CN112326636A - High-sensitivity ECL kit and preparation method thereof - Google Patents
High-sensitivity ECL kit and preparation method thereof Download PDFInfo
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- CN112326636A CN112326636A CN202011159016.9A CN202011159016A CN112326636A CN 112326636 A CN112326636 A CN 112326636A CN 202011159016 A CN202011159016 A CN 202011159016A CN 112326636 A CN112326636 A CN 112326636A
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- 238000002360 preparation method Methods 0.000 title claims description 13
- 239000007800 oxidant agent Substances 0.000 claims abstract description 24
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical group O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 22
- GZFGOTFRPZRKDS-UHFFFAOYSA-N 4-bromophenol Chemical group OC1=CC=C(Br)C=C1 GZFGOTFRPZRKDS-UHFFFAOYSA-N 0.000 claims abstract description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000000891 luminescent agent Substances 0.000 claims abstract description 18
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical group OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 230000001590 oxidative effect Effects 0.000 claims abstract description 16
- 239000012744 reinforcing agent Substances 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 230000035945 sensitivity Effects 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000007853 buffer solution Substances 0.000 claims description 9
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid group Chemical group C(CC(O)(C(=O)O)CC(=O)O)(=O)O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 9
- 239000003381 stabilizer Substances 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000003623 enhancer Substances 0.000 claims description 6
- QYYMDNHUJFIDDQ-UHFFFAOYSA-N 5-chloro-2-methyl-1,2-thiazol-3-one;2-methyl-1,2-thiazol-3-one Chemical compound CN1SC=CC1=O.CN1SC(Cl)=CC1=O QYYMDNHUJFIDDQ-UHFFFAOYSA-N 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 239000007979 citrate buffer Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 239000000645 desinfectant Substances 0.000 claims description 2
- 238000011161 development Methods 0.000 abstract description 14
- 229960002163 hydrogen peroxide Drugs 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract description 5
- 238000004020 luminiscence type Methods 0.000 abstract description 5
- 230000002708 enhancing effect Effects 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 230000027756 respiratory electron transport chain Effects 0.000 abstract description 3
- 239000000203 mixture Substances 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 102000003992 Peroxidases Human genes 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 241000283707 Capra Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- RKTYLMNFRDHKIL-UHFFFAOYSA-N copper;5,10,15,20-tetraphenylporphyrin-22,24-diide Chemical compound [Cu+2].C1=CC(C(=C2C=CC([N-]2)=C(C=2C=CC=CC=2)C=2C=CC(N=2)=C(C=2C=CC=CC=2)C2=CC=C3[N-]2)C=2C=CC=CC=2)=NC1=C3C1=CC=CC=C1 RKTYLMNFRDHKIL-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
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Abstract
The invention provides a high-sensitivity ECL kit, which comprises a luminescent agent and an oxidant, wherein the luminescent agent is a luminol solution, the oxidant is a hydrogen peroxide solution, the ECL kit also comprises a reinforcing agent, the reinforcing agent is a p-bromophenol solution, and the luminescent agent, the oxidant and the reinforcing agent are mixed according to the volume ratio of 100: 100: 1, mixing and using the mixture, wherein the enhancing agent is 100mM para-bromophenol dissolved in DMSO; according to the invention, the p-bromophenol is used as a reinforcing agent, and the efficiency of electron transfer in the reaction process is greatly improved in the reaction process of carrying out chemiluminescence on the p-bromophenol in the luminol oxydol oxide, so that the luminous efficiency in unit time is improved, the fluorescent light with enough intensity can be emitted by using less enzyme to realize development and color development, the purpose of prolonging the platform period of luminescence and maintaining stronger luminous intensity for a longer time is realized.
Description
Technical Field
The invention relates to the technical field of chemiluminescence immunoassay, in particular to a high-sensitivity ECL kit and a preparation method thereof.
Background
Chemiluminescence analysis is a novel labeling analysis technology for detecting trace substances, is applied to the field of immunology, and is mainly used for determining the content of an antigen or an antibody according to light radiation generated by chemical reaction. Due to the advantages of high sensitivity, wide linear range, simple instrument, convenient operation, no radiation hazard, easy automation and the like, the method becomes an important direction of labeled immunity in recent years. Among them, chemiluminescence immunoassay using horseradish peroxidase (HRP) and Alkaline Phosphatase (AP) as labels is the main development trend.
Luminol, which was a luminescent substance used in the early days of chemiluminescence, has the chemical name 3-aminophthalic hydrazine, which is oxidized to give an intermediate of alpha-hydroxyperoxide, which subsequently decomposes to release light energy. The luminol-hydrogen peroxide-horseradish peroxidase luminescent system is a common chemiluminescence analysis system, but the luminol-hydrogen peroxide-horseradish peroxidase luminescent system has low sensitivity and is easy to attenuate when being directly used for measuring biomacromolecules, and the luminol-hydrogen peroxide-horseradish peroxidase luminescent system also has the common defects of luminescent substrates on the market at present. In addition, the problems of poor stability and high background of the reagent after the luminescent agent and the oxidant are mixed exist.
Disclosure of Invention
In view of the above, the invention provides a high-sensitivity ECL kit with high detection sensitivity, good color development stability and low cost, and a preparation method thereof.
The technical scheme of the invention is realized as follows: the invention provides a high-sensitivity ECL kit which comprises a luminescent agent and an oxidant, wherein the luminescent agent is a luminol solution, the oxidant is a hydrogen peroxide solution, and the ECL kit also comprises a reinforcing agent which is a p-bromophenol solution.
On the basis of the technical scheme, preferably, the luminescent agent, the oxidant and the reinforcing agent are mixed according to the volume ratio of 100: 100: 1 and mixing and using.
Based on the technical scheme, the enhancer is 100mM of p-bromophenol dissolved in DMSO.
Based on the technical scheme, the luminous agent is preferably a buffer solution with pH of 8.5-9.5 and containing 0.5-10mmol/L of luminol or luminol derivative and 1mM EDTA.
Based on the above technical solution, preferably, the oxidizing agent is a citric acid buffer solution with pH 4.5-5.5 containing hydrogen peroxide at a final concentration of 0.5-10mmol/L and 1mM EDTA.
In addition to the above technical solution, it is preferable that the oxidizing agent further comprises a stabilizer, the stabilizer is added into the oxidizing agent, and the stabilizer is ProClin 300 of 0.1%.
On the other hand, a method for preparing an ECL kit with high sensitivity is provided, which comprises the following steps,
s1 preparing a luminescent agent: dissolving luminol in 0.1M NaOH solution, adding into 50mM Tris buffer solution until the final concentration of the luminol is 1mM, adding 1mM EDTA, and finally adjusting pH to 9.5 with hydrochloric acid;
s2 preparation of oxidant: addition of H to 50mM citrate buffer2O2Up to H2O2The final concentration is 1mM, then 1mM EDTA is added, and the pH is adjusted to 5.0;
s3 preparation of enhancer: para-bromophenol was dissolved in DMSO to a final concentration of 100mM para-bromophenol.
Compared with the prior art, the high-sensitivity ECL kit and the preparation method thereof have the following beneficial effects:
(1) according to the invention, the p-bromophenol is used as a reinforcing agent, and the efficiency of electron transfer in the reaction process is greatly improved in the reaction process of carrying out chemiluminescence on the p-bromophenol in the luminol oxydol oxide, so that the luminous efficiency in unit time is improved, the fluorescent light with enough intensity can be emitted by using less enzyme to realize development and color development, the purpose of prolonging the platform period of luminescence and maintaining stronger luminous intensity for a longer time is realized.
(2) The raw material reagents used in the invention have low cost, the components used in a luminescent system are relatively simple, the luminescent background is low, the signal-to-noise ratio can be obviously improved when the fluorescent material is applied to HRP enzymatic reaction, and the detection sensitivity is higher.
Drawings
FIG. 1 is a graph showing the development effect of the luminescent liquid of example 1 of the present invention and the luminescent liquid of the comparative example when they are simultaneously exposed for 10 seconds;
FIG. 2 is a graph showing the development effect of the luminescent liquid of example 1 of the present invention and the luminescent liquid of the comparative example after exposure for 20min and then exposure for 10 seconds.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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 obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1:
the invention discloses a high-sensitivity ECL kit, which comprises a luminescent agent, an oxidizing agent, an enhancing agent and a stabilizing agent, wherein the enhancing agent is 100mM para-bromophenol dissolved in DMSO, the luminescent agent is a buffer solution with the pH value of 9.5 and containing 10mmol/L of luminol or luminol derivative and 1mM of EDTA, the oxidizing agent is a citric acid buffer solution with the final concentration of 10mmol/L of hydrogen peroxide and 1mM of EDTA and the pH value of 5.0, and the luminescent agent, the oxidizing agent and the enhancing agent are mixed according to the volume ratio of 100: 100: 1, adding a stabilizer into an oxidizing agent, wherein the stabilizer is ProClin 300 of 0.1%.
The mixed solution of the luminescent system in the preparation example 1 is placed for 1 day, the background value is not increased, and the signal value is stable.
In another aspect, a method for preparing the same comprises the steps of,
s1 preparing a luminescent agent: dissolving luminol in 0.1M NaOH solution, adding into 50mM Tris buffer solution until the final concentration of the luminol is 1mM, adding 1mM EDTA, and finally adjusting pH to 9.5 with hydrochloric acid;
s2 preparation of oxidant: addition of H to 50mM citrate buffer2O2Up to H2O2The final concentration is 1mM, then 1mM EDTA is added, and the pH is adjusted to 5.0;
s3 preparation of enhancer: para-bromophenol was dissolved in DMSO to a final concentration of 100mM para-bromophenol.
Example 2:
compared with example 1, the luminescent agent is replaced by a buffer solution with pH 9.5 and containing 0.5mmol/L of luminol or luminol derivative and 1mM EDTA, and the oxidizing agent is replaced by a buffer solution with pH 5.0 and containing hydrogen peroxide with final concentration of 0.5mmol/L and 1mM EDTA, and other conditions are not changed.
Example 3:
in comparison with example 1, no stabilizer was added to the oxidizing agent, and the other conditions were not changed.
The main component of the p-bromophenol is 4-BOP, which is named as 4-bromophenol in Chinese. The crystal is precipitated from chloroform or ether to form a tetragonal biconical crystal, is an organic synthesis intermediate, is commonly used as a disinfectant, a pesticide and a medical raw material, and has low cost and simple acquisition.
The invention utilizes the principle that p-bromophenol is used as a reinforcing agent, luminol is oxidized by hydrogen peroxide to generate an intermediate of alpha-hydroxy peroxide, and then is decomposed to release light energy, and p-bromophenol can improve the efficiency of electron transfer in the process of oxidizing luminol by hydrogen peroxide, so that more light is emitted in unit time, and the luminous intensity is improved; further, since the luminescence intensity is increased, fluorescence of sufficient intensity can be emitted with a smaller amount of enzyme, and a band is developed on a film, thereby achieving the object of increasing the detection sensitivity.
Test methods and analysis of results:
the chemiluminescence system in example 1 was prepared, a commercially available ECL kit of a certain brand was used as a control example, and applied to WB detection of anti-CD34, and the luminescence intensity of the target strip and the stability of the luminescence intensity after 20min were respectively compared. The antibody is a commercially available antibody.
The test procedure is as follows:
(1) electrophoresis of a sample: sequentially spotting the denatured samples into lanes, performing laminated gel electrophoresis at 60V, and performing gel separation electrophoresis at 120V;
(2) film transfer: assembling a transfer film sandwich structure, putting a transfer clip into a transfer tank filled with a transfer buffer solution in advance, placing a film on the positive electrode and a glue on the negative electrode, putting the whole transfer tank into an ice water bath, and carrying out constant-current transfer for 1.5 hours at 150 mA;
(3) and (3) sealing: immersing the nitrocellulose membrane in 5% skimmed milk powder, and shaking at room temperature for 1 hour;
(4) washing the membrane and hybridizing:
4-1) primary antibody incubation: the primary antibody is anti-CD34 rabbit polyclonal antibody, and is directly added into the sealing solution, shaken at 4 deg.C overnight, and the concentration of the primary antibody is 1 ug/ml;
4-2) washing the membrane: washing with TBST for 10min for 3 times;
4-3) incubation with secondary antibody: the secondary antibody is horseradish peroxidase (HRP) labeled goat anti-rabbit IgG antibody, and the dilution ratio is 1: 7000, shaking for 1 hour at room temperature;
4-4) washing the membrane: washing with TBST for 10min for 3 times;
(5) chemiluminescence and exposure: the film was cut in half from the middle, the pre-prepared luminescent liquid of example 1 and the luminescent liquid of the comparative example were dropped on two films, respectively, the excess luminescent liquid was decanted off, and the development was carried out with X-ray sensitive film by simultaneous exposure for 10 seconds, the results are shown in FIG. 1; after 20min, development was again carried out by simultaneous exposure for 10 seconds, and the results are shown in FIG. 2.
As is obvious from the figure, the color development effect of the embodiment 1 is more clear and obvious, and the color development stability is still kept high after 20 min; the color development effect of the comparative example was less blurred than that of example 1, and the color development effect was seriously deteriorated after 20 min.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (7)
1. The high-sensitivity ECL kit comprises a luminescent agent and an oxidant, wherein the luminescent agent is luminol solution, and the oxidant is hydrogen peroxide solution, and is characterized in that: the disinfectant also comprises an enhancer which is a p-bromophenol solution.
2. A high sensitivity ECL kit as defined in claim 1 wherein: the luminescent agent, the oxidant and the reinforcing agent are mixed according to the volume ratio of 100: 100: 1 and mixing and using.
3. A high sensitivity ECL kit as defined in claim 1 wherein: the enhancer is 100mM para-bromophenol dissolved in DMSO.
4. A high sensitivity ECL kit as defined in claim 1 wherein: the luminescent agent is a buffer solution with pH of 8.5-9.5 and containing 0.5-10mmol/L of luminol or luminol derivative and 1mM EDTA.
5. A high sensitivity ECL kit as defined in claim 1 wherein: the oxidant is a citric acid buffer solution with pH 4.5-5.5 containing hydrogen peroxide and 1mM EDTA at final concentration of 0.5-10 mmol/L.
6. A high sensitivity ECL kit as defined in claim 1 wherein: the stabilizer is added into the oxidant, and the stabilizer is 0.1% of ProClin 300.
7. A preparation method of a high-sensitivity ECL kit is characterized by comprising the following steps: comprises the following steps of (a) carrying out,
s1 preparing a luminescent agent: dissolving luminol in 0.1M NaOH solution, adding into 50mM Tris buffer solution until the final concentration of the luminol is 1mM, adding 1mM EDTA, and finally adjusting pH to 9.5 with hydrochloric acid;
s2 preparation of oxidant: addition of H to 50mM citrate buffer2O2Up to H2O2The final concentration is 1mM, then 1mM EDTA is added, and the pH is adjusted to 5.0;
s3 preparation of enhancer: para-bromophenol was dissolved in DMSO to a final concentration of 100mM para-bromophenol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113567679A (en) * | 2021-06-19 | 2021-10-29 | 百美特(上海)生物科技有限公司 | Hypersensitive ECL luminescent liquid reagent and using method thereof |
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