CN107632159B - Immunofluorescence chromatography detection test strip, immunofluorescence chromatography detection system and method for determining content of substance to be detected in sample - Google Patents
Immunofluorescence chromatography detection test strip, immunofluorescence chromatography detection system and method for determining content of substance to be detected in sample Download PDFInfo
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Abstract
The invention provides an immunofluorescence chromatography detection test strip, an immunofluorescence chromatography detection system and a method for determining the content of an object to be detected in a sample. The immunofluorescence chromatography detection test strip comprises: the device comprises a body, a sample area, a binding area, a detection area and an adsorption area, wherein the sample area, the binding area, the detection area and the adsorption area are sequentially connected; the first antibody is labeled by fluorescent microspheres, coats the binding region and specifically recognizes an object to be detected; the detection line and the quality control line are arranged in the detection area; a second antibody coated on the detection line, the second antibody specifically recognizing the test substance; and the third antibody is coated on the quality control line, and specifically recognizes the first antibody, wherein the fluorescent microsphere is a near-infrared II-region high-molecular fluorescent microsphere. The immunofluorescence chromatography detection test strip has the advantages of strong detection accuracy, high sensitivity and high precision.
Description
Technical Field
The present invention relates to the field of biology. Specifically, the invention relates to an immunofluorescence chromatography detection test strip, an immunofluorescence chromatography detection system and a method for determining the content of an object to be detected in a sample.
Background
The polymer fluorescent microspheres are used as special functional microspheres, each microsphere can be wrapped by tens of thousands or even hundreds of thousands of fluorescent molecules, so that the marking efficiency of fluorescence can be greatly improved, the photobleaching resistance of the fluorescent molecules is enhanced, and the analysis sensitivity is greatly improved; meanwhile, the surface of the fluorescent microsphere can be flexibly modified with various functional groups (such as carboxyl, amino, aldehyde and the like), thereby facilitating covalent coupling with protein or antibody and improving the stability and labeling efficiency of the marker. At present, fluorescent microspheres have been widely used for labeling, tracing, detecting, imaging, immobilized enzyme, immune medicine, high-throughput drug screening, and the like. However, the emission of the traditional fluorescent microspheres is in a visible light region (the emission wavelength is less than 780nm), and when the traditional fluorescent microspheres are applied to the fields of biological living bodies, cell or tissue imaging, in-vitro diagnosis and the like, the poor fluorescence penetration performance and the high background fluorescence intensity are easily caused. Meanwhile, due to the fact that the fluorescence quantum efficiency is generally low, excitation and emission wavelengths are relatively close (generally about 20 nm), and the like, the analysis sensitivity is reduced, and the requirement on a color filter required by fluorescence detection is high.
Therefore, the polymeric fluorescent microspheres are in need of further research and improvement.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide an immunofluorescence chromatography detection test strip, an immunofluorescence chromatography detection system, and a method for determining the content of an analyte in a sample. The immunofluorescence chromatography detection test strip is coated with the antibody marked by the fluorescent microspheres, the fluorescent microspheres have high fluorescence quantum efficiency, the wavelengths of excitation (365nm or 740nm) and emission (900-1400nm) are high, and the fluorescent microspheres have the characteristics of strong penetrating power and small background interference during fluorescence detection, so that the detection result is high in accuracy, sensitivity and precision.
The present invention is accomplished mainly based on the following findings: the traditional fluorescent microsphere has poor fluorescent penetration performance and strong background fluorescence, and causes low analysis sensitivity and high requirements on color filters due to low fluorescent quantum efficiency, relatively close excitation and emission wavelengths and the like. The near-infrared fluorescent microspheres, especially the fluorescent microspheres in the near-infrared II region, have the characteristics of strong penetrating power, small background interference and the like, and have good application prospects in the aspects of living body imaging, biomarker detection and the like. However, the current method for preparing the near-infrared region II fluorescent microsphere still has limitations, for example, for a fluorescent dye with strong hydrophobicity, the fluorescent dye cannot be directly applied to living body imaging, and hydrophilic modification must be performed first, the modification process is not only complicated, but also the quantum efficiency of the modified fluorescent dye is greatly reduced after the modified fluorescent dye is dispersed in an aqueous solution, and the sensitivity of fluorescence detection is seriously affected.
In view of the above, the inventor finds that the swelling method can be used to wrap the fluorescent dye inside the polymer microsphere, the effect is very ideal, the fluorescent quantum efficiency of the microsphere can reach more than 20%, the microsphere can be well dispersed in an aqueous solution, the labeling of various biomacromolecules is convenient, and the microsphere has wide applicability for different fluorescent dyes. Furthermore, the obtained fluorescent microspheres are marked on the antibody, and the antibody is coated on the binding area of the test strip, so that the test strip has the advantages of high detection accuracy, high sensitivity and high precision.
To this end, in one aspect of the present invention, an immunofluorescence chromatography test strip is provided. According to an embodiment of the present invention, the test strip comprises: the device comprises a body, a sample area, a binding area, a detection area and an adsorption area, wherein the sample area, the binding area, the detection area and the adsorption area are sequentially connected; the first antibody is labeled by fluorescent microspheres, coats the binding region and specifically recognizes an object to be detected; the detection line and the quality control line are arranged in the detection area, and the detection line is close to the binding area; a second antibody coated on the detection line, the second antibody specifically recognizing the test substance; and a third antibody, wherein the third antibody is coated on the quality control line, and the third antibody specifically recognizes the first antibody, wherein the fluorescent microsphere is a near-infrared II-region high-molecular fluorescent microsphere, and the near-infrared II-region high-molecular fluorescent microsphere is obtained by the following method:
(1) dissolving a fluorescent dye by using a water-immiscible organic solvent so as to obtain a fluorescent dye solution;
(2) dispersing the polymer microspheres in a sodium dodecyl sulfate aqueous solution to obtain a microsphere carrier solution;
(3) mixing the fluorescent dye solution with the microsphere carrier solution and carrying out ultrasonic treatment so as to obtain an emulsified mixed solution;
(4) swelling the emulsified mixed solution so as to enable the fluorescent dye solution to be immersed into the nano holes generated in the swelling process of the polymer microspheres; and
(5) and (4) heating the mixture obtained in the step (4), and with the volatilization of the organic solvent, crystallizing and separating out the fluorescent dye and wrapping the fluorescent dye in the nano hole so as to obtain the near-infrared II-region macromolecular fluorescent microsphere.
According to the preparation method of the near-infrared region II high-molecular fluorescent microsphere provided by the embodiment of the invention, firstly, the fluorescent dye is dissolved by using the organic solvent to obtain the fluorescent dye solution, then the fluorescent dye solution and the microsphere carrier solution are mixed and then are subjected to ultrasonic treatment, swelling and heating in sequence, so that the fluorescent dye is successfully wrapped in the high-molecular fluorescent microsphere, and finally the near-infrared region II high-molecular fluorescent microsphere is obtained. The preparation method of the near-infrared region II high-molecular fluorescent microsphere of the embodiment of the invention has simple and rapid process, and the prepared near-infrared region II high-molecular fluorescent microsphere has better dispersibility in aqueous solution, quantum efficiency up to 25 percent, and wavelengths of excitation (365nm or 740nm) and emission (900-1400 nm). Therefore, the preparation method of the near-infrared II-region high-molecular fluorescent microspheres provided by the embodiment of the invention has wide applicability for different fluorescent dyes, and the prepared near-infrared II-region high-molecular fluorescent microspheres have the characteristics of strong penetration capability and small background interference during fluorescence detection, can greatly amplify fluorescent signals, improve detection sensitivity, and have good application prospects in aspects of living body imaging, biological labeling and the like.
Furthermore, the obtained fluorescent microspheres are marked on the antibody, and the antibody is coated on the binding area of the test strip, so that the test strip has the advantages of high detection accuracy, high sensitivity and high precision.
According to an embodiment of the present invention, the immunofluorescence chromatography detection test strip may further have the following additional technical features:
according to an embodiment of the present invention, the fluorescent dye has a structure represented by formula I
According to the embodiment of the present invention, the analyte is cardiac troponin, and the first antibody is cardiac troponin antibody 1; the second antibody is a cardiac troponin antibody 2, the sites of the first antibody and the second antibody which specifically recognize the object to be detected are different, and the third antibody is a second antibody, preferably a goat anti-mouse antibody. Therefore, the immunofluorescence chromatography detection test strip provided by the embodiment of the invention has the advantages of strong detection accuracy, high sensitivity and high precision.
According to an embodiment of the present invention, the concentration of the fluorescent dye in the fluorescent dye solution is 1-50 mg/mL. Therefore, the polymer microspheres can wrap more fluorescent dye, and the detection sensitivity is further improved.
According to an embodiment of the present invention, the organic solvent is at least one selected from the group consisting of ethyl acetate, dichloromethane, chloroform, 1, 2-dichloroethane and aromatic hydrocarbons, preferably dichloromethane.
According to the embodiment of the invention, the polymer microsphere is at least one of polystyrene microsphere, polymethyl methacrylate microsphere, polyformaldehyde microsphere and polylactic acid-glycolic acid copolymer microsphere, and the microspheres can be well dispersed in an aqueous solution, and meanwhile, the surface can flexibly modify various groups, thereby facilitating subsequent coupling. Therefore, the near-infrared II-region high-molecular fluorescent microspheres with strong penetrating power, small background interference and strong dispersibility in aqueous solution can be effectively prepared.
According to the embodiment of the invention, the particle size of the polymer microsphere is 20nm-1000 nm. Therefore, the capability of wrapping the fluorescent dye by the polymer microspheres can be further improved, and the detection sensitivity can be further improved.
According to the embodiment of the invention, in the step (2), the polymer microspheres are dispersed in the sodium dodecyl sulfate aqueous solution according to the mass-to-volume ratio of 10-200 mg/ml. Therefore, the polymer microspheres can be stably dispersed in the solution, and can be fully contacted with dichloromethane in the subsequent swelling process, so that the polymer microspheres can be swelled to the maximum extent.
According to an embodiment of the present invention, the volume ratio of the fluorescent dye solution to the microsphere carrier solution is 1: (5-20). Therefore, the appropriate amount of dichloromethane can enable the polymer microspheres to be fully swelled, so that the encapsulation rate of the fluorescent dye is improved, and the quality of the near-infrared II region polymer fluorescent microspheres is improved.
According to the embodiment of the invention, the mass ratio of the fluorescent dye to the polymer microsphere is (0.1-30): 100. Therefore, hundreds of thousands to millions of fluorescent molecules can be wrapped in each microsphere, and the detection sensitivity is greatly improved.
According to an embodiment of the present invention, step (4), the swelling is performed by stirring at 10 to 50 degrees Celsius for 1 to 10 hours. Therefore, the polymer fluorescent microspheres can be fully swelled under the action of dichloromethane, so that the fluorescent dye can be smoothly immersed into nanopores generated in the swelling process of the polymer microspheres.
According to the embodiment of the present invention, in the step (5), the heating temperature is 50 to 90 degrees celsius. Thus, methylene chloride can be completely volatilized in a short time.
In another aspect of the invention, an immunofluorescence chromatography detection system is provided. According to an embodiment of the invention, the system comprises: a fluorescence immunoassay analyzer; and the fluorescence immunoassay test strip. The test strip can generate a fluorescence detection signal, and the signal is analyzed by a fluorescence immunoassay analyzer so as to realize qualitative/quantitative detection of an object to be detected. As mentioned above, the immunofluorescence chromatography detection system provided by the embodiment of the invention has the advantages of high detection accuracy, high sensitivity and high precision.
In yet another aspect, the present invention provides a method for determining the amount of an analyte in a sample. According to an embodiment of the invention, the method comprises: (1) applying said sample to the sample area of said immunofluorescence detection test strip as described previously; (2) determining a fluorescence signal of the immunofluorescence detection test strip; and (3) determining the content of the substance to be detected in the sample based on the fluorescence signal of the immunofluorescence detection test strip. Therefore, the method for determining the content of the substance to be detected in the sample according to the embodiment of the invention has the advantages of strong accuracy, high sensitivity, high precision and simple and convenient operation.
According to an embodiment of the invention, the sample is a serum sample.
According to an embodiment of the invention, the fluorescence signal is determined using a fluorescence immunoassay.
According to an embodiment of the invention, the fluoroimmunoassay analyzer employs a 800nm long pass filter. Thereby, strong fluorescence can be generated, facilitating observation and determination of fluorescence intensity.
According to the embodiment of the invention, in the step (2), the fluorescence signal of the detection line and the fluorescence signal of the quality control line are determined simultaneously, and in the step (3), the content of the analyte in the sample is determined based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the quality control line.
According to the embodiment of the invention, the content of the analyte in the sample is determined based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the quality control line by using a standard curve, wherein the standard curve is obtained by adopting: the contents are respectively determined by standards of 50, 25, 12.5, 6.25, 3.2, 2.0, 1.0, 0.5, 0.2 and 0ng/mL cardiac troponin. Therefore, the accuracy of the detection result is improved.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a schematic structural diagram of an immunofluorescent chromatography test strip according to one embodiment of the present invention;
FIG. 2 is a flow chart of a method for preparing near-infrared region II polymeric fluorescent microspheres according to an embodiment of the present invention;
FIG. 3 is a flow chart of a method for preparing near-infrared region II polymeric fluorescent microspheres according to another embodiment of the present invention;
FIG. 4 is a graph showing the absorption spectrum and emission spectrum of a fluorescent dye used to prepare a near-infrared region II polymeric fluorescent microsphere according to an embodiment of the present invention;
FIG. 5 shows a scanning electron micrograph of a carboxylated polystyrene particle and a carboxylated polystyrene fluorescent microsphere, according to one embodiment of the present invention;
FIG. 6 shows a dispersion of carboxy polystyrene microspheres, a dye solution, and a carboxy polystyrene fluorescent microsphere solution according to one embodiment of the present invention;
FIG. 7 shows a fluorescence photograph and a fluorescence spectrum of a carboxylic polystyrene fluorescent microsphere under the irradiation of 740nm light according to an embodiment of the invention;
FIG. 8 shows a schematic structural diagram of an immunofluorescent-chromatography detection test strip according to another embodiment of the present invention;
FIG. 9 shows a schematic structural diagram and an analysis diagram of a near-infrared fluorescent immune test strip reader according to another embodiment of the invention;
FIG. 10 shows a graph of fluorescence signals according to one embodiment of the invention;
FIG. 11 shows a plot of fluorescence intensity according to one embodiment of the present invention;
FIG. 12 shows a standard curve graph according to an embodiment of the invention; and
FIG. 13 shows an analysis of the results of an immunochromatographic assay according to an embodiment of the present invention together with the results of a clinical assay.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
The invention provides an immunofluorescence chromatography detection test strip, an immunofluorescence chromatography detection system and a method for determining the content of an object to be detected in a sample, which are respectively described in detail below.
Immunofluorescence chromatography detection test paper strip
In one aspect of the invention, the invention provides an immunofluorescence chromatography detection test strip. The immunofluorescence chromatography test strip has the advantages of strong detection accuracy, high sensitivity and high precision. For ease of understanding, the following detailed description will be made in conjunction with fig. 1.
According to an embodiment of the present invention, the immunofluorescence chromatography detection test strip includes:
a body 1, which defines a sample area 100, a binding area 200, a detection area 300 and an adsorption area 400 which are connected in sequence;
the first antibody is labeled by the fluorescent microspheres, the first antibody is coated on the binding region 200, and the first antibody specifically recognizes an object to be detected;
a detection line 310 and a quality control line 320, the detection line 310 and the quality control line 320 being disposed in the detection zone 200, and the detection line 310 being adjacent to the binding zone 200;
a second antibody coated on the detection line 310, wherein the second antibody specifically recognizes the object to be detected; and
and a third antibody coated on the quality control line 320, wherein the third antibody specifically recognizes the first antibody.
For convenience of understanding, the detection principle of the immunofluorescence chromatography detection test strip of the present invention is briefly described below:
the first antibody marked by the fluorescent microspheres on the binding area and the second antibody coated on the detection line (also called T line) are respectively bound with two different sites of the object (antigen) to be detected. For example, for a positive sample, under the capillary force of the adsorption region, the analyte dropped on the sample region is firstly specifically bound with the first antibody labeled by the fluorescent microsphere on the binding region, the fluorescent microsphere-first antibody-antigen passes through the detection line, and the second antibody attached to the detection line is then bound with another site on the antigen surface, so as to form a sandwich structure. The analyte gradually increases with the time, and the fluorescence intensity on the detection line is higher. The fluorescent microsphere labeled first antibody which is not bound on the detection line continues chromatography, and because the third antibody on the quality control line (also called C line) does not interact with the antigen, when the fluorescent microsphere labeled first antibody reaches the quality control line, the fluorescent microsphere labeled first antibody is specifically bound with the third antibody coated on the quality control line and continuously accumulates until color development is realized. The positive results are shown as follows: the detection line and the quality control line are colored; negative results were: the detection line becomes light or disappears, and the quality control line develops color; failure of the quality control line due to no color development.
According to the embodiment of the invention, the object to be tested is cardiac troponin, and the first antibody is cardiac troponin antibody 1(anti-CTNI 1); the second antibody is cardiac troponin antibody 2(anti-CTNI2), the sites of the first antibody and the second antibody which specifically recognize the object to be detected are different, and the third antibody is a second antibody, preferably a goat anti-mouse antibody.
According to the embodiment of the present invention, the fluorescent microsphere is a near-infrared II-region polymeric fluorescent microsphere, and referring to fig. 2 and 3, the near-infrared II-region polymeric fluorescent microsphere is obtained by the following method:
s100: preparation of fluorescent dye solution
According to an embodiment of the present invention, a fluorescent dye is dissolved using a water-immiscible organic solvent to obtain a fluorescent dye solution.
It should be noted that the type of the fluorescent dye is not limited in the present invention, as long as the fluorescent dye can generate a detectable signal in the near-infrared region II. According to a specific embodiment of the present invention, the fluorescent dye has a structure represented by formula I
The inventors have unexpectedly found that the fluorescent dye having the structure shown in formula I has strong absorption at 365nm and 740nm, and can emit fluorescence at 900-1400nm under the excitation light, and the absorption spectrum and the emission spectrum are shown in FIG. 4(A) and FIG. 4(B), respectively. Due to the long excitation (740nm) and emission (900-. However, it is hydrophobic and cannot be directly applied to in vivo imaging. Furthermore, the swelling method is utilized to wrap the fluorescent dye inside the polymer microsphere, the effect is very ideal, the fluorescent quantum efficiency of the microsphere can reach more than 20%, the microsphere can be well dispersed in aqueous solution, and the labeling of various biomacromolecules is facilitated. The near-infrared II-region high-molecular fluorescent microsphere prepared by adopting the fluorescent dye has the characteristics of strong penetrating power and small background interference, and has good application prospects in aspects of living body imaging, biological labeling and the like. Furthermore, the obtained fluorescent microspheres are marked on the antibody, and the antibody is coated on the binding area of the test strip, so that the test strip has the advantages of high detection accuracy, high sensitivity and high precision.
According to an embodiment of the present invention, the concentration of the fluorescent dye in the first prepared fluorescent dye solution may be 1
-50 mg/mL. Therefore, the polymer microspheres can wrap more fluorescent dye, so that the prepared near-infrared II-region polymer fluorescent microspheres can have higher fluorescence intensity during fluorescence detection, and the detection sensitivity is further improved.
According to an embodiment of the present invention, the organic solvent may be at least one selected from the group consisting of ethyl acetate, dichloromethane, trichloromethane, 1, 2-dichloroethane and aromatic hydrocarbons. According to a specific example of the present invention, the organic solvent is preferably dichloromethane. Therefore, the methylene dichloride is adopted to further promote the swelling of the polymer microspheres, so that the encapsulation efficiency of the fluorescent dye is improved.
S200: preparation of microsphere Carrier solution
According to the embodiment of the invention, the polymer microspheres are dispersed in the sodium dodecyl sulfate aqueous solution so as to obtain the microsphere carrier solution.
According to an embodiment of the present invention, the polymer microspheres may be at least one of polystyrene microspheres, polymethyl methacrylate microspheres, polyoxymethylene microspheres, and polylactic acid-glycolic acid copolymer microspheres. Because the interior of the polymer microsphere is hydrophobic, the hydrophobic fluorescent dye can be wrapped in the interior of the polymer microsphere without leakage. And one polymer particle can wrap tens of thousands or even hundreds of thousands of fluorescent molecules, and the outside of the polymer particle can be well dispersed in the aqueous solution due to the action of charges or hydrophilic groups. Therefore, the near-infrared II-region high-molecular fluorescent microsphere with strong penetrating power, small background interference and strong dispersibility in aqueous solution can be effectively prepared by adopting the high-molecular microsphere. According to the embodiment of the present invention, the particle size of the polymer microsphere may be 20nm to 1000 nm. Therefore, the capability of the polymer microspheres to wrap the fluorescent dye can be further improved, so that the prepared near-infrared II-region polymer fluorescent microspheres can have higher fluorescence intensity during fluorescence detection, and the detection sensitivity is further improved. According to an embodiment of the present invention, the concentration of the aqueous solution of sodium dodecyl sulfonate may be 0.25%. The invention adopts the sodium dodecyl sulfate aqueous solution as the emulsifier, so that the polymer microspheres can be better dispersed in the sodium dodecyl sulfate aqueous solution by selecting the sodium dodecyl sulfate aqueous solution with the concentration, and the uniform emulsified mixed solution can be obtained in the subsequent ultrasonic treatment.
According to the specific embodiment of the invention, the polymer microspheres can be dispersed in the sodium dodecyl sulfate aqueous solution according to the mass-to-volume ratio of 10-200 mg/ml. The inventor finds that when the polymer microspheres are dispersed in a sodium dodecyl sulfate aqueous solution according to the mass-to-volume ratio of 10-200mg/ml, the yield of the near-infrared II polymer fluorescent microspheres can be effectively improved, the polymer microspheres can be swelled to the maximum extent in the subsequent swelling process, the capability of the polymer microspheres for wrapping fluorescent dye is further improved, the prepared near-infrared II region polymer fluorescent microspheres can have higher fluorescence intensity during fluorescence detection, and the detection sensitivity can be further improved.
S300: ultrasonic treatment to obtain emulsified mixed liquid
According to an embodiment of the present invention, a fluorochrome solution is mixed with a microsphere carrier solution and subjected to ultrasonic treatment to obtain an emulsified mixed solution.
According to a specific embodiment of the present invention, the volume ratio of the fluorescent dye solution to the microsphere carrier solution may be 1: (5-20). Therefore, the proper amount of dichloromethane in the fluorescent dye solution can enable all the polymer microspheres in the microsphere carrier solution to be fully swelled, so that the fluorescent dye solution can fully enter into the nano holes generated in the swelling process of the polymer microspheres, the wrapping capacity of the polymer microspheres on the fluorescent dye is improved, and finally the high-quality near-infrared II macromolecule fluorescent microspheres are obtained, so that the prepared near-infrared II macromolecule fluorescent microspheres can have higher fluorescence intensity during fluorescence detection, and the detection sensitivity can be further improved.
According to the specific embodiment of the invention, the mass ratio of the fluorescent dye to the polymer microsphere is (0.1-30): 100. Therefore, the fluorescent dye can be further matched with the using amount of the polymer microspheres, the utilization rate of raw materials is improved, and waste of the raw materials is avoided. In addition, the fluorescent dye solution can fully enter into nanopores generated in the subsequent polymer microsphere swelling process by adopting the mass ratio, so that the wrapping capacity of the polymer microspheres on the fluorescent dye is further improved, the prepared near-infrared II-region polymer fluorescent microspheres can have higher fluorescence intensity during fluorescence detection, and the detection sensitivity can be further improved.
S400: swelling the emulsified mixed solution
According to the embodiment of the invention, the emulsified mixed solution is swelled, so that the fluorescent dye solution is immersed into the nano-pores generated in the swelling process of the polymer microspheres.
The inventors found that a fluorescent dye with strong hydrophobicity cannot be directly applied to living body imaging, and hydrophilic modification is required. The modification process is complicated in steps, and the quantum efficiency of the modified fluorescent dye is greatly reduced after the modified fluorescent dye is dispersed in an aqueous solution. The interior of the polymer microsphere is hydrophobic, the hydrophobic fluorescent dye can be stably wrapped in the interior of the polymer microsphere and cannot leak out, one polymer particle can wrap tens of thousands or even hundreds of thousands of fluorescent molecules, and the exterior of the polymer particle can be well dispersed in an aqueous solution due to the action of charges or hydrophilic groups. Meanwhile, various functional groups can be flexibly modified outside the kit, so that the labeling of molecules such as protein, DNA and the like is facilitated. Therefore, the inventor wraps the fluorescent microspheres in the polymer microspheres by adopting a swelling method to obtain the near-infrared II-region polymer fluorescent microspheres, the fluorescent quantum efficiency of the fluorescent microspheres can reach more than 20%, the fluorescent microspheres can be well dispersed in aqueous solution, and various biomacromolecules can be conveniently marked.
According to a specific embodiment of the present invention, the swelling may be accomplished by stirring at 10-50 deg.C for 1-10 hours. Therefore, the polymer fluorescent microspheres can be fully swelled under the action of dichloromethane, so that the fluorescent dye can be smoothly immersed into the nano-pores generated in the swelling process of the polymer microspheres, and the prepared near-infrared II-region polymer fluorescent microspheres can have higher fluorescence intensity during fluorescence detection.
S500: heating to volatilize the organic solvent
According to the embodiment of the invention, the mixture obtained by swelling is heated, and the fluorescent dye is crystallized and separated out and is wrapped in the nanometer hole along with the volatilization of the organic solvent, so that the near-infrared II-region high-molecular fluorescent microsphere is obtained.
According to the specific embodiment of the invention, in the heating process, the organic solvent is slowly volatilized, the nano-pores on the surface of the polymer microsphere are shrunk along with the organic solvent, and meanwhile, the hydrophobic fluorescent dye is crystallized and separated out to form hydrophobic particles which are wrapped in the microsphere. And after the organic solvent is completely volatilized, the near-infrared II-region high-molecular fluorescent microspheres are obtained, and the fluorescent dye wrapped in the near-infrared II-region high-molecular fluorescent microspheres hardly leaks in the aqueous solution.
According to an embodiment of the present invention, the temperature of heating may be 50-90 degrees celsius. Therefore, the organic solvent, preferably dichloromethane, can be quickly and completely volatilized from the solution, and the preparation efficiency of the near-infrared II-region high-molecular fluorescent microspheres is improved.
According to a specific embodiment of the present invention, the heating may be performed by magnetic stirring in a water bath at 50-90 degrees Celsius. Therefore, the volatilization rate of the dichloromethane can be further improved, the dichloromethane can be completely volatilized, and the preparation efficiency of the near-infrared II-region high-molecular fluorescent microspheres is improved.
According to the specific embodiment of the present invention, the preparation method of the near-infrared II-region polymeric fluorescent microsphere may further comprise: and (3) ultrasonically cleaning the obtained near-infrared II-region macromolecular fluorescent microspheres by sequentially adopting ethanol and water.
According to the specific embodiment of the present invention, a specific amount of the near-infrared II-region polymeric fluorescent microspheres obtained by the preparation method of the above embodiment of the present invention is dissolved in dichloromethane, the fluorescence intensity is measured, and the number of the fluorescent dye molecules contained in each particle is calculated according to a standard curve to be about 8 ten thousand. Therefore, compared with the small molecular fluorescent dye, the fluorescent dye can greatly amplify fluorescent signals by using the near-infrared II-region high-molecular fluorescent microspheres as the markers, and the detection sensitivity is improved.
Immunofluorescence chromatography detection system
According to an embodiment of the invention, the system comprises: a fluorescence immunoassay analyzer; and the fluorescence immunoassay test strip. The test strip can generate a fluorescence detection signal, and the signal is analyzed by a fluorescence immunoassay analyzer so as to realize qualitative/quantitative detection of an object to be detected. As mentioned above, the immunofluorescence chromatography detection system provided by the embodiment of the invention has the advantages of high detection accuracy, high sensitivity and high precision.
It will be understood by those skilled in the art that the features and advantages described above for the immunofluorescent chromatography test strip are equally applicable to the immunofluorescent chromatography test system and will not be described in detail herein.
Method for determining content of substance to be detected in sample
In yet another aspect, the present invention provides a method for determining the amount of an analyte in a sample. According to an embodiment of the invention, the method comprises: (1) applying a sample to the sample area of the immunofluorescence detection test strip described previously; (2) determining a fluorescence signal of the immunofluorescence detection test strip; and (3) determining the content of the substance to be detected in the sample based on the fluorescence signal of the immunofluorescence detection test strip. Therefore, the method for determining the content of the substance to be detected in the sample according to the embodiment of the invention has the advantages of strong accuracy, high sensitivity, high precision and simple and convenient operation.
According to an embodiment of the invention, the sample is a serum sample. Therefore, the sampling is convenient, and the accuracy of the detection result is strong.
According to an embodiment of the invention, the fluorescence signal is determined using a fluorescence immunoassay analyzer.
According to an embodiment of the present invention, the fluoroimmunoassay analyzer employs a 800nm long pass filter. Thereby, strong fluorescence can be generated, facilitating observation and determination of fluorescence intensity.
According to the embodiment of the invention, in the step (2), the fluorescence signal of the detection line and the fluorescence signal of the quality control line are determined simultaneously, and in the step (3), the content of the substance to be detected in the sample is determined based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the quality control line.
According to the embodiment of the invention, the content of the substance to be detected in the sample is determined based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the quality control line by using a standard curve, wherein the standard curve adopts the following steps: the contents are respectively determined by standards of 50, 25, 12.5, 6.25, 3.2, 2.0, 1.0, 0.5, 0.2 and 0ng/mL cardiac troponin. Therefore, the accuracy of the detection result is improved.
It will be understood by those skilled in the art that the features and advantages described above for the immunofluorescence chromatography test strip are also applicable to the method for determining the content of the analyte in the sample, and will not be described herein again.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
(1) Synthesis of carboxy polystyrene microsphere
190mL of water was added to a 500mL round bottom flask and stirred at 350rpm for half an hour in a 70 degree water bath. Then 16mg SDS was added as an emulsifier and 0.05g sodium bicarbonate as a buffer. After stirring for an additional 10 minutes, 8mL of styrene and 0.8mL of acrylic acid were added to the flask. After one hour, 0.2g of potassium persulfate was added and the polymerization was carried out for 18 hours under nitrogen. After the reaction is finished, the reaction is carried out by using a reaction mixture of 2: 1(v/v) ethanol and water solution are centrifugally washed for 3 times to obtain the carboxyl polystyrene microsphere, and the scanning electron microscope image of the carboxyl polystyrene microsphere is shown in figure 5 (A). Then, the carboxylated polystyrene microspheres were dispersed in an aqueous SDS solution having a concentration of 0.25% (w/v) to prepare a 30mg/ml carboxylated polystyrene microsphere dispersion, as shown in FIG. 6(A), and stored in a 4-degree refrigerator.
(2) Synthesis of near-infrared II-region carboxyl polystyrene fluorescent microspheres
Dissolving 40mg of near-infrared region II fluorescent dye in 2mL of dichloromethane to obtain a dye solution with the concentration of 20mg/mL, as shown in FIG. 6 (B); taking 20mL of the carboxyl polystyrene microsphere dispersion prepared in the step (1) into a 50mL flat-bottom conical flask, and carrying out ultrasonic treatment for 5min to uniformly mix; adding the 2mL of dye solution into a flat-bottom conical flask, ultrasonically mixing the dye solution with 20mL of carboxyl polystyrene microsphere dispersion liquid, and magnetically stirring the mixture for 6 hours at 40 ℃ so as to fully swell the carboxyl polystyrene microspheres, wherein the dye solution enters micropores generated after the carboxyl polystyrene microspheres are swelled; magnetically stirring the mixture solution in a water bath at 50 ℃ overnight to completely volatilize dichloromethane in the mixture solution; and (3) carrying out centrifugal separation on the obtained product, then carrying out ultrasonic cleaning for 3 times by using ethanol, and then carrying out ultrasonic cleaning for a plurality of times by using water until the supernatant solution after centrifugation almost does not contain fluorescent dye, thus obtaining the carboxyl polystyrene fluorescent microsphere product in the near-infrared region II. The near-infrared II-region carboxyl polystyrene fluorescent microsphere product was dispersed in water (5% w/v) and stored in a 4-degree refrigerator, as shown in FIG. 6 (C).
(3) Evaluation of the carboxyl polystyrene fluorescent microsphere product obtained
1) FIG. 6(A) shows that the carboxylated polystyrene particles dispersed in an aqueous SDS solution appear white; FIG. 6(B) shows that the fluorescent dye dissolved in methylene chloride is cyan; and FIG. 6(C) shows that the carboxyl polystyrene fluorescent microsphere product wrapped with the fluorescent dye is cyan. Thus, the change in color indicates that the fluorescent dye has been successfully encapsulated within the interior of the microsphere without a large change in the properties of the particle surface. And it can be seen from fig. 6(C) that the synthesized near-infrared II-region carboxy polystyrene fluorescent microspheres can be stably dispersed in an aqueous solution.
2) And scanning the carboxyl polystyrene fluorescent microsphere product by an electron microscope, wherein the scanning electron microscope image is shown as a figure 5 (B). As can be seen from FIG. 5, the morphology of the particles before and after the carboxyl polystyrene particles wrap the fluorescent dye is not significantly changed, and the synthesized fluorescent microspheres have uniform size and no aggregation phenomenon.
3) The fluorescence photograph and the fluorescence spectrogram obtained by irradiating the carboxyl polystyrene fluorescent microsphere product with 740nm excitation light are respectively shown in fig. 7(A) and 7 (B). The figure shows that the fluorescent microsphere emits strong fluorescence at 800-1400nm, and the quantum efficiency is up to 25% by measurement.
(4) Application of carboxyl polystyrene fluorescent microsphere product
1) Near-infrared II-region carboxyl polystyrene fluorescent microsphere coupled antibody
The near-infrared II-region carboxypolystyrene fluorescent microspheres prepared above were dispersed with MES (10mM pH 6.2) as a 1% (w/v) homogeneous dispersion. EDC (5mg/mL) and sulfo-NHS (5mg/mL) were added to the dispersion and activated for 15 min. The supernatant was discarded by centrifugation, redispersed in MES (10mM pH 6.5), and 0.4mg/mL anti-CTNI1 murine mab was added to the solution and the reaction stirred for 2 h. The particles were dispersed in 20mM PBS (0.5% casein, 2.5% BSA, 1% sucrose, 2% PEG-2000 and 0.03 wt% NaN) by ultrasonic dispersion after centrifugation and discarding the supernatant3pH 8.0), the antibody-bound fluorescent microsphere dispersion was prepared at a solid content of 1% (w/v) and stored in a 4-degree refrigerator for later use.
2) Preparation of immunofluorescence chromatography test paper strip
The immunofluorescence chromatography test strip mainly comprises five parts: comprising a plastic support shell, a sample pad, a conjugate pad, an adsorption pad and a nitrocellulose membrane, as shown in figure 8. Before the test paper is assembled into a complete test paper, all parts of the test paper need to be pretreated correspondingly: first, the sample pad was soaked in sample pad buffer (1% BSA, 2% Triton X-100, 2% PVP 40,20mM Tris-HCl,50mM NaAc) for 1h and then dried in a 37 deg.C vacuum oven overnight. And spraying the marked fluorescent microsphere (marked anti-CTNI1) dispersion liquid on the sample pad through a special nozzle for immunochromatography to prepare a bonding pad, and then freeze-drying for 10h for later use. Then, anti-CTNI2(1.0mg/mL, 75. mu.L) and goat anti-mouse (0.5mg/mL, 75. mu.L) solutions were uniformly sprayed on the nitrocellulose membrane by a dedicated streaker for immunochromatography, forming a detection line (T-line) and a control line (C-line), respectively, and treated in a 37 ℃ oven overnight. Then, the sample pad, the combination pad, the nitrocellulose membrane and the adsorption pad are fixed on the hard pasting paperboard along the shaft in order, wherein the adsorption pad and the NC membrane, the NC membrane and the combination pad, and the combination pad and the sample pad are mutually overlapped to be in close contact, and the smooth liquid flow in the detection process is ensured. Finally, the assembled chromatographic plate is cut into test strips with the width of 4mm by an immunochromatographic test strip cutting machine, and the test strips are stored in a sealed aluminum packaging bag for storage.
3) Establishment of fluorescence immunoassay instrument
The fluorescent signal generated by the test strip is observed and analyzed by a near-infrared fluorescence immune test strip reader, the structure of which is shown in fig. 9(A), and the analysis method is shown in fig. 9 (B).
Specifically, the light source adopts 365nm 1W LED, and divergent light of the LED is collimated through the combined lens. Fluorescence is excited and collected by a confocal optical system, when the immunochromatography test paper is driven by a stepping motor conveyor belt to scan through an excitation light source, fluorescence emitted by rare earth fluorescent microspheres on a C line and a T line on the immunochromatography test paper passes through a long-wave pass color filter (800nm) and then is focused on a photoelectric photosensitive panel, and the silicon photoelectric cell has high response sensitivity to light waves of 500nm-1100 nm. The A/D chip collects the voltage signal of the photocell and finally obtains a scanning fluorescence curve. And the lower computer processor respectively calculates the integral areas of the corresponding peaks of the C line and the T line, then calculates the ratio of the peak areas of the T line and the C line, fits the concentration of the standard sample to obtain a standard curve, and stores the standard curve into the immunoassay analyzer. In the process of detecting the actual sample, the instrument measures the peak area ratio of the T line to the C line, and the concentration of the cardiac troponin in the sample is reversely deduced according to the standard curve.
4) Detection of
In order to preliminarily evaluate the detection effect of the prepared immunofluorescence test strip, firstly, a cardiac troponin sample prepared by primary bovine serum is used for detection, after 0ng/mL and 20ng/mL of the cardiac troponin sample are respectively added, strong fluorescence emitted by a C line and a T line on the immunochromatography test strip can be clearly seen through an InGaAs camera under the irradiation of laser light with 808nm (as shown in figure 10). When the concentration of cardiac troponin is 0, the fluorescence on the T line is very weak; when the concentration of the cardiac troponin reaches 20ng/mL, strong fluorescence is displayed on a T line, which can preliminarily prove the effectiveness of the prepared fluorescent test strip.
Drawing a standard curve: the newborn cattle blood relative is used for preparing 50, 25, 12.5, 6.25, 3.2, 2.0, 1.0, 0.5, 0.2 and 0ng/mL cardiac troponin. A75 μ L sample was applied to the sample pad and the standard sample and control sample were moved towards the adsorption pad by capillary force. After 15min, the reagent card is placed into a fluorescence immunoassay analyzer to read the fluorescence signal, and each sample is detected three times. And performing curve fitting on the obtained fluorescence signal and the concentration value of the standard sample, and inputting the curve to an analyzer for storage. The fluorescence intensity is shown in FIG. 11, and the standard curve is shown in FIG. 12.
And (3) serum detection: to the sample pad, 75 μ L of human serum was added, and the standard sample and the control sample were moved toward the adsorption pad by capillary force. After 15min, the reagent card is placed into a fluorescence immunoassay analyzer to read the concentration of the cardiac troponin in the sample. Fig. 13 shows a comparison between the test strip of the present invention and the clinical test results, and it can be seen that the test results of the test strip of the present invention are substantially the same as the clinical test results, thereby indicating that the test results of the test strip of the present invention are accurate.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. An immunofluorescence chromatography test strip, which is characterized by comprising:
the device comprises a body, a sample area, a binding area, a detection area and an adsorption area, wherein the sample area, the binding area, the detection area and the adsorption area are sequentially connected;
the first antibody is labeled by fluorescent microspheres, coats the binding region and specifically recognizes an object to be detected;
the detection line and the quality control line are arranged in the detection area, and the detection line is close to the binding area;
a second antibody coated on the detection line, the second antibody specifically recognizing the test substance; and
a third antibody coated on the quality control line, the third antibody specifically recognizing the first antibody,
the fluorescent microspheres are near-infrared II-region high-molecular fluorescent microspheres, and the near-infrared II-region high-molecular fluorescent microspheres are obtained by the following method:
(1) dissolving a fluorescent dye in dichloromethane to obtain a dye solution with the concentration of 20mg/mL,
the fluorescent dye has a structure shown in formula I
(2) Adding 190mL of water into a flask, carrying out 70-degree water bath, adding 16mg of SDS (sodium dodecyl sulfate) serving as an emulsifier and 0.05g of sodium bicarbonate serving as a buffer reagent, stirring, adding 8mL of styrene and 0.8mL of acrylic acid into the flask, adding 0.2g of potassium persulfate after one hour, carrying out polymerization reaction for 18 hours under the protection of nitrogen, carrying out centrifugal washing by using ethanol and an aqueous solution after the reaction is finished to obtain carboxyl polystyrene microspheres, and dispersing the carboxyl polystyrene microspheres in the SDS aqueous solution to obtain a microsphere carrier solution, wherein the particle size of the carboxyl polystyrene microspheres is 20-1000 nm;
(3) adding 2mL of the fluorescent dye solution with the concentration of 20mg/mL into a flask, ultrasonically mixing the fluorescent dye solution with 20mL of the microsphere carrier solution with the concentration of 30mg/mL, and magnetically stirring the mixture for 6 hours at 40 ℃; magnetically stirring the mixture solution in a water bath at 50 ℃ to completely volatilize dichloromethane in the mixture solution; and carrying out centrifugal separation on the obtained product, then carrying out ultrasonic cleaning by using ethanol, and then carrying out ultrasonic cleaning by using water so as to obtain the near-infrared II-region macromolecular fluorescent microsphere.
2. The immunofluorescence chromatography detection test strip according to claim 1, wherein, the object to be detected is cardiac troponin,
the first antibody is cardiac troponin antibody 1;
the second antibody is a cardiac troponin antibody 2,
the third antibody is a secondary antibody.
3. The immunofluorescence chromatography detection test strip of claim 1, wherein the third antibody is a goat anti-mouse antibody.
4. An immunofluorescence chromatography detection system, comprising:
a fluorescence immunoassay analyzer; and
the immunofluorescence chromatography detection test strip of any one of claims 1 to 3.
5. A method for determining the amount of a test agent in a sample for non-diagnostic purposes, comprising:
(1) applying the sample to a sample zone of the immunofluorescent chromatographic test strip of any one of claims 1 to 3;
(2) determining a fluorescence signal of the immunofluorescence chromatography detection test strip; and
(3) and determining the content of the substance to be detected in the sample based on the fluorescence signal of the immunofluorescence chromatography detection test strip.
6. The method of claim 5, wherein the sample is a serum sample.
7. The method of claim 5, wherein the fluorescent signal is determined using a fluorescence immunoassay.
8. The method of claim 7, wherein the fluoroimmunoassay analyzer employs a 800nm long pass filter.
9. The method of claim 5, wherein in step (2), the fluorescence signal of the detection line and the fluorescence signal of the quality control line are determined simultaneously,
in the step (3), the content of the analyte in the sample is determined based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the quality control line.
10. The method of claim 9, wherein the amount of the analyte in the sample is determined based on the ratio of the fluorescence signal of the detection line to the fluorescence signal of the quality control line using a standard curve.
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| CN201710608436.2A CN107632159B (en) | 2017-07-24 | 2017-07-24 | Immunofluorescence chromatography detection test strip, immunofluorescence chromatography detection system and method for determining content of substance to be detected in sample |
| US16/632,889 US11668706B2 (en) | 2017-07-24 | 2017-11-06 | Near-infrared II polymer fluorescent microsphere and method for preparing same |
| US16/632,890 US20200166501A1 (en) | 2017-07-24 | 2017-11-06 | Test strip for short-wave near infrared immunofluorescence chromatographic detection and use thereof |
| PCT/CN2017/109555 WO2019019471A1 (en) | 2017-07-24 | 2017-11-06 | Test strip for short-wave near infrared immunofluorescence chromatographic detection and use thereof |
| EP17918876.8A EP3658916A4 (en) | 2017-07-24 | 2017-11-06 | Test strip for short-wave near infrared immunofluorescence chromatographic detection and use thereof |
| EP17918703.4A EP3658647A4 (en) | 2017-07-24 | 2017-11-06 | Near-infrared ii polymer fluorescent microsphere and method for preparing same |
| PCT/CN2017/109556 WO2019019472A1 (en) | 2017-07-24 | 2017-11-06 | Near-infrared ii polymer fluorescent microsphere and method for preparing same |
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| CN109884294B (en) * | 2019-04-03 | 2022-09-13 | 深圳无微华斯生物科技有限公司 | Preparation method of high-precision fluorescent immune test strip |
| CN110596378A (en) * | 2019-08-05 | 2019-12-20 | 天津一安生物技术有限公司 | Multichannel universal chromatography method for detecting small molecules, test strip and kit |
| CN110455766A (en) * | 2019-09-03 | 2019-11-15 | 莎穆(上海)生物科技有限公司 | Detection method based on two area's fluorescence imaging of near-infrared for the strip of lateral flow immune detection, detection device and non-diagnostic purpose |
| WO2021128065A1 (en) * | 2019-12-25 | 2021-07-01 | 广州菲康生物技术有限公司 | Human fgf-23 fluorescence immunochromatographic test paper and human fgf-23 fluorescence immunochromatographic test kit |
| CN114231401B (en) * | 2022-02-21 | 2022-05-03 | 北京芯迈微生物技术有限公司 | Nucleic acid detection kit and detection method |
| CN115932249A (en) * | 2022-12-30 | 2023-04-07 | 杭州纽太生物科技有限公司 | Real-time calibration immunochromatography detection method |
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