CN111426827A - Long-afterglow material-based labeling solution, immunochromatography test paper and application thereof - Google Patents
Long-afterglow material-based labeling solution, immunochromatography test paper and application thereof Download PDFInfo
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- CN111426827A CN111426827A CN202010186358.3A CN202010186358A CN111426827A CN 111426827 A CN111426827 A CN 111426827A CN 202010186358 A CN202010186358 A CN 202010186358A CN 111426827 A CN111426827 A CN 111426827A
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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
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
Abstract
The invention provides a long afterglow material-based marker, immunochromatographic test paper and application thereof, wherein the long afterglow material-based marker solution is prepared by the following steps of preparing a long afterglow material/water soluble high molecular surfactant/carboxymethylcellulose (CMC) solution as a marker, and detecting CRP antigen and SAA antigen.
Description
Technical Field
The invention relates to the technical field of immunochromatography, in particular to a long-afterglow-material-based labeling solution, immunochromatography test paper and application thereof.
Background
Since the 90 s of the 20 th century, immunochromatography has been rapidly developed due to its characteristics of rapidness, simplicity, convenience, economy, single-person detection and the like, and has been widely applied to medical detection, food quality monitoring, drug detection and environmental monitoring. At present, the immunochromatography technology is developed towards high sensitivity, quantitative and multiplex detection, and is likely to become an early effective and sensitive screening and diagnosis technology for pathogens, malignant tumors, cardiovascular diseases and the like.
At present, the most common test paper is colloidal gold test paper, which can generally read the detection result within 15 minutes, but the test paper has relatively low sensitivity, complex manufacturing process and poor stability and repeatability. Due to the photo-electromagnetic property and the unique structure of various fluorescent nano materials, the fluorescent nano materials become very promising colloidal gold substitute tracers. The fluorescent test paper can quantitatively detect trace or trace substances in a sample to be detected according to the change of the fluorescence intensity, and more meets the requirements of accurate quantification, multiple markers and high flux of biomacromolecule detection in the field of in vitro diagnosis at present. The fluorescent microsphere adopted by the fluorescent microsphere immunochromatographic test paper detection technology is generally realized by labeling fluorescent substances on the surface of the microsphere or chelating rare earth elements and other micro particles in the microsphere by physical or chemical methods and the like, and the particle diameter of the fluorescent microsphere is generally in the range from nano-scale to micron-scale. The fluorescent microspheres can realize the purposes of multi-labeling and detecting different objects to be detected by labeling fluorescent substances with different colors and combining specific monoclonal antibodies. Compared with the traditional organic dye, the quantum dot immunochromatographic test paper detection technology has obvious advantages, such as larger Stokes shift, wider excitation wavelength and narrower emission wavelength of the quantum dot, stable fluorescence emitted by the quantum dot, high quantum yield, fluorescence bleaching resistance and the like. Compared with fluorescent dye and quantum dot nano particles, the up-conversion luminescent particles adopted by the up-conversion luminescent immunochromatographic test paper detection technology have the advantages of low toxicity, high sensitivity, good luminescent signal stability, small external background interference and no influence of a carrier or a substrate with a fluorescent characteristic in a sample. There are also new time-resolved fluorescence immunochromatographic test strip detection technology and nanometer enzyme immunochromatographic test strip detection technology in recent years.
Although the detection sensitivity of the fluorescent microsphere immunochromatographic test paper detection technology and the fluorescent microsphere immunochromatographic test paper detection technology is improved by a lot compared with the colloidal gold test paper detection technology under the same condition, the external background interference is large, and the application of the fluorescent microsphere immunochromatographic test paper detection technology and the fluorescent microsphere immunochromatographic test paper detection technology is limited due to the large influence of a carrier or a matrix with fluorescence characteristics in a sample. The quantum dot immunochromatographic test paper detection technology is restricted by the technical level, including factors such as material preparation, equipment precision and the like, so that the detection cost is high and the market popularization is difficult.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a long afterglow material-based labeling solution, immunochromatography test paper and application thereof.
In contrast, the technical scheme adopted by the invention is as follows:
a long persistence material based marking solution comprising long persistence organophosphorous molecules.
The preparation method of the marking solution based on the long afterglow material comprises the following steps:
step S1, preparing a solution of long afterglow material/water soluble surfactant/carboxymethyl cellulose (CMC), wherein the long afterglow material contains long afterglow organophosphorus optical molecules; the long-afterglow organic phosphorus molecules are a series of organic compounds which can continuously emit light after an excitation light source such as an ultraviolet lamp is removed under the excitation of the ultraviolet lamp.
Step S2, adding MES (2- (N-morpholino) ethanesulfonic acid) into the solution of the long afterglow material/the water soluble surfactant/the CMC, centrifuging, pouring out the supernatant, adding MES, and performing ultrasonic pulverization to obtain a pulverized long afterglow material/water soluble surfactant/CMC solution;
step S3, adding MES into a mixed solution of EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide, mixing, adding into the long afterglow material/water soluble surfactant/CMC/MES solution obtained in the step S2, mixing uniformly, centrifuging, pouring out the supernatant, adding Phosphate Buffer Saline (PBS), and performing ultrasound;
step S4, adding CRP or SAA antibody, mixing uniformly, centrifuging, pouring out supernatant, adding PBS, and performing ultrasonic treatment;
step S5, adding MEA (Malt Extract Agar), mixing uniformly, centrifuging, pouring out supernatant, adding Bovine Serum Albumin (BSA), and performing ultrasonic treatment to obtain a marking solution based on the long afterglow material.
The technical scheme adopts the long-afterglow organic phosphorescent compound as the tracer, can combine the long-afterglow organic forest phosphorescent labeling technology with high sensitivity and high-specificity immunoreaction, can be used in detection and analysis technologies of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, medicines and the like, has higher sensitivity, and is quicker and more reliable in detection.
As a further improvement of the invention, the water-soluble surfactant is at least one of Polyacrylamide (PAM), Hydrolyzed Polyacrylamide (HPAM) and polyvinylpyrrolidone (PVP).
As a further improvement of the invention, the long afterglow material is long afterglow organic phosphorescent nanoparticles. Further, the long afterglow material is at least one of PDBCz (9, 9'- (2,5-dibromo-1,4-phenylene) bis [9H-carbazole ], 9,9' - (2,5-dibromo-1,4-phenylene) bis [9H carbazole ]), CPhCz (carbazol-9-yl- (4-chlorophenylene) methanone, carbazol-9-yl- (4-chlorophenyl) methanone), and the molecular formula is as follows:
as a further improvement of the invention, in step S1, the concentration of the long afterglow material in the long afterglow material/water soluble surfactant/CMC solution is 1-5mg/m L.
As a further improvement of the present invention, the parameters of the centrifugation are: 4 ℃ 13000 rmp.
As a further improvement of the invention, the blending is carried out by adopting an oscillating or rotating disc type blender.
The invention also discloses immunochromatographic test paper based on the long afterglow material, which is prepared by the following steps:
spraying gold on a glass fiber with a marking solution, wherein the marking solution is the marking solution based on the long afterglow material as described in any one of the above items;
drying the glass fiber;
respectively sticking absorbent paper and an NC membrane on a bottom card, then respectively spraying gold on the NC membrane by adopting CRP or SAA and goat anti-mouse IgG, and respectively scribing a T line and a C line on the NC membrane;
after the scribing is finished, drying the bottom card;
and (3) sticking the dried glass fiber on a bottom card, and cutting to obtain the immunochromatographic test paper based on the long afterglow material.
By adopting the technical scheme, long-afterglow organic phosphorus molecules are used as markers, a test strip assembled by a plurality of different antibodies is marked to detect the CRP antigen/SAA antigen, and the two antigens are qualitatively detected by giving out long-afterglow color differences to the different organic phosphorus molecules; the two antigens to be detected are quantitatively detected by the long afterglow with the same color of different phosphorescent molecules, and the detection sensitivity of the test strip is superior to that of the traditional colloidal gold test strip.
As a further improvement of the invention, the drying method comprises the following steps: drying in a drying oven at 37 deg.C for not less than 12 hr.
The invention also discloses application of the immunochromatographic test paper based on the long-afterglow material in CRP or SAA antigen detection.
The invention also discloses application of the long-afterglow organophosphorus photolysis molecules in immunochromatographic test paper. The method comprises the following steps of (1) adopting long-afterglow organic phosphorus molecules as markers, marking a test strip assembled by a plurality of different antibodies to detect CRP antigen and SAA antigen, and qualitatively detecting the two antigens by giving out long-afterglow color differences to the different organic phosphorus molecules; two antigens to be detected are quantitatively detected through the long afterglow with the same color but the afterglow service life difference (caused by the concentration difference of the two antigens) of different phosphorescent molecules. Due to the advantages of quantitative and multi-label detection and the characteristic of high-flux detection, the long-afterglow organophosphorus photo molecules become a very potential phosphorescent probe label.
Compared with the prior art, the invention has the beneficial effects that:
the long-afterglow organic phosphorescent nanoparticles prepared by the technical scheme of the invention have low toxicity, high sensitivity, good stability of luminescent signals and small interference of external background, can almost eliminate the influence even if the carrier or the sample contains more fluorescent characteristic matrixes, and has strong anti-fluorescent interference capability. Due to the advantages of quantitative and multi-label detection, the long-afterglow organic phosphorus molecule is hopeful to become a phosphorescent probe label with great potential. Compared with the traditional fluorescent dye and quantum dot nano-particles, the chromatographic technology can realize rapid, simple, sensitive, accurate and stable qualitative and quantitative detection, and is most likely to become an ideal probe marker in the future.
Detailed Description
Preferred embodiments of the present invention are described in further detail below.
An immunochromatographic test paper based on a long afterglow material is prepared by firstly preparing a labeling solution, wherein the labeling solution is prepared by the following steps:
1) the long afterglow material/water soluble surfactant and carboxymethyl cellulose are mixed according to a certain volume ratio to prepare a solution of 1-5mg/m L, i.e. the concentration of the long afterglow material is 1-5mg/m L. the water soluble surfactant is at least one of polyacrylamide, hydrolyzed polyacrylamide and polyvinylpyrrolidone, and polyvinylpyrrolidone is used in this embodiment.
2) Taking 1-5mg/m L of long afterglow material/water soluble surfactant and carboxymethyl cellulose in a No. 1 centrifugal tube (1.5m L);
3) weighing MES 0.5-1.5m L in centrifuge tube No. 1, centrifuging with centrifuge at 4 deg.C 13000rmp for 5 min;
4) pouring out the supernatant in the No. 1 centrifuge tube, adding 0.5-1.5m of MES L, and performing ultrasonic treatment by using an ultrasonic cell crusher;
5) weighing 1-2mg of EDC and 1-1.5mg of NHS in a No. 2 centrifuge tube (1.5m L) by a balance, adding 200u of MES L, and oscillating;
6) after oscillation, pouring the solution in the No. 2 centrifugal tube into the No. 1 centrifugal tube, and placing the centrifugal tube on a rotating disc type mixer to rotate for 1 hour;
7) after 1h of rotation, trim and centrifuge (6min, 4 ℃, 13000 rmp);
8) after centrifugation, the supernatant was decanted, 1m L of PBS was added, and sonication was carried out;
9) after ultrasonic treatment, placing CRP or SAA in a No. 1 centrifugal tube, oscillating, and rotating the turntable for 2-6 h;
10) after 2-6h, balancing and centrifuging (6min, 4 ℃, 13000 rmp);
11) after centrifugation, the supernatant was decanted, 1m L of PBS was added, and sonication was carried out;
12) after ultrasonic treatment, adding 100u L MEA, oscillating, and rotating the turntable for 1-2 h;
13) after 1-2h of rotation, trimming and centrifuging (6min, 4 ℃, 13000 rmp);
14) after centrifugation, the supernatant was decanted, 1m L of BSA was added, and the labeling solution was prepared by shaking and sonication.
The immunochromatographic test paper was produced as follows:
1) spraying gold on the glass fiber by using a three-dimensional film-scribing gold spraying instrument;
2) after the gold spraying is finished, putting the glass fiber into an electric heating air blast drying oven for drying (staying overnight at 37 ℃);
3) respectively sticking absorbent paper and an NC membrane on a bottom card, and spraying gold on the NC membrane by using a three-dimensional membrane-scribing gold spraying instrument, namely respectively scribing a T line and a C line on the NC membrane by using CRP or SAA and goat anti-mouse IgG;
4) after the scribing is finished, putting the bottom card into a drying oven to dry (at 37 ℃ overnight);
5) and (3) sticking the dried glass fibers on the bottom card, and cutting the bottom card into test strips by using a test strip numerical control cutting machine.
The immunochromatographic test paper prepared by the embodiment adopts long-afterglow organophosphorus molecules as markers, and is used for detecting CRP antigen and SAA antigen by using test paper assembled by a plurality of different antibodies, so that the immunochromatographic test paper can be used for semi-quantitative and quantitative detection of two antigens to be detected.
And (3) quantitative detection: and (3) dropwise adding a sample to be detected onto the sample pad, carrying out chromatographic expansion on the sample to be detected along the directions of the combination pad, the nitrocellulose membrane and the water absorption pad under the action of the water absorption pad, wherein a red line appearing on the detection line is positive, otherwise, the detection line is negative, and if the quality control line does not show red, the detection is invalid.
A series of CRP antigen and SAA antigen standard solutions, namely 0ng/m L, 0.5ng/m L, 0.8ng/m L, 1ng/m L, 5ng/m L, 10ng/m L, 50ng/m L, 100ng/m L, 200ng/m L and 400ng/m L, are prepared and are respectively dripped on the sample pads of the immunochromatographic test strips of the long-afterglow materials of the examples 1 and 2 for detection, the average time of the detection is 5 times at each concentration, and the color development is observed, wherein the color development results are shown in the following table 1.
TABLE 1
According to the results of Table 1, the lowest detection limit of the CRP antigen and SAA antigen of the present invention is 0.8ng/m L and 0.5ng/m L, respectively, and the sensitivity is very high.
The immunochromatography test of the embodiment comprises the long-afterglow organic phosphorescent nanoparticles, the toxicity is low, the sensitivity is high, the stability of a luminescent signal is good, the interference of the external background is small, and even if the carrier or a sample contains more fluorescent characteristic matrixes, the influence can be almost eliminated.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (9)
1. A marker of an immunochromatographic test strip based on a long afterglow material is characterized by being prepared by the following steps:
step S1, preparing a solution of long afterglow material/water soluble surfactant/CMC, wherein the long afterglow material contains long afterglow organic phosphorus photon;
step S2, adding MES into the solution of the long afterglow material/the water soluble surfactant/the CMC, centrifuging, pouring out the supernatant, adding MES, and carrying out ultrasonic pulverization to obtain a pulverized long afterglow material/the water soluble surfactant/the CMC/MES solution;
step S3, adding MES into the mixed solution of EDC and NHS, mixing, adding into the long afterglow material/water soluble surfactant/CMC/MES solution obtained in step S2, mixing, centrifuging, pouring out the supernatant, adding PBS, and performing ultrasound;
step S4, CRP or SAA is added, the mixture is evenly mixed and then centrifuged, the supernatant is poured out, PBS is added, and ultrasound is carried out;
and step S5, adding MEA, mixing uniformly, centrifuging, pouring out supernatant, adding BSA, and performing ultrasonic treatment to obtain the marking solution based on the long afterglow material.
2. The long persistent material-based marking solution of claim 1, wherein: the long afterglow material is long afterglow organic phosphorescent nano particles.
3. The long persistent material-based marking solution of claim 2, wherein: the long afterglow material comprises at least one of CPhCz and PDBCz.
4. The long-afterglow-material-based marking solution as claimed in claim 2, wherein the concentration of the long-afterglow material in the long-afterglow material/water-soluble surfactant/CMC solution is 1-5mg/m L in step S1.
5. The long persistent material-based marking solution of claim 4, wherein: the parameters of the centrifugation are: 4 ℃ 13000 rmp.
6. The marking solution based on long afterglow materials of claims 1 to 5, wherein: the water-soluble surfactant is at least one of polyacrylamide, hydrolyzed polyacrylamide and polyvinylpyrrolidone.
7. The immunochromatographic test paper based on the long afterglow material is characterized in that: the preparation method comprises the following steps:
spraying gold on glass fibers by using a marking solution, wherein the marking solution is the marking solution based on the long afterglow material as defined in any one of claims 1 to 6;
drying the glass fiber;
respectively sticking absorbent paper and an NC membrane on a bottom card, then respectively spraying gold on the NC membrane by adopting CRP or SAA and goat anti-mouse IgG, and respectively scribing a T line and a C line on the NC membrane;
after the scribing is finished, drying the bottom card;
and (3) sticking the dried glass fiber on a bottom card, and cutting to obtain the immunochromatographic test paper based on the long afterglow material.
8. The immunochromatographic test paper based on a long-lasting phosphor according to claim 7, characterized in that: the drying method comprises the following steps: drying in a drying oven at 37 deg.C for not less than 12 hr.
9. The use of the long persistence-based material immunochromatographic strip of claim 7 for the detection of CRP or SAA antigens.
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| CN114163996A (en) * | 2021-12-13 | 2022-03-11 | 成都云芯医联科技有限公司 | Preparation method of hydrophilic functionalized luminous bead-like organic material |
| CN114200131A (en) * | 2021-12-13 | 2022-03-18 | 成都云芯医联科技有限公司 | Detection system for luminous characteristics of luminous pearl-like material and preparation method of matched detection test paper |
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Application publication date: 20200717 |