CN117607430A - Preparation method and application of zinc germanate long afterglow nano probe detection test strip - Google Patents
Preparation method and application of zinc germanate long afterglow nano probe detection test strip Download PDFInfo
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- CN117607430A CN117607430A CN202311642495.3A CN202311642495A CN117607430A CN 117607430 A CN117607430 A CN 117607430A CN 202311642495 A CN202311642495 A CN 202311642495A CN 117607430 A CN117607430 A CN 117607430A
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- Urology & Nephrology (AREA)
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Abstract
The invention discloses a preparation method and application of a zinc germanate long afterglow nanometer probe detection test strip, wherein the probe comprises Zn 2 GeO 4 :xMn 2+ The test paper comprises a base plate, a detection film arranged on the base plate, a sample pad and absorbent paper, wherein the sample pad is lapped on the detection film, probe lines are arranged on the sample pad, and a detection line and a quality control line are arranged on the detection film; the probe line is internally coated with a first biological marker containing long-afterglow nano probe marks and a quality control substance containing long-afterglow nano probe marks; the detection line is coated with a second biomarker, and the quality control line is coated with a quality control marker; the application adopts Zn 2 GeO 4 :xMn 2+ As a probe, the emitted light is about 537nm, and the high-concentration sample test strip can emit light under the irradiation of an ultraviolet lampThe bright green color was observed by naked eyes; after the excitation light source is turned off, the continuous light-emitting time reaches 30min, and the test strip can be observed by naked eyes and tested by a CCD camera in the test process, and has a dual-mode test function.
Description
Technical Field
The invention relates to a preparation method and application of a zinc germanate long afterglow nanometer probe detection test strip, belonging to the technical field of immunochromatography detection.
Background
The immunochromatography test paper based on the signal label is a convenient measurement mode, the main method is a colloidal gold method, the main method is based on color observation, and qualitative detection can only be met in most cases. The immunochromatography test paper based on the fluorescent label has higher sensitivity than colloidal gold, and the fluorescent label commonly used at present mainly comprises up-conversion, rare earth doped microspheres, quantum dots and the like, but background fluorescence interference generally exists, and the sensitivity is often reduced due to the background interference, so that the fluorescent label is difficult to be used for detecting low-content biological analytes. Therefore, development of immunochromatography techniques for high-sensitivity quantitative detection without background interference has become an attractive research direction.
Heparin Binding Protein (HBP) is a functional protein produced by neutrophils. It is considered to be one of the indicators of the extent of inflammatory response and the severity of infection. Especially in severe infections, sepsis and organ failure, the level of heparin-binding protein is significantly increased. By monitoring the change of heparin binding protein, the activity degree of inflammatory reaction can be estimated, and the severity and prognosis of infection can be judged in an auxiliary way, and the detection requirement of the binding protein is extremely high in view of the wide application scene. Aiming at the problem of HBP detection, the current common methods are a specific protein meter, an HBP reagent (rate scattering turbidimetry) and an HBP biochemical reagent (immunoturbidimetry), which are both required to be put into a special full-automatic biochemical meter, so that the detection cost is high, and the large-scale popularization is not facilitated.
Based on the background, how to develop a quantitative detection method without background fluorescence interference and with high sensitivity, and effectively reduce equipment investment at the same time, becomes one of the problems to be solved.
Disclosure of Invention
In order to solve the technical problems of insufficient fluorescence interference and test sensitivity in the background technology, the invention provides a long afterglow nano probe dual-mode immunochromatography test paper.
The technical scheme for realizing the aim of the invention is as follows:
an immunochromatography test strip based on zinc germanate long afterglow nano probe comprises a bottom plate, wherein a sample pad, a detection membrane and a water absorption pad which are sequentially connected are arranged on the bottom plate;
the sample pad contains a first biological marker marked by a zinc germanate long afterglow nano probe and a quality control substance marked by the zinc germanate long afterglow nano probe;
the zinc germanate long afterglow nano probe is a manganese doped zinc germanate nano material;
the detection membrane is provided with a detection line and a quality control line, the detection line is coated with a second biomarker, and the quality control line is coated with a quality control marker;
the first biomarker and the second biomarker can be specifically combined with a target in a sample to be detected, and the combining sites are different;
the quality control substance and the quality control marker are specifically combined.
The molar ratio of zinc to germanium to manganese is (1-2): (0.001-0.01), wherein specific point values in (1-2) can be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 and the like; wherein the specific point value in (0.001-0.01) can be selected from 0.001, 0.002, 0.08, 0.009, 0.01, etc.
The invention adopts Zn 2 GeO 4 :xMn 2+ As a probe, the luminous component emits light of about 537nm, and is used as a luminous label of a test strip to detect HBP, and by utilizing an immunochromatography technology, not only can macroscopic qualitative detection be carried out under the excitation of a light source, but also the afterglow intensity after excitation can be detected, so that the quantitative detection without excitation and background fluorescence interference can be realized, the HBP with lower concentration can be detected, and the double-mode high-sensitivity quantitative detection capability can be realized.
Further, the molar ratio of zinc to germanium to manganese is (1-2): (0.001-0.01), and the dual-mode detection can be realized within the range through detection.
Further, the particle size of the zinc germanate long afterglow nano probe is 90-130 nm;
further, the target is a heparin binding protein.
The radius of the zinc germanate long afterglow nano probe comprises, but is not limited to, 100nm, 110nm, 115nm or 128nm, etc., and is not limited to the examples.
Further, the quality control substance is selected from rabbit IgG antibodies, and the quality control marker is selected from goat anti-rabbit IgG antibodies;
the first biomarker and the second biomarker are monoclonal antibodies.
The application also provides an immunochromatographic test strip based on the zinc germanate long afterglow nano probe, which is characterized in that the preparation method comprises the following steps:
preparing zinc germanate long afterglow nano probes;
amination of the surface of the zinc germanate long afterglow nano probe;
carboxylation of the surface of zinc germanate long afterglow nano probe;
the carboxylated zinc germanate long afterglow nano probe surface antibody is coupled;
preparing a sample pad;
preparing a detection film;
and sequentially and mutually overlapping and pasting a sample pad, a detection film and a water absorption pad on the bottom plate to obtain a test paper board, and cutting the test paper board to obtain the test paper strip.
Specifically, the preparation process comprises the following steps:
(1) Preparation of zinc germanate long afterglow nano probe
Synthesizing zinc germanate long afterglow nano probe by hydrothermal method. 1 to 3mmol Zn (NO) is stirred vigorously 3 ) 2 、0.001～0.01mmol Mncl 2 And 100 to 500 mu L of concentrated HNO 3 Adding into 5-20 mL deionized water. Then, slowly adding 0.1-2 mmol Na into the solution 2 GeO 3 . Immediately thereafter, ammonium hydroxide (28 wt) was added to adjust the pH of the solution to 7-8. The resulting reaction system was stirred at room temperature for 0.5 to 2 hours. The solution was then transferred to a teflon-lined autoclave and allowed to react at 150-300 ℃ for 6-15 hours. Collecting the obtained long afterglow nano probe of the amino zinc germanate through centrifugationAnd washing with deionized water for three times, freeze-drying the washed zinc germanate long afterglow nano probe, and collecting.
(2) Surface modified zinc germanate long afterglow nano probe
Amination of zinc germanate long afterglow nanoprobe: first, zinc germanate long afterglow nanoprobe (25-75 mg) is dissolved in DMF (20-50 mL), and zinc germanate long afterglow nanoprobe is dispersed in DMF solution under ultrasound. Then, the mixture was put into an oil bath at 80℃and APTES was added in an amount of 100 to 400. Mu.L with stirring, followed by stirring for 18 to 28 hours. The product was collected by centrifugation and washed once with DMF and twice with absolute ethanol, and after freeze-drying overnight, the resulting zinc germanate amide long persistence nanoprobe was collected.
Carboxylating the zinc germanate long afterglow nano probe: firstly, PAA (20-80 mg) is added into MES solution, ultrasonic is carried out to dissolve the PAA, EDC (5-20 mg) is added, NHS (5-20 mg) is added after dissolution, stirring is carried out for 10-40 min at room temperature to completely dissolve the PAA, PH is adjusted to 7-8, then aminated zinc germanate long afterglow nano probe (10-50 mg) is added, stirring is carried out at room temperature overnight, the obtained carboxylated zinc germanate long afterglow nano probe is collected through centrifugation, washed three times by deionized water, and freeze-dried overnight for collection.
The carboxylated zinc germanate long afterglow nano probe is coupled with an antibody: preparing a solution of 8-15 mg/mL of carboxylated zinc germanate long afterglow nano probe by using water, carrying out ultrasonic treatment for 2-5 min, taking 80-200 mu L of nano probe solution, carrying out high-speed centrifugation at 9000-12000 rpm for 5-15 min, washing precipitate to 300-1000 mu L by using MES solution with pH value of 5.0-7.0, and carrying out ultrasonic treatment for 2-5 min; adding 10-100 mu L of 20-100 mg/mL carbodiimide, uniformly mixing for 5-10 min, adding 50-200 mu L of 20-100 mg/mLN-hydroxy thiosuccinimide, uniformly mixing for 10-20 min, performing high-speed centrifugation at 9000-12000 rpm for 5-15 min, washing the precipitate to 300-1000 mu L by using MES solution with pH of 5.0-7.0, performing ultrasonic treatment for 2-5 min, respectively adding a first biomarker and a quality control substance according to 150-250 mu L of the solution, uniformly mixing for 1-3 h, sealing by using 10-50 mM containing 0.5-2% BSA and 0.1-1% glycine, sealing by using Tris-HCl sealing solution with pH of 7.5-8.5 rpm for 0.5-1 h, performing high-speed centrifugation at 9000-12000 rpm for 5-15 min, washing by using 10-50 mM containing 0.5-2% NaCl, 0.1% BSA, 0.1-1% sucrose, 0.01-0.5% Tween20, and re-8.5 mM pH of pH 7-1 to obtain a long-suspended antibody, and washing by using a long-suspended solution of HCl to obtain a long-suspended antibody;
(3) Preparation of sample pad
Uniformly spraying two lines in parallel on one side close to the sample pad by using sample pad treatment liquid, wherein the dosage is 2-4 mu L of liquid amount/cm of the sample pad, diluting a first biomarker marked by a zinc germanate long afterglow nano probe and a quality control object by 1-25 times by using diluent on one side of the sample pad close to a detection membrane, and uniformly spraying one line serving as probe line, wherein the dosage is 2-4 mu L of liquid amount/cm of the sample pad;
(4) Preparation of detection film
Adjusting the concentration of the second biomarker and the quality control marker to 0.5-3 mg/mL by using a coating buffer solution, wherein the coating liquid amount/cm membrane is 0.5-1.5 mu L, and the second biomarker and the quality control marker are respectively used as a detection line and a quality control line to be marked on a nitrocellulose membrane in parallel for coating, and the distance between the quality control line, the detection line and a probe line is 3-7 mm;
(5) And sequentially and mutually overlapping and pasting a sample pad, a detection film and absorbent paper on the bottom plate to obtain a test paper board, and cutting to obtain the test paper strip.
The preparation method has simple steps, is easy to operate and is easy for mass production.
The application also provides an immunochromatography test strip based on the zinc germanate long afterglow nano probeIn the detectionUse of heparin binding proteins.
The present application also provides a method of detecting heparin-binding proteins for the purpose of non-disease diagnosis, the method comprising the steps of:
adding a sample to be detected to a sample pad of the double-mode detection immunochromatography test strip based on the zinc germanate long afterglow nano probe;
for a high-concentration sample, the test strip emits bright green under the irradiation of an ultraviolet lamp, and qualitative observation is carried out by naked eyes; after the excitation light source is turned off, the afterglow luminescence of the test strip is captured by the CCD camera, the average luminescence intensity of the detection line and the quality control line is read by combining with image visual analysis software, and the average intensity ratio of the detection line and the quality control line is calculated for quantitative detection.
By adopting the technical scheme, the invention has the following beneficial effects:
according to the method, the zinc germanate long afterglow nano probe is connected with the antibody, so that the product is stable, the content of a target object to be detected can be detected by high-efficiency immunochromatography, and for a high-concentration sample, the sample can be observed with naked eyes under the excitation of ultraviolet light, so that qualitative detection is realized; after the excitation light source is turned off, the long-time afterglow luminescence is utilized, the afterglow luminescence is captured by a CCD camera and the luminous intensity is calculated by combining with visual image analysis software, so that the high-sensitivity quantitative detection without excitation and background fluorescence interference is realized. Compared with the existing chromatographic test paper, the fluorescent quantitative detection method has the advantages that the macroscopic qualitative detection is carried out under the excitation of the light source, the afterglow intensity after the excitation can be detected, the quantitative detection without excitation and background fluorescence interference is realized, the target with lower concentration can be detected, and the fluorescent quantitative detection method has the double-mode and high-sensitivity quantitative detection capability.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which
Fig. 1 is a schematic structural view of the present invention.
FIG. 2 shows the zinc germanate long afterglow nanoprobe and ZnGe of the present invention 2 O 4 X-ray diffraction contrast plot of standard lines of (2);
FIG. 3 is a transmission electron microscope image of the zinc germanate long afterglow nanoprobe of the present invention, the length of which is about 100 nm;
FIG. 4 is an excitation spectrum of the zinc germanate long afterglow nanoprobe under 537nm monitoring and an emission spectrum under 254nmUV excitation of the zinc germanate long afterglow nanoprobe of the present invention;
FIG. 5 is an afterglow emission spectrum of the zinc germanate long afterglow nanoprobe of the present invention measured after excitation of an ultraviolet 254nm light source for 5min;
FIG. 6 is a graph showing the afterglow attenuation of the zinc germanate long afterglow nanoprobe of the present invention after excitation of an ultraviolet 254nm light source for 5min;
FIG. 7 is an infrared view of the zinc germanate long persistence nanoprobe and the zinc germanate long persistence nanoprobe of the present invention labeled with a first biomarker and the zinc germanate long persistence nanoprobe labeled with a quality control substance;
FIG. 8 shows the photoluminescence image of HBP under excitation of ultraviolet 254nm light source (upper) and the fluorescence image after excitation of ultraviolet 254nm light source for 5min (lower)
The reference numerals in the drawings are:
the device comprises a bottom plate 1, a detection membrane 2, a sample pad 3, absorbent paper 4, a probe wire 5, a detection wire 6 and a quality control wire 7.
Detailed Description
In order to better understand the above technical solutions, the following detailed description will refer to the accompanying drawings and specific embodiments.
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. The components of the embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
It should be noted that: like reference numerals and letters denote like items in the following figures, and thus once an item is defined in one figure, no further definition or explanation thereof is necessary in the following figures.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
The detection principle of the test strip in the specific embodiment of the invention is a double-antibody sandwich method, and the HBP content in human serum, plasma and whole blood samples is detected. The blood sample dilution containing HBP is dripped in a sample loading area, is chromatographed to a sample pad through capillary action, is combined with a first biomarker marked by a zinc germanate long afterglow nano probe and HBP antigen to form a compound, is chromatographed to a detection area on a nitrocellulose membrane, the compound is captured by a second biomarker coated by a detection line, and the zinc germanate long afterglow nano probe marked with a quality control object is continuously chromatographed forwards and is directly combined with the quality control object fixed on the quality control line. After chromatography is finished, the test strip emits bright green under the irradiation of an ultraviolet lamp for a high-concentration sample, and qualitative observation can be carried out by naked eyes; after the excitation light source is turned off, the afterglow luminescence of the zinc germanate long afterglow nano probe exceeds thirty minutes, the afterglow luminescence of the test strip is captured by a CCD camera, the average luminescence intensity of the detection line and the quality control line is read by combining image visual analysis software, and the average intensity ratio of the detection line and the quality control line is calculated for quantification, so that a sample with lower concentration is detected, and the high-sensitivity quantitative detection without excitation and background fluorescence interference is realized.
Example 1
The embodiment prepares the dual-mode detection immunochromatography test paper based on the zinc germanate long afterglow nano probe.
A dual-mode immunochromatography test paper based on zinc germanate long afterglow nano probes adopts the principle of double-antibody sandwich immunochromatography to detect HBP in human serum, plasma and whole blood samples.
As shown in fig. 1, a sample pad 3, a detection film 2 and a water absorbing paper 4 are sequentially lapped on a bottom plate 1, wherein the sample pad 3 is a sample adding area for absorbing a detection sample to be detected, a probe line 5 is arranged, and a detection line 6 and a quality control line 7 are arranged on the detection film 2.
The zinc germanate long afterglow nano probe Zn with specific excitation light (254 nm)/emission light (537 nm) wavelength is used on the probe line on the sample pad 3 2 GeO 4 :Mn 0.005 (length about 100 nm) labeled first biomarker and quality control (200. Mu.g antibody/100. Mu.L probe solution), the detection line coated with second biomarker (2 mg/mL), and the quality control line coated with quality control marker at a concentration of 2 mg/mL. The probe line was used in an amount of 4. Mu.L of coating liquid per cm of sample pad, the detection line and the quality control line were used in an amount of 1. Mu.L of coating liquid per cm of membrane.
In this embodiment, the preparation of the dual-mode detection immunochromatographic test paper based on the zinc germanate long afterglow nanoprobe comprises the following steps:
(1) Preparation method of zinc germanate long afterglow nano probe
Synthesizing zinc germanate long afterglow nanometer probe by a hydrothermal method. Briefly, 2mmol Zn (NO 3 ) 2 、0.005mmol Mncl 2 And 300. Mu.L of concentrated HNO 3 Added to 11mL of deionized water. Then, 1mmol Na was slowly added to the above solution 2 GeO 3 . Immediately thereafter, ammonium hydroxide (28 wt) was added to adjust the pH of the solution to 7.5. The resulting reaction system was stirred at room temperature for 1 hour. The solution was then transferred to a teflon-lined autoclave and allowed to react at 220 ℃ for 10 hours. Collecting the obtained zinc germanate long afterglow nanoprobe by centrifugation, washing with deionized water for three times, and freezing the washed zinc germanate long afterglow nanoprobeDrying and collecting. X-ray diffraction (XRD) analysis of zinc germanate long afterglow nano probe shows that its diffraction pattern is as shown in figure 2, and compared with standard card 2 GeO 4 The standard lines of the (2) are well matched, which shows that the long afterglow nano probe needed by us is successfully prepared. As a result of observation by a transmission electron microscope, the sample was rod-shaped and had a length of about 100nm, and the sample was uniformly dispersed, as shown in FIG. 3, and good chromatography was performed. The excitation spectrum of the zinc germanate long afterglow nano probe under 537nm monitoring and the emission spectrum under 254nm UV excitation are shown as figure 4, and afterglow emission (figure 5) and afterglow attenuation (figure 6) are detected after an ultraviolet 254nm light source is excited for 5min, so that the result shows that the prepared zinc germanate long afterglow nano probe has a photoinduced emission peak and an afterglow emission peak at 537nm and has afterglow exceeding 30min, thus not only endowing the zinc germanate long afterglow nano probe with dual-mode detection capability, but also realizing high-sensitivity quantitative detection without excitation and background fluorescence interference. Has great potential in the field of immunochromatography and even in-vitro diagnosis application.
Amination of zinc germanate long afterglow nanoprobe: first, a zinc germanate long afterglow nanoprobe (50 mg) was dissolved in DMF (30 mL), and a zinc germanate long afterglow nanoprobe was dispersed in DMF solution under ultrasound. Then, the mixture was placed in an oil bath at 80℃and 300. Mu.L of APTES was added thereto with stirring, followed by stirring for 24 hours. The product was collected by centrifugation and washed once with DMF and twice with absolute ethanol, and after freeze-drying overnight, the resulting zinc germanate amide long persistence nanoprobe was collected.
Carboxylating the zinc germanate long afterglow nano probe: firstly, PAA (50 mg) is added into MES solution (30 mL PH=6.5), ultrasonic is carried out to dissolve the PAA, EDC (10 mg) is added, NHS (10 mg) is added after dissolution, stirring is carried out for 30min at room temperature to completely dissolve the PAA, PH is adjusted to 7-8, then aminated zinc germanate long afterglow nano probe (20 mg) is added, stirring is carried out at room temperature overnight, the obtained carboxylated zinc germanate long afterglow nano probe is collected through centrifugation and washed three times by deionized water, and after freeze drying of the washed carboxylated zinc germanate long afterglow nano probe is carried out, the constant volume is 10mg/mL by water.
The carboxylated zinc germanate long afterglow nano probe is coupled with an antibody: after carrying out ultrasonic treatment on the carboxylated zinc germanate long afterglow nano probe for 2min, taking 300 mu l of the carboxylated zinc germanate long afterglow nano probe (10 mg/mL), centrifuging at 11000rpm for 10min at high speed, washing the precipitate to 1mL by using MES solution with pH of 5.0, and carrying out ultrasonic treatment for 2min; adding 25 mu l of 100mg/ml carbodiimide, uniformly mixing for 5min, adding 75 mu l of 100mg/ml N-hydroxysulfosuccinimide, uniformly mixing for 15min to activate carboxyl groups on the surface of the carboxylated zinc germanate long afterglow nanoprobe, centrifuging at 10000rpm for 10min at a high speed, and washing the precipitate to 1ml by using MES solution with pH of 5.0; the first biomarker (200. Mu.g) and the quality control substance (200. Mu.g) were mixed with carboxylated zinc germanate long afterglow nanoprobe at room temperature for 2 hours and coupled to form a complex (the first biomarker, i.e. HBP antibody, was purchased from Boyue Biotechnology Co., hangzhou, and the quality control substance, i.e. rabbit IgG, and the quality control marker, i.e. goat anti-rabbit IgG, were purchased from Luoyang Baiott laboratory materials center), followed by addition of 250. Mu. LpH Tris-HCl blocking solution of 8.5 for 1 hour. Finally, the complexes were collected by centrifugation at 10000rpm for 10 minutes, washed with Tris-HCl preservation solution at pH 8.5 and resuspended to 100. Mu.l for preservation at 4℃in the dark. As shown in fig. 7, the zinc germanate long afterglow nanoprobe and the zinc germanate long afterglow nanoprobe connected with the first biomarker and the quality control substance are subjected to infrared spectrum test, and the infrared peak is obviously changed, which indicates that the first biomarker and the quality control substance are successfully connected to the surface of the zinc germanate long afterglow nanoprobe.
(3) Preparation of sample pad
Two lines were sprayed in parallel and uniformly on the side of the sample pad near the sample pad with a solution of 4. Mu.L of liquid/cm of sample pad, and a zinc germanate long afterglow nanoprobe with a second biomarker and a quality control marker attached thereto was diluted 4-fold with a dilution solution (20 mM Tris-HCl buffer containing 0.5% BSA, 25% sucrose) for a long afterglow nanoprobe with a solution of 4-fold and uniformly sprayed on the side of the sample pad near the coating film, with a solution of 4. Mu.L of liquid/cm of sample pad, with a solution of 1mg/mL anti-RBC, 0.5% NaCl, 0.5% Tween, 20mM of 0.1% BSA, tris-HCl buffer of pH 8.0. Placed in an oven and dried overnight at 37 ℃.
(4) Preparation of coating film
The concentration of the second biomarker and the quality control marker (the second biomarker is HBP antibody purchased from Boyue biotechnology Co., hangzhou, and the quality control marker is sheep anti-rabbit IgG purchased from Luoyang Baiao laboratory materials center) was adjusted to 2mg/mL with a coating buffer (10 mM PBS buffer containing 2.5% sucrose) to 1 μl of coating liquid volume/cm membrane, and the coating, quality control line, detection line and probe line were drawn on nitrocellulose membrane in parallel for coating, with a line spacing of 4mM, and the coated membrane was placed in an oven and dried overnight at 37 ℃.
(5) And sequentially and mutually bonding a sample pad, a coating film and absorbent paper on the substrate in an overlapping manner to obtain a test paper board, and cutting to obtain the test paper strip.
(6) 30 mu L of negative serum sample is taken and added into 70 mu L of sample diluent, 90 mu L of the mixture is taken and added into a sample adding area of a sample pad after uniform mixing, and after chromatography for 20min, high-concentration sample can be observed with naked eyes under the excitation of ultraviolet light to realize qualitative detection; after the excitation light source is turned off, the long-time afterglow luminescence is utilized, the afterglow luminescence is captured by a CCD camera and the luminous intensity is calculated by combining visual image analysis software, so that the excitation-free high-sensitivity quantitative detection without background fluorescence interference is realized, a sample with lower concentration can be detected, and the method has the characteristics of dual-mode detection, high detection sensitivity, rapidness, convenience and the like.
Example 2
The procedure was the same as in example 1 except that the negative plasma sample in step (6) was replaced with 10ng/mL HBP serum sample.
Example 3
The procedure was the same as in example 1 except that the negative plasma sample in step (6) was replaced with 25ng/mL HBP serum sample.
Example 4
The procedure was the same as in example 1 except that the negative plasma sample in step (6) was replaced with 50ng/mL HBP serum sample.
Example 5
The procedure was the same as in example 1 except that the negative plasma sample in step (6) was replaced with 100ng/mL HBP serum sample.
Example 6
The procedure was the same as in example 1 except that the negative plasma sample in step (6) was replaced with 200ng/mL HBP serum sample.
The detection result is shown in figure 8, and the result shows that the double-mode detection immunochromatography test paper based on the zinc germanate long afterglow nano probe is successfully prepared, heparin binding proteins with different concentrations are detected, and under 254nm ultraviolet excitation, the detection can be performed by naked eyes in a fluorescence mode, and the lowest concentration which can be seen is 25ng/mL; after the excitation light source is turned off, the afterglow mode is utilized, the afterglow luminescence is captured by a CCD camera, and the rest of the glow luminescence intensity is quantitatively calculated by combining image vision analysis software, so that the concentration of HBP is quantitatively detected, the detection limit under the afterglow mode is calculated to be 4.7ng/mL, and the detection sensitivity is higher. The difference of the bands presented in the two detection modes is obvious, so that the method can be effectively used for detecting heparin binding proteins, can be used for naked eye qualitative observation and high-sensitivity quantitative detection, and has great application potential in the technical field of immunochromatography.
In conclusion, for the high-concentration sample, the zinc germanate long-afterglow nanoprobe emits strong green light under the irradiation of the excitation light source, so that the test paper can be directly and qualitatively observed by naked eyes; after the excitation light source is turned off, the afterglow imaging is carried out by utilizing the afterglow luminescence of the zinc germanate long afterglow nano probe, so that the excitation-free quantitative detection without background interference is realized, and the sensitivity is higher. The feasibility of the dual-mode detection is verified by taking HBP as a detection analyte, and the dual-mode detection immunochromatography test paper based on the zinc germanate long afterglow nano probe can be used for simple qualitative detection and high-sensitivity quantitative detection, and has great potential in the immunochromatography technology and even in-vitro diagnosis fields.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. An immunochromatography test strip based on a zinc germanate long afterglow nano probe is characterized by comprising a bottom plate, wherein a sample pad, a detection membrane and a water absorption pad which are sequentially connected are arranged on the bottom plate;
the sample pad contains a first biological marker marked by a zinc germanate long afterglow nano probe and a quality control substance marked by the zinc germanate long afterglow nano probe;
the zinc germanate long afterglow nano probe is a manganese doped zinc germanate nano material;
the detection membrane is provided with a detection line and a quality control line, the detection line is coated with a second biomarker, and the quality control line is coated with a quality control marker;
the first biomarker and the second biomarker can be specifically combined with a target in a sample to be detected, and the combining sites are different;
the quality control substance and the quality control marker are specifically combined.
2. The immunochromatographic test strip based on zinc germanate long afterglow nanoprobe according to claim 1, which is characterized in that: the molar ratio of zinc to germanium to manganese is (1-2): (0.001-0.01).
3. The immunochromatographic test strip based on zinc germanate long afterglow nanoprobe according to claim 1, which is characterized in that: it is characterized in that the particle size of the zinc germanate long afterglow nano probe is 90-130 nm.
4. The immunochromatographic test strip based on zinc germanate long persistence nanoprobe according to claim 3, wherein the immunochromatographic test strip is characterized in that:
the target is heparin binding protein.
The quality control object is selected from rabbit IgG antibodies, and the quality control marker is selected from goat anti-rabbit IgG antibodies;
the first biomarker and the second biomarker are monoclonal antibodies.
5. The immunochromatographic test strip based on the zinc germanate long afterglow nanoprobe according to any one of claims 1 to 4, which is characterized by comprising the following steps:
preparing zinc germanate long afterglow nano probes;
amination of the surface of the zinc germanate long afterglow nano probe;
carboxylation of the surface of zinc germanate long afterglow nano probe;
the carboxylated zinc germanate long afterglow nano probe surface antibody is coupled;
preparing a sample pad;
preparing a detection film;
and sequentially and mutually overlapping and pasting a sample pad, a detection film and a water absorption pad on the bottom plate to obtain a test paper board, and cutting the test paper board to obtain the test paper strip.
6. The immunochromatographic test strip based on the zinc germanate long persistence nanoprobe according to claim 5, which is characterized in that the preparation method comprises the following steps:
the hydrothermal method comprises the following steps: according to the proportion of the raw materials, zn (NO 3 ) 2 、Mncl 2 And concentrated HNO 3 Adding Na into deionized water 2 GeO 3 The method comprises the steps of carrying out a first treatment on the surface of the Immediately adding ammonium hydroxide, regulating the pH value of the solution, and stirring; placing the mixture into a Teflon-lined autoclave for reaction after stirring;
the amination step is as follows: ultrasonically dissolving a zinc germanate long afterglow nano probe in DMF (dimethyl formamide), adding APTES (ammonium paratungstate) under the stirring of an oil bath, stirring, washing with DMF once, washing with absolute ethyl alcohol twice, and performing freeze drying treatment;
the carboxylation step is as follows: adding PAA into MES solution for ultrasonic dissolution, adding EDC for dissolution, adding NHS for dissolution, adjusting pH at room temperature, and adding aminated zinc germanate long afterglow nano probe; stirring overnight at room temperature, centrifuging, washing with deionized water, and lyophilizing;
the antibody coupling step is as follows: dissolving carboxylated zinc germanate long afterglow nano probe in water, centrifuging, washing a centrifugal precipitate by adopting MES solution, performing ultrasonic treatment, adding carbodiimide, mixing uniformly, adding N-hydroxy thiosuccinimide, mixing uniformly, centrifuging, washing the centrifugate by adopting MES solution, performing ultrasonic treatment, adding a first biological marker and a quality control substance, adding a sealing liquid, performing sealing, performing centrifugal treatment, washing by adopting NaCl solution, BSA solution, sucrose, tween20 solution and Tris-HCl preservation solution, and performing resuspension treatment.
7. The immunochromatographic test strip based on the zinc germanate long persistence nanoprobe according to claim 6, which is characterized in that the preparation method comprises the following steps:
the hydrothermal reaction temperature in the hydrothermal step is 50-300 ℃;
the oil bath temperature in the amination step is 80 ℃;
the pH value in the carboxylation step is 7-8;
the ultrasonic treatment time in the antibody coupling step is 2-5 min, and the pH of the MES solution for washing is 5.0-7.0.
8. The immunochromatographic test strip based on a zinc germanate long afterglow nanoprobe according to claim 5, wherein the material of the sample pad is glass fiber;
preferably, the material of the detection film is nitrocellulose film;
preferably, the material of the water absorbing pad is water absorbing paper.
9. The immunochromatographic test strip based on zinc germanate long-afterglow nanoprobe according to any one of claims 1 to 4In the detectionUse of heparin binding proteins.
10. The immunochromatographic test strip based on zinc germanate long afterglow nanoprobe according to claim 9In-check MeasuringHeparin knotUse of a synthetic protein, characterized in that the method comprises the steps of:
adding a sample to be detected to a sample pad of the zinc germanate-based long afterglow nanoprobe dual mode detection immunochromatographic test strip according to any one of claims 1 to 4;
for a high-concentration sample, the test strip emits bright green under the irradiation of an ultraviolet lamp, and qualitative observation is carried out by naked eyes; after the excitation light source is turned off, the afterglow luminescence of the test strip is captured by the CCD camera, the average luminescence intensity of the detection line and the quality control line is read by combining with image visual analysis software, and the average intensity ratio of the detection line and the quality control line is calculated for quantitative detection.
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