Test Strip For Short-wave Near Infrared Immunofluorescence Chromatographic Detection And Use Thereof

Abstract

Provided are a test strip for short-wave near infrared immunofluorescence chromatographic detection, a system for immunofluorescence chromatographic detection and a method for quantifying an analyte in a sample. The test strip for short-wave near infrared immunofluorescence chromatographic detection includes: a body, defining a sample region, a binding region, a detecting region and an adsorbing region connected with one another sequentially; a first antibody, labeled with a fluorescent microsphere, coated on the binding region and configured to specifically recognize an analyte; a detecting line and a quality control line, located in the detecting region, wherein the detecting line closes to the binding region; a second antibody, coated on the detecting line and configured to specifically recognize the analyte; and a third antibody, coated on the quality control line and configured to specifically recognize the first antibody, in which the fluorescent microsphere is a near-infrared II polymer fluorescent microsphere.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priorities to and benefits of Chinese Patent Application Nos. 201710607567.9, 201710607934.5 and 201710608436.2, all filed with the State Intellectual Property Office of P. R. China on Jul. 24, 2017, the entire contents of which are incorporated herein by reference.

FIELD

[0002] The present disclosure relates to the field of biotechnology, in particular, to a test strip for short-wave near infrared immunofluorescence chromatographic detection, a system for immunofluorescence chromatographic detection and a method for quantifying an analyte in a sample.

BACKGROUND

[0003] A polymer fluorescent microsphere, as a special function microsphere, is capable of encapsulating tens of thousands to hundreds of thousands of fluorescent molecules for one microsphere, such that labeling efficiency and resistance to photobleaching of the fluorescent molecules are both enhanced, thereby improving sensitivity of fluorescence detection greatly. Besides, the polymer fluorescent microsphere is allowed to be modified from the outside with one or more functional groups (such as a carboxy group, an amino group and an aldehyde group) in a very flexibly way, which benefits for covalent coupling to a protein (such as an antibody) and improvement of stability and labeling efficiency of an labeling agent. At present, the polymer fluorescent microsphere has been widely used in labeling, tracing, detecting, imaging, enzyme immobilizing, medical immunology, high-throughput drug screening and so on. However, the traditional polymer fluorescent microsphere emits lights merely in a visible region with an emitting wavelength below 780 nm, resulting in poor penetrability and intense background fluorescence when applied in imaging of living organisms, cells or tissues, diagnosing in vitro, and so on. Meantime, since the traditional polymer fluorescent microsphere is generally with low quantum efficiency and relative closer interval (around 20 nm) between an exciting wavelength and an emitting wavelength, which results in poor analysis sensitivity, there exits high demand on a color filter for the fluorescence detection.

[0004] Therefore, there is still a need to improve the polymer fluorescent microsphere.

SUMMARY

[0005] Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent.

[0006] An object of the present disclosure is to provide a test strip for short-wave near infrared immunofluorescence chromatographic detection, a system for immunofluorescence chromatographic detection and a method for quantifying an analyte in a sample. The test strip for short-wave near infrared immunofluorescence chromatographic detection proposed by the present disclosure includes an antibody labeled with a fluorescent microsphere which has advantageous characteristics of high quantum efficiency, an exciting wavelength less than 1000 nm, such as at 365 nm or 740 nm, and an emitting wavelength at 1000 nm to 1700 nm, and strong penetrability and low background interference during fluorescence detection, thus improving accuracy, sensitivity and precision of detected results.

[0007] In a first aspect, the present disclosure provides in embodiments a test strip for short-wave near infrared immunofluorescence chromatographic detection, including:

[0008] a body, defining a sample region, a binding region, a detecting region and an adsorbing region connected with one another sequentially;

[0009] a first antibody, labeled with a fluorescent microsphere emitting a wavelength in a range of 1000 nm to 1700 nm under an excitation light less than 1000 nm, coated on the binding region and configured to specifically recognize an analyte;

[0010] a testing line and a quality control line, located in the detecting region, in which the testing line closes to the binding region;

[0011] a second antibody, coated on the testing line and configured to specifically recognize the analyte; and

[0012] a third antibody, coated on the quality control line and configured to specifically recognize the first antibody,

[0013] wherein the fluorescent microsphere is a near-infrared II polymer fluorescent microsphere prepared by the following steps:

[0014] 1) dissolving a fluorochrome in a water-immiscible organic solvent, thus obtaining a fluorochrome solution;

[0015] 2) distributing a polymer microsphere into a sodium dodecyl sulfonate solution, thus obtaining a microsphere solution with the polymer microsphere as a carrier for the fluorochrome;

[0016] 3) subjecting a first mixture of the fluorochrome solution and the microsphere solution to ultrasonic treatment, thus obtaining an emulsion;

[0017] 4) swelling the emulsion such that the fluorochrome solution enters nanopores formed during swelling of the polymer microsphere, thus obtaining a second mixture; and

[0018] 5) heating the second mixture to volatilize the organic solvent, such that the fluorochrome is crystallized out and encapsulated in the nanopores, thus obtaining the near-infrared II polymer fluorescent microsphere.

[0019] In some embodiments of the present disclosure, the fluorochrome is selected from the group consisting of organic molecules including those shown as formula (I), formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earth nanoparticles.

[0020] In embodiments of the present disclosure, the formula (I) is 4,8-(5-(9,9-di(6-bromohexyl)-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,- 4]dioxine)-1H,5H-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole); the formula (II) is 4,8-(5-(9,9-dihexyl-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,4- ]dioxine)-1H,5H-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole); and the formula (III) is 2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c']bis([1,2,5- ]thiadiazole), thus improving accuracy, sensitivity and precision of the result detected by the present test strip

##STR00001##

[0021] In some embodiments of the present disclosure, the analyte is cardiac troponin, the first antibody is an antibody I against cardiac troponin, the second antibody is an antibody II against cardiac troponin, and the third antibody is a secondary antibody, preferably a goat-anti-mouse antibody, in which the first antibody recognizes the analyte at a first site different from a second site recognized by the second antibody, thus improving accuracy, sensitivity and precision of the result detected by the present test strip.

[0022] In some embodiments of the present disclosure, the fluorochrome in the fluorochrome solution has a concentration of 1 mg/mL to 50 mg/mL, so that the polymer microsphere is capable of encapsulating more fluorochrome, thereby further improving sensitivity of fluorescence detection.

[0023] In some embodiments of the present disclosure, the organic solvent is at least one selected from the group consisting of ethyl acetate, dichloromethane, trichloromethane, 1,2-dichloroethane and aromatic hydrocarbons, preferably dichloromethane.

[0024] In some embodiments of the present disclosure, the polymer microsphere is at least one selected from the group consisting of polystyrene microspheres, poly (methyl methacrylate) microspheres, polyformaldehyde microspheres and poly (lactic acid-co-glycolic acid) microspheres which can be well distributed in an aqueous solution and allows to be modified at its surface with different radicals in a flexible way, thereby facilitating to subsequent coupling. Therefore, the near-infrared II polymer fluorescent microsphere can be obtained effectively with strong penetrability, low background interference and excellent dispersibility in the aqueous solution.

[0025] In some embodiments of the present disclosure, the polymer microsphere has a particle size of 20 nm to 1000 nm, so that the polymer microsphere is capable of encapsulating more fluorochrome, thereby further improving sensitivity of fluorescence detection.

[0026] In some embodiments of the present disclosure, in the step 2), the polymer microsphere is distributed into the sodium dodecyl sulfonate solution in a mass/volume ratio of 10 mg/mL to 200 mg/mL, thereby not only guaranteeing the polymer microsphere to well disperse in the sodium dodecyl sulfonate solution, but also allowing the polymer microsphere to be fully in contact with dichloromethane during the subsequent swelling, such that the polymer microsphere can be swelled to a maximal extent.

[0027] In some embodiments of the present disclosure, the first mixture includes the fluorochrome solution and the microsphere solution in a volume ratio of 1:5 to 1:20, such that dichloromethane is in a proper amount for swelling the polymer microspheres thoroughly, thus further increasing encapsulation efficiency and enhancing weight of the near-infrared II polymer fluorescent microsphere.

[0028] In some embodiments of the present disclosure, in the step 3), the first mixture includes the fluorochrome and the polymer microsphere in a mass ratio of 0.1:100 to 30:100. Therefore, each microsphere is capable of encapsulating tens of thousands to hundreds of thousands of fluorescent molecules, thereby improving sensitivity of fluorescence detection greatly.

[0029] In some embodiments of the present disclosure, in the step 4), the emulsion is swelled at 10.degree. C. to 50.degree. C. under stirring for 1 hour to 10 hours, such that the polymer microsphere can be swelled sufficiently in the presence of dichloromethane, which ensures the fluorochrome entering nanopores formed during swelling of the polymer microsphere successfully.

[0030] In some embodiments of the present disclosure, in the step 5), the second mixture is heated at a temperature of 50.degree. C. to 90.degree. C., such that dichloromethane can be volatilized completely in a short time period.

[0031] In another aspect, the present disclosure provides in embodiments a system for immunofluorescence chromatographic detection, including a fluorescence immunity analyzer, and a test strip for short-wave near infrared immunofluorescence chromatographic detection described in the first aspect. As the present test strip can provide a fluorescence signal which can be analyzed by the fluorescence immunity analyzer, an analyte applied to the test strip can be detected qualitatively or quantitatively. Therefore, use of the system for the immunofluorescence chromatographic detection according to embodiments of the present embodiments contributes to obtaining a detection result with high accuracy, sensitivity and precision.

[0032] In still another aspect, the present disclosure provides in embodiments a method for quantifying an analyte in a sample, including 1) applying the sample to a sampling region of a test strip for short-wave near infrared immunofluorescence chromatographic detection described in the first aspect; 2) determining a fluorescence signal generated in the test strip for the immunofluorescence chromatographic detection; and 3) quantifying the analyte in the sample based on the fluorescence signal determined, thus contributing to obtaining a detection result with high accuracy, sensitivity and precision, in a simple way.

[0033] In an embodiment of the present disclosure, the sample is serum.

[0034] In an embodiment of the present disclosure, the fluorescence signal is determined by a fluorescence immunity analyzer.

[0035] In an embodiment of the present disclosure, the fluorescence immunity analyzer is equipped with a color filter at a wavelength of 800 nm, thus benefiting for obtaining fluorescence with high intensity, and facilitating to observing and quantifying the fluorescence obtained.

[0036] In an embodiment of the present disclosure, the method for quantifying the analyte in the sample further includes determining fluorescence signals in both a testing line and a quality control line in the step 2); and quantifying the analyte in the sample based on a ratio of the fluorescence signal generated in the testing line to the fluorescence signal generated in the quality control line.

[0037] In an embodiment of the present disclosure, the method for quantifying the analyte in the sample further includes quantifying the analyte in the sample by means of a standard curve, based on the ratio of the fluorescence signal generated in the testing line to the fluorescence signal generated in the quality control line, in which the standard curve is created with serial cardiac troponin standards in concentrations of 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL, thus contributing to improving accuracy of a detected result.

DESCRIPTION OF DRAWINGS

[0038] The foregoing and/or additional aspects and advantages of the present disclosure will become apparent and be readily understood by combining the description of the embodiments with the drawings.

[0039] FIG. 1 is a structural representation showing a test strip for short-wave near infrared immunofluorescence chromatographic detection according to embodiments of the present disclosure.

[0040] FIG. 2 is a flow chart showing a method for preparing a near-infrared II polymer fluorescent microsphere according to embodiments of the present disclosure.

[0041] FIG. 3 is a flow chart showing a method for preparing a near-infrared II polymer fluorescent microsphere according to other embodiments of the present disclosure.

[0042] FIG. 4 shows absorption spectrums of and fluorescence spectrums emitted respectively by fluorochrome represented by formula (I), (II) or (III) which is contained in near-infrared II polymer fluorescent microspheres according to embodiments of the present disclosure.

[0043] FIG. 5 shows scanning electron microscope photographs of carboxylic polystyrene pellets and carboxylic polystyrene fluorescent microspheres according to embodiments of the present disclosure.

[0044] FIG. 6 are schematic graphs showing a carboxylic polystyrene microsphere dispersion, a fluorochrome solution and a carboxylic polystyrene fluorescent microsphere solution respectively.

[0045] FIG. 7 shows fluorescent photographs and fluorescent spectrums of carboxylic polystyrene fluorescent microspheres under irradiation with an excitation light at a wavelength of 740 nm according to embodiments of the present disclosure.

[0046] FIG. 8 is a structural representation showing a test strip for short-wave near infrared immunofluorescence chromatographic detection according to other embodiments of the present disclosure.

[0047] FIG. 9 is a structural representation showing a system for immunofluorescence chromatographic detection according to embodiments of the present disclosure and a fluorescent spectrum of an analyte in a sample detected by the system.

[0048] FIG. 10 are fluorescent photographs of cardiac troponin respectively at concentrations of 0 ng/mL and 20 ng/mL detected by test strips for immunofluorescence chromatographic detection according to embodiments of the present disclosure.

[0049] FIG. 11 are fluorescent spectrums of cardiac troponin at concentrations from 0 ng/mL to 50 ng/mL in a testing line and in a quality control line respectively according to an embodiment of the present disclosure.

[0050] FIG. 12 are fluorescent spectrums of cardiac troponin at concentrations from 0 ng/mL to 80 ng/mL in a testing line and in a quality control line respectively according to another embodiment of the present disclosure.

[0051] FIG. 13 is illustrative graphs showing standard curves of cardiac troponin according to embodiments of the present disclosure.

[0052] FIG. 14 is illustrative graphs showing comparison between results obtained by the present test strips for short-wave near infrared immunofluorescence chromatographic detection according to embodiments of the present disclosure and results obtained from clinical detections.

DETAILED DESCRIPTION

[0053] The exemplary embodiments of the present disclosure are described in detail below, which are intended to be illustrative of the disclosure and are not to be construed to limit the disclosure.

[0054] It should be noted that the terms "first" and "second" are just intended to be illustrative and are not to be construed as indicating or anticipating a relative importance or the number of technical features indicated. Thus, a feature that is defined as "first" or "second" may expressly or implicitly include one or more features. Further, throughout the description of the present disclosure, term "plural" includes two or more, unless otherwise specified.

[0055] The present disclosure is accomplished by the present inventors based on the following discoveries.

[0056] Because of disadvantageous characteristics of poor penetrability and intense background fluorescence, low quantum efficiency and relative closer interval between an exciting wavelength and an emitting wavelength, the existing method for fluorescence detection using the traditional polymer fluorescent microsphere is in low sensitivity and with high demand on a color filter used. The near-infrared fluorescent microsphere, especially the near-infrared II fluorescent microsphere has advantageous characteristics of strong penetrability and low background interference during the fluorescence detection, thereby having promising prospects in terms of live imaging, biolabeling detection and so on. However, there still exist limitations on the existing method for preparing the near-infrared II fluorescent microsphere, for example, a hydrophobic fluorochrome cannot be directly applied in live imaging because of strong hydrophobicity per se, and thus requiring hydrophilic modification in advance, which not only involves tedious processes, but also provides a modified fluorochrome with significantly decreased quantum efficiency after dissolved in the aqueous solution, thus causing an adverse effect to sensitivity of the fluorescence detection.

[0057] In order to overcome the above disadvantages, the present inventors surprisingly find that the method including encapsulating the fluorochrome selected from the group consisting of organic molecules including those shown as formula (I), formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earth nanoparticles within the polymer microsphere by swelling achieves a desirable effect with high quantum efficiency of 25% or more and good dispersibility in an aqueous solution, thereby facilitating labeling detection of various biological macromolecule, with wide applicability for different fluorochrome. Further, the present inventors also find that when a binding region of a test strip is coated with an antibody labeled with the near-infrared II fluorescent microsphere prepared by the above method, the result detected by the test strip can be highly accurate, sensitive and precise

##STR00002##

[0058] According to some embodiments of the present disclosure, provided are a test strip for short-wave near infrared immunofluorescence chromatographic detection, a system for immunofluorescence chromatographic detection and a method for quantifying an analyte in a sample, which will be described in detail in the following description.

[0059] A Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0060] In one aspect of the present disclosure, provided is a test strip for short-wave near infrared immunofluorescence chromatographic detection, by which an analyte in a sample can be detected accurately, sensitively and precisely. To facilitate understanding, the test strip for short-wave near infrared immunofluorescence chromatographic detection is described by reference to FIG. 1.

[0061] In embodiments of the present disclosure, the test strip for short-wave near infrared immunofluorescence chromatographic detection includes:

[0062] a body 1, defining a sample region 100, a binding region 200, a detecting region 300 and an adsorbing region 400 connected with one another sequentially;

[0063] a first antibody, labeled with a fluorescent microsphere emitting a wavelength in a range of 1000 nm to 1700 nm under an excitation light less than 1000 nm, coated on the binding region 200 and configured to specifically recognize an analyte;

[0064] a testing line 310 and a quality control line 320, located in the detecting region 300, in which the testing line 310 closes to the binding region 200;

[0065] a second antibody, coated on the testing line 310 and configured to specifically recognize the analyte; and

[0066] a third antibody, coated on the quality control line 320 and configured to specifically recognize the first antibody.

[0067] To facilitate understanding, the principle of detection by the present test strip for short-wave near infrared immunofluorescence chromatographic detection is described as follows.

[0068] The first antibody labeled with the fluorescent microspheres which is coated on the binding region and the second antibody which is coated on the testing line (refer to a T line) bind to two different sites in an analyte (refer to an antigen) respectively. On the above basis, a sample containing an analyte moves toward the direction of the adsorbing region under capillary force after applied to the sample region and arrives at the bonding region, at which the analyte is specifically bound to the first antibody firstly, thereby obtaining a first complex composed of fluorescent microsphere-first antibody-antigen. Next, the first complex obtained continues to move toward the direction of the adsorbing region under capillary force and arrives at the testing line in the detecting region, at which the first complex is specifically bound to the second antibody, thereby obtaining a second complex composed of fluorescent microsphere-first antibody-antigen-second antibody in a sandwich structure, in which the antigen contains two different sites bound by the second antibody and the first antibody respectively. As the second complex formed at the testing line accumulates over time, the fluorescence generated in the testing line is of an increased intensity. On the other hand, those first antibody labeled with the fluorescent microspheres which is not bound to the analyte also moves toward the direction of the adsorbing region under capillary force and arrives at the quality control line (refer to a C line), at which the first antibody is specifically bound to the third antibody (a secondary antibody specifically recognizing the first antibody) since there exists no interaction between the analyte and the third antibody, thereby obtaining a third complex between the first antibody and the third antibody. As the third complex formed at the quality control line accumulates over time, the fluorescence generated in the quality control line is of an increased intensity till color development. A positive result is demonstrated by color development at both the testing line and the quality control line. A negative result is demonstrated by weak or none color development at the testing line but color development at the quality control line. None color development at the quality control line indicates that the test trip for the immunofluorescence chromatographic detection is invalid.

[0069] In embodiments of the present disclosure, the analyte is cardiac troponin, the first antibody is an antibody I against cardiac troponin (anti-CTNI1) (19C7, HyTest Ltd.), the second antibody is an antibody II against cardiac troponin (anti-CTNI2) (16A11, HyTest Ltd.), and the third antibody is a secondary antibody, preferably a goat-anti-mouse antibody, in which the first antibody recognizes the analyte at a first site different from a second site recognized by the second antibody.

[0070] In embodiments of the present disclosure, the fluorescent microsphere labeled to the first antibody is a near-infrared II polymer fluorescent microsphere prepared by the following steps:

[0071] 1) dissolving a fluorochrome in a water-immiscible organic solvent, thus obtaining a fluorochrome solution; 2) distributing a polymer microsphere into a sodium dodecyl sulfonate solution, thus obtaining a microsphere solution with the polymer microsphere as a carrier for the fluorochrome; 3) subjecting a first mixture of the fluorochrome solution and the microsphere solution to ultrasonic treatment, thus obtaining an emulsion; 4) swelling the emulsion such that the fluorochrome solution enters nanopores formed during swelling of the polymer microsphere, thus obtaining a second mixture; and 5) heating the second mixture to volatilize the organic solvent, such that the fluorochrome is crystallized out and encapsulated in the nanopores, thus obtaining the near-infrared II polymer fluorescent microsphere.

[0072] According to the method for preparing the near-infrared II polymer fluorescent microsphere in embodiments described above, the fluorochrome is dissolved in the organic solvent at first, thus obtaining the fluorochrome solution; meanwhile the polymer microsphere is distributed into the sodium dodecyl sulfonate solution, thus obtaining a microsphere solution with the polymer microsphere as a carrier for the fluorochrome; subsequently the first mixture of the fluorochrome solution and the microsphere solution is subjected to ultrasonic treatment, swelling and heating, such that the fluorochrome can be successfully encapsulated in the microsphere, thus obtaining the near-infrared II polymer fluorescent microsphere.

[0073] According to the embodiments described above, in a simple and rapid way, the near-infrared II polymer fluorescent microsphere is prepared with good dispersibility in an aqueous solution, high quantum efficiency up to 25%, and relative broader interval between an exciting wavelength less than 1000 nm, such as at 365 nm or 740 nm, and an emitting wavelength at 1000 nm to 1700 nm. As a result, such a method not only can be widely used for various different fluorochrome, but also provides the near-infrared II polymer fluorescent microsphere with strong penetrability and low background interference during the fluorescence detection, which can extremely magnify the fluorescent signal and thus improve the sensitivity of the fluorescence detection, thereby having promising prospects in terms of live imaging, biolabeling detection and so on.

[0074] In embodiments of the present disclosure, the preparation of the near-infrared II polymer fluorescent microsphere is described in detail with reference to FIG. 2 and FIG. 3.

[0075] S100: Preparation of a Fluorochrome Solution

[0076] In embodiments of the present disclosure, a fluorochrome is dissolved in a water-immiscible organic solvent, thus obtaining a fluorochrome solution. In embodiments of the present disclosure, the fluorochrome is organic molecules including those shown as formula (I), formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earth nanoparticles

##STR00003##

[0077] In embodiments of the present disclosure, the formula (I) is 4,8-(5-(9,9-di(6-bromohexyl)-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,- 4]dioxine)-1H,5H-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole); the formula (II) is 4,8-(5-(9,9-dihexyl-9H-fluoren-2-yl)-2,3-dihydrothieno[3,4-b][1,4- ]dioxine)-1H,5H-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole); and the formula (III) is 2,3-dihydrothieno[3,4-b][1,4]dioxine)-1H,5H-benzo[1,2-c:4,5-c']bis([1,2,5- ]thiadiazole).

[0078] In embodiments of the present disclosure, the fluorochrome selected from the group consisting of organic molecules including those shown as formula (I), formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earth nanoparticles is used to prepare a near-infrared II polymer fluorescent microsphere. As shown in FIGS. 4 (a) to (c), absorption spectrums of and fluorescence spectrums emitted respectively by fluorochrome of formula (I) to (III), the inventors find that the fluorochrome has strong absorption to lights less than 1000 nm, such as at 365 nm and 740 nm, and emits fluorescence between 1000 nm and 1700 nm. Accordingly, because of the relative broader interval between the exciting wavelength and the emitting wavelength, the fluorochrome has many advantageous characteristics such as strong penetrability (penetration distance of several millimeters) and low background interference in the live imaging application, thus can replace the conventional near-infrared fluorescent microsphere for application in imaging of living organisms, cells or tissues, diagnosing in vitro, and so on. Nevertheless, the fluorochrome as indicated above cannot be directly applied in live imaging because of strong hydrophobicity per se, and thus requiring hydrophilic modification in advance. The present inventors also find that the method including encapsulating the fluorochrome as indicated above within the polymer microsphere by swelling achieves a desirable effect with high quantum efficiency of 25% or more and good dispersibility in an aqueous solution, thereby facilitating labeling detection of various biological macromolecule. Therefore, the present near-infrared II polymer fluorescent microsphere prepared with the fluorochrome has advantageous characteristics such as strong penetrability and low background interference during the fluorescence detection, thereby having promising prospects in terms of live imaging, biolabeling detection and so on. Further, the present inventors find that when such a near-infrared II fluorescent microsphere prepared by the above method is applied in labeling an antibody which is further coated on a binding region of a test strip, the result detected by the test strip can be highly accurate, sensitive and precise.

[0079] In a specific embodiment of the present disclosure, the fluorochrome in the fluorochrome solution has a concentration between 1 mg/mL to 50 mg/mL, specifically 1 mg/mL to 10 mg/mL, 10 mg/mL to 20 mg/mL, 20 mg/mL to 30 mg/mL, 30 mg/mL to 40 mg/mL or 40 mg/mL to 50 mg/mL, for example, 1 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL or 50 mg/mL, preferably 20 mg/mL, so that the polymer microsphere is capable of encapsulating more fluorochrome, and thus the present near-infrared II polymer fluorescent microsphere prepared emits the fluorescence with high intensity during the fluorescence detection, thereby further improving sensitivity of the fluorescence detection.

[0080] In embodiments of the present disclosure, the organic solvent is at least one selected from the group consisting of ethyl acetate, dichloromethane, trichloromethane, 1,2-dichloroethane and aromatic hydrocarbons, in an example of the present disclosure, dichloromethane, so that dichloromethane can further facilitate swelling of the polymer microsphere, thereby further increasing encapsulating efficiency of the fluorochrome.

[0081] S200: Preparation of a Microsphere Solution

[0082] In embodiments of the present disclosure, a polymer microsphere is distributed into a sodium dodecyl sulfonate solution, thus obtaining the microsphere solution with the polymer microsphere as a carrier for the fluorochrome.

[0083] In some embodiments of the present disclosure, the polymer microsphere is at least one selected from the group consisting of polystyrene microspheres, poly (methyl methacrylate) microspheres, polyformaldehyde microspheres and poly (lactic acid-co-glycolic acid) microspheres. Such a polymer microsphere is capable of encapsulating tens of thousands to hundreds of thousands of fluorescent molecules in one microsphere and preventing the hydrophobic fluorochrome mentioned above from leaking out owing to a hydrophobic moiety inside the polymer microsphere; and is capable of well dispersing in the aqueous solution due to a charge or hydrophilic moiety outside the polymer microsphere. Therefore, the near-infrared II polymer fluorescent microsphere, with advantageous characteristics such as strong penetrability and low background interference during the fluorescence detection, can be prepared with such the polymer microsphere.

[0084] In embodiments of the present disclosure, the polymer microsphere has a particle size of 20 nm to 1000 nm, specifically 20 nm to 100 nm, 100 nm to 200 nm, 200 nm to 300 nm, 300 nm to 400 nm, 400 nm to 500 nm, 500 nm to 600 nm, 600 nm to 700 nm, 700 nm to 800 nm, 800 nm to 900 nm or 900 nm to 1000 nm, for example, 20 nm, 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm or 1000 nm, preferably 850 nm. Accordingly, the polymer microsphere is capable of encapsulating more fluorochrome, such that the near-infrared II polymer fluorescent microsphere prepared can have high intensity of the fluorescence, thereby further improving sensitivity of fluorescence detection.

[0085] In embodiments of the present disclosure, the sodium dodecyl sulfonate (SDS) solution is of a concentration of 0.1% to 0.8%, for example 0.2% to 0.6%, eg., 0.25%, such that the SDS solution as an emulsifier not only guarantees the polymer microspheres to be better dispersed in the SDS solution, but also facilitates obtaining a uniform emulsion during the subsequent ultrasonic treatment.

[0086] In embodiments of the present disclosure, the present polymer microsphere is distributed into the sodium dodecyl sulfonate solution in a mass/volume ratio of 10 mg/mL to 200 mg/mL, specifically 10 mg/mL to 50 mg/mL, 50 mg/mL to 100 mg/mL, 100 mg/mL to 150 mg/mL or 150 mg/mL to 200 mg/mL, for example, 10 mg/mL, 30 mg/mL, 50 mg/mL, 70 mg/mL, 100 mg/mL, 120 mg/mL, 150 mg/mL, 170 mg/mL or 200 mg/mL, preferably 30 mg/mL, which not only enhances yield of the near-infrared II polymer fluorescent microsphere effectively, but also allows the polymer microspheres to be swelled to a maximal extent in the next step, such that the polymer microsphere is capable of encapsulating more fluorochrome, and thus the present near-infrared II polymer fluorescent microsphere prepared emits the fluorescence with high intensity during the fluorescence detection, thereby further improving sensitivity of fluorescence detection.

[0087] S300: Ultrasonic Treatment

[0088] In embodiments of the present disclosure, a first mixture of the fluorochrome solution and the microsphere solution is subjected to ultrasonic treatment, thus obtaining an emulsion.

[0089] In embodiments of the present disclosure, the first mixture includes the fluorochrome solution and the microsphere solution in a volume ratio of 1:5 to 1:20, specifically 1:5, 1:8, 1:10, 1:12, 1:15, 1:17 or 1:20, preferably 1:10, such that dichloromethane is in a proper amount for swelling all of the polymer microspheres thoroughly, and the fluorochrome solution is allowed to enter the nanopores formed during swelling of the polymer microsphere completely, thus the polymer microsphere is capable of encapsulating more fluorochrome and the high quality of near-infrared II polymer fluorescent microsphere prepared by the present method emits the fluorescence with high intensity during the fluorescence detection, thereby further improving sensitivity of fluorescence detection.

[0090] In embodiments of the present disclosure, the first mixture includes the fluorochrome and the polymer microsphere in a mass ratio of 0.1:100 to 30:100, specifically 0.1:100, 0.5:100, 1:100, 5:100, 7:100, 10:100, 12:100, 15:100, 20:100, 22:100, 25:100, 27:100 or 30:100, preferably 1:15. Thus, the fluorochrome and the polymer microsphere are in such a proper matching ratio that availability of these raw material is improved and the fluorochrome solution is allowed to enter the nanopores formed during swelling of the polymer microsphere completely, thus the polymer microsphere is capable of encapsulating more fluorochrome and the near-infrared II polymer fluorescent microsphere prepared by the present method emits the fluorescence with high intensity during the fluorescence detection, thereby further improving sensitivity of fluorescence detection.

[0091] S400: Swelling of the Emulsion

[0092] In embodiments of the present disclosure, the emulsion is swelled such that the fluorochrome solution enters nanopores formed during swelling of the polymer microsphere, thus obtaining a second mixture.

[0093] The present inventors find that a hydrophobic fluorochrome cannot be directly applied in live imaging, thus requiring hydrophilic modification in advance, which not only involves tedious processes, but also provides a modified fluorochrome with significantly decreased quantum efficiency after dissolved in the aqueous solution. However, the polymer microsphere used in the present disclosure is capable of encapsulating tens of thousands to hundreds of thousands of fluorescent molecules in one microsphere and preventing the hydrophobic fluorochrome mentioned above from leaking out owing to a hydrophobic moiety inside the polymer microsphere; and is capable of well dispersing in the aqueous solution due to a charge or hydrophilic moiety outside the polymer microsphere. Besides, such a polymer microsphere is allowed to be modified from the outside with various functional groups in a flexible way, which facilitates labeling of molecules such as proteins and DNAs. Accordingly, the present inventors provide the method including encapsulating the fluorochrome selected from the group consisting of organic molecules including those shown as formula (I), formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earth nanoparticles within the polymer microsphere by swelling to obtain the near-infrared II polymer fluorescent microsphere with high quantum efficiency of 25% or more and good dispersibility in an aqueous solution, thereby facilitating labeling detection of various biological macromolecules.

[0094] In embodiments of the present disclosure, the emulsion is swelled at 10.degree. C. to 50.degree. C. under stirring for 1 hour to 10 hours, specifically, at 10.degree. C., 15.degree. C., 20.degree. C., 25.degree. C., 30.degree. C., 35.degree. C., 40.degree. C., 45.degree. C. or 50.degree. C., for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours, preferably, at 40.degree. C. for 6 hours, such that the polymer microsphere can be swelled sufficiently in the presence of dichloromethane, which ensures the fluorochrome entering nanopores formed during swelling of the polymer microsphere successfully, and thus the present near-infrared II polymer fluorescent microsphere prepared emits the fluorescence with high intensity during the fluorescence detection.

[0095] S500: Volatilization of the Organic Solvent

[0096] In embodiments of the present disclosure, the second mixture is heated to volatilize the organic solvent, such that the fluorochrome is crystallized out and encapsulated in the nanopores, thus obtaining the near-infrared II polymer fluorescent microsphere.

[0097] With slow volatilization of the organic solvent during heating, exterior of the polymer microsphere is shrunk and the hydrophobic fluorochrome is crystallized out (forming hydrophobic pellets), so that the fluorochrome is encapsulated inside the polymer microsphere. After the organic solvent is totally volatilized, the near-infrared II polymer fluorescent microsphere is thus obtained, with the fluorochrome encapsulated inside barely with leakage.

[0098] In embodiments of the present disclosure, the second mixture is heated at a temperature of 50.degree. C. to 90.degree. C., specifically 50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C., 70.degree. C., 75.degree. C., 80.degree. C., 85.degree. C. or 90.degree. C., preferably 50.degree. C., so that the organic solvent, such as dichloromethane can be volatilized quickly and completely in a short time period, so as to improve the efficiency of the present method.

[0099] In embodiments of the present disclosure, the second mixture is heated under magnetic stirring in a water bath at a temperature of 50.degree. C. to 90.degree. C., so that the dichloromethane in the microsphere is volatilized in a high volatilizating rate, which improves the efficiency of the present method.

[0100] In embodiments of the present disclosure, the method for preparing the near-infrared II polymer fluorescent microsphere further includes subjecting the near-infrared II polymer fluorescent microsphere obtained to ultrasonic cleaning with ethanol and water successively.

[0101] In embodiments of the present disclosure, after dissolved in a certain quantity in dichloromethane, the near-infrared II polymer fluorescent microsphere prepared by the present method is detected with its fluorescence intensity based on a standard curve, with a calculated result of around 80,000 fluorescent molecules encapsulated in each polymer microsphere. Therefore, the near-infrared II polymer fluorescent microsphere prepared can emit a greatly magnified fluorescence signal when applied in labeling detection as compared with small molecule fluorochrome, thereby improving sensitivity of the fluorescence detection.

[0102] A System for Immunofluorescence Chromatographic Detection

[0103] In embodiments of the present disclosure, the present system for immunofluorescence chromatographic detection includes a fluorescence immunity analyzer, and a test strip for short-wave near infrared immunofluorescence chromatographic detection described above, so that a fluorescence signal can be generated in the test strip and then analyzed by the fluorescence immunity analyzer, thus achieving to qualitatively or quantitatively detect an analyte, and improving accuracy, sensitivity and precision of the detected results as described above.

[0104] It should be understood by those skilled in the art that the features and advantages described above with respect to the test strip for short-wave near infrared immunofluorescence chromatographic detection are equally applicable to the system for immunofluorescence chromatographic detection, which will not be elaborated herein

[0105] A Method for Quantifying an Analyte in a Sample

[0106] In embodiments of the present disclosure, the method for quantifying the analyte in the sample includes 1) applying the sample to a sampling region of a test strip for short-wave near infrared immunofluorescence chromatographic detection described in the first aspect; 2) determining a fluorescence signal generated in the test strip of the immunofluorescence chromatographic detection; and 3) quantifying the analyte in the sample based on the fluorescence signal determined, thus improving accuracy, sensitivity and precision of the detected results in a simple way.

[0107] In embodiments of the present disclosure, the sample is serum, thus facilitating to sampling and obtaining a detection result with high accuracy.

[0108] In embodiments of the present disclosure, the fluorescence signal is determined by a fluorescence immunity analyzer.

[0109] In embodiments of the present disclosure, the fluorescence immunity analyzer is equipped with a color filter at a wavelength of 800 nm, thus benefiting for obtaining fluorescence with high intensity, thereby facilitating to observing and quantifying the fluorescence obtained.

[0110] In embodiments of the present disclosure, the method for quantifying the analyte in the sample further includes determining fluorescence signals in both a testing line and a quality control line in the step 2); and quantifying the analyte in the sample based on a ratio of the fluorescence signal generated in the testing line to the fluorescence signal generated in the quality control line.

[0111] In embodiments of the present disclosure, the method for quantifying the analyte in the sample further includes quantifying the analyte in the sample by means of a standard curve, based on the ratio of the fluorescence signal in the testing line to the fluorescence signal generated in the quality control line, in which the standard curve is created with serial cardiac troponin standards in concentrations of 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL, thus contributing to improving accuracy of the detected result.

[0112] It should be understood by those skilled in the art that the features and advantages described above with respect to the test strip for short-wave near infrared immunofluorescence chromatographic detection are equally applicable to the method for quantifying the analyte in the sample, which will not be elaborated herein.

[0113] The embodiments of the present disclosure will be described in detail by reference to the following examples. It would be appreciated by those skilled in the art that the following examples are explanatory, and cannot be construed to limit the scope of the present disclosure. If the specific technology or conditions are not specified in the examples, a step will be performed in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. If the manufacturers of reagents or instruments are not specified, the reagents or instruments may be commercially available.

Example 1

[0114] (1) Synthesis of a Carboxyl Polystyrene Microsphere

[0115] 190 mL of water was added into a 500 mL round bottom flask and then incubated in a water bath with a temperature of 70.degree. C. under stirring at a speed of 350 rpm for half an hour. 16 mg of sodium dodecyl sulfate (SDS) as an emulsifier and 0.05 g of sodium bicarbonate as a buffer reagent were added and then incubated for another 10 minutes under the stirring. To the mixture, 8 mL of styrene and 0.8 mL of acrylic acid were further added. After one hour, 0.2 g of potassium persulfate was added and an obtained reaction mixture was subjected to polymerization reaction under nitrogen atmosphere for 18 hours. After completion of the reaction, the resulting product was centrifuged with a mixture of ethanol and water in a volume ratio of 2:1 (v/v) three times, thus obtaining the carboxyl polystyrene microsphere. The scanning electron microscopy of such a carboxyl polystyrene microsphere is shown in panel A of FIG. 5 (a). The carboxyl polystyrene microsphere was distributed into a SDS solution at a concentration of 0.25% (w/v), thus obtaining a 30 mg/mL carboxyl polystyrene microsphere dispersion as shown in FIG. 6 (A) which was stored in a refrigerator under 4.degree. C. for the next step.

[0116] (2) Synthesis of a Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere

[0117] 40 mg of the near-infrared II fluorochrome represented by formula (I) was dissolved in 2 mL dichloromethane, thus obtaining a fluorochrome solution at a concentration of 20 mg/mL as shown in FIG. 6 (B). 20 mL of the carboxyl polystyrene microsphere dispersion obtained in (1) was added into a 500 mL conical flask and subjected to ultrasonic treatment for 5 minutes, after which 2 mL fluorochrome solution obtained above was added and subjected to ultrasonic treatment, thus obtaining an emulsion. Then the emulsion was subjected to magnetic stirring at a temperature of 40.degree. C. for 6 hours, such that the carboxyl polystyrene microsphere swelled sufficiently and the fluorochrome solution entered nanopores formed during swelling of the polymer microsphere. The mixture swelled was then subjected to magnetic stirring in a water bath at a temperature of 50.degree. C. overnight, so as to volatilize the dichloromethane in the mixture completely. The product obtained was centrifuged, and then subjected to ultrasonic cleaning with ethanol three times and with water for several times until the supernatant of the product centrifuged contained no fluorochrome, thus obtaining the near-infrared II carboxyl polystyrene fluorescent microsphere which was distributed into water in a weight/volume of 5% and stored in a refrigerator under 4.degree. C. for the next step (FIG. 6 (C)).

[0118] (3) Evaluation of the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere Obtained in (2)

[0119] 3.1 In FIG. 6, FIG. 6 (A) shows that the solution of carboxyl polystyrene in SDS is white, FIG. 6 (B) shows that the solution of fluorochrome represented by formula (I) in dichloromethane is cyan, and FIG. 6 (C) shows that the solution of carboxyl polystyrene fluorescent microsphere encapsulating the fluorochrome represented by formula (I) is also cyan. Thus, it is demonstrated that the fluorochrome was successfully encapsulated in the microsphere and properties on the surface of the microsphere have not been changed significantly according to the color change. It also can be seen from the FIG. 6 (C) that the near-infrared II carboxyl polystyrene fluorescent microsphere obtained can be distributed into water uniformly and stably.

[0120] 3.2 Panel B of FIG. 5 (a) shows a scanning electron microscope photograph of the near-infrared II carboxyl polystyrene fluorescent microsphere obtained in (2). It can be seen from FIG. 5 (a) that morphology of the carboxyl polystyrene microsphere has not been changed significantly before and after encapsulation of the fluorochrome, and the fluorescent microspheres obtained are uniform in size and are not aggregated together.

[0121] 3.3 Panel A of FIG. 7 (a) shows a fluorescent photograph of the carboxylic polystyrene fluorescent microsphere obtained in (2) under irradiation with an excitation light at a wavelength of 740 nm, and panel B of FIG. 7 (a) shows its fluorescent spectrum. It can be seen from the FIG. 7 (a) that the carboxylic polystyrene fluorescent microsphere can emit fluorescence in a wavelength between 800 nm and 1700 nm nm with high fluorescence intensity, and its fluorescence quantum yield reaches 25% based on measurement.

[0122] (4) Application of the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere Obtained in (2)

[0123] 4.1 Coupling an Antibody to the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere

[0124] The near-infrared II carboxyl polystyrene fluorescent microsphere obtained in (2) was distributed into a 2-morpholinoethanesulfonic acid (MES) buffer (10 mM, pH 6.2), thus obtaining a uniform dispersion in a weight/volume ratio of 1%. To the dispersion, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) buffer (5 mg/mL) and sulfo-(N-hydroxysulfosuccinimide) (sulfo-NHS) buffer (5 mg/mL) were added for activation for 15 minutes. After centrifugation, the supernatant was discarded and the remaining mixture was distributed into the IVIES buffer (10 mM, pH 6.5) again. And then 0.4 mg/mL of Mouse Monoclonal Antibody (anti-CTNI1 (19C7, HyTest Ltd.)) was added and incubated for 2 hours under stirring. The resulting mixture was centrifuged again, the supernatant was discarded and the remaining pellets were distributed into 20 mM PBS containing 0.5% casein, 2.5% BSA, 1% sugar, 2% PEG-2000 and 0.03 wt % NaN.sub.3 (pH 8.0) under ultrasonic treatment, thus obtaining a fluorescent microsphere dispersion coupled with antibody anti-CTNI1 (1%, w/v) which was stored in a refrigerator under 4.degree. C. for the next step.

[0125] 4.2 Preparation of a Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0126] The test strip for short-wave near infrared immunofluorescence chromatographic detection includes five parts, a supporting plastic board, a sample pad, a binding pad, an adsorbing pad and a nitrocellulose membrane, as shown in FIG. 8. Before assembled into the test strip for short-wave near infrared immunofluorescence chromatographic detection completely, each part has to be pretreated. Specifically, the sample pad was soaked in a buffer containing 1% BSA, 2% TritonX-100, 2% PVP 40, 20 mM Tris-HCl and 50 mM NaAc for 1 hour, and then incubated at a temperature of 37.degree. C. in an oven overnight; the fluorescent microsphere dispersion coupled with antibody anti-CTNI1 obtained in the above step was sprayed uniformly onto the binding pad by a sprayer specialized for an immunofluorescence chromatographic test, subjected to lyophilization for 10 hours and stored for future use; and 75 .mu.L of anti-CTNI2 solution (1.0 mg/mL) (16A11, HyTest Ltd.) was sprayed uniformly onto a part of the nitrocellulose membrane by a scriber specialized for an immunofluorescence chromatographic detection, thus obtaining a testing line (that is a T line); in a similar way, 75 .mu.L of goat-anti-mouse antibody solution (0.5 mg/mL) (Hangzhou Qitai biotechnology Co., LTD) was sprayed uniformly onto another part of the nitrocellulose membrane by the scriber, thus obtaining a quality control line (that is a C line), after which the nitrocellulose membrane was incubated at a temperature of 37.degree. C. in an oven overnight. Then the sample pad, the binding pad, the nitrocellulose membrane and the adsorbing pad together were fixed neatly onto a hard cardboard along the axis direction, in which the left margin of the adsorbing pad was overlapped with the right margin of the nitrocellulose membrane, the left margin of the nitrocellulose membrane was overlapped with the right margin of the binding pad and the left margin of the binding pad was overlapped with the right margin of the sample pad, such that each part was in close contact with its neighbor part, thus ensuring that the sample can move from the sample pad to the adsorbing pad smoothly. Finally, the cardboard assembled was cut into the test strips in a width of 4 mm by a cutter for the test strip for short-wave near infrared immunofluorescence chromatographic detection. The strip obtained was then packaged into an aluminium bag for storage.

[0127] 4.3 Assembly of a System for Immunofluorescence Chromatographic Detection

[0128] The fluorescence signal generated in the test strip for short-wave near infrared immunofluorescence chromatographic detection was observed and analyzed by a fluorescence immunity analyzer shown in FIG. 9 (A); and a fluorescent spectrum of an analyte detected by the detection system is shown in FIG. 9 (B).

[0129] The light source of the fluorescence immunity analyzer is a light-emitting diode (LED) (365 nm, 1W); the divergent lights of LED were collimated by combined lenses; and Laser confocal optical system was used for irradiation and collection of fluorescence. When the test strip for short-wave near infrared immunofluorescence chromatographic detection applied with a sample was transported on a conveyor belt driven by a stepper motor to expose under an excitation light, fluorescence was emitted at both the C line and the T line in the detection region of the test strip. After passing through a color filter at a wavelength of 800 nm, the fluorescence emitted was focused to a photosensitive panel of a silicon photocell sensitive to a light between 500 nm to 1100 nm. An A/D (analog to digital) chip was configured to transform the voltage signal from the silicon photocell into a digital signal, thus obtaining a fluorescent spectrum of the sample. A lower machine was configured to integrate peak areas corresponding to the C line and the T line respectively, calculate the ratio of the peak area of the C line to the peak area of the T line, and fit the concentrations of standard samples, thus obtaining a standard curve which was saved in the fluorescence immunity analyzer. During detection of a working sample, the ratio of the peak area of the C line to the peak area of the T line was determined by the fluorescence immunity analyzer, and the concentration of cardiac troponin in the working sample was quantified according to the standard curve.

[0130] 4.4 Evaluation of the Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0131] In order to preliminarily evaluate efficacy of the test strip or the immunofluorescence chromatographic detection, cardiac troponin standards dissolved in newborn calf serum at concentrations of 0 ng/mL and 20 ng/mL were detected. Specifically, the cardiac troponin standards were applied to the sample region respectively; after 15 min, intense fluorescence was observed by a InGaAs camera at both the C line and the T line in the detection region of the test strip for short-wave near infrared immunofluorescence chromatographic detection under irradiation with an excitation light at 808 nm (as shown in FIG. 10 (a)). For the cardiac troponin standard with a concentration of 0 ng/mL, very weak fluorescence was observed at the T line; while for the cardiac troponin standard with a concentration of 20 ng/mL, intense fluorescence was observed at the T lime, indicating validity of the test strip or the immunofluorescence chromatographic detection.strip for immunofluorescence.

[0132] A standard curve of cardiac troponin was created as the following steps. Cardiac troponin standards in serial concentrations of 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL were formulated with newborn calf serum. Then 75 .mu.L of the cardiac troponin standard at each concentration was applied to the sample region of the test strip and moved toward the direction of the adsorbing region of the test strip under capillary force. After 15 minutes, the test strip was detected by the fluorescence immunity analyzer in triplicate, thus obtaining a mean value of fluorescence intensity for each cardiac troponin standard. According, all mean values of fluorescence intensity obtained were curve-fitted with corresponding concentrations of the cardiac troponin standards, with the standard curve generated and saved in the analyzer. The fluorescent spectrums of the cardiac troponin standards were shown in FIG. 11, and the standard curve was shown in FIG. 13(a).

[0133] A serum sample was detected by the test strip for short-wave near infrared immunofluorescence chromatographic detection. 75 .mu.L of a human serum sample was applied to the sample region of the test strip and moved toward the direction of the adsorbing region of the test strip under capillary force. After 15 minutes, the test strip was detected by the fluorescence immunity analyzer, and then the concentration of the cardiac troponin in the human serum sample was quantified base on the standard curve created. The detection results obtained by the present test strip were compared with those from clinical detection, and there exists consistency between them, as shown in FIG. 14 (a), thus demonstrating the results detected by the present test strips are accurate.

Example 2

[0134] (1) Synthesis of a Carboxyl Polystyrene Microsphere

[0135] All steps for synthesizing a carboxyl polystyrene microsphere are same as those in Example 1. The scanning electron microscopy of the carboxyl polystyrene microsphere obtained is shown in panel A of FIG. 5 (b) and a carboxyl polystyrene microsphere dispersion obtained is same as that shown in FIG. 6 (A).

[0136] (2) Synthesis of a Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere

[0137] All steps for synthesizing a near-infrared II carboxyl polystyrene fluorescent microsphere are similar with those in Example 1, except that the near-infrared II fluorochrome is represented by formula (II).

[0138] (3) Evaluation of the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere Obtained in (2)

[0139] All steps for evaluating the near-infrared II carboxyl polystyrene fluorescent microsphere obtained are same as those in Example 1.

[0140] 3.1 The solution of fluorochrome represented by formula (II) in dichloromethane is also cyan, as shown in FIG. 6 (B); and the solution of carboxyl polystyrene fluorescent microsphere encapsulating the fluorochrome represented by formula (II) is also cyan, as shown in FIG. 6 (C). It also can be seen from the FIG. 6 (C) that the near-infrared II carboxyl polystyrene fluorescent microsphere obtained can be distributed into water uniformly and stably.

[0141] 3.2 Panel B of FIG. 5 (b) shows a scanning electron microscope photograph of the near-infrared II carboxyl polystyrene fluorescent microsphere obtained in (2). It can be seen from FIG. 5 (b) that morphology of the carboxyl polystyrene microsphere has not been changed significantly before and after encapsulation of the fluorochrome represented by formula (II), and the fluorescent microspheres obtained are uniform in size and are not aggregated together.

[0142] 3.3 A fluorescent photograph of the carboxylic polystyrene fluorescent microsphere obtained in (2) and its fluorescent spectrum are shown in FIG. 7 (b), which indicates that the carboxylic polystyrene fluorescent microsphere can emit fluorescence in a wavelength between 800 nm to 1700 nm nm with high fluorescence intensity, and its fluorescence quantum yield reaches 25% based on measurement.

[0143] (4) Application of the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere Obtained in (2)

[0144] 4.1 Coupling an Antibody to the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere

[0145] All the steps for coupling an antibody to the near-infrared II carboxyl polystyrene fluorescent microsphere obtained in (2) are same as those in Example 1.

[0146] 4.2 Preparation of a Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0147] All the steps for preparing a test strip for short-wave near infrared immunofluorescence chromatographic detection are same as those in Example 1.

[0148] 4.3 Assembly of a System for Immunofluorescence Chromatographic Detection

[0149] All the steps for assembling a system for immunofluorescence chromatographic detection are same as those in Example 1.

[0150] 4.4 Evaluation of the Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0151] All the steps for evaluating the test strip for short-wave near infrared immunofluorescence chromatographic detection are similar with those in Example 1, except that a standard curve of cardiac troponin was created by using cardiac troponin standards in serial concentrations of 80 ng/mL, 40 ng/mL, 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL and 0 ng/mL, and the fluorescent spectrums of the cardiac troponin standards were shown in FIG. 12, and the standard curve was shown in FIG. 13 (b).

[0152] As shown in FIG. 10 (b), intense fluorescence was observed by a InGaAs camera at both the C line and the T line in the detection region of the test strip for short-wave near infrared immunofluorescence chromatographic detection under irradiation with an excitation light at 808 nm. For the cardiac troponin standard with a concentration of 0 ng/mL, very weak fluorescence was observed at the T line; while for the cardiac troponin standard with a concentration of 20 ng/mL, intense fluorescence was observed at the T lime, indicating validity of the test strip or the immunofluorescence chromatographic detection strip for immunofluorescence.

[0153] As shown in FIG. 14 (b), there exists consistency between the detection results obtained by the present test strip and results obtained from a clinical detection, thus demonstrating the results detected by the present test strips are accurate.

Example 3

[0154] (1) Synthesis of a Carboxyl Polystyrene Microsphere

[0155] All steps for synthesizing a carboxyl polystyrene microsphere are same as those in Example 1. The scanning electron microscopy of the carboxyl polystyrene microsphere obtained is shown in panel A of FIG. 5 (c) and a carboxyl polystyrene microsphere dispersion obtained is same as that shown in FIG. 6 (A).

[0156] (2) Synthesis of a Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere

[0157] All steps for synthesizing a near-infrared II carboxyl polystyrene fluorescent microsphere are similar with those in Example 1, except that the near-infrared II fluorochrome is represented by formula (III).

[0158] (3) Evaluation of the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere Obtained in (2)

[0159] All steps for evaluating the near-infrared II carboxyl polystyrene fluorescent microsphere obtained are same as those in Example 1.

[0160] 3.1 The solution of fluorochrome represented by formula (III) in dichloromethane is also cyan, as shown in FIG. 6 (B); and the solution of carboxyl polystyrene fluorescent microsphere encapsulating the fluorochrome represented by formula (III) is also cyan, as shown in FIG. 6 (C). It also can be seen from the FIG. 6 (C) that the near-infrared II carboxyl polystyrene fluorescent microsphere obtained can be distributed into water uniformly and stably.

[0161] 3.2 Panel B of FIG. 5 (c) shows a scanning electron microscope photograph of the near-infrared II carboxyl polystyrene fluorescent microsphere obtained in (2). It can be seen from FIG. 5 (c) that morphology of the carboxyl polystyrene microsphere has not been changed significantly before and after encapsulation of the fluorochrome represented by formula (III), and the fluorescent microspheres obtained are uniform in size and are not aggregated together.

[0162] 3.3 A fluorescent spectrum of the carboxylic polystyrene fluorescent microsphere obtained in (2) is shown in FIG. 7 (c), which indicates that the carboxylic polystyrene fluorescent microsphere can emit fluorescence in a wavelength between 800 nm to 1700 nm nm with high fluorescence intensity, and its fluorescence quantum yield reaches 25% based on measurement.

[0163] (4) Application of the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere Obtained in (2)

[0164] 4.1 Coupling an Antibody to the Near-Infrared II Carboxyl Polystyrene Fluorescent Microsphere

[0165] All the steps for coupling an antibody to the near-infrared II carboxyl polystyrene fluorescent microsphere obtained in (2) are same as those in Example 1.

[0166] 4.2 Preparation of a Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0167] All the steps for preparing a test strip for short-wave near infrared immunofluorescence chromatographic detection are same as those in Example 1.

[0168] 4.3 Assembly of a System for Immunofluorescence Chromatographic Detection

[0169] All the steps for assembling a system for immunofluorescence chromatographic detection are same as those in Example 1.

[0170] 4.4 Evaluation of the Test Strip for Short-Wave Near Infrared Immunofluorescence Chromatographic Detection

[0171] All the steps for evaluating the test strip for short-wave near infrared immunofluorescence chromatographic detection are same as those in Example 1, and the standard curve was shown in FIG. 13 (c).

[0172] As shown in FIG. 14 (c), there exists consistency between the detection results obtained by the present test strip and results obtained from a clinical detection, thus demonstrating the results detected by the present test strips are accurate.

[0173] Throughout this specification, reference to "an embodiment", "some embodiments", "one embodiment", "another example", "an example", "a specific example" or "some examples" means 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 present disclosure. Thus, the appearances of the phrases such as "in some embodiments", "in one embodiment", "in an embodiment", "in another example", "in an example", "in a specific example" or "in some examples" in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials or characteristics may be combined in any suitable manner in one or more embodiments or examples. In addition, it will be apparent to those skilled in the art that different embodiments or examples as well as features of the different embodiments or examples described in this specification may be combined without contradictory.

[0174] Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

1. A test strip for short-wave near infrared immunofluorescence chromatographic detection, comprising: a body, defining a sample region, a binding region, a detecting region and an adsorbing region connected with one another sequentially; a first antibody, labeled with a fluorescent microsphere emitting a wavelength in a range of 1000 nm to 1700 nm under an excitation light less than 1000 nm, coated on the binding region and configured to specifically recognize an analyte; a detecting line and a quality control line, located in the detecting region, wherein the detecting line closes to the binding region; a second antibody, coated on the detecting line and configured to specifically recognize the analyte; and a third antibody, coated on the quality control line and configured to specifically recognize the first antibody, wherein the fluorescent microsphere is a near-infrared II polymer fluorescent microsphere prepared by the following steps: 1) dissolving a fluorochrome in a water-immiscible organic solvent, thus obtaining a fluorochrome solution; 2) distributing a polymer microsphere into a sodium dodecyl sulfonate solution, thus obtaining a microsphere solution with the polymer microsphere as a carrier for the fluorochrome; 3) subjecting a first mixture of the fluorochrome solution and the microsphere solution to ultrasonic treatment, thus obtaining an emulsion; 4) swelling the emulsion such that the fluorochrome solution enters nanopores formed during swelling of the polymer microsphere, thus obtaining a second mixture; and 5) heating the second mixture to volatilize the organic solvent, such that the fluorochrome is crystallized out and encapsulated in the nanopores, thus obtaining the near-infrared II polymer fluorescent microsphere.

2. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein the analyte is cardiac troponin, the first antibody is an antibody I against cardiac troponin, the second antibody is an antibody II against cardiac troponin, and the third antibody is a secondary antibody, preferably a goat-anti-mouse antibody, wherein the first antibody recognizes the analyte at a first site different from a second site recognized by the second antibody.

3. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein the fluorochrome is organic molecules including those shown as formula (I), formula (II) or formula (III), or carbon nanotubes, or PbS, PbSe or InAs quantum dots, or rare earth nanoparticles ##STR00004##

4. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein the fluorochrome in the fluorochrome solution has a concentration of 1 mg/mL to 50 mg/mL.

5. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein the organic solvent is at least one selected from the group consisting of ethyl acetate, dichloromethane, trichloromethane, 1,2-dichloroethane and aromatic hydrocarbons, preferably dichloromethane.

6. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein the polymer microsphere is at least one selected from the group consisting of polystyrene microspheres, poly (methyl methacrylate) microspheres, polyformaldehyde microspheres and poly (lactic acid-co-glycolic acid) microspheres.

7. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein the polymer microsphere has a particle size of 20 nm to 1000 nm.

8. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein in the step 2), the polymer microsphere is distributed into the sodium dodecyl sulfonate solution in a mass/volume ratio of 10 mg/mL to 200 mg/mL.

9. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein in the step 3), the first mixture comprises the fluorochrome solution and the microsphere solution in a volume ratio of 1:5 to 1:20.

10. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein in the step 3), the first mixture comprises the fluorochrome and the polymer microsphere in a mass ratio of 0.1:100 to 30:100.

11. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein in the step 4), the emulsion is swelled at 10.degree. C. to 50.degree. C. under stirring for 1 hour to 10 hours.

12. The test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1, wherein in the step 5), the second mixture is heated at a temperature of 50.degree. C. to 90.degree. C.

13. A system for immunofluorescence chromatographic detection, comprising: a fluorescence immunity analyzer; and a test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1.

14. A method for quantifying an analyte in a sample, comprising: 1) applying the sample to a sampling region of a test strip for short-wave near infrared immunofluorescence chromatographic detection according to claim 1; 2) determining a fluorescence signal generated in the test strip for the immunofluorescence chromatographic detection; and 3) quantifying the analyte in the sample based on the fluorescence signal determined.

15. The method according to claim 14, wherein the sample is serum.

16. The method according to claim 14, wherein the fluorescence signal is determined by a fluorescence immunity analyzer.

17. The method according to claim 16, wherein the fluorescence immunity analyzer is equipped with a color filter at a wavelength of 800 nm.

18. The method according to claim 14, further comprising: determining fluorescence signals in both a detecting line and a quality control line in the step 2); and quantifying the analyte in the sample based on a ratio of the fluorescence signal generated in the detecting line to the fluorescence signal generated in the quality control line.

19. The method according to claim 18, further comprising: quantifying the analyte in the sample by means of a standard curve, based on the ratio of the fluorescence signal generated in the detecting line to the fluorescence signal generated in the quality control line, wherein the standard curve is created with serial cardiac troponin standards in concentrations of 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.2 ng/mL, 2.0 ng/mL, 1.0 ng/mL, 0.5 ng/mL, 0.2 ng/mL and 0 ng/mL.

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