Thesit) and mixed well.
3) The test was performed using the immuno-layer test strips prepared in example 2: 100 mu L of mixed solution which is uniformly mixed is added to a sample adding hole (the position corresponding to a sample adding area on the plastic card shell) of the immunochromatography test strip, and the mixed solution passes through the sample area, the detection area and the water absorption area in sequence through the capillary action. When the detection sample contains the human anti-SARS-CoV-2 IgM and IgG antibodies, the antigen is firstly combined with the fluorescent probe in the sample area to form an immune complex, then the immune complex is electrophoresed to the T1 and T2 line along with the liquid to form a sandwich immune complex with the mouse anti-human IgM polyclonal antibody and the mouse anti-human IgG polyclonal antibody, and the fluorescent probe coupled with the goat anti-chicken IgY is electrophoresed to the C line to be combined with the chicken IgY. And when no antigen exists in the detection sample, the fluorescent probe coupled with the goat anti-chicken IgY is driven to directly swim to the C line to be combined with the chicken IgY.
4) After the reaction is carried out for 11 minutes, the immunochromatographic test strip is inserted into a near-infrared detector, cut-off values, A1 and A2 of a blank sample are obtained through measurement, and then B1 and B2 of a positive mixed sample are obtained through calculation. The experimental results are shown in FIG. 5, and the results show that the method can detect the human anti-SARS-CoV-2 IgM and IgG antibodies after the positive mixed sample is diluted by 1000 times.
5) The detection sensitivity of the fluorescent microspheres is tested, meanwhile, the conventional colloidal gold is used as a control, as shown in figure 5, the prepared fluorescent spheres can detect the human anti-SARS-CoV-2 IgM and IgG antibodies after the positive mixed sample is diluted by 1000 times, the colloidal gold immunochromatographic test strip can detect the human anti-SARS-CoV-2 IgM and IgG antibodies after the positive mixed sample is diluted by 10 times, and the experimental result shows that the immunochromatographic test strip prepared by coupling the fluorescent microspheres doped with the near-infrared fluorescent dye with the recombinant SARS-CoV-2 spinous-process protein and the fluorescent microspheres doped with the near-infrared fluorescent dye with the goat anti-chicken IgY is 100 times higher than the detection sensitivity of the colloidal gold and has ultrahigh detection sensitivity.
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim the description of selected embodiments in the best possible combination contemplated. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims. The scope is broad and the examples presented herein are based on all possible implementations.
Claims (10)
1. A near-infrared fluorescent dye-doped fluorescent microsphere is characterized by comprising a high-molecular polymer microsphere and a near-infrared fluorescent dye embedded in the high-molecular polymer microsphere; wherein the structure of the near-infrared fluorescent dye is as follows:
2. the near-infrared fluorochrome-doped fluorescent microsphere of claim 1, wherein the polymeric microspheres are carboxylated polystyrene spheres.
3. The near-infrared fluorescent dye-doped fluorescent microsphere of claim 2, wherein the particle size of the carboxylated polystyrene sphere is 150-350nm, and the coefficient of variation is less than 5%.
4. A method for preparing fluorescent microspheres doped with near-infrared fluorescent dye according to any one of claims 1 to 3, comprising at least the steps of:
(1) dispersing the near-infrared fluorescent dye in an organic solvent to form an organic phase;
(2) dispersing the high molecular polymer microspheres in an aqueous solution, and performing ultrasonic treatment to form a high molecular polymer microsphere aqueous solution;
(3) adding a surfactant into a high-molecular polymer microsphere aqueous solution to obtain a solution A;
(4) mixing the organic phase and the solution A, heating and continuously stirring for reaction, and carrying out aftertreatment to obtain the catalyst.
5. The method of claim 4, wherein in step (1), the concentration of the NIR fluorescent dye in the organic phase is 10-25 mg/mL.
6. The method for preparing fluorescent microspheres doped with near-infrared fluorescent dye according to claim 4, wherein in the step (3), the concentration of the polymer microspheres in the solution A is 30-50mg/mL, and the concentration of the surfactant is 2-8 mg/mL.
7. The method for preparing fluorescent microspheres doped with near-infrared fluorescent dye according to claim 4, wherein the volume ratio of the organic phase to the solution A in the step (4) is 1: (5-10).
8. A near-infrared fluorescence immunochromatographic test strip for detecting a human anti-SARS-CoV-2 antibody is characterized by comprising a bottom plate; the bottom plate is sequentially provided with a sample area, a detection area and an adsorption area which are connected;
the sample area contains a fluorescent probe; the fluorescent probe comprises the near-infrared fluorescent dye-doped fluorescent microsphere as defined in any one of claims 1-3 in the preparation process; one part of the fluorescent probe is coupled with recombinant SARS-CoV-2 spinous process protein, the other part of the fluorescent probe is coupled with anti-chicken IgY, and the number of microspheres coupled with the recombinant SARS-CoV-2 spinous process protein and the number of microspheres coupled with the anti-chicken IgY are preferably 1: 1-5: 1;
and a detection line and a quality control line are arranged in the detection area.
9. The near-infrared fluorescence immunochromatographic test strip of claim 8, wherein the detection line is sprayed with a mouse anti-human IgM polyclonal antibody and a mouse anti-human IgG polyclonal antibody.
10. The near-infrared fluorescence immunochromatographic test strip of claim 8, wherein the quality control line is sprayed with chicken IgY.
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