Light-emitting signal probe and light-emitting adjusting medium thereof, immunochromatography test strip and preparation method thereof, kit and immunochromatography detection method

Abstract

The invention provides a light-emitting signal probe and a light-emitting adjusting medium thereof, an immunochromatographic test strip and a preparation method thereof, a kit and an immunochromatographic detection method, the light-emitting signal probe comprises at least two metal oxide nanoparticles, and when the at least two metal oxide nanoparticles are mixed with the same light-emitting adjusting medium, the light-emitting signal probe can be used for detecting the light-emitting signal. Different light-emitting complexes are respectively formed; and when the same exciting light is adopted for irradiation, different light-emitting complexes emit light with different wavelength ranges and different service lives. The light-emitting signal probe has the light-emitting characteristics of single-wavelength excitation and multi-wavelength multi-lifetime emission, can realize simultaneous detection of different disease markers, and is particularly suitable for simultaneous detection of disease markers with large concentration difference.

Description

Technical Field

[0001] The present invention relates to the field of luminescence detection technology, and in particular to a luminescence signal probe and a luminescence regulating medium thereof, an immunochromatographic test strip and a preparation method thereof, a kit and an immunochromatographic detection method. Background Art

[0002] Lateral flow immunoassay (LFIA) has been widely used in various rapid testing occasions. It uses the principle of antigen-antibody binding and can perform qualitative or semi-quantitative analysis of various biomarkers without the need for complex equipment. LFIA technology is widely used in medical testing, food safety testing, environmental monitoring and other fields because of its simple operation, low cost and no need for complex laboratory equipment.

[0003] Immunochromatographic test strips usually consist of multiple parts, including a sample application area, a binder pre-coated area, a test line area (T line area) and a control line area (or quality control line area, C line area). After the analyte in the sample binds to the antibody pre-coated on the test strip, it migrates to the T line area under the promotion of chromatography and binds to the antibody fixed there to form a visible line. The unbound analyte continues to migrate to the C line area and binds to the control antibody to form a second visible line, proving that the test is effective. This binding effect is usually achieved by visual recognition of markers such as metal nanoparticles (such as gold or silver), fluorescent particles or dye particles.

[0004] Despite the above advantages of LFIA, it has encountered some challenges in practical applications, especially when facing the need to detect multiple target analytes at the same time. In existing luminescence detection technologies, a single wavelength excitation light source is usually used to excite the marker, and then the emitted light is recorded. This single wavelength excitation emission (SWEE) method has efficiency problems when simultaneously detecting multiple markers with different excitation and emission wavelengths. In order to detect multiple analytes, each requires a separate excitation and emission process, which is not only time-consuming, but also increases the cost accordingly. At the same time, each analyte requires an independent excitation source and detection channel, which makes the equipment more complex and expensive, thereby limiting its application in resource-limited environments.

[0005] Multiplex detection also brings the problem of signal interference due to the reliance on multiple markers during the detection process. The spectra emitted by different markers may be close or overlap, which makes it difficult for existing SWEE methods to distinguish different signals. In this case, cross-reaction of signals may lead to misjudgment, reducing the accuracy and reliability of the results. This limitation is particularly serious in applications with extremely high requirements for detection sensitivity and specificity, such as clinical diagnosis. In addition, ease of operation is a major advantage of rapid diagnostic technology. However, in order to adapt to the needs of multiple detection, traditional luminescence detection technology must perform multiple tests or use multiple excitation sources, which not only increases the complexity of the operation, but also prolongs the overall detection time and increases the technical requirements of the operator. The limitations of this method are particularly significant in situations where rapid response is required, such as rapid screening of multiple pathogens during an epidemic outbreak.

[0006] Therefore, an innovative solution is urgently needed to overcome these limitations and improve the capabilities of LFIA technology in multiplexing. Such a solution should be able to improve the sensitivity and specificity of the detection, reduce signal interference, and simplify the operation process to make it more user-friendly and reduce costs. Summary of the invention

[0007] To solve the above problems in the prior art, the present application provides a luminescent signal probe and a luminescent regulating medium thereof, an immunochromatographic test strip and a preparation method thereof, a kit and an immunochromatographic detection method, which can simultaneously perform rapid detection of at least two disease markers with a large difference in concentration.

[0008] The first aspect of the present application provides a luminescent signal probe, which includes at least two metal oxide nanoparticles. When the at least two metal oxide nanoparticles are mixed with the same luminescent regulating medium, different luminescent complexes are formed respectively; and when irradiated with the same excitation light, the different luminescent complexes emit light of different wavelength ranges and different lifetimes. Since the luminescent signal probe of the present application has the above-mentioned luminescent characteristics of single-wavelength excitation and multi-wavelength and multi-lifetime emission, when the above-mentioned at least two metal oxide nanoparticles are respectively used as signal tags of at least two disease markers, the simultaneous detection of these disease markers can be achieved, which is particularly suitable for the simultaneous detection of disease markers with large concentration differences.

[0009] In some embodiments, one of the metal oxide nanoparticles is Eu2O3 nanoparticles, and the other metal oxide nanoparticles is ZnO nanoparticles. When the Eu2O3 nanoparticles and the luminescence regulating medium are mixed, a europium complex is formed, and when the ZnO nanoparticles and the luminescence regulating medium are mixed, a zinc complex is formed. The europium complex and the zinc complex can emit light of different wavelength ranges and different lifespans under the same excitation light.

[0010] In some preferred embodiments, the excitation light uses 365nm ultraviolet light. Under the irradiation of the 365nm ultraviolet light, the luminescent complex formed by the Eu2O3 nanoparticles can excite red light with a central wavelength of 613nm and a lifetime in the microsecond level, while the luminescent complex formed by the ZnO nanoparticles can excite green light with a central wavelength of 520nm and a lifetime in the nanosecond level. Although the luminescent spectra of the red light and the green light overlap, the luminescent lifetimes of the two are quite different, so that the two optical signals can be extracted separately, thereby realizing the simultaneous detection of different disease markers.

[0011] In some embodiments, the luminescent signal probe includes at least two luminescent microspheres, one of which includes a first carrier medium, and the first carrier medium is loaded with one of the metal oxide nanoparticles, and the other of which includes a second carrier medium, and the second carrier medium is loaded with another metal oxide nanoparticle.

[0012] In some embodiments, the first support medium and the second support medium are both mesoporous silica.

[0013] In some embodiments, the surface of the luminescent microspheres also has carboxyl functional groups for coupling with antibodies.

[0014] In some embodiments, the luminescent signal probe further comprises at least two antibodies coupled to the at least two luminescent microspheres, one of which is a first antibody and the corresponding antigen is a first antigen, and the other of which is a second antibody and the corresponding antigen is a second antigen; the concentration of the first antigen in the sample to be tested is less than the concentration of the second antigen in the sample to be tested, and the detection linear range of the first antigen is narrower than the detection linear range of the second antigen. For example, the first antigen is procalcitonin (PCT), the first antibody is a procalcitonin antibody, the second antigen is C-reactive protein (CRP), and the second antibody is a C-reactive protein antibody. The procalcitonin is a highly sensitive infectious factor, and its concentration in the blood is very low, and its Cut-off value is 0.25ng / mL, so it places high demands on the sensitivity of the detection technology. The concentration of C-reactive protein in the blood is much higher, with a Cut-off value of 10μg / mL, which is nearly 4 orders of magnitude higher than the Cut-off value of procalcitonin, and the detection linear range of C-reactive protein is very wide, and clinically, samples with high C-reactive protein values ​​can be greater than 1000μg / mL. If the two are detected together, the linear range spans nearly 7 orders of magnitude. Therefore, for immunochromatographic analysis technology, it is a huge challenge to perform joint detection of procalcitonin and C-reactive protein on the same test strip. However, the present application can effectively achieve simultaneous joint detection of the two by designing the luminescent signal probe.

[0015] In some embodiments, the preparation method of the luminescent signal probe includes: providing at least two luminescent microspheres, one of which includes a first carrier medium, and the first carrier medium is loaded with one of the metal oxide nanoparticles, and the second luminescent microsphere includes a second carrier medium, and the second carrier medium is loaded with another metal oxide nanoparticle; at least two antibodies are respectively coupled to the at least two luminescent microspheres. Preferably, the covalent coupling between the luminescent microspheres and the corresponding antibodies is achieved by a cross-linking agent such as 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and / or N-hydroxysuccinimide (NHS). The luminescent microspheres can be prepared by any existing method.

[0016] The second aspect of the present application provides a luminescence regulating medium for the above-mentioned luminescent signal probe, wherein the luminescence regulating medium comprises at least two luminescent ligands, a surfactant and a buffer solution, wherein the at least two luminescent ligands are used to form at least two luminescent complexes with at least two metal oxide nanoparticles.

[0017] In some preferred embodiments, one of the luminescent ligands includes β-naphthoyltrifluoroacetone (β-NTA) and topoisomerase (TOPO), and another luminescent ligand includes 8-hydroxyquinoline (8-HQ). The β-NTA, the TOPO and the Eu2O3 nanoparticles can form a luminescent complex Eu(β-NTA)3(TOPO)2, and the 8-HQ and the ZnO nanoparticles can form a luminescent complex ZnQ2. The luminescent complex Eu(β-NTA)3(TOPO)2 and the luminescent complex ZnQ2 can respectively excite two lights with different central wavelengths and different lifespans under the irradiation of ultraviolet light of the same wavelength.

[0018] In some preferred embodiments, the luminescence regulating medium further satisfies at least one of the following conditions:

[0019] (1) The surfactant includes an amphoteric surfactant and / or a nonionic surfactant; preferably, the nonionic surfactant includes Triton X-100 and / or Tween 20 (polysorbate 20), and the amphoteric surfactant includes BS12 (dodecyl dimethyl betaine) and / or CAB35 (cocamidopropyl betaine); most preferably, the surfactant is CAB35;

[0020] (2) The mass percentage concentration of the surfactant is 0.05% to 0.5%. When the mass percentage concentration of the surfactant is within this range, the luminescence intensity of different luminescent complexes can be higher; most preferably, the mass percentage concentration of the surfactant is 0.2%;

[0021] (3) The buffer solution is a glycine-hydrochloric acid buffer solution, and the glycine-hydrochloric acid buffer system has the characteristics of coordination dissociation for Eu2O3 nanoparticles and ZnO nanoparticles;

[0022] (4) The pH value of the luminescence regulating medium is 4 to 8. The pH value will have a complex effect on the coordination dissociation kinetics of Eu nanomaterials, including fluorescence intensity and reaction rate. A slightly alkaline environment is conducive to the luminescence enhancement of Zn complexes, while a weakly acidic environment is conducive to the luminescence enhancement of Eu complexes. Considering that Zn complexes have a wider peak band and are easier to extract signals, and the signals of Eu complexes will be used for high-sensitivity detection projects with low target analyte content in samples, the selection of pH value will give priority to conditions that are conducive to the luminescence of Eu complexes; most preferably, the pH value of the luminescence regulating medium is 5;

[0023] (5) The molar ratio of the β-naphthoyl trifluoroacetone, the topoisomerase and Eu is (1.2-4.5):(0.8-3):1, and the molar ratio of Zn and the 8-hydroxyquinoline is 1:(0.8-3); when the molar ratio of the luminescent ligand is within this range, the luminescent complex has a high luminescence intensity. Most preferably, the molar ratio of the β-naphthoyl trifluoroacetone, the topoisomerase and Eu is 3:2:1, and the molar ratio of Zn and the 8-hydroxyquinoline is 1:2.

[0024] The third aspect of the present application provides an immunochromatographic test strip comprising any of the luminescent signal probes described above.

[0025] In some embodiments, the immunochromatographic test strip comprises a sample pad, a conjugate pad, a test line and a quality control line arranged in sequence, wherein the conjugate pad is provided with the luminescent signal probe, and the test line is fixed with at least two antibodies.

[0026] In some preferred embodiments, one of the antibodies is a procalcitonin antibody, another of the antibodies is a C-reactive protein antibody, and the quality control line is fixed with goat anti-chicken IgY.

[0027] In some preferred embodiments, the immunochromatographic test strip further comprises a detection pad, wherein the test line and the quality control line are arranged on the detection pad.

[0028] In some preferred embodiments, the immunochromatographic test strip further comprises a water absorbent pad, and the water absorbent pad is located on a side of the detection pad away from the conjugate pad.

[0029] The fourth aspect of the present application provides a method for preparing the above-mentioned immunochromatographic test strip, comprising: sticking a sample pad, a binding pad, and a detection pad provided with a test line and a quality control line on a bottom plate.

[0030] In some preferred embodiments, the preparation method of the conjugate pad includes: coating the surface of the conjugate pad with a dilution of the luminescent signal probe described in any one of the above and drying; further preferably, in the dilution, the concentration of one luminescent microsphere is 50μg / mL to 150μg / mL, and the concentration of another luminescent microsphere is 40μg / mL to 100μg / mL. When the concentration of the luminescent microsphere is within this range, it has a stronger detection signal (fluorescent signal), and thus has a higher detection sensitivity.

[0031] In some embodiments, the preparation method of the detection pad includes: on the NC membrane, coating at least two antibodies in the form of lines on the same test line or different test lines, and coating goat anti-chicken IgY in the form of lines on the quality control line.

[0032] The fifth aspect of the present application provides a kit, comprising the luminescent signal probe described in any of the above items, or the immunochromatographic test strip described in any of the above items, or the immunochromatographic test strip prepared by the preparation method of the immunochromatographic test strip described in any of the above items, and the luminescent regulating medium described in any of the above items.

[0033] The sixth aspect of the present application provides an immunochromatographic detection method, comprising: providing a luminescent signal probe as described in any of the above items, or an immunochromatographic test strip as described in any of the above items, or an immunochromatographic test strip prepared according to the preparation method of the immunochromatographic test strip as described in any of the above items, or the above-mentioned test kit; first contacting the luminescent signal probe with a sample to be tested, and then treating the luminescent signal probe with a luminescent regulating medium as described in any of the above items; irradiating the luminescent signal probe after treatment with excitation light, and reading the light signal emitted by the luminescent signal probe.

[0034] In some preferred embodiments, the contact time between the luminescent signal probe and the sample to be tested is 6 minutes to 15 minutes. When the sample to be tested with an antigen contacts the luminescent signal probe, an immune reaction of the antigen and antibody will occur. After the antigen and antibody are fully reacted and combined, the test is performed. In addition to obtaining more stable and accurate test results, the sensitivity is further improved due to the improvement of the signal-to-noise ratio. Therefore, a suitable immune reaction time is conducive to having a higher sensitivity in a wide linear range.

[0035] In some preferred embodiments, the time for treating the luminescent signal probe with the luminescent regulating medium is 2 min to 5 min.

[0036] In some preferred embodiments, the excitation light is 365nm ultraviolet light, and the luminescent signal probe emits two different light signals with central wavelengths of 613nm and 520nm and lifetimes in microseconds and nanoseconds, respectively.

[0037] In some preferred embodiments, the method for reading the light signal emitted by the luminescent signal probe includes: collecting one of the light signals at a time by time-gated fluorescence technology, and sequentially delaying the collection of other light signals. For example, the green fluorescence of ZnQ2 at 520nm is collected at zero time, and the red afterglow of Eu(β-NTA)3(TOPO)2 at 613nm is collected with a delay, so as to realize the joint detection of different inflammatory markers CRP (local inflammatory response protein marker) and PCT (systemic outbreak infection inflammatory marker).

[0038] Compared with the prior art, the technical solution of the present application has the following beneficial effects:

[0039] The luminescent signal probe of the present application has the characteristics of single-wavelength excitation, multi-wavelength emission and multi-lifetime luminescence, so multiple probes can be labeled in two dimensions: emission wavelength and luminescence lifetime, effectively expanding the throughput of immunochromatographic detection, and realizing the simultaneous detection of multiple disease markers, especially the simultaneous detection of disease markers with large concentration differences. Based on the expansion of the luminescence lifetime dimension, the performance of wide linear range and high sensitivity is taken into account under a single channel, and the simultaneous detection of different items with large differences in sample concentration content is realized, laying the foundation for the development of multiple combined detection of point-of-care testing (POCT). Furthermore, Eu2O3 nanoparticles and ZnO nanoparticles are used as the combined labeling of enhanced luminescent probes, which can detect PCT antigens with very low concentrations in blood samples, and CRP antigens with very high concentrations in blood samples, achieving the compatibility of high sensitivity and wide linearity.

[0040] Different from the current principle that acidic media promote the luminescence enhancement of metal complexes, the present application found during the research process that the luminescence enhancement phenomenon is closely related to the coordination process of metal ions, and therefore proposed a new mechanism of coordination-enhanced luminescence. The luminescence regulation medium of the present application adopts a glycine-hydrochloric acid buffer system with a weak acidity to neutrality or even weak alkalinity (pH value of 4 to 8), which subverts the existing technology that must use a strong acidic medium (pH value of 2 to 3), thereby bringing many conveniences to the treatment of waste liquid after the detection is completed, such as no need to neutralize the waste liquid, and it is easier to maintain without volatile liquids such as acetic acid.

[0041] The immunochromatographic test strip of the present application can realize the simultaneous detection of multiple disease markers with very different concentrations on a single card and a single detection line, thereby realizing high-throughput detection of a single-card immunochromatographic test strip, and solving the problem that the window length of the classic immunochromatographic test strip is limited (about 18 mm) and multiple immune reaction areas (i.e., multiple T lines) cannot be arranged, resulting in a very limited detection throughput of the single-card immunochromatographic test strip.

[0042] The immunochromatographic detection method of the present application has developed "Coordination-enhanced Fluorescence-based Lateral Flow Immunoassay" (CELFIA) and "Coordination-enhanced Time-resolved Fluorescence-based Lateral Flow Immunoassay" (TR-CELFIA) based on the new coordination-enhanced luminescence mechanism. Through time-gated fluorescence technology and spectral splitting technology, it can realize the simultaneous detection of multiple disease markers, and can also meet the requirements of wide linear range and high sensitivity compatibility, thereby enabling immunochromatographic instant detection to meet more application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The following drawings describe in detail the exemplary embodiments disclosed in this application. The same reference numerals represent similar structures in several views of the drawings. Those skilled in the art will understand that these embodiments are non-limiting, exemplary embodiments, and the drawings are only used for the purpose of illustration and description, and are not intended to limit the scope of this application. Other embodiments may also accomplish the inventive intent in this application. It should be understood that the drawings are not drawn to scale. Among them:

[0044] Figure 1 This is a schematic diagram of the single wavelength excitation dual wavelength emission and dual lifetime luminescence multiple detection principle of Example 1 of the present application;

[0045] Figure 2 The emission spectra when ZnO solution and Eu2O3 solution are added separately or simultaneously to the luminescence regulating medium of Example 4 of the present application;

[0046] Figure 3 The enhanced luminescence spectrum of the luminescence regulating medium of Examples 4 to 7 of the present application;

[0047] Figure 4 The enhanced luminescence spectrum of the luminescence regulating medium of Examples 8 to 12 of the present application;

[0048] Figure 5 This is a comparison chart of the luminescence intensity of the luminescence regulating media of Examples 13 to 19 of the present application at 520 nm and 613 nm;

[0049] Figure 6 This is the correlation between the test kit in Example 52 of the present application and the CRP test results of the Siemens nephelometry method, as well as the correlation with the PCT test results of the Siemens chemiluminescence method. DETAILED DESCRIPTION

[0050] The following description provides specific application scenarios and requirements of the present application, with the purpose of enabling those skilled in the art to make and use the content in the present application. It will be apparent to those skilled in the art that various local modifications to the disclosed embodiments are apparent, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present application. Therefore, the present application is not limited to the embodiments shown, but to the widest scope consistent with the claims.

[0051] Multi-item joint detection with high sensitivity and wide linear range is urgently needed in clinical practice. However, current luminescent immunochromatography methods and detection systems are usually difficult to take both into account, which limits the clinical application scope of chromatography technology based on fluorescence quantitative detection to a certain extent. For example, the detection level of CRP is mg level, while the detection level of PCT is pg level, and the sensitivity differs by two orders of magnitude. Based on this, the present application provides a luminescent signal probe with single-wavelength excitation, multi-wavelength emission and multi-lifetime luminescence characteristics, including at least two metal oxide nanoparticles, each of which can form a corresponding luminescent complex when mixed with a luminescent regulating medium. Different luminescent complexes can emit light of different wavelengths and different lifespans under the irradiation of excitation light, which can achieve simultaneous detection of disease markers with large concentration differences.

[0052] Furthermore, considering that the high luminescence brightness of the probe is beneficial to improving the detection signal-to-noise ratio, the present application selects the high-brightness Eu 3+ The luminescent signal is adapted to the high-sensitivity detection project, and combined with Eu 3+ The long lifespan of ZnQ2 can further improve the sensitivity by eliminating background fluorescence interference using time gating technology, thus achieving high sensitivity detection of PCT. 3+ The emission peak is wide, so it is still easy to extract the signal in the low value area of ​​CRP, and the range can be widened to the high value area of ​​CRP without producing a hook effect.

[0053] Based on the above analysis, the long-life narrow peak and high-brightness fluorescence in the fluorescence detection system can adapt to the high-sensitivity detection of low-concentration target analytes, and the low-brightness wide peak can adapt to the wide-range detection of high-concentration target analytes. Based on the special luminescence properties of the above two probes, the fluorescence immunoassay instrument can distinguish different fluorescence signals, further improve the detection sensitivity through time resolution technology, and ultimately achieve a wide range of multiple quantitative detection targets for different target detection objects with large concentration differences.

[0054] During the test, the steady-state luminescence signal of 520nm wavelength can be collected under 365nm ultraviolet light, and the 613nm Eu3+ Finally, the two collected fluorescence signals are substituted into the pre-stored instant and delayed standard curves, respectively, to obtain the concentration values ​​of the two targets to be tested in the same sample. It can be seen that the signal amplification strategy of coordination-enhanced luminescence proposed in this application, combined with the characteristics of single-wavelength excitation and dual-lifetime dual-wavelength emission of the probe, can achieve highly sensitive immunoassays with a wide linear range.

[0055] The following examples will be described using luminescent signal probes including Eu2O3 nanoparticles and ZnO nanoparticles as examples. Unless otherwise specified, the reagents and raw materials used can be purchased through commercial channels. The experimental methods in the following examples that do not specify specific conditions are selected according to conventional methods and conditions, or according to the product specifications.

[0056] Table 1 lists the main instruments and materials involved in the following examples.

[0057] Table 1 Main instruments and materials

[0058]

[0059]

[0060] Luminescent signal probe

[0061] Example 1

[0062] The luminescent signal probe of this embodiment includes Eu2O3 nanoparticles and ZnO nanoparticles.

[0063] The luminescent signaling probe is mixed with a luminescence regulating medium comprising β-NTA, TOPO and 8-HQ.

[0064] Figure 1 The schematic diagram of single wavelength excitation dual wavelength emission and dual lifetime luminescence multiple detection. After Eu2O3 nanoparticles are mixed with their luminescent ligands (β-NTA and TOPO), the system can be excited by 365nm ultraviolet light to emit bright red light. After ZnO nanoparticles are mixed with their luminescent ligands (8-HQ), the system can be excited by 365nm ultraviolet light to emit bright green light. Therefore, the excitation wavelength of both systems can be selected as 365nm.

[0065] Specifically, under the excitation of 365nm ultraviolet light, after Eu2O3 nanoparticles are mixed with their luminescent ligands, they emit green light with a central wavelength of 613nm and a half-peak width of 12nm. After ZnO nanoparticles are mixed with their luminescent ligands, they emit green light with a central wavelength of 520nm and a half-peak width of 80nm. The emission peaks of the two systems overlap. Therefore, when Eu2O3 nanoparticles and ZnO nanoparticles are mixed with their luminescent ligands at the same time, the luminescence spectra of the two systems overlap, and the luminescence of the formed ZnO2 complex will interfere with the luminescence of Eu2O3 nanoparticles. 3+ The measurement of luminous intensity can be processed by spectrum unmixing.

[0066] Due to Eu 3+ The luminescence lifetime of the complex (Eu(β-NTA)3(TOPO)2) is long (in microseconds), while the luminescence lifetime of ZnQ2 is only in nanoseconds. Therefore, the existing time-gated fluorescence technology can be used to 3+ The luminescence signal of the complex is extracted from the mixed spectrum of the two.

[0067] Example 2

[0068] The luminescent signal probe of the embodiment of the present application includes a first luminescent microsphere (Eu2O3@DMSNs microsphere) and a second luminescent microsphere (ZnO@DMSNs microsphere), wherein the Eu2O3@DMSNs microsphere includes mesoporous silica and Eu2O3 nanoparticles loaded on the mesoporous silica. The ZnO@DMSNs microsphere includes mesoporous silica and ZnO nanoparticles loaded on the mesoporous silica.

[0069] The preparation method of the Eu2O3@DMSNs microspheres comprises:

[0070] Surface hydrophilic modification of Eu2O3 nanoparticles: Eu2O3 nanoparticles were dispersed in 10 mL of toluene, stirred magnetically in a water bath, and 1 mL of 3-aminopropyltriethoxysilane (APTES) toluene solution (0.1 M) and 1 mL of TMAH methanol solution (0.1 M) were added, and stirred at 85°C for 30 minutes. Centrifuged, washed with toluene 3 times to remove oleic acid and oleylamine. The precipitate was dispersed again with toluene, and 1 mL of APTES toluene solution (0.1 M) and 1 mL of TMAH methanol solution (0.1 M) were added, and stirred at 85°C for 30 minutes. Washed with n-hexane by centrifugation twice, and washed with ethanol by centrifugation twice, and finally the precipitate was added with 10 mL of pure water for ultrasonic dispersion and preservation.

[0071] Dendritic mesoporous silica nanoparticles were prepared in one step using the CTAB template method: 68 mg of triethanolamine (TEA) was added to 25 mL of water and magnetically stirred at 80 °C for 30 minutes. 168 mg of sodium salicylate (NaSal) and 380 mg of CTAB were added, and stirring continued for 1 hour. Under stirring at 300 rpm, 4 mL of TEOS was added dropwise, and stirring continued at 300 rpm for 2 hours. Then stirring was stopped and the mixture was allowed to stand at 80 °C for 6 hours. The product was collected by centrifugation at 20,000 rpm for 15 minutes, and washed several times with water and ethanol. Finally, V (HCl) :V (ethanol) = 1:9 mixture, stirred at 80°C for 2 hours, then washed at 20000 rpm for 15 min. Repeat the previous step three times, stirring for 2 hours each time, to remove CTAB.

[0072] Preparation of Eu2O3@DMSNs microspheres: Take a hexane solution of hydrophilically modified Eu2O3 nanoparticles (containing about 80 mg of Eu2O3), centrifuge and re-dissolve with 10 mL of toluene by ultrasound. Take dendritic mesoporous silica nanoparticles (DMSNs, 200 mg) and centrifuge and add the aforementioned toluene containing hydrophilically modified Eu2O3 nanoparticles and 100 μL of oleylamine, sonicate for 10 minutes, and stir magnetically at 50°C for 10 hours. Then add ethanol, centrifuge, wash twice with ethanol and hexane by centrifugation, and finally disperse the Eu2O3@DMSNs microspheres in toluene for storage.

[0073] The preparation method of the ZnO@DMSNs microspheres can refer to the preparation method of Eu2O3@DMSNs microspheres.

[0074] ZnO@DMSNs microspheres have broad peaks that are easy to extract, while Eu2O3@DMSNs microspheres have bright narrow peaks. 3+ The emission peaks of Eu2O3 and Eu3O3 overlap, but because it is a short-lived fluorescence, when the time-gated fluorescence technique (set delay 200ms) is used to collect Eu2O3, 3+ When the emission peak of ZnQ3 is reached, the emission peak of ZnQ3 can no longer be detected by the detector. 3+ The luminescent signal eliminates the interference of background signals and further improves the sensitivity.

[0075] In order to further verify the feasibility of the dual-luminescence system, the luminescent ligands were added interchangeably, that is, Eu2O3@DMSNs microspheres were mixed with 8-HQ, and ZnO@DMSNs microspheres were mixed with β-NTA and TOPO. The emission spectra of the two mixed systems were tested respectively under 365nm ultraviolet light excitation using FS5 fluorescence spectrometer to study their mutual cross-influence on the emission spectra.

[0076] The results showed that after 8-HQ was mixed with excess Eu2O3@DMSNs microspheres, no Eu 3+ The emission peak of 8-HQ was not significantly enhanced, indicating that Eu2O3@DMSNs microspheres would not react with 8-HQ; after mixing β-NTA, TOPO and excess ZnO, no obvious luminescence enhancement effect was observed. Therefore, the two luminescent ligands have good specificity for their respective ion coordination, which also shows that there is no mutual interference in the dual-wavelength luminescence system, verifying the feasibility of the system.

[0077] Furthermore, the luminescence lifetimes of the Eu(β-NTA)3(TOPO)2 complex formed by mixing Eu2O3@DMSNs microspheres with β-NTA and TOPO and the ZnQ3 complex formed by mixing ZnO@DMSNs microspheres with 8-HQ were tested and compared. The results showed that the lifetime of ZnQ3 was only in the nanosecond range, while the lifetime characterization results of Eu(β-NTA)3(TOPO)2 were consistent with the luminescence lifetime of Eu2O3, both showing that 3 + Lifetime of microseconds. The long-life luminescence of microseconds not only creates conditions for time-gated fluorescence technology, but also lays the foundation for distinguishing on the same carrier through the dimension of lifetime.

[0078] Example 3

[0079] The luminescent signal probe of this embodiment is based on the embodiment 2, and the Eu2O3@DMSNs microspheres and PCT antibody are covalently coupled by cross-linking agents EDC and NHS to form Eu2O3@DMSNs- PCT ZnO@DMSNs microspheres were covalently coupled with CRP antibody to form ZnO@DMSNs- CRP .

[0080] Eu2O3@DMSNs microspheres and PCT antibody were covalently coupled. The specific steps are as follows:

[0081] 1) Surface carboxylation of Eu2O3@DMSNs microspheres: Take a certain amount of Eu2O3@DMSNs nanoparticles, centrifuge and remove the supernatant, add 20mL toluene and ultrasonically dissolve, add 2mL of 0.1M Silane-PEG2000-COOH toluene solution under magnetic stirring, and continue stirring for 5 minutes. Then, add 2mL of 0.1M TMAH methanol solution, heat to 85℃, and react for 45 minutes. After cooling to room temperature, add n-hexane for centrifugal separation, wash with n-hexane by centrifugation 3 times, and then wash with ethanol by centrifugation 2 times. Finally, add pure water and ultrasonically disperse.

[0082] 2) Activation: 100 μL of carboxylated Eu2O3@DMSNs microspheres (10 mg / mL) were added to 900 μL of MES (25 mM, pH = 6.0) buffer, followed by 20 μL of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) solutions, mixed quickly and activated at room temperature for 15 minutes. After centrifugation and washing, the microspheres were ultrasonically dispersed in 950 μL of BBS (0.01 M, pH = 7.4) buffer.

[0083] 3) Labeling: Add 0.2 mg PCT labeled antibody to the activated microsphere solution, rotate and mix at room temperature, and incubate for 3 hours.

[0084] 4) Blocking: 10 wt % BSA (final concentration of 1 wt %) was added as a blocking agent to the labeled microsphere solution. After rotating and mixing at room temperature for 2 hours, the solution was centrifuged and washed to obtain BSA-blocked nanoparticles.

[0085] 5) Storage: The above nanoparticles were dispersed into 1 mL of BBS buffer (0.05 M BBS, pH=7.0, 0.5 wt % BSA and 0.05 wt % proclin 300), and stored in a refrigerator at 4° C. for future use.

[0086] The method for covalently coupling ZnO@DMSNs microspheres with CRP antibody is as follows:

[0087] 1) Activation: 100 μL ZnO@DMSNs microspheres (10 mg / mL) were added to 900 μL MES (25 mM, pH = 6.0), and 20 μL NHS (10 mg / mL) and 20 μL EDC (10 mg / mL) were added in sequence. After activation for 15 minutes, the microspheres were centrifuged and washed to remove excess cross-linking reagents, and then ultrasonically dispersed and redissolved in 950 μL BBS (0.01 M, pH = 7.4) buffer.

[0088] 2) Labeling: Add 0.2 mg of CRP labeled antibody to the activated microsphere solution and incubate at room temperature for 3 hours with 360-degree rotation.

[0089] 3) Blocking: Add 50 μL of 1% BSA solution to the labeled solution for blocking. After reacting for 2 hours, centrifuge and remove the supernatant. Add 1 mL of BBS stock solution (0.01 M, pH=7.4, containing 1 wt% BSA and 0.05 wt% PC300) to the centrifuged product and re-dissolve it by ultrasonication.

[0090] 4) Storage: The coupled microspheres are stored in the reserve solution and placed in a 4°C refrigerator for future use.

[0091] Luminescence Modulating Medium

[0092] Example 4

[0093] ZnQ3 emits strong light in an environment with a pH greater than 5.0, and the emission peak width is easy to extract. In a weak acid environment, it is conducive to the reaction of Eu2O3 with its ligand β-NTA and TOPO (for example, pH = 4). However, it has been experimentally verified that it can be selected in the pH range of 3 to 7. However, the emission peak of Eu(β-NTA)3(TOPO)2 is narrow and not easy to extract in the two-color peak spectrum. Therefore, the luminescence regulating medium is temporarily more inclined to favor Eu 3+ Weakly acidic environment for reaction with β-NTA and TOPO.

[0094] Based on this, this embodiment prepares a luminescence regulating medium containing 0.8mM 8-HQ, 15μM β-NTA, 10μM TOPO, with glycine-hydrochloric acid at pH=5.0 as the buffer system, and containing 0.1wt% Triton X-100, and ultrasonically dispersed for use.

[0095] The prepared luminescence regulating medium was added to a cuvette, and the feasibility of ZnO and 8-HQ, Eu2O3 and β-NTA, and TOPO dual-coordinated components in a reaction system was verified by adding ZnO nanoparticles in n-hexane solution (10mM, 100μL) and Eu2O3 nanoparticles in n-hexane solution (1.25mM, 50μL) separately and adding ZnO nanoparticles in n-hexane solution (10mM, 100μL) and Eu2O3 nanoparticles in n-hexane solution (1.25mM, 50μL) at the same time. After mixing and standing for 5 minutes, the fluorescence spectrum (λex=365nm) was tested on an FS5 fluorescence spectrometer.

[0096] refer to Figure 2 The emission peak positions and emission peak intensities of the n-hexane solution of ZnO nanoparticles or the n-hexane solution of Eu2O3 nanoparticles added alone, and the n-hexane solution of ZnO nanoparticles and the n-hexane solution of Eu2O3 nanoparticles added simultaneously are almost completely overlapped, which proves the feasibility of the luminescence regulating medium of this embodiment.

[0097] Example 5

[0098] The only difference from the luminescence regulating medium of Example 4 is that the surfactant Triton X-100 is replaced by BS12.

[0099] Example 6

[0100] The only difference from the luminescence regulating medium of Example 4 is that the surfactant Triton X-100 is replaced by CAB35.

[0101] Example 7

[0102] The only difference from the luminescence regulating medium of Example 4 is that the surfactant Triton X-100 is replaced by Tween20.

[0103] Comparative Example 1

[0104] The only difference from the luminescence regulating medium of Example 4 is that the surfactant Triton X-100 is replaced by cetyltrimethylammonium bromide (CTAB).

[0105] Comparative Example 2

[0106] The only difference from the luminescence regulating medium of Example 4 is that the surfactant Triton X-100 is replaced by sodium dodecyl sulfate (SDS).

[0107] According to the reaction system with molar ratio (NTA:TOPO):Eu=(3:2):1 and 8-HQ:Zn=2:1, samples were added respectively to test the effect of different surfactant types on luminescence intensity (fluorescence spectrum), and the afterglow life of ZnO and Eu2O3 was tested at the same time. The test method is as follows: the luminescence regulating medium prepared in Examples 4 to 7 and Comparative Examples 1 to 2 was added to the cuvette, and then a n-hexane solution (10 mM) of ZnO nanoparticles and a n-hexane solution (1.25 mM) of Eu2O3 nanoparticles were added, and after rapid mixing and standing for 3 minutes, the fluorescence spectrum was tested using an FS5 fluorescence spectrometer, λex=365nm.

[0108] The results show that the coordination enhancement effects of anionic surfactants and cationic surfactants on Eu2O3 and NTA, TOPO and ZnO and 8-HQ are not ideal, while the coordination enhancement effects of non-ionic surfactants and amphoteric surfactants on Eu2O3 and NTA, TOPO and ZnO and 8-HQ are relatively ideal. Figure 3The figure shows the coordination enhancement effect of nonionic surfactants and amphoteric surfactants. Although nonionic surfactants (Triton X-100, Tween 20) have a relatively good effect on enhancing luminescence, they are still slightly inferior to amphoteric surfactants (BS12, CAB35). When TritonX-100 is added, the emission peak at 520nm enhanced by the coordination dissociation of ZnO and 8-HQ is the weakest, while for Eu 3+ The luminescence at 613nm has a stronger enhancement effect; similarly, after adding BS12, although the emission peak at 520nm is the strongest, the luminescence at 613nm is the weakest among the four, that is to say, the addition of these two surfactants will have a trade-off effect on the two emission peaks in the dual-luminescence system. After adding the luminescence regulating medium containing CAB35, the emission peaks at 520nm and 613nm are stronger than the luminescence of the luminescence regulating medium with Tween 20 added. Therefore, CAB35 is the most preferred surfactant.

[0109] Example 8

[0110] In this embodiment, a luminescence regulating medium is prepared, which contains 0.8 mM 8-HQ, 15 μM β-NTA, 10 μM TOPO, a glycine-hydrochloric acid buffer system with pH = 4.0, and 0.1 wt % CAB35, and is dispersed by ultrasound for use.

[0111] Example 9

[0112] The only difference from Example 8 is that pH=5.0.

[0113] Example 10

[0114] The only difference from Example 8 is that pH=6.0.

[0115] Embodiment 11

[0116] The only difference from Example 8 is that pH=7.0.

[0117] Example 12

[0118] The only difference from Example 8 is that pH=8.0.

[0119] The luminescence regulating medium prepared in Examples 8 to 12 was added to the cuvette, and then a n-hexane solution (10 mM) of ZnO nanoparticles and a n-hexane solution (1.25 mM) of Eu2O3 nanoparticles were added. After rapid mixing and standing for 3 min, the fluorescence spectrum was tested using an FS5 fluorescence spectrometer, λex = 365 nm, and photographed under a 365 nm ultraviolet lamp. Figure 4 To enhance the luminescence spectrum.

[0120] refer to Figure 4 As the pH value of the luminescence regulating medium increases, the emission peak intensity of ZnQ2 at 520 nm gradually increases, while the emission peak intensity of Eu(β-NTA)3(TOPO)2 at 613 nm gradually increases. 3+ The emission peak of ZnQ2 shows the strongest fluorescence signal at pH 5.0 (its fluorescence intensity is not only greater than pH 6.0-8.0, but also higher than pH 4.0). In other words, a slightly alkaline environment is conducive to the enhancement of the luminescence of ZnQ2, while a weakly acidic environment is conducive to the enhancement of the luminescence of Eu(β-NTA)3(TOPO)2. Considering that ZnQ2 has a wider peak band, it is easier to extract signals, while Eu 3+ The signal will be used for high-sensitivity detection of target analytes with low content in samples, so the selection of pH value will give priority to the signal that favors Eu 3+ Therefore, the most preferred condition is pH=5.0.

[0121] Example 13

[0122] In this embodiment, a luminescence regulating medium is prepared, which contains 0.8 mM 8-HQ, 15 μM β-NTA, 10 μM TOPO, with glycine-hydrochloric acid at pH = 5.0 as the buffer system, and contains 0.05 wt % CAB35, and is dispersed by ultrasound for use.

[0123] Embodiment 14

[0124] The only difference from Example 13 is that CAB35 is 0.1 wt%.

[0125] Embodiment 15

[0126] The only difference from Example 13 is that CAB35 is 0.2 wt%.

[0127] Example 16

[0128] The only difference from Example 13 is that CAB35 is 0.3 wt%.

[0129] Embodiment 17

[0130] The only difference from Example 13 is that CAB35 is 0.4 wt%.

[0131] Embodiment 18

[0132] The only difference from Example 13 is that CAB35 is 0.5 wt%.

[0133] Embodiment 19

[0134] The only difference from Example 12 is that CAB35 is 0.0 wt %.

[0135] The luminescence regulating media prepared in Examples 13 to 19 were added to the cuvette respectively, and then the n-hexane solution of ZnO nanoparticles and the n-hexane solution of Eu2O3 nanoparticles were added. After rapid mixing and standing for 3 minutes, the fluorescence spectrum was tested using an FS5 fluorescence spectrometer, λex = 365nm.

[0136] refer to Figure 5 When the concentration of CAB35 reaches 0.2wt%, the luminescence intensity at 613nm has reached the highest. As the concentration of CAB35 further increases, the luminescence signal at 613nm begins to weaken; while the luminescence of ZnQ2 at 520nm begins to maintain a high luminescence intensity after the concentration of CAB35 reaches 0.2wt%. Therefore, the concentration of the surfactant is preferably 0.2wt%.

[0137] Embodiment 20

[0138] In this example, β-NTA and TOPO were added to a glycine-HCl buffer system (50 mM, containing 0.1 wt% CAB35) at pH 5.0 at a concentration of 0.4 times the ligand (molar ratio (β-NTA:TOPO):Eu=(3:2):1 for 1 times the ligand), and 0 times the ligand (molar ratio Zn 2+ :8-HQ=1:2 is 1 times ligand) add 8-HQ, and prepare the luminescence regulating medium by ultrasonic dispersion. Molar ratio (β-NTA:TOPO):Eu=(1.2:0.8):1, molar ratio Zn 2+ :8-HQ=1:2.

[0139] Embodiment 21

[0140] The only difference from Example 20 is that β-NTA and TOPO are added at a concentration of 0.6 times that of the ligand, and the molar ratio (β-NTA:TOPO):Eu=(1.8:1.2):1.

[0141] Embodiment 22

[0142] The only difference from Example 20 is that β-NTA and TOPO are added at a concentration of 0.8 times that of the ligand, and the molar ratio (β-NTA:TOPO):Eu=(2.4:1.6):1.

[0143] Embodiment 23

[0144] The only difference from Example 20 is that β-NTA and TOPO are added at a concentration of 1.0 times that of the ligand, and the molar ratio (β-NTA:TOPO):Eu=(3:2):1.

[0145] Embodiment 24

[0146] The only difference from Example 20 is that β-NTA and TOPO are added at a concentration 1.5 times that of the ligand, and the molar ratio (β-NTA:TOPO):Eu=(4.5:3):1.

[0147] Embodiment 25

[0148] In this embodiment, in a glycine-hydrochloric acid buffer system (50 mM, containing 0.1 wt% CAB35) at pH = 5.0, 8-HQ was added at a concentration of 1 times the ligand, and β-NTA and TOPO were added at a concentration of 0.4 times the ligand, and the luminescence regulating medium was prepared by ultrasonic dispersion. 2+ :8-HQ=1:2, according to the molar ratio (β-NTA:TOPO):Eu=(3:2):1.

[0149] Embodiment 26

[0150] The only difference from Example 25 is that 8-HQ is added at a concentration 10 times that of the ligand, and the molar ratio of Zn 2+ :8-HQ=1:20.

[0151] Embodiment 27

[0152] The only difference from Example 25 is that 8-HQ is added at a concentration 100 times that of the ligand, and the molar ratio of Zn 2+ :8-HQ=1:200.

[0153] Embodiment 28

[0154] The only difference from Example 25 is that 8-HQ is added at a concentration 1000 times that of the ligand, and the molar ratio of Zn 2+ :8-HQ=1:2000.

[0155] Embodiment 29

[0156] The only difference from Example 26 is that 8-HQ is added at a concentration of 10,000 times that of the ligand, and the molar ratio of Zn 2+ :8-HQ=1:20000.

[0157] Embodiment 30

[0158] In this example, β-NTA, TOPO and 8-HQ were added to a glycine-hydrochloric acid buffer system (50 mM, containing 0.1 wt% CAB35) at pH 5.0 at a concentration of 0.4 times that of the ligand, and a luminescence regulating medium was prepared by ultrasonic dispersion.

[0159] Embodiment 31

[0160] The only difference from Example 30 is that β-NTA, TOPO and 8-HQ were added at a concentration of 0.6 times that of the ligand.

[0161] Embodiment 32

[0162] The only difference from Example 30 is that β-NTA, TOPO and 8-HQ were added at a concentration of 0.8 times that of the ligand.

[0163] Embodiment 33

[0164] The only difference from Example 30 is that β-NTA, TOPO and 8-HQ were added at a concentration of 1.0 times that of the ligand.

[0165] Embodiment 34

[0166] The only difference from Example 30 is that β-NTA, TOPO and 8-HQ were added at a concentration 1.5 times that of the ligands.

[0167] The luminescence spectra of ZnO and 8-HQ in the luminescence regulating media of Examples 20 to 24 at 520 nm after coordination were tested using an FS5 fluorescence spectrometer, and the luminescence regulating media of Examples 25 to 29 and the luminescence regulating media in which 8-HQ was added at a concentration of 0 times the ligand were tested. 3+ The luminescence spectra of ZnO and Eu2O3 at 613nm after coordination with β-NTA and TOPO, as well as the luminescence spectra of ZnO and Eu2O3 in the luminescence regulating medium of test examples 30 to 34, λex = 365nm. The results show that with the increase of β-NTA and TOPO ligand concentrations, Zn 2+ The luminescence intensity of the ligand formed by 8-HQ will weaken; with the increase of 8-HQ ligand concentration, Eu 3+ The luminescence intensity will also weaken after coordination with β-NTA and TOPO, and the luminescence is already very weak when the 8-HQ concentration ratio reaches 1000 times. When the 8-HQ concentration ratio reaches 10000 times, the instrument cannot detect Eu 3+ of emitted light.

[0168] Immunochromatographic test strips

[0169] Embodiment 35

[0170] The immunochromatographic test strip of this embodiment comprises: a bottom plate and a sample pad, a conjugation pad, a test line and a quality control line arranged in sequence on the bottom plate, wherein the conjugation pad is provided with the Eu2O3@DMSNs- PCT and ZnO@DMSNs- CRP There is also a Eu2O3@DMSNs- 鸡IgYThe test line and the quality control line are arranged on the test pad, wherein the test line is fixed with PCT capture antibody and C-reactive protein antibody, and the quality control line is fixed with goat anti-chicken IgY. A water-absorbing pad is also arranged on the side of the test pad away from the binding pad.

[0171] The preparation method of the immunochromatographic test strip of this embodiment comprises the following steps:

[0172] 1) Provide sample pad and absorbent pad.

[0173] 2) Preparation of conjugate pads: Dilute the luminescent signal probes prepared in Example 3 with 25 mM glycine buffer at pH 8.0 to a concentration of 0.1 mg / mL Eu2O3@DMSNs- PCT and 0.05 mg / mL ZnO@DMSNs- CRP The luminescent signal probe dilution was evenly sprayed on the conjugate pad (glass fiber) at a speed of 2.0 μL / cm by non-contact air spraying and dried in a 37°C oven overnight.

[0174] 3) Preparation of detection pad: On the Bio Dot XYZ3210 film-scraping platform, PCT antibody and CRP antibody with a concentration of 0.8 mg / mL were streaked in the form of lines (scraping speed was 1.5 μL / cm) on the same T line (i.e., test line) of the NC membrane, and 1 mg / mL of goat anti-chicken IgY was streaked on the C line (i.e., quality control line) position, and placed in a 37°C oven to dry overnight.

[0175] 4) Large plate preparation: Paste the sample pad, conjugate pad, detection pad and absorbent paper onto the PVC base plate in sequence;

[0176] 5) Cut into strips and put into plastic cases: Cut the large board into test strips of a certain width and put them into plastic cases.

[0177] Reagent test kit

[0178] Embodiment 36

[0179] This embodiment provides a kit, comprising the immunochromatographic test strip of Example 35 and the luminescence regulating medium of Example 4.

[0180] Embodiment 37

[0181] This embodiment provides a kit, which is different from the embodiment 36 only in that when preparing the conjugate pad of the immunochromatographic test strip, ZnO@DMSNs- CRP The concentration of Eu2O3@DMSNs- PCT The concentration is 80μg / mL.

[0182] Embodiment 38

[0183] This embodiment provides a kit, which is different from the embodiment 37 only in that when preparing the conjugate pad, ZnO@DMSNs- CRP The concentration is 20μg / mL.

[0184] Embodiment 39

[0185] This embodiment provides a kit, which is different from the embodiment 37 only in that when preparing the conjugate pad, ZnO@DMSNs- CRP The concentration is 30μg / mL.

[0186] Embodiment 40

[0187] This embodiment provides a kit, which is different from the embodiment 37 only in that when preparing the conjugate pad, ZnO@DMSNs- CRP The concentration is 40μg / mL.

[0188] Embodiment 41

[0189] This embodiment provides a kit, which is different from the embodiment 37 only in that when preparing the conjugate pad, ZnO@DMSNs- CRP The concentration is 50μg / mL.

[0190] Embodiment 42

[0191] This embodiment provides a kit, which is different from the embodiment 37 only in that when preparing the conjugate pad, ZnO@DMSNs- CRP The concentration is 60μg / mL.

[0192] Embodiment 43

[0193] This embodiment provides a kit, which is different from the embodiment 37 only in that when preparing the conjugate pad, ZnO@DMSNs- CRP The concentration is 70μg / mL.

[0194] Embodiment 44

[0195] This embodiment provides a kit, which is different from the embodiment 36 only in that when preparing the conjugate pad, Eu2O3@DMSNs- PCT The concentration of ZnO@DMSNs- CRP The concentration is 50μg / mL.

[0196] Embodiment 45

[0197] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCTThe concentration is 20μg / mL.

[0198] Embodiment 46

[0199] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCT The concentration is 40μg / mL.

[0200] Embodiment 47

[0201] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCT The concentration is 60μg / mL.

[0202] Embodiment 48

[0203] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCT The concentration is 80μg / mL.

[0204] Embodiment 49

[0205] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCT The concentration is 100 μg / mL.

[0206] Embodiment 50

[0207] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCT The concentration is 120μg / mL.

[0208] Embodiment 51

[0209] This embodiment provides a kit, which is different from the embodiment 44 only in that Eu2O3@DMSNs- PCT The concentration is 140μg / mL.

[0210] Since there are two detection antibodies solidified on the T line, the optimization of the dosage of the two signal probes on the binding pad needs to consider the complexity of the presence of a single target analyte or multiple target analyses in the sample. The following immunochromatographic test strips of Examples 37 to 51 will be used to detect negative samples (0 mg / mL) and positive samples (0.5 ng / mL) of PCT, negative samples (0 mg / mL) and positive samples (10 mg / mL) of CRP, and negative samples (CRP: 0 mg / mL, PCT: 0 mg / mL) and positive samples (CRP: 10 mg / mL, PCT: 0.5 ng / mL) of CRP and PCT, respectively. During the test, drop the sample on the sample pad, wait for 10 minutes, and then drop 15 μL of the luminescence adjustment medium directly above the detection pad. After 2 minutes, use the FS5 spectrometer to read the fluorescence intensity values ​​​​I of the T line and C line of the test strip. T and I C and I T / I C Ratio, calculate the I of positive samples and negative samples T / I C ratio.

[0211] The results showed that when only CRP was present in the target analyte, the I T / I C I T / I C The ratio between them increases with the increase of ZnO@DMSNs- CRP The I of PCT positive samples with a concentration of 0.5 ng / mL increased with the increase of probe dosage and reached a balance at 50 μg / mL. T / I C I T / I C The ratio between Eu2O3@DMSNs- PCT The ratio of ZnO@DMSNs- CRP The dosage of the probe is preferably 50 μg / mL; Eu2O3@DMSNs- PCT The amount of the probe used is preferably 80 μg / mL.

[0212] After the antigen and antibody have fully reacted and combined, the test is performed. In addition to obtaining more stable and accurate test results, the sensitivity is also further improved due to the improvement of the signal-to-noise ratio. Therefore, by optimizing the immune reaction time, it is beneficial for the immunochromatographic test strip to have a higher sensitivity in a wide linear range. The immune reaction time will be explored below.

[0213] Embodiment 52

[0214] This embodiment provides a kit, including an immunochromatographic test strip and a luminescence regulating medium.

[0215] The difference between the immunochromatographic test strip of this embodiment and that of embodiment 35 is that when preparing the conjugate pad, ZnO@DMSNs- CRP The dosage of the probe is preferably 50 μg / mL, Eu2O3@DMSNs- PCT The amount of the probe used is preferably 80 μg / mL.

[0216] The composition of the luminescence regulating medium of this embodiment is as follows: 0.8 mM 8-HQ, 15 μM β-NTA, 10 μM TOPO, glycine-hydrochloric acid with pH=5.0 as the buffer system, and 0.2 wt% CAB35.

[0217] Test sample preparation: 10 μL PCT standard solution (20 mg / mL) was added to 90 μL sample diluent (0.1 MBBS buffer (borate buffer), pH = 8.0, 0.9% NaCl by mass, 1% BSA (pure bovine serum) by mass, 0.5% Tween-20 by mass and 0.05% PC300 by mass) and mixed.

[0218] The test kit of this embodiment is used for testing. The test method is as follows: the test sample is dripped into the sample well of the sample pad. After reacting for 1 minute, 15 μL of luminescence regulating medium is dripped on the detection pad. After 2 minutes, the fluorescence intensity is read by the fluorescence immunoassay analyzer and the time-resolved immunoassay analyzer. The test is performed every 1 minute, and the fluorescence intensity values ​​of the T line and the C line within 15 minutes are recorded. T and I C and I T / I C The test method of CRP is the same as above, and the concentration of CRP standard is 100mg / L.

[0219] The results showed that when detecting target analytes including CRP (100 mg / L) and PCT (5 ng / mL), I T / I CThe ratios all showed an upward trend and basically leveled off after 10 minutes, so the detection time when using the kit of the present application for detection is preferably 10 minutes.

[0220] Performance Evaluation

[0221] (1) Establishing a standard curve

[0222] The prepared CRP and PCT series concentration calibrants were tested using the kit of Example 52. Each concentration point was tested three times in parallel, and the CRP and PCT concentrations were used as the horizontal axes, and the I T / I C The mean is taken as the vertical axis, and two standard curves are established.

[0223] Test method: After diluting the calibrator of each concentration 10 times, take 10 μL and drop it into the sample well of the sample pad. Let it react for 10 minutes. Then add 15 μL of luminescence regulating medium just above the detection pad. After 3 minutes, use the fluorescence immunoassay analyzer and time-resolved immunoassay analyzer to test respectively. Record the fluorescence intensity values ​​of T line and C line. T and I C and I T / I C ratio.

[0224] For the CRP calibration curve (y = 0.00674 + 0.01309X - 5.46386E - 5X 2 +1.005E-7X 3 ) and PCT calibration curve (y = 0.0042 + 0.11305X - 6.79018X 2 -6.43676E-5X 3 ) were used to fit the trinomial equation and draw the CRP calibration curve and PCT calibration curve.

[0225] (2) Linear range and sensitivity test

[0226] The high-value samples of PCT and CRP were selected and diluted to a series of gradient concentrations with serum matrix diluent, and then the CRP and PCT samples were tested in a series of gradient tests using the kit of Example 52 of the present application. Three groups of tests were repeated for each concentration to analyze the linear correlation between the actual measured values ​​of each concentration of PCT and CRP and the theoretical concentration. Linear fitting showed that the CRP concentration was in the range of 1 mg / L to 320 mg / L and the PCT concentration was in the range of 0.05 ng / mL to 20 ng / mL, which had good linear correlation with the theoretical concentration, and R 2 All >0.99.

[0227] Furthermore, the negative samples of PCT and CRP were further diluted and tested to obtain I T / I CAfter repeated testing, the average value (xB) plus three times the standard deviation (sB) was substituted into the standard curve to calculate the detection limit (LoD). The detection limit (LoD) of the kit for CRP was 0.37ng / mL, and the detection limit for PCT was 0.05ng / mL, both of which were lower than the corresponding clinical reference values ​​(CRP <10mg / L and PCT <0.25ng / mL).

[0228] (3) Specificity test

[0229] The kit of Example 52 was used to detect SAA antigen at a concentration of 1000 ng / mL, MYO antigen at 1000 ng / mL, PCT antigen at 5 ng / mL, and CRP antigen at 10 mg / L, all of which were serum matrices. SAA antigen and MYO antigen were used as interfering protein experimental groups, the blank group was negative serum without any interfering substance added, and the positive group was added with PCT antigen and CRP antigen, respectively.

[0230] Test method: Take 10μL of the sample to be tested and drop it into the sample well of the sample pad. Let it react for 10 minutes. Then drop 15μL of the luminescence regulating medium just above the detection pad. After 3 minutes, use the fluorescence immunoassay analyzer and time-resolved immunoassay analyzer to test respectively. Record the fluorescence intensity values ​​of the T line and C line. T and I C and I T / I C ratio.

[0231] The results showed that there was almost no difference in the measured values ​​between the addition of SAA antigen and MYO antigen to the negative serum and the negative serum without adding interference. Only when the sample contained CRP antigen and PCT antigen, the measured values ​​would increase significantly. The experimental results show that the kit of Example 52 has good specificity and can specifically detect CRP antigen and PCT antigen in serum samples without being interfered by other proteins.

[0232] (4) Clinical comparison

[0233] 10 PCT and CRP serum samples measured by Siemens nephelometry and Siemens chemiluminescence were selected respectively, one sample was selected from each group according to the serial number and mixed into 20 mixed samples, which were then tested using the prepared kit of Example 52, and the correlation between the kit and the clinical test results was compared.

[0234] Through clinical comparison experiments, the correlation between the dual-color coordination enhanced luminescence immunochromatography (CELFIA) of the kit of Example 52 and the Siemens nephelometry for detecting CRP was preliminarily evaluated ( Figure 6 ), and the correlation between the dual-color coordination enhanced luminescence immunochromatography (CELFIA) of the kit of Example 52 and the Siemens chemiluminescence method for detecting PCT ( Figure 6 20 clinical samples of CRP measured by Siemens nephelometry and 20 clinical samples of PCT customized by Siemens chemiluminescence were selected, and the same samples were tested with the kit of Example 52. The results showed that there was no significant difference between the measured values. The correlation coefficients r of CRP and PCT were both > 0.99, indicating that the dual-color coordination enhanced luminescence immunochromatography method of the present application for detecting CRP and PCT had a good correlation with the Siemens nephelometry method for detecting CRP and the Siemens chemiluminescence method for detecting PCT.

[0235] The above description of the embodiments is to facilitate those of ordinary skill in the art to understand and apply the present application. It is obvious that those familiar with the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without having to pay creative labor. Therefore, the present application is not limited to the embodiments herein, and improvements and modifications made by those skilled in the art based on the contents disclosed in the present application without departing from the scope and spirit of the present application are within the scope of the present application.

Claims

1. A luminescent signal probe, characterized in that: Comprising at least two metal oxide nanoparticles; wherein: When the at least two metal oxide nanoparticles are mixed with the same luminescence regulating medium, different luminescent complexes are formed respectively; Furthermore, when irradiated with the same excitation light, different luminescent complexes emit light with different wavelength ranges and different lifespans.

2. The luminescent signal probe according to claim 1, characterized in that One of the metal oxide nanoparticles is Eu2O3 nanoparticles, and the other metal oxide nanoparticles is ZnO nanoparticles; preferably, the excitation light is 365nm ultraviolet light, and the luminescent complex formed by the Eu2O3 nanoparticles emits red light with a central wavelength of 613nm and a lifetime in microseconds, and the luminescent complex formed by the ZnO nanoparticles emits green light with a central wavelength of 520nm and a lifetime in nanoseconds.

3. The luminescent signal probe according to claim 1 or 2, characterized in that The luminescent signal probe comprises at least two luminescent microspheres, one of which comprises a first carrier medium, and the first carrier medium is loaded with one of the metal oxide nanoparticles, and the other of which comprises a second carrier medium, and the second carrier medium is loaded with another metal oxide nanoparticle; preferably, the first carrier medium and the second carrier medium are both mesoporous silica.

4. The luminescent signal probe according to claim 3, characterized in that The luminescent signal probe further comprises at least two antibodies coupled to the at least two luminescent microspheres, one of which is a first antibody and the corresponding antigen is a first antigen, and the other of which is a second antibody and the corresponding antigen is a second antigen; The concentration of the first antigen in the sample to be tested is lower than the concentration of the second antigen in the sample to be tested, and the detection linear range of the first antigen is narrower than the detection linear range of the second antigen; preferably, the first antibody is a procalcitonin antibody, and the second antibody is a C-reactive protein antibody.

5. A luminescence regulating medium for a luminescent signal probe according to any one of claims 1 to 4, characterized in that: include: At least two luminescent ligands, a surfactant and a buffer solution, wherein the at least two luminescent ligands are used to form at least two luminescent complexes with at least two metal oxide nanoparticles.

6. The luminescence regulating medium according to claim 5, characterized in that: One of the luminescent ligands includes β-naphthoyltrifluoroacetone and topoisomerase, and another of the luminescent ligands includes 8-hydroxyquinoline.

7. The luminescence regulating medium according to claim 6, characterized in that: The luminescence regulating medium also satisfies at least one of the following conditions: (1) The surfactant includes an amphoteric surfactant and / or a nonionic surfactant; (2) The mass percentage concentration of the surfactant is 0.05% to 0.5%; (3) The buffer solution is a glycine-hydrochloric acid buffer; (4) The pH value of the luminescence regulating medium is 4 to 8; (5) The molar ratio of the β-naphthoyltrifluoroacetone, the topoisomerase and Eu is (1.2-4.5):(0.8-3):1, and the molar ratio of Zn to the 8-hydroxyquinoline is 1:(0.8-3).

8. An immunochromatographic test strip, characterized in that: The invention comprises the luminescent signal probe according to any one of claims 1 to 4.

9. The immunochromatographic test strip according to claim 8, characterized in that: The immunochromatographic test strip comprises a sample pad, a conjugation pad, a test line and a quality control line which are arranged in sequence, wherein the conjugation pad is provided with the luminescent signal probe, and the test line is fixed with at least two antibodies; preferably, one of the antibodies is a procalcitonin antibody, and the other antibody is a C-reactive protein antibody, and the quality control line is fixed with sheep anti-chicken IgY.

10. A method for preparing the immunochromatographic test strip according to claim 8 or 9, characterized in that: include: The sample pad, the binding pad and the detection pad provided with the test line and the quality control line are pasted on the bottom plate.

11. The method for preparing the immunochromatographic test strip according to claim 10, characterized in that: The preparation method of the conjugate pad comprises: coating the surface of the conjugate pad with a diluent of the luminescent signal probe according to any one of claims 1 to 4 and drying the conjugate pad; preferably, in the diluent, the concentration of one luminescent microsphere is 50 μg / mL to 150 μg / mL, and the concentration of another luminescent microsphere is 40 μg / mL to 100 μg / mL.

12. A kit, characterized in that: It comprises the luminescent signal probe according to any one of claims 1 to 4, or the immunochromatographic test strip according to claim 8 or 9, or the immunochromatographic test strip prepared by the preparation method of the immunochromatographic test strip according to claim 10 or 11, and the luminescent regulating medium according to any one of claims 5 to 7.

13. An immunochromatographic detection method, characterized in that: include: Providing the luminescent signal probe according to any one of claims 1 to 4, or the immunochromatographic test strip according to claim 8 or 9, or the immunochromatographic test strip prepared according to the method for preparing the immunochromatographic test strip according to claim 10 or 11, or the kit according to claim 12; Firstly, the luminescent signal probe is brought into contact with the sample to be tested, and then the luminescent signal probe is treated with the luminescent regulating medium according to any one of claims 5 to 7; The luminescent signal probe treated with excitation light is irradiated, and the light signal emitted by the luminescent signal probe is read.

14. The immunochromatographic detection method according to claim 13, characterized in that: The immunochromatographic detection method also satisfies at least one of the following conditions: (1) The contact time between the luminescent signal probe and the sample to be tested is 6 min to 15 min; (2) the time for treating the luminescent signal probe with the luminescent regulating medium is 2 min to 5 min; (3) The excitation light is 365 nm ultraviolet light, and the luminescent signal probe emits two different light signals with central wavelengths of 613 nm and 520 nm and lifetimes in the microsecond and nanosecond levels, respectively; (4) The method for reading the light signal emitted by the luminescent signal probe includes: collecting one of the light signals at a time through time-gated fluorescence technology, and collecting other light signals in sequence with a delayed delay.

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