CN105738616A - Preparation method and application of dual-amplifying fluorescent immune labeling probe and method for preparing fluorescent immune chromatography reagent strip from probe - Google Patents
Preparation method and application of dual-amplifying fluorescent immune labeling probe and method for preparing fluorescent immune chromatography reagent strip from probe Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a dual-amplifying fluorescent immune labeling probe and a method for preparing a fluorescent immune chromatography reagent strip from the probe, and belongs to the field of fluorescent labeling in fluorescent immune chromatography in-vitro diagnosis.The preparation method of the dual-amplifying fluorescent immune labeling probe comprises the following steps of 1 preparation of biotinylated detecting molecules for a substance to be detected and biotinylated carriers, 2 preparation of fluorescent microspheres modified with biotin and micromolecular fluorescent dye and 3 preparation of the dual-amplifying fluorescent immune labeling probe.According to the dual-amplifying fluorescent immune labeling probe, the mode of enhancing the fluorescent intensity and the mode of increasing the detecting molecule combining weight of the fluorescent microspheres are effectively combined, one-time amplification is achieved through the combination effect between biotin and avidin, second-time amplification is achieved by enhancing the fluorescent intensity outside the fluorescent microspheres through the carriers at the same time, and the fluorescent signal intensity of fluoroimmunoassay is greatly improved through the dual amplifying mode.
Description
Technical field
The invention belongs to the fluorescent labeling field in fluorescence immune chromatography in-vitro diagnosis, the method be specifically related to the preparation method of a kind of dual amplification fluorescence immunoassay label probe and application, preparing fluorescence immune chromatography reagent strip with it.
Background technology
Immunoassay technology be the fields such as current medical science, biology, food safety most basic be also one of the most frequently used detection means, determinand is carried out qualitative and quantitative analysis based on specific reaction principle between antibody and antigen by this technology, is widely used in the detection of Medical Biology basic research, clinical medicine, environmental monitoring, medical diagnosis on disease, disease progression prediction or even food and microorganism pollution.
Fluorescence immunoassay technology is to utilize fluorescent material labelled antigen or antibody molecule, by detecting the Strength Changes of fluorescence signal with determinand molecule-specific immune after being combined, it is achieved to the qualitative of target analytes and rational judgment.
In carrying out immune detection or fluorescence immunoassay process, the detection of the antigen of low concentration is still that the significant problem of clinical diagnosis and inspection and quarantine field, above-mentioned analysis method is when the material tackling very trace detects, be frequently present of that signal is low, noise jamming big, traditional method is difficult to the difficult problem that detects, therefore, develop new immunolabelling technique or new detection means, improve an urgent demand that detection sensitivity is the development of present analysis detection field.
At present, in fluorescence immunoassay field, conventional amplification means include biotin～Avidin amplification system, 1981, Hsu etc. demonstrates an affinity element can in combination with four biotin molecules, and the method that both are applied in immunology is discussed in detail, by the bridge joint effect of streptavidin～biotin, respectively in connection with antigen-antibody, mark substance or fluorescent microsphere etc., can greatly strengthen antibodies amount or fluorescence binding capacity, play the effect of amplification.
Chinese patent CN201310470907X describes a kind of signal scale-up version fluorescent probe and preparation method and application, one of which is amplified mode and is namely utilized biotin and affinity element directly combination to realize, but the method is only for the increase of antibodies amount and amplification, may not detect at detection low value when the Indexs measure of some more trace.
What general biotin, affinity element amplification system all adopted is a kind of amplification mode, it is utilized only to the amplification effect between biotin and affinity element, effectively biotin and affinity element amplification mode can not be fully utilized, often can seem at a loss what to do when in the face of some trace detection.
Summary of the invention
The method that it is an object of the invention to overcome the deficiencies in the prior art and the preparation method of a kind of dual amplification fluorescence immunoassay label probe and application are provided, prepare fluorescence immune chromatography reagent strip with it, this dual amplification fluorescence immunoassay label probe utilizes and strengthens fluorescence intensity and increase by two kinds of amplification modes of molecular detection binding capacity, make fluorescence immunoassay detection kit fluorescence intensity have bigger amplification more in the past, detection low value is substantially reduced, it is achieved trace detection.
Technical solution of the present invention is as follows:
The preparation method of dual amplification fluorescence immunoassay label probe, comprises the steps:
Step one: take the activated biotin purchased, wherein the activated biotin of 1/4th carries out hybrid reaction with test substance molecular detection, the activated biotin of 3/4ths carries out hybrid reaction with carrier, stir 1～2 hour under equal room temperature, then dialyse 48～72 hours in PBS bag filter, obtaining biotinylation test substance molecular detection and biotinylation carrier, wherein the molar concentration of PBS is 10mM, pH is 7.4；
Step 2: take the biotinylation carrier that step one prepares, use NaHCO3After regulating pH to 8～9, add small molecule fluorescent dyestuff, reaction is stirred at room temperature 1～2 hour, after reaction terminates, remove unreacted small molecule fluorescent dyestuff with Purification Resin, add fluorescent microsphere, reaction is stirred at room temperature 1～2 hour, it is centrifuged 3～5 minutes under 1100g, collects centrifugal rear solution, it is thus achieved that the plain fluorescent microsphere with small molecule fluorescent dyestuff of modified biological；
Step 3: modified biological element step 2 prepared and the fluorescent microsphere of small molecule fluorescent dyestuff mix with streptavidin, room temperature reaction 1～2 hour, add the biotinylation test substance molecular detection that step one prepares, continue reaction 1～2 hour, obtain described dual amplification fluorescence immunoassay label probe.
Further, the preparation method of described dual amplification fluorescence immunoassay label probe, wherein test substance described in step one is antigen or antibody class protein or polypeptide, and described carrier is bovine serum albumin, casein or dissaving polymer class material.
Further, the preparation method of described dual amplification fluorescence immunoassay label probe, wherein described in step one activation biotin, test substance molecular detection, carrier mol ratio be 4:1:3.
Further, the preparation method of described dual amplification fluorescence immunoassay label probe, wherein biotinylation carrier described in step 2, fluorescent microsphere mol ratio be 1:1.
Further, the preparation method of described dual amplification fluorescence immunoassay label probe, wherein NaHCO described in step 23The mass concentration of solution is 10%, and described small molecule fluorescent dyestuff is identical with fluorescent microsphere fluorescence spectrum.
Further, the preparation method of above-mentioned dual amplification fluorescence immunoassay label probe, wherein modified biological described in step 3 element and the fluorescent microsphere of small molecule fluorescent dyestuff, streptavidin, biotinylation test substance molecular detection mol ratio be 1:5:5.
The method detecting the fluorescence immune chromatography reagent strip of cardiac muscle troponin I (cTnI) with described dual amplification fluorescence immunoassay label probe preparation, including being made by step:
Step one: the preparation of sample pad: use buffer solution albumen, add surfactant, wherein, every 100mL buffer adds 0.01～0.05g surfactant, regulating pH is 6～8, using the ratio of 2～5ml aforesaid liquid with the long sample pad of every 30cm, be uniformly coated in sample pad by above-mentioned solution, 20～25 DEG C dry 8～12 hours；
Step 2, the preparation of pad: the method processing sample pad with step one is identical, first pad is processed, after the dual amplification fluorescence immunoassay label probe of preparation is diluted 3000 times with PBS, be sprayed on uniformly on pad, dry 4～8 hours in 20～25 DEG C, wherein, the molar concentration of described PBS is 10mM, pH is 7.4, containing 2%BSA and 2% sucrose；
Step 3, the process of nitrocellulose filter:
A. the preparation of line is detected: the PB buffer catching monoclonal antibody or polyclonal antibody 20mmol/L, pH7.2 corresponding for cardiac muscle troponin I is diluted to the concentration of 2.0mg/mL, 0.8ul/cm rules on nitrocellulose filter and to obtain detection line, 20～25 DEG C of forced air drying 8～12h and get final product in drying baker；
B. the preparation of nature controlling line: rabbit anti-mouse igg antibody presses the concentration of 4mg/mL, 0.8ul/cm draws nature controlling line on nitrocellulose filter, and this line is spaced and parallel with detection line, 20～25 DEG C of forced air drying 8～12h and get final product in drying baker；
Step 4, the preparation of adsorptive pads: absorbent paper is cut into 30*2.7cm every,；
Step 5, assemble: described sample pad, pad, nitrocellulose filter and adsorptive pads are sequentially attached on base plate, wherein, overlapping 1～2mm is needed between sample pad, pad, nitrocellulose filter and adsorptive pads each several part, the reagent strip of one fixed width it is cut into afterwards with cutting machine, 20～25 DEG C of forced air drying 8～12h, obtain described fluorescence immune chromatography reagent strip.
Further, the method of the fluorescence immune chromatography reagent strip of described use dual amplification fluorescence immunoassay label probe preparation detection cardiac muscle troponin I (cTnI), wherein buffer described in step one is 10mM, pH is the PBS of 7.2～7.4, Tris or glycine buffer, with the BSA of described buffer solution 5mg/mL or casein.
Further, the method of the fluorescence immune chromatography reagent strip of described use dual amplification fluorescence immunoassay label probe preparation detection cardiac muscle troponin I (cTnI), wherein surfactant described in step one is polysorbas20 (Tween20) or Triton X-100 (TritonX-100).
The application in fluorescence immunoassay detects of the described dual amplification fluorescence immunoassay label probe.
Described fluorescent microsphere connects carrier can arbitrarily select physical absorption, covalent coupling and a kind of realization in conjunction with centering.
Beneficial effect: dual amplification fluorescence immunoassay label probe of the present invention, diagnosis immune detection will strengthen effective combination of the molecular detection binding capacity two ways of fluorescence intensity and increase fluorescent microsphere in vitro, combination between biotin affinity element is utilized to realize once amplifying, utilize carrier to add fluorescence intensity outside fluorescent microsphere simultaneously and realize another amplification, dual amplification pattern is greatly improved fluorescence immunoassay fluorescence signal intensity, and the immunological assay reagents performance such as detection sensitivity and detection range been significantly enhanced.
Accompanying drawing explanation
Fig. 1 is the dual amplification principle schematic of dual amplification fluorescence immunoassay label probe of the present invention；
Fig. 2 is the structural representation of the fluorescence immune chromatography reagent strip that the present invention prepares；
Fig. 3 is the detection range schematic diagram of the fluorescence immune chromatography reagent strip that the present invention prepares；
The dependency diagram of the fluorescence immune chromatography reagent strip that Fig. 4 present invention prepares；
Wherein, 1, base plate；2, sample pad；3, pad；4, nitrocellulose filter；5, adsorptive pads；6, detection line 7, nature controlling line.
Detailed description of the invention
Further the present invention being explained by the following examples, but be not limited thereto system, any those skilled in the art are changed or are modified as the Equivalent embodiments of equivalent variations possibly also with the technology contents of the disclosure above.But everything is without departing from technical solution of the present invention content, according to the technical spirit of the present invention to any amendment made for any of the above embodiments, equivalent variations and remodeling, still fall within the protection domain of technical solution of the present invention.
Embodiment one: the preparation of dual amplification fluorescent microsphere
1.1 biotin taking the 20mmol activation purchased, wherein biotin and the 5mmol test substance molecular detection hybrid reaction (the present embodiment for cardiac muscle troponin I (cTnI) antibody) of 5mmol activation, the biotin of 15mmol activation and 15mmol bovine serum albumin hybrid reaction, it is stirred at room temperature 1～2 hour, then put in the bag filter filling PBS (10mMpH7.4) and dialyse 48～72 hours, obtain biotinylation cTnI antibody and biotinylation bovine serum albumin.
1.2 take the biotinylation bovine serum albumin that 20 μ L above-mentioned steps prepare, pH to 8～9 is regulated with 10%NaHCO3, add the small molecule fluorescent dyestuff identical with fluorescent microsphere fluorescent spectroscopic properties, gentle agitation room temperature reaction 1～2 hour, after reaction terminates, unreacted fluorescent dye is gone out with Purification Resin, fluorescent microsphere is added again with 1:1 mol ratio, reaction is stirred at room temperature 1～2 hour, centrifugal 3～5 minutes of 1100g, collect centrifugal rear solution, it is thus achieved that the fluorescent microsphere of modified biological element and small molecule fluorescent dyestuff.
Modified biological element and the fluorescent microsphere of small molecule fluorescent dyestuff that 1.3 prepare 1.2 press the mixed in molar ratio of 1:5 with streptavidin, room temperature reaction 1～2 hour, add the biotinylation cTnI antibody of preparation in 1.1, continue reaction 1～2 hour, wherein biotinylation fluorescent microsphere, streptavidin and biotinylation cTnI antibody molar ratio example are 1:5:5, after reaction terminates, it is thus achieved that strengthen fluorescence intensity and increase the dual amplification fluorescent microsphere of antibodies amount.
Embodiment two: the preparation of fluorescence immune chromatography diagnostic reagent srip
As in figure 2 it is shown, a kind of fluorescence immune chromatography reagent strip detecting cardiac muscle troponin I, including base plate 1, base plate sequentially sticks sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 5；The wherein each parts junction overlap 1～2mm of sample pad 2, pad 3, nitrocellulose filter 4 and adsorptive pads 5, it is ensured that detection sample arrives detection zone from sample area by pad smoothly, and the preparation method of each parts is as described below:
The preparation of 2.1 sample pad 2: dissolve the BSA albumen of 0.5g with the PBS 100mL of 10mM, pH7.2～7.4, is subsequently adding the surfactant Tween20 of 0.01～0.05g, regulates pH to 6～8；Sample pad can be selected for glass fibre or polyester material, the long sample pad of 30cm is placed in the mixed solution 2～5ml of above-mentioned buffer, surfactant and albumen and soaks 2～4 hours, after taking-up, 20～25 DEG C dry can obtain for 8～12 hours, wherein, PBS also can replace with Tris or glycine buffer, BSA albumen also can replace with casein, and surfactant Tween20 also can replace with TritonX100；
The preparation of 2.2 pads 3: the method same first with above-mentioned sample pad processes pad, the dual amplification fluorescent microsphere PBS (10mM again prepared by embodiment one, pH7.4, containing 2%BSA and 2% sucrose) dilute 3000 times, it is coated on pad uniformly, is placed in 20～25 DEG C dry 4～8 hours；
The process of 2.3 nitrocellulose filters 4:
A. the preparation of line 6 is detected: cTnI is caught accordingly monoclonal antibody or polyclonal antibody 20mmol/L, the PB buffer of pH7.2 is diluted to the concentration of 2.0mg/mL, 0.8 μ L/cm rules on nitrocellulose filter and is coated with to obtain detection line, 20～25 DEG C of forced air drying 8～12h in drying baker；
B. the preparation of nature controlling line 7: rabbit anti-mouse igg antibody presses the concentration of 4mg/mL, 0.8 μ L/cm draws nature controlling line on nitrocellulose filter, and this line is parallel with detection line, and detection line interval 5mm, 20～25 DEG C of forced air drying 8～12h in drying baker；
The preparation of 2.4 adsorptive pads 5: absorbent paper is cut into 30*2.7cm every；
2.5 assemble: plastic bottom board 1, sample pad 2 and adsorptive pads 5 are parts generally in the art, above-mentioned nitrocellulose filter 4, adsorptive pads 5, sample pad 3 are pasted onto on plastic bottom board 1, the intermedium cutting machine posted is cut into the reagent strip of one fixed width, and namely 20～25 DEG C of forced air drying 8～12h obtain described fluorescence immunoassay reagent strip.
Embodiment three: fluorescence immune chromatography diagnostic reagent detects
3.1 tradition polystyrene mark fluorescent chromatography reagent and reagent of the present invention detection performance comparison
Sample pad adds variable concentrations cTnI antigen standard (take 10 variable concentrations, respectively 0,0.01,0.02,0.04,0.08,0.16,0.32,0.64,1.28,2.56,5.12ng/mL, each concentration of specimens sets three times and repeats) drip 120 μ L respectively in two reagent strip wells, reading signal by the fluorescence immunoassay quantitative analysis instrument Getein1100 of Ji Dan biotech inc after 15min, experimental result is shown in Fig. 3:
As it is shown on figure 3, the detection range lower limit of tradition polystyrene fluorescent microsphere system is 0.08ng/mL.After utilizing double; two enhancing fluorescent microsphere of the present invention, detection range lower limit is 0.01ng/mL, and compared with traditional gold colloidal and polystyrene microsphere, Monitoring lower-cut is lower, and fluorescence intensity has had the raising of more than 10～20 times.This result confirms that system of the present invention achieves fluorescence immunoassay labelling and amplifies enhancing performance, and sensitivity is greatly improved.
3.2 reagent strip relevance evaluations of the present invention
Taking many parts of serum specimens respectively with after Roche Related product and system reagent bar of the present invention test cTnI content, pick out that wherein gradient is suitable 15 parts, mapping compares dependency as seen from Figure 4.Diagram of system reveals dependency well, R2Value is up to 0.9992, it was demonstrated that system of the present invention and according to the test kit of this system except having hypersensitivity, outside broader detection range, also have splendid accuracy.
3.3 test strips times of the present invention development property assessment
Compound concentration is the detection sample of 1.00ng/mL, drip 120 μ L respectively in reagent strip well of the present invention, respectively at 1min, 3min, 5min, 10min, 20min and 30min uses GT1100 line fluorescent immune quantitative analyser replication, the relatively difference of different time testing result, calculates relative deviation (with 3min for benchmark), and result is as shown in table 1.It is all less that result is shown in 3～30min the pattern detection concentration difference opposite sex, time development property < 5%.Confirm that system of the present invention and the test kit according to this system detect time development property little, better embody measurement accuracy.
The different time development property of table 1 reagent strip
| Time | 3min | 5min | 8min | 10min | 20min | 30min |
| Concentration | 0.98 | 0.99 | 0.98 | 0.10 | 0.10 | 0.99 |
| Relative deviation | 0 | 1.02% | 0% | 2.04% | 2.04% | 1.02% |
3.4 reagent strip repeatability of the present invention assessments
The cTnI standard solution of preparation 1.0ng/mL and 0.2ng/mL, adopts system reagent bar of the present invention to be measured concentration, respectively replication 10 times, calculates respectively and measures average and standard deviation, CV value such as table 2.Being repeated property is investigated, and result shows two kinds of concentration repeatability respectively 9.7%, 9.3%, is fully able to and meets clinical requirement.
Table 2 reagent strip repeatability
| Sequence number | Standard value 1.00ng/mL | Standard value 0.20ng/mL |
| 1 | 0.893 | 0.178 |
| 2 | 1.218 | 0.234 |
| 3 | 1.110 | 0.204 |
| 4 | 0.980 | 0.210 |
| 5 | 0.974 | 0.207 |
| 6 | 1.020 | 0.184 |
| 7 | 1.010 | 0.189 |
| 8 | 0.925 | 0.176 |
| 9 | 0.905 | 0.219 |
| 10 | 1.115 | 0.224 |
| Meansigma methods | 1.015 | 0.203 |
| Standard deviation | 0.099 | 0.019 |
| CV | 9.70% | 9.30% |
Claims (10)
1. the preparation method of dual amplification fluorescence immunoassay label probe, it is characterised in that comprise the steps:
Step one: take the activated biotin purchased, wherein the activated biotin of 1/4th carries out hybrid reaction with test substance molecular detection, the activated biotin of 3/4ths carries out hybrid reaction with carrier, stir 1～2 hour under equal room temperature, then dialyse 48～72 hours in PBS bag filter, obtaining biotinylation test substance molecular detection and biotinylation carrier, wherein the molar concentration of PBS is 10mM, pH is 7.4；
Step 2: take the biotinylation carrier that step one prepares, use NaHCO3After regulating pH to 8～9, add small molecule fluorescent dyestuff, reaction is stirred at room temperature 1～2 hour, after reaction terminates, remove unreacted small molecule fluorescent dyestuff with Purification Resin, add fluorescent microsphere, reaction is stirred at room temperature 1～2 hour, it is centrifuged 3～5 minutes under 1100g, collects centrifugal rear solution, it is thus achieved that the plain fluorescent microsphere with small molecule fluorescent dyestuff of modified biological；
Step 3: modified biological element step 2 prepared and the fluorescent microsphere of small molecule fluorescent dyestuff mix with streptavidin, room temperature reaction 1～2 hour, add the biotinylation test substance molecular detection that step one prepares, continue reaction 1～2 hour, obtain described dual amplification fluorescence immunoassay label probe.
2. the preparation method of dual amplification fluorescence immunoassay label probe according to claim 1, it is characterized in that, test substance described in step one is antigen or antibody class protein or polypeptide, and described carrier is bovine serum albumin, casein or dissaving polymer class material.
3. the preparation method of dual amplification fluorescence immunoassay label probe according to claim 1, it is characterised in that described in step one activation biotin, test substance molecular detection, carrier mol ratio be 4:1:3.
4. the preparation method of dual amplification fluorescence immunoassay label probe according to claim 1, it is characterised in that biotinylation carrier described in step 2, fluorescent microsphere mol ratio be 1:1.
5. the preparation method of dual amplification fluorescence immunoassay label probe according to claim 1, it is characterised in that NaHCO described in step 23The mass concentration of solution is 10%, and described small molecule fluorescent dyestuff is identical with fluorescent microsphere fluorescence spectrum.
6. the preparation method of the dual amplification fluorescence immunoassay label probe according to any one of claim 1 to 5, it is characterized in that, modified biological described in step 3 element and the fluorescent microsphere of small molecule fluorescent dyestuff, streptavidin, biotinylation test substance molecular detection mol ratio be 1:5:5.
7. the method for the fluorescence immune chromatography reagent strip of dual amplification fluorescence immunoassay label probe preparation detection cardiac muscle troponin I (cTnI) prepared by claim 1, it is characterised in that include being made by step:
Step one: the preparation of sample pad: use buffer solution albumen, add surfactant, wherein, every 100mL buffer adds 0.01～0.05g surfactant, regulating pH is 6～8, using the ratio of 2～5ml aforesaid liquid with the long sample pad of every 30cm, be uniformly coated in sample pad by above-mentioned solution, 20～25 DEG C dry 8～12 hours；
Step 2, the preparation of pad: the mode processing sample pad with step one is identical, first pad is processed, after the dual amplification fluorescence immunoassay label probe of preparation is diluted 3000 times with PBS, be sprayed on uniformly on pad, dry 4～8 hours in 20～25 DEG C, wherein, the molar concentration of described PBS is 10mM, pH is 7.4, containing 2%BSA and 2% sucrose；
Step 3, the process of nitrocellulose filter:
A. the preparation of line is detected: the PB buffer catching monoclonal antibody or polyclonal antibody 20mmol/L, pH7.2 corresponding for cardiac muscle troponin I is diluted to the concentration of 2.0mg/mL, 0.8ul/cm rules on nitrocellulose filter and to obtain detection line, 20～25 DEG C of forced air drying 8～12h and get final product in drying baker；
B. the preparation of nature controlling line: rabbit anti-mouse igg antibody presses the concentration of 4mg/mL, 0.8ul/cm draws nature controlling line on nitrocellulose filter, and this line is spaced and parallel with detection line, 20～25 DEG C of forced air drying 8～12h and get final product in drying baker；
Step 4, the preparation of adsorptive pads: absorbent paper is cut into 30*2.7cm every,；
Step 5, assemble: described sample pad, pad, nitrocellulose filter and adsorptive pads are sequentially attached on base plate, wherein, overlapping 1～2mm is needed between sample pad, pad, nitrocellulose filter and adsorptive pads each several part, the reagent strip of one fixed width it is cut into afterwards with cutting machine, 20～25 DEG C of forced air drying 8～12h, obtain described fluorescence immune chromatography reagent strip.
8. the method for the fluorescence immune chromatography reagent strip of dual amplification fluorescence immunoassay label probe according to claim 7 preparation detection cardiac muscle troponin I (cTnI), it is characterized in that, buffer described in step one is 10mM, pH is the PBS of 7.2～7.4, Tris or glycine buffer, with the BSA of described buffer solution 5mg/mL or casein.
9. the method for the fluorescence immune chromatography reagent strip of dual amplification fluorescence immunoassay label probe according to claim 7 preparation detection cardiac muscle troponin I (cTnI), it is characterized in that, surfactant described in step one is polysorbas20 or Triton X-100.
10. the application in fluorescence immunoassay detects of the dual amplification fluorescence immunoassay label probe described in claim 1.
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| CN111323576A (en) * | 2020-02-27 | 2020-06-23 | 四川新健康成生物股份有限公司 | Method for enhancing signal of antibody-fluorescent microsphere conjugate and application of method in troponin I detection |
| CN113671171A (en) * | 2021-07-06 | 2021-11-19 | 安徽惠邦生物工程有限公司 | Signal amplification quantum dot fluorescence immunoassay probe and preparation method and application thereof |
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