CN111735957A - Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology - Google Patents

Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology Download PDF

Info

Publication number
CN111735957A
CN111735957A CN202010586396.8A CN202010586396A CN111735957A CN 111735957 A CN111735957 A CN 111735957A CN 202010586396 A CN202010586396 A CN 202010586396A CN 111735957 A CN111735957 A CN 111735957A
Authority
CN
China
Prior art keywords
pad
psa
ucnps
solution
monoclonal antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202010586396.8A
Other languages
Chinese (zh)
Inventor
李智椋
何皓
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Guanghe Biotechnology Co Ltd
Original Assignee
Shenzhen Guanghe Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Guanghe Biotechnology Co Ltd filed Critical Shenzhen Guanghe Biotechnology Co Ltd
Priority to CN202010586396.8A priority Critical patent/CN111735957A/en
Publication of CN111735957A publication Critical patent/CN111735957A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/77—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals
    • C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
    • C09K11/7772—Halogenides
    • C09K11/7773—Halogenides with alkali or alkaline earth metal
    • 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
    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Materials Engineering (AREA)
  • Organic Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Inorganic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nanotechnology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention belongs to the technical field of wastewater treatment devices, and particularly relates to an immunochromatography detection card for quantitatively detecting PSA (pressure swing adsorption) based on an up-conversion luminescence technology, which comprises a sample pad, a combination pad, a reaction membrane and a water absorption pad, wherein the reaction membrane is provided with a detection line and a quality control line; an anti-PSA monoclonal antibody 1 is fixed on the detection line, and a secondary goat anti-mouse IgG is fixed on the quality control line; the combination pad is fixedly provided with an up-conversion luminescent nanoparticle-anti-PSA monoclonal antibody 2 combination. Compared with the prior art, the invention has the following advantages: the kit disclosed by the invention has the advantages that based on the unique up-conversion luminescence characteristic of the up-conversion luminescence immunochromatography technology, the detection is free from background interference, the sensitivity can reach about 100 of that of colloidal gold, the luminescence property is stable, the quenching is not easy, the kit can be used for multiple and quantitative analysis, and the kit has important significance for quantitatively detecting the concentration of Prostate Specific Antigen (PSA) in serum and early diagnosis of prostate cancer.

Description

Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology
Technical Field
The invention belongs to the technical field of wastewater treatment devices, and particularly relates to an immunochromatography detection card for quantitatively detecting PSA (pressure-sensitive adhesive) based on an up-conversion luminescence technology.
Background
Up-Conversion luminescent nanoparticles (UCNPs) are rare earth metal compositions that can Up-convert energy, i.e., UCNPs can absorb low-energy (long wavelength) infrared light and emit high-energy (short wavelength) visible light. UCNPs are composed of several rare earth metal elements doped in the crystal lattice of certain crystals, and there are three main components in this material: host matrix, absorber and emitter, the ion pairs of which are suitably spatially oriented and spaced within the host matrix lattice, are the basis for producing up-converted luminescence.
Prostate cancer accounts for 10% -20% of all types of cancer in men, is the most common cancer of men, progresses slowly, and has a violent incidence in men older than 65 years old, so that prostate cancer is the main cancer threatening the life of men older than 50 years old, and accounts for the second place of mortality in the west. Epidemiological investigation shows that the incidence of the prostate cancer is increasing along with the improvement of the living standard of China, the westernization of pollution and dietary structure, and the advanced attention of the tumor clinic is required. PSA detection is widely used for diagnosing prostate cancer and is a good tumor marker, and the FDA in the United states has approved the PSA detection as an index for the general investigation of men over 50 years old. The PSA test is performed in conjunction with a digital rectal exam to increase the detection rate of prostate cancer. Recent studies have shown that PSA exceeds digital rectal examination in its role in early diagnosis of prostate cancer when FSA assays are performed with digital rectal examination.
Prostate Specific Antigen (PSA), a specific marker for prostate cancer. PSA is of great interest for the diagnosis of early asymptomatic prostate cancer. Normally, PSA is a serine protease secreted by prostate epithelial cells, a glycoprotein, secreted directly into the prostatic ductal system, and tissue-specific. A blood-epithelial barrier exists around the normal prostatic ductal system, preventing PSA produced by the prostatic epithelium from directly entering the blood, thereby maintaining a low concentration of PSA in the blood. Serum PSA less than 4.0 ng/ml is considered normal, and PSA greater than 10ng/ml is considered at increased risk for prostate cancer. When the prostate is cancerous, the blood-epithelial barrier is destroyed, and the cancer secretes too much PSA, so that PSA enters directly into the blood, the more malignant the cancer is, and the more destroyed the normal prostate tissue, the higher the PSA in the serum is.
Research shows that the combination of the up-conversion luminescent material UCNPs and the immunochromatography technology can bring the following breakthrough changes to the traditional immunochromatography technology:
the characteristics of the UCNPs luminescent marker enable the immunochromatography detection using the UCNPs luminescent marker as the marker to be combined with an instrument, thereby realizing the high-sensitivity quantitative detection of the detected object;
the UCNPs have various characteristic spectra (including excitation spectra and emission spectra), so that the immunochromatography detection based on the up-conversion luminescence technology can carry out multiple analyses with extremely high sensitivity, namely, the detection of multiple target detected objects in a biological sample is carried out at one time;
the unique upconversion luminescence phenomenon of the UCNPs ensures that the possibility of interference caused by autofluorescence of a biological sample to be detected is eliminated in the detection process by taking the UCNPs as a marker, the signal-to-noise ratio is improved, and the sensitivity and the stability of detection are improved;
4. the biological active molecules are crosslinked in a covalent mode, so that the reliability and stability of the system are improved on the premise of ensuring the detection sensitivity;
5. the rare earth high-doped up-conversion nanoparticles can emit stronger emitted light under the irradiation of sufficient exciting light.
Based on the unique up-conversion luminescence characteristic of the up-conversion luminescence immunochromatography technology, the detection has no background interference, and the sensitivity can reach 100 times of that of colloidal gold in terms of qualitative determination; meanwhile, the luminescent property of the fluorescent material is stable, the fluorescent material is not easy to quench, and the fluorescent material can be used for multiple and quantitative analysis. Therefore, the development of immunochromatography detection based on the upconversion luminescence technology for quantitatively detecting the concentration of Prostate Specific Antigen (PSA) in serum has important significance for early diagnosis of prostate cancer.
Disclosure of Invention
The invention aims to provide an immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on an up-conversion luminescence technology, aiming at the problems of low sensitivity and poor stability of the existing detection method.
The invention is realized by the following technical scheme: an immunochromatography detection card for quantitatively detecting PSA (pressure-sensitive adhesive) based on an up-conversion luminescence technology comprises a sample pad, a combination pad, a reaction membrane and a water absorption pad, wherein the sample pad and the combination pad are glass fiber membranes, the reaction membrane is a nitrocellulose membrane, and the reaction membrane is provided with a detection line and a quality control line; an anti-PSA monoclonal antibody 1 is fixed on the detection line, and a secondary goat anti-mouse IgG is fixed on the quality control line; the sample pad, the combination pad, the reaction membrane and the water absorption pad are partially overlapped in sequence and then adhered to the surface of the viscous bottom plate, wherein the sample pad at the overlapped part of the sample pad and the combination pad is positioned above the combination pad, the combination pad at the overlapped part of the combination pad and the reaction membrane is positioned above the reaction membrane, and the water absorption pad at the overlapped part of the reaction membrane and the water absorption pad is positioned above the reaction membrane; cutting the processed bottom plate into narrow test strips by using a cutting machine, and mounting the test strips in a customized card shell to obtain a test card; an up-conversion luminescent nanoparticle-anti-PSA monoclonal antibody 2 conjugate is fixedly arranged on the conjugate pad;
the up-conversion luminescent nanoparticle-PSA monoclonal antibody 2 conjugate is the PSA monoclonal antibody 2 modified on the surface of the up-conversion luminescent nanoparticle; the up-conversion nano particles are rare earth high-doped up-conversion luminescent nano particles.
Specifically, the laser diode is 790nm, 980nm or 1550nm, and the light emitted by the laser diode reaches more than 0.001mw/cm under the action of the hemispherical lens3An energy density; when the result of the detection card is observed, the detection line and the quality control line are intensively irradiated by a laser diode under the action of the hemispherical lens and can be collected by a signal collector.
Specifically, the sample pad, the combination pad, the reaction membrane and the water absorption pad are partially overlapped at intervals of 1-2mm in sequence and then are adhered to the surface of the sticky bottom plate, and the width of the test strip is 3-5 mm; further, the width of the test strip is preferably 3.5 mm.
Further, the upconversion nanoparticles are NaYF4、NaGdF4、CaF2、LiYF4、NaLuF4、LiLuF4、KMnF3、Y2O3The surface of the nano-particle is modified by carboxylation after a sensitizer and an activator are co-doped as a luminescent substrate.
Furthermore, the content of the rare earth highly doped up-conversion luminescence nanoparticle sensitizer can be selected from: 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol%, 75mol%, and the activator content may be selected from: 2.5mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8 mol%.
Further, the sensitizer may be selected from Yb3+、Nd3+、Gd3+、Ce3+Or a mixture thereof, the activator being selected from Yb3+、Er3+、Tm3+、Sm3+、Dy3+、Ho3+、Eu3+、Tb3+、Pr3+Or mixtures thereof.
Furthermore, the particle size of the upconversion nanoparticles is 25-200 mm.
The preparation process of the immunochromatography detection card for quantitatively detecting PSA based on the up-conversion luminescence technology comprises the following steps:
a) preparation of upconversion luminescent nanoparticles
1mmol of RECl was taken3Mixing the solution, 6mL of oleic acid and 15mL of octadecene, adding the mixture into a 100mL three-neck round-bottom flask, stirring and heating the mixture to 160 ℃ under the protection of argon flow, and maintaining the temperature for 20min to obtain a clear primary mixed solution; RE in RECl3 solution3+Comprising Y3+A sensitizer, an activator;
cooling the primary mixed solution to 50 ℃, adding 10mL of methanol solution containing 4mmol of ammonium fluoride and 2.5mmol of sodium hydroxide, then heating to 150 ℃, and maintaining for 20min to remove methanol to obtain a secondary mixed solution;
heating the secondary mixed solution to 310 ℃ and maintaining for 90min, thermally injecting an oleic acid/octadecene solution containing sodium trifluoroacetate and yttrium trifluoroacetate into a reaction system, wherein the volume ratio of oleic acid to octadecene is 1:1, maintaining for 60min at 310 ℃, cooling the liquid to room temperature after the reaction is finished, centrifugally washing with ethanol and cyclohexane, and drying at 52-58 ℃ to obtain UCNPs;
b) surface modification of upconversion luminescent nanoparticles
Adding 1mL of 10mM UCNPs cyclohexane solution into 1mL of 0.3mol/L hydrochloric acid solution, performing ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5-8 times to obtain acid-washed UCNPs, and dispersing the acid-washed UCNPs into 1mL of deionized water to obtain an acid-washed UCNPs dispersion liquid;
mixing the obtained acid-washed UCNPs dispersion with a proper amount of PEG-COOH, stirring for 24 hours at room temperature, centrifuging to obtain a second precipitate, washing the second precipitate with water for 3-5 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;
c) preparation of UCNPs-PSA monoclonal antibody 2 conjugate
Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain a dispersion of UCNP @ PEG-COOH, adding the dispersion of UCNP @ PEG-COOH into a PB buffer solution with the pH value of 7.2 and the concentration of 0.2mol/L to prepare a suspension with the concentration of 1-2mg/mL, then adding NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL into the suspension in sequence to ensure that the mass ratio of the NHS to the luminescent substrate is 1:2-5 and the mass ratio of the EDC to the UCNPs is 1:2-5, then reacting for 20-60min at the temperature of 4-30 ℃, centrifuging to remove the redundant NHS and EDC, then adding 0.2mol/L of PB buffer solution to obtain a second suspension, adding a monoclonal antibody 2 into the second suspension, reacting for 1-3 hours at the temperature of 4-30 ℃, wherein the mass ratio of the labeled nucleic acid fragments to the UCNPs is 1:100-1000, adding BSA solution with the mass concentration of 0.5-2% after the reaction is finished, and sealing for 1-2 hours; after the washing, carrying out centrifugal washing for 1-2 times at the temperature of 3-5 ℃ to obtain a UCNPs-PSA monoclonal antibody 2 conjugate, storing the UCNPs-PSA monoclonal antibody 2 conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 2-8 ℃ for later use;
d) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in a sample pad treatment solution for 10min, wherein the sample pad treatment solution is a PB (phosphate buffer) slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the sample pad treatment solution contains 0.1wt% of Tween-20, and drying the strips at 37 ℃ for later use after soaking;
e) preparation of the conjugate pad
Selecting a glass cellulose membrane as a bonding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips for 10 minutes by using a bonding pad treatment solution, wherein the bonding pad treatment solution is a PB slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the bonding pad treatment solution contains 0.1wt% of BSA, 2wt% of sucrose and 0.1wt% of Tween-20, drying the strips at 37 ℃, and uniformly spraying the UCNPs-PSA monoclonal antibody 2 conjugate prepared in the step a) on the bonding pad by using a gold spraying instrument and drying the conjugate at 37 ℃ for later use;
f) preparation of the reaction film
Cutting the nitrocellulose membrane into strips with the specification of 2.5cm x 30cm, sticking the obtained strips in the middle of the viscous bottom plate, scribing PSA monoclonal antibody 2 on different positions of the nitrocellulose membrane by using a scribing instrument as a detection line, scribing goat anti-mouse IgG antibody as a quality control line, and drying at 37 ℃ for later use;
g) assembly of immunochromatographic test strip
Respectively sticking the sample pad, the combination pad and the water absorption pad on two sides of the reaction film prepared in the step f), ensuring that the distance between the component pads is 1-2mm, and then, cutting the components into test strips with the width of 3.5mm and installing the test strips in a customized PSA (pressure swing adsorption) card shell to obtain the detection card.
Compared with the prior art, the invention has the following advantages: the kit disclosed by the invention has the advantages that based on the unique up-conversion luminescence characteristic of the up-conversion luminescence immunochromatography technology, the detection is free from background interference, the sensitivity can reach about 100 of that of colloidal gold, the luminescence property is stable, the quenching is not easy, the kit can be used for multiple and quantitative analysis, and the kit has important significance for quantitatively detecting the concentration of Prostate Specific Antigen (PSA) in serum and early diagnosis of prostate cancer.
Drawings
FIG. 1 is a schematic diagram of the structure of the detection card of the present invention.
FIG. 2 is an electron microscope image and a core shell of erbium ion doped upconversion luminescent nanoparticle.
FIG. 3 is an electron micrograph and a core-shell of a thulium ion doped up-conversion luminescent nanoparticle.
FIG. 4 is a diagram of a quantitative detection optical path.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The most key concept of the invention is as follows: according to the technology of combining the up-conversion luminescent material UCNPs and the immunochromatography technology, the detection card is applied based on the unique up-conversion luminescent characteristic of the up-conversion luminescent immunochromatography technology, so that the detection has no background interference, the sensitivity can reach about 100 of colloidal gold, the luminescent property is stable, quenching is not easy, and the detection card can be used for multiple and quantitative analysis and for quantitatively detecting the concentration of prostate specific antigen PSA in serum.
As shown in fig. 1, an immunochromatographic detection card for quantitatively detecting PSA based on an up-conversion luminescence technology includes a sample pad 2, a binding pad 3, a reaction membrane 4 and a water absorption pad 5, wherein the sample pad 2 and the binding pad 3 are glass fiber membranes, the reaction membrane 4 is a nitrocellulose membrane, and the reaction membrane 4 is provided with a detection line 41 and a quality control line 42; an anti-PSA monoclonal antibody 1 is fixed on the detection line 41, and a secondary goat anti-mouse IgG is fixed on the quality control line 42; the sample pad 2, the combination pad 3, the reaction membrane 4 and the water absorption pad 5 are partially overlapped in sequence and then adhered to the surface of the viscous bottom plate 1, wherein the sample pad 2 at the overlapped part of the sample pad 2 and the combination pad 3 is positioned above the combination pad 3, the combination pad 3 at the overlapped part of the combination pad 3 and the reaction membrane 4 is positioned above the reaction membrane 4, and the water absorption pad 5 at the overlapped part of the reaction membrane 4 and the water absorption pad 5 is positioned above the reaction membrane 4; cutting the processed bottom plate 1 into narrow test strips by using a cutting machine, and mounting the test strips in a customized card shell to obtain a detection card; an up-conversion luminescent nanoparticle-anti-PSA monoclonal antibody 2 conjugate is fixedly arranged on the conjugate pad 3;
the up-conversion luminescent nanoparticle-PSA monoclonal antibody 2 conjugate is the PSA monoclonal antibody 2 modified on the surface of the up-conversion luminescent nanoparticle; the up-conversion nano particles are rare earth high-doped up-conversion luminescent nano particles.
Specifically, the laser diode is 790nm, 980nm or 1550nm, and the light emitted by the laser diode reaches more than 0.001mw/cm under the action of the hemispherical lens3An energy density; when the result of the detection card is observed, the detection line and the quality control line are intensively irradiated by a laser diode under the action of the hemispherical lens and can be collected by a signal collector.
The sample pad, the combination pad, the reaction membrane and the water absorption pad are partially overlapped at intervals of 1-2mm in sequence and then are adhered to the surface of the viscous bottom plate, and the width of the test strip is 3-5 mm; the test strip preferably has a width of 3.5 mm.
Wherein the upconversion nanoparticles are NaYF4、NaGdF4、CaF2、LiYF4、NaLuF4、LiLuF4、KMnF3、Y2O3The surface of the luminescent substrate is modified by carboxylation after a sensitizer and an activator are co-doped; the content of the rare earth highly-doped up-conversion luminescence nanoparticle sensitizer can be selected as follows: 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol%, 75mol%, and the activator content may be selected from: 2.5mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8 mol%; the sensitizer may be selected from Yb3+、Nd3+、Gd3+、Ce3+Or a mixture thereof, the activator being selected from Yb3+、Er3+、Tm3+、Sm3+、Dy3+、Ho3+、Eu3+、Tb3+、Pr3+Or mixtures thereof; the particle size of the up-conversion nanoparticles is 25-200 mm.
The preparation process of the immunochromatography detection card for quantitatively detecting PSA based on the up-conversion luminescence technology comprises the following steps:
a) preparation of upconversion luminescent nanoparticles
1mmol of RECl was taken3Mixing the solution, 6mL of oleic acid and 15mL of octadecene, adding the mixture into a 100mL three-neck round-bottom flask, stirring and heating the mixture to 160 ℃ under the protection of argon flow, and maintaining the temperature for 20min to obtain a clear primary mixed solution; RE in RECl3 solution3+Comprising Y3+A sensitizer, an activator;
cooling the primary mixed solution to 50 ℃, adding 10mL of methanol solution containing 4mmol of ammonium fluoride and 2.5mmol of sodium hydroxide, then heating to 150 ℃, and maintaining for 20min to remove methanol to obtain a secondary mixed solution;
heating the secondary mixed solution to 310 ℃ and maintaining for 90min, thermally injecting an oleic acid/octadecene solution containing sodium trifluoroacetate and yttrium trifluoroacetate into a reaction system, wherein the volume ratio of oleic acid to octadecene is 1:1, maintaining for 60min at 310 ℃, cooling the liquid to room temperature after the reaction is finished, centrifugally washing with ethanol and cyclohexane, and drying at 52-58 ℃ to obtain UCNPs;
b) surface modification of upconversion luminescent nanoparticles
Adding 1mL of 10mM UCNPs cyclohexane solution into 1mL of 0.3mol/L hydrochloric acid solution, performing ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5-8 times to obtain acid-washed UCNPs, and dispersing the acid-washed UCNPs into 1mL of deionized water to obtain an acid-washed UCNPs dispersion liquid;
mixing the obtained acid-washed UCNPs dispersion with a proper amount of PEG-COOH, stirring for 24 hours at room temperature, centrifuging to obtain a second precipitate, washing the second precipitate with water for 3-5 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;
c) preparation of UCNPs-PSA monoclonal antibody 2 conjugate
Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain a dispersion of UCNP @ PEG-COOH, adding the dispersion of UCNP @ PEG-COOH into a PB buffer solution with the pH value of 7.2 and the concentration of 0.2mol/L to prepare a suspension with the concentration of 1-2mg/mL, then adding NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL into the suspension in sequence to ensure that the mass ratio of the NHS to the luminescent substrate is 1:2-5 and the mass ratio of the EDC to the UCNPs is 1:2-5, then reacting for 20-60min at the temperature of 4-30 ℃, centrifuging to remove the redundant NHS and EDC, then adding 0.2mol/L of PB buffer solution to obtain a second suspension, adding a monoclonal antibody 2 into the second suspension, reacting for 1-3 hours at the temperature of 4-30 ℃, wherein the mass ratio of the labeled nucleic acid fragments to the UCNPs is 1:100-1000, adding BSA solution with the mass concentration of 0.5-2% after the reaction is finished, and sealing for 1-2 hours; after the washing, carrying out centrifugal washing for 1-2 times at the temperature of 3-5 ℃ to obtain a UCNPs-PSA monoclonal antibody 2 conjugate, storing the UCNPs-PSA monoclonal antibody 2 conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 2-8 ℃ for later use;
d) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in a sample pad treatment solution for 10min, wherein the sample pad treatment solution is a PB (phosphate buffer) slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the sample pad treatment solution contains 0.1wt% of Tween-20, and drying the strips at 37 ℃ for later use after soaking;
e) preparation of the conjugate pad
Selecting a glass cellulose membrane as a bonding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips for 10 minutes by using a bonding pad treatment solution, wherein the bonding pad treatment solution is a PB slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the bonding pad treatment solution contains 0.1wt% of BSA, 2wt% of sucrose and 0.1wt% of Tween-20, drying the strips at 37 ℃, and uniformly spraying the UCNPs-PSA monoclonal antibody 2 conjugate prepared in the step a) on the bonding pad by using a gold spraying instrument and drying the conjugate at 37 ℃ for later use;
f) preparation of the reaction film
Cutting the nitrocellulose membrane into strips with the specification of 2.5cm x 30cm, sticking the obtained strips in the middle of the viscous bottom plate, scribing PSA monoclonal antibody 2 on different positions of the nitrocellulose membrane by using a scribing instrument as a detection line, scribing goat anti-mouse IgG antibody as a quality control line, and drying at 37 ℃ for later use;
g) assembly of immunochromatographic test strip
Respectively sticking the sample pad, the combination pad and the water absorption pad on two sides of the reaction film prepared in the step f), ensuring that the distance between the component pads is 1-2mm, and then, cutting the components into test strips with the width of 3.5mm and installing the test strips in a customized PSA (pressure swing adsorption) card shell to obtain the detection card.
Example 1
The preparation process of the immunochromatography detection card for quantitatively detecting PSA based on the up-conversion luminescence technology comprises the following steps:
a) preparation of erbium ion doped up-conversion luminescent nano-particles
1mmol of RECl was taken3(RE = Y, Yb, Er) solution, 6mL of oleic acid and 15mL of octadecene are mixed and added into a 100mL three-neck round-bottom flask, and the mixture is stirred and heated to 160 ℃ under the protection of argon flow and is maintained for 20min to obtain a clear primary mixed solution;
cooling the primary mixed solution to 50 ℃, adding 10mL of methanol solution containing 4mmol of ammonium fluoride and 2.5mmol of sodium hydroxide, then heating to 150 ℃, and maintaining for 20min to remove methanol to obtain a secondary mixed solution;
heating the secondary mixed solution to 310 ℃ and maintaining for 90min, thermally injecting an oleic acid/octadecene solution containing sodium trifluoroacetate and yttrium trifluoroacetate into a reaction system, wherein the volume ratio of oleic acid to octadecene is 1:1, maintaining for 60min at 310 ℃, cooling the liquid to room temperature after the reaction is finished, centrifugally washing with ethanol and cyclohexane, and drying at 52-58 ℃ to obtain UCNPs; NaYF4 for different rare earth ion contents: er and Yb can be adjusted by YbCl3And ErCl3The ratio of NaYF4 of different shell thicknesses is realized: er, Yb @ NaYF4 can be realized by adjusting the heat injection amount of the shell layer precursor solution, and the erbium ion doped up-conversion luminescent nano-particles are obtained as shown in figure 2;
b) surface modification of erbium ion doped up-conversion luminescent nanoparticles
Adding 1mL of UCNPs cyclohexane solution with the concentration of 10mM into 1mL of hydrochloric acid solution with the concentration of 0.3mol/L, carrying out ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, then centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 8 times to obtain acid-washed UCNPs, and dispersing the acid-washed UCNPs into 1mL of deionized water to obtain an acid-washed UCNPs dispersion liquid;
mixing the obtained acid-washed UCNPs dispersion with a proper amount of PEG-COOH, stirring for 24 hours at room temperature, centrifuging to obtain a second precipitate, washing the second precipitate with water for 3 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;
c) preparation of UCNPs-PSA monoclonal antibody 2 conjugate
Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain a UCNP @ PEG-COOH dispersion liquid, adding the UCNP @ PEG-COOH dispersion liquid into a PB buffer solution with the pH value of 7.2 and the concentration of 0.2mol/L to prepare a suspension with the concentration of 1.5mg/mL, then, NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL are added into the suspension in sequence, so that the mass ratio of the NHS to the luminescent substrate is 1:3, the mass ratio of the EDC to the UCNPs is 1:5, then reacting for 40min at 22 ℃, centrifuging to remove redundant NHS and EDC, adding 0.2mol/L PB buffer solution to obtain suspension II, adding PSA monoclonal antibody 2 into the suspension II, reacting for 1-3 hours at the temperature of 4-30 ℃, wherein the mass ratio of the labeled nucleic acid fragments to the UCNPs is 1:320, and adding BSA solution with the mass concentration of 1.5% for blocking for 2 hours after the reaction is finished; after the washing, carrying out centrifugal washing for 1 time at the temperature of 3 ℃ to obtain a UCNPs-PSA monoclonal antibody 2 conjugate, storing the UCNPs-PSA monoclonal antibody 2 conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 6 ℃ for later use;
d) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in a sample pad treatment solution for 10min, wherein the sample pad treatment solution is a PB (phosphate buffer) slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the sample pad treatment solution contains 0.1wt% of Tween-20, and drying the strips at 37 ℃ for later use after soaking;
e) preparation of the conjugate pad
Selecting a glass cellulose membrane as a bonding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips for 10 minutes by using a bonding pad treatment solution, wherein the bonding pad treatment solution is a PB slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the bonding pad treatment solution contains 0.1wt% of BSA, 2wt% of sucrose and 0.1wt% of Tween-20, drying the strips at 37 ℃, and uniformly spraying the UCNPs-PSA monoclonal antibody 2 conjugate prepared in the step a) on the bonding pad by using a gold spraying instrument and drying the conjugate at 37 ℃ for later use;
f) preparation of the reaction film
Cutting a nitrocellulose membrane into strips with the specification of 2.5cm x 30cm, pasting the obtained strips in the middle of an adhesive bottom plate, diluting PSA monoclonal antibody 2 into 1mg/mL by using PBS with the concentration of 0.01mol/L, diluting goat anti-mouse IgG antibody into 0.5mg/mL by using 0.02mol/L PB +2% sucrose, scribing PSA monoclonal antibody 2 and goat anti-mouse IgG antibody on a certain position of an NC membrane (the nitrocellulose membrane) at the speed of 0.8ul/mm by using a scribing instrument as a detection line and a quality control line, and drying for 12 hours at 37 ℃ for later use;
g) assembly of immunochromatographic test strip
Respectively sticking the sample pad, the combination pad and the water absorption pad on two sides of the reaction film prepared in the step f), ensuring that the distance between the component pads is 1.5mm, and then, alternately laminating, cutting into test strips with the width of 3.5mm, and installing the test strips in a customized PSA (pressure swing adsorption) card shell to obtain the detection card.
The operation of quantitatively detecting PSA using the immunochromatographic test paper card for detecting PSA by using the prepared immunochromatographic detection card amount is as follows:
1) 1mg/mL PSA standard was used as a diluent to prepare a series of concentration standards: 0ng/mL, 2ng/mL, 5 ng/mL, 10ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 400 ng/mL, 600ng/mL, 800 ng/mL, 1000 ng/mL, 1200 ng/mL, 1400 ng/mL, 1500 ng/mL, 1800 ng/mL, 2000 ng/mL;
2) dropping the concentration series standard substance into the prepared detection cards respectively, detecting the ratio of the fluorescence intensity of the T line 41 of the detection line and the fluorescence intensity of the C line of the quality control line by a fluorescence measuring instrument after 15min (each sample is detected by the detection card prepared in the embodiment for 3 times respectively, and taking an average value), and drawing a standard curve by linear fitting of T/C detection data of each concentration standard sample;
3) and (3) detecting the clinical serum sample by using the standard curve, and calculating the concentration of PSA in the clinical serum sample by using the curve through judging the T/C value.
As shown in FIG. 4, the light emitted from the 980nm laser diode 10 during the above operation, the 980nm laser diode 10 reaches more than 0.001mw/cm by the hemispherical lens 93An energy density; fluorescence intensity information in the detection card 6 is collected through the signal collector 7, and a KG-3 heat absorption sheet 8 and a hemispherical lens 9 are arranged between the detection card 6 and the signal collector 7 so as to ensure the reliability and stability of the detection information.
Example 2
On the basis of the embodiment 1, erbium ion doped up-conversion luminescent nanoparticles are replaced by thulium ion doped up-conversion luminescent nanoparticles, and the obtained thulium ion doped up-conversion luminescent nanoparticles are shown in fig. 3; the rest is the same as in example 1.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (9)

1. An immunochromatography detection card for quantitatively detecting PSA (pressure-sensitive adhesive) based on an up-conversion luminescence technology comprises a sample pad, a combination pad, a reaction membrane and a water absorption pad, wherein the sample pad and the combination pad are glass fiber membranes, the reaction membrane is a nitrocellulose membrane, and the reaction membrane is provided with a detection line and a quality control line; an anti-PSA monoclonal antibody 1 is fixed on the detection line, and a secondary goat anti-mouse IgG is fixed on the quality control line; the sample pad, the combination pad, the reaction membrane and the water absorption pad are partially overlapped in sequence and then adhered to the surface of the viscous bottom plate, wherein the sample pad at the overlapped part of the sample pad and the combination pad is positioned above the combination pad, the combination pad at the overlapped part of the combination pad and the reaction membrane is positioned above the reaction membrane, and the water absorption pad at the overlapped part of the reaction membrane and the water absorption pad is positioned above the reaction membrane; cutting the processed bottom plate into narrow test strips by using a cutting machine, and mounting the test strips in a customized card shell to obtain a test card; the kit is characterized in that an up-conversion luminescent nanoparticle-anti-PSA monoclonal antibody 2 conjugate is fixedly arranged on the conjugate pad;
the up-conversion luminescent nanoparticle-PSA monoclonal antibody 2 conjugate is the PSA monoclonal antibody 2 modified on the surface of the up-conversion luminescent nanoparticle; the up-conversion nano particles are rare earth high-doped up-conversion luminescent nano particles.
2. The immunochromatographic detection card for quantitatively detecting PSA based on the upconversion luminescence technology according to claim 1, wherein the laser diode is a 790nm, 980nm or 1550nm laser diode, and the emission light of the laser diode reaches more than 0.001mw/cm under the action of the hemispherical lens3An energy density; when the result of the detection card is observed, the detection line and the quality control line are intensively irradiated by a laser diode under the action of the hemispherical lens and can be collected by a signal collector.
3. The immunochromatographic detection card for quantitatively detecting PSA based on the upconversion luminescence technology according to claim 1, wherein the sample pad, the conjugate pad, the reaction membrane and the water absorption pad are partially overlapped in sequence at a distance of 1-2mm and then adhered to the surface of the adhesive base plate, and the test strip has a width of 3-5 mm.
4. The immunochromatographic test card for quantitatively detecting PSA based on the upconversion luminescence technology according to claim 3, wherein the test strip has a width of 3.5 mm.
5. The immunochromatographic detection card for quantitatively detecting PSA based on the upconversion luminescence technology according to claim 1, wherein the upconversion nanoparticles are NaYF4、NaGdF4、CaF2、LiYF4、NaLuF4、LiLuF4、KMnF3、Y2O3The surface of the nano-particle is modified by carboxylation after a sensitizer and an activator are co-doped as a luminescent substrate.
6. The immunochromatographic detection card for quantitative detection of PSA based on the upconversion luminescence technology according to claim 5, wherein the content of the rare earth highly doped upconversion luminescence nanoparticle sensitizer is selected from the group consisting of: 15mol%, 20mol%, 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, 55mol%, 60mol%, 65mol%, 70mol%, 75mol%, and the activator content may be selected from: 2.5mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8 mol%.
7. The immunochromatographic test card for quantitative detection of PSA based on the upconversion luminescence technology according to claim 5, wherein the sensitizer is Yb3+、Nd3+、Gd3+、Ce3+Or a mixture thereof, the activator being selected from Yb3+、Er3+、Tm3+、Sm3+、Dy3+、Ho3+、Eu3+、Tb3+、Pr3+Or mixtures thereof.
8. The immunochromatographic detection card for quantitatively detecting PSA based on the upconversion luminescence technology according to claim 5, wherein the particle size of the upconversion nanoparticles is 25 to 200 mm.
9. The immunochromatographic detection card for quantitatively detecting PSA based on the upconversion luminescence technology according to claim 5, which is prepared by the following process:
a) preparation of upconversion luminescent nanoparticles
1mmol of RECl was taken3Mixing the solution, 6mL of oleic acid and 15mL of octadecene, adding the mixture into a 100mL three-neck round-bottom flask, stirring and heating the mixture to 160 ℃ under the protection of argon flow, and maintaining the temperature for 20min to obtain a clear primary mixed solution; RE in RECl3 solution3+Comprising Y3 +A sensitizer, an activator;
cooling the primary mixed solution to 50 ℃, adding 10mL of methanol solution containing 4mmol of ammonium fluoride and 2.5mmol of sodium hydroxide, then heating to 150 ℃, and maintaining for 20min to remove methanol to obtain a secondary mixed solution;
heating the secondary mixed solution to 310 ℃ and maintaining for 90min, thermally injecting an oleic acid/octadecene solution containing sodium trifluoroacetate and yttrium trifluoroacetate into a reaction system, wherein the volume ratio of oleic acid to octadecene is 1:1, maintaining for 60min at 310 ℃, cooling the liquid to room temperature after the reaction is finished, centrifugally washing with ethanol and cyclohexane, and drying at 52-58 ℃ to obtain UCNPs;
b) surface modification of upconversion luminescent nanoparticles
Adding 1mL of 10mM UCNPs cyclohexane solution into 1mL of 0.3mol/L hydrochloric acid solution, performing ultrasonic treatment for 10min, stirring for 3h to obtain a mixed solution, centrifuging the mixed solution at a high speed, removing a supernatant to obtain a first precipitate, washing the first precipitate for 5-8 times to obtain acid-washed UCNPs, and dispersing the acid-washed UCNPs into 1mL of deionized water to obtain an acid-washed UCNPs dispersion liquid;
mixing the obtained acid-washed UCNPs dispersion with a proper amount of PEG-COOH, stirring for 24 hours at room temperature, centrifuging to obtain a second precipitate, washing the second precipitate with water for 3-5 times to remove the redundant PEG-COOH, and obtaining UCNP @ PEG-COOH;
c) preparation of UCNPs-PSA monoclonal antibody 2 conjugate
Dispersing UCNP @ PEG-COOH into 1mL of deionized water to obtain a dispersion of UCNP @ PEG-COOH, adding the dispersion of UCNP @ PEG-COOH into a PB buffer solution with the pH value of 7.2 and the concentration of 0.2mol/L to prepare a suspension with the concentration of 1-2mg/mL, then adding NHS with the concentration of 50mg/mL and EDC with the concentration of 50mg/mL into the suspension in sequence to ensure that the mass ratio of the NHS to the luminescent substrate is 1:2-5 and the mass ratio of the EDC to the UCNPs is 1:2-5, then reacting for 20-60min at the temperature of 4-30 ℃, centrifuging to remove the redundant NHS and EDC, then adding 0.2mol/L of PB buffer solution to obtain a second suspension, adding a monoclonal antibody 2 into the second suspension, reacting for 1-3 hours at the temperature of 4-30 ℃, wherein the mass ratio of the labeled nucleic acid fragments to the UCNPs is 1:100-1000, adding BSA solution with the mass concentration of 0.5-2% after the reaction is finished, and sealing for 1-2 hours; after the washing, carrying out centrifugal washing for 1-2 times at the temperature of 3-5 ℃ to obtain a UCNPs-PSA monoclonal antibody 2 conjugate, storing the UCNPs-PSA monoclonal antibody 2 conjugate in a PB buffer solution with the concentration of 0.02mol/L, and storing at the temperature of 2-8 ℃ for later use;
d) preparation of sample pad
Selecting a glass cellulose membrane as a sample pad material, cutting the sample pad material into strips with the specification of 1.5cm x 30cm, soaking the strips in a sample pad treatment solution for 10min, wherein the sample pad treatment solution is a PB (phosphate buffer) slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the sample pad treatment solution contains 0.1wt% of Tween-20, and drying the strips at 37 ℃ for later use after soaking;
e) preparation of the conjugate pad
Selecting a glass cellulose membrane as a bonding pad material, cutting the glass cellulose membrane into strips with the specification of 1cm x 30cm, soaking the strips for 10 minutes by using a bonding pad treatment solution, wherein the bonding pad treatment solution is a PB slow-release solution with the pH of 7.2 and the concentration of 0.02mol/L, and the bonding pad treatment solution contains 0.1wt% of BSA, 2wt% of sucrose and 0.1wt% of Tween-20, drying the strips at 37 ℃, and uniformly spraying the UCNPs-PSA monoclonal antibody 2 conjugate prepared in the step a) on the bonding pad by using a gold spraying instrument and drying the conjugate at 37 ℃ for later use;
f) preparation of the reaction film
Cutting the nitrocellulose membrane into strips with the specification of 2.5cm x 30cm, sticking the obtained strips in the middle of the viscous bottom plate, scribing PSA monoclonal antibody 2 on different positions of the nitrocellulose membrane by using a scribing instrument as a detection line, scribing goat anti-mouse IgG antibody as a quality control line, and drying at 37 ℃ for later use;
g) assembly of immunochromatographic test strip
Respectively sticking the sample pad, the combination pad and the water absorption pad on two sides of the reaction film prepared in the step f), ensuring that the distance between the component pads is 1-2mm, and then, cutting the components into test strips with the width of 3.5mm and installing the test strips in a customized PSA (pressure swing adsorption) card shell to obtain the detection card.
CN202010586396.8A 2020-06-24 2020-06-24 Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology Pending CN111735957A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010586396.8A CN111735957A (en) 2020-06-24 2020-06-24 Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010586396.8A CN111735957A (en) 2020-06-24 2020-06-24 Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology

Publications (1)

Publication Number Publication Date
CN111735957A true CN111735957A (en) 2020-10-02

Family

ID=72650886

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010586396.8A Pending CN111735957A (en) 2020-06-24 2020-06-24 Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology

Country Status (1)

Country Link
CN (1) CN111735957A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113702632A (en) * 2021-09-02 2021-11-26 深圳市光与生物科技有限公司 Immunochromatography detection card for rapidly detecting preeclampsia and preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101551388A (en) * 2009-05-22 2009-10-07 上海师范大学 Immunochromatographic test paper for quantitatively detecting AFP based on up-converting phosphor technology
CN106053806A (en) * 2016-07-23 2016-10-26 苏州东尼生物技术有限公司 HPV (human papillomavirus) immune colloidal gold diagnostic test strip, method for preparing same and detection method implemented by HPV immune colloidal gold diagnostic test strip
CN107632161A (en) * 2017-09-14 2018-01-26 湖南大学 Transgene protein CP4EPSPS upper conversion immuno-chromatographic test paper strip and detection method
CN107632156A (en) * 2017-09-14 2018-01-26 湖南大学 Detect Escherichia coli O 157:H7 upper conversion immuno-chromatographic test paper strip and detection method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101551388A (en) * 2009-05-22 2009-10-07 上海师范大学 Immunochromatographic test paper for quantitatively detecting AFP based on up-converting phosphor technology
CN106053806A (en) * 2016-07-23 2016-10-26 苏州东尼生物技术有限公司 HPV (human papillomavirus) immune colloidal gold diagnostic test strip, method for preparing same and detection method implemented by HPV immune colloidal gold diagnostic test strip
CN107632161A (en) * 2017-09-14 2018-01-26 湖南大学 Transgene protein CP4EPSPS upper conversion immuno-chromatographic test paper strip and detection method
CN107632156A (en) * 2017-09-14 2018-01-26 湖南大学 Detect Escherichia coli O 157:H7 upper conversion immuno-chromatographic test paper strip and detection method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAO HE ET AL.: "Quantitative Lateral Flow Strip Sensor Using Highly Doped Upconversion Nanoparticles", 《ANALYTICAL CHEMISTRY》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113702632A (en) * 2021-09-02 2021-11-26 深圳市光与生物科技有限公司 Immunochromatography detection card for rapidly detecting preeclampsia and preparation method and application thereof

Similar Documents

Publication Publication Date Title
US7560284B2 (en) Fine particles containing rare earth element and fluorescent probe using the same
CN111334282B (en) PTH rare earth detection kit, PTH rare earth detection card, microsphere and preparation and detection methods thereof
CN110514825B (en) Immunochromatography detection method based on near-infrared luminescent rare earth nano material
Xu et al. 2.05 µm emission properties and energy transfer mechanism of germanate glass doped with Ho3+, Tm3+, and Er3+
CN104804741A (en) Single-emission up-conversion nano fluorescent probe and synthetic method thereof
CN111735964A (en) Single-molecule immunodetection method based on up-conversion fluorescent probe
CN107356570B (en) Solid-state up-conversion fluorescent probe and preparation method and application thereof
CN111735957A (en) Immunochromatography detection card for quantitatively detecting PSA (prostate specific antigen) based on up-conversion luminescence technology
CN107748147B (en) White luminous up-conversion nano-particles and test strip based on same and capable of simultaneously realizing detection of multi-component tumor markers
US20110306065A1 (en) Use of an antibody and a rare-earth based crystal
Zaitoun et al. Observation of Electron− Hole Carrier Emission in the Eu3+-Doped Silica Xerogel
JP2012032263A (en) Reagent for measuring immune containing fluorescent fine particle
Wen et al. Effect of Al powder on Tb3+-doped borogermanate glass for X-ray detection
El Jouad et al. Structural, optical behavior and Judd-Ofelt analysis of europium-doped silicophosphate glasses for red-emitting device applications
Song et al. Handheld NIR-to-NIR Platform for on-site evaluating protective neutralizing antibody against SARS-CoV-2 ancestral strain and Omicron variant after vaccination or infection
CN114675026A (en) Dissolution-enhanced long afterglow luminescence detection method
CN105754600B (en) A kind of core-shell structure copolymer-shell structural nano fluorescence probe of europium ion activation and its preparation method and application
EP3857231B1 (en) Analyte quantitation
CN111733215A (en) Rapid nucleic acid detection method based on up-conversion fluorescent probe
CN115572602A (en) Near-infrared two-region fluorescent rare earth nanoprobe test strip and preparation method thereof
JP2009084576A (en) Fine particle containing rare earth element and fluorescent probe using the same
CN111044493B (en) Near infrared excitation and emission-based lateral flow test strip for luminescent detection of serum marker and preparation and use methods thereof
JPH11265033A (en) Image recording and reading system
CN112763715A (en) CHI3L1/PC III detection card, and preparation method and application thereof
CN100538340C (en) A kind of reader with sandwich construction

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20201002