CN104020162A - Chemiluminiscence sensitization liquid and preparation method thereof - Google Patents
Chemiluminiscence sensitization liquid and preparation method thereof Download PDFInfo
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- CN104020162A CN104020162A CN201410302196.XA CN201410302196A CN104020162A CN 104020162 A CN104020162 A CN 104020162A CN 201410302196 A CN201410302196 A CN 201410302196A CN 104020162 A CN104020162 A CN 104020162A
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- luminol
- bromophenol
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- mother liquor
- chemiluminescence
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Abstract
The invention relates to the technical field of chemiluminescence immune assay, and in particular relates to a chemiluminiscence sensitization liquid and a preparation method thereof. The chemiluminiscence sensitization liquid comprises 0.1-100 mmol/L para-bromophenol, 0.05-100 mmol/L hydrogen peroxide, 0.05-100 mmol/L luminol or luminol derivative, 0.01-10 mol/L alkali, an organic solvent with the volume percentage of 0.01%-20%, and a buffer solution with the ion strength of 0.01-10 mol/L and the pH value of 7-11. Experiments prove that the chemiluminiscence sensitization liquid is capable of remarkably improving the luminous intensity of a reaction system and greatly prolonging the light duration time of the reaction system; the reaction system has the maximum light intensity (or chemiluminiscence value) reaching up to 2.5*10<7> and the light duration time reaching up to 25 minutes.
Description
Technical field
The present invention relates to chemiluminescence immunoassay technology field, particularly, particularly a kind of chemiluminescence enhanced sensitivity liquid and preparation method thereof.
Background technology
Chemiluminescence is at normal temperatures, the transmitting of the light being produced by chemical reaction.Its luminescence mechanism is: the Cucumber molecule in reaction system, as reactant, intermediate or fluorescent material absorbed energy that reaction discharges and by ground state transition to excited state, the photon of energy level such as in the time that intermediate is got back to ground state by excited state, can discharge, photon is measured and realized quantitative test.1977, the people such as Halman, by the antigen-antibody reaction of high specific and highly sensitive chemiluminescence reaction are combined, created chemiluminescence immune analysis method (Chemiluminescence Immunoassay, CLIA).
Chemiluminescence immune analysis method is the product that chemiluminescence is combined with immune response, because chemiluminescence has the specificity of fluorescence, does not need exciting light, thereby has avoided the impact of exciting light parasitic light in fluorescence analysis simultaneously.Luminol is widely used mark substance in chemiluminescence immune assay, and in alkaline solution, luminol can be by many oxygenant oxyluminescences, wherein H
2o
2the most conventional.In the process of chemiluminescence immune assay, slower because of luminescence-producing reaction speed, therefore, often need to add some enzyme or organic catalyst.Enzyme is mainly horseradish peroxidase (HRP), and mineral-type comprises O
3, halogen and Fe
3+, Cu
2+, Co
2+or their complex.
Chemiluminescence enzyme immunoassay (Chemiluminescent enzyme immunoassay, CLELA) be to carry out immune response with enzyme labeling bioactivator, in the process of reaction, enzyme acts on luminous substrate (as luminol), luminous under signal reagent effect, recycling luminous signal analyzer carries out luminescence assays.Conventional marker enzyme is horseradish peroxidase (HRP) and alkaline phosphatase (ALP) at present, and both have luminous substrate separately.
In correlation technique, the most frequently used luminous substrate of horseradish peroxidase (HRP) is luminol.In chemiluminescence enzyme immunoassay (CLEIA), use Horseradish Peroxidase Conjugates, carry out, after immune response, utilizing luminol as luminous substrate, at horseradish peroxidase (HRP) and starting luminescence reagent (NaOH and H
2o
2) under effect, luminous substrate (luminol) is luminous.In the process of reaction, this chemiluminescence reaction system (HRP-H
2o
2-luminol) be generally transient flash in several seconds, there is the defect that luminous intensity is low, the duration is short.
Summary of the invention
The object of the present invention is to provide a kind of chemiluminescence enhanced sensitivity liquid, to overcome chemiluminescence reaction system (HRP-H of the prior art
2o
2-luminol) due to the technical matters that luminous intensity is low, the duration is short existing.Another object of the present invention is to provide a kind of preparation method of above-mentioned chemiluminescence enhanced sensitivity liquid.
This chemiluminescence enhanced sensitivity liquid that the embodiment of the present invention provides, comprising: the alkali of the p bromophenol of 0.1-100mmol/L, the hydrogen peroxide of 0.05-100mmol/L, 0.05-100mmol/L luminol or Derivative of Luminol, 0.01-10mol/L, percent by volume are that 0.01%-20% organic solvent, ionic strength are the damping fluid that 0.01-10mol/L, pH value are 7-11.
This chemical sensitization liquid provided by the invention, it is by p bromophenol, hydrogen peroxide, and luminol or Derivative of Luminol, alkali, organic solvent and damping fluid form.In chemiluminescence enzyme immunoassay (CLEIA), after this enhanced sensitivity liquid is joined in the chemical luminous system that contains horseradish peroxidase (HRP), this enhanced sensitivity liquid is taking p bromophenol as reinforcing agent, its preferential and horseradish peroxidase effect formation free radical, and improve the productive rate of luminol or Derivative of Luminol free radical as electronics transfer intermediate, and then realize the effect of sensitized chemiluminescence reaction, found through experiments, this enhanced sensitivity liquid can significantly intensified response system luminous intensity, extend greatly its lighting time interval, its maximum emission intensity (values of chemiluminescence) can reach 2.5 × 10
7, lighting time interval can reach 25 minutes.Therefore, can be widely used in chemistry taking chemiluminescence as detection means, biology, immunity etc. and detect in analytic product, in the process detecting, because luminous enhancing effect is remarkable, its lighting time interval is long, thereby is convenient to duplicate measurements, and then can improve and detect sensitivity and the accuracy analyzed.
Optionally, described chemiluminescence enhanced sensitivity liquid comprises: the alkali of the p bromophenol of 0.1-50mmol/L, the hydrogen peroxide of 0.05-50mmol/L, 0.05-50mmol/L luminol or Derivative of Luminol, 0.01-5mol/L, percent by volume are the damping fluid that 0.01%-10% organic solvent, ionic strength are 0.01-5mol/L.
Optionally, described Derivative of Luminol is luminol sodium salt, different luminol or 7-amino-6-sulfydryl phthalylhydrazine.
Optionally, described alkali is NaOH, potassium hydroxide or calcium hydroxide.
Optionally, described organic solvent is dimethyl formamide, acetone, methyl alcohol or acetonitrile; And/or; Described damping fluid is phosphate buffer or Tris-HCl damping fluid.
The preparation method of this chemical sensitization liquid provided by the invention, comprises the following steps:
(1), in aqueous slkali, add luminol or Derivative of Luminol, obtain the luminol of set concentration or the mother liquor of Derivative of Luminol;
(2), p bromophenol is joined in organic solvent, obtain the p bromophenol mother liquor of set concentration;
(3), according to set volume ratio, the mother liquor of described luminol or Derivative of Luminol is mixed with described p bromophenol mother liquor after, add successively therein damping fluid and superoxol, mix, obtain chemiluminescence enhanced sensitivity liquid.
Optionally, in step (1), specifically comprise: take 111mg luminol, join in 0.1mol/LNaOH aqueous solution, and be settled to 100mL, the luminol mother liquor that acquisition concentration is 6.25mmol/L.
Optionally, in step (2), specifically comprise: take 86.5mg p bromophenol, join in 2%DMF aqueous solution, and be settled to 100mL, the p bromophenol mother liquor that acquisition concentration is 5mmol/L.
Optionally, in described step (3), the volume ratio of described luminol mother liquor and described p bromophenol mother liquor is 1:1.
Brief description of the drawings
In order to be illustrated more clearly in the specific embodiment of the invention or technical scheme of the prior art, to the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described below, apparently, accompanying drawing in the following describes is some embodiments of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
The preparation method of the chemiluminescence enhanced sensitivity liquid that Fig. 1 provides for the present embodiment five;
After the chemiluminescence enhanced sensitivity liquid that Fig. 2 provides for the embodiment of the present invention joins in the immune response system that contains horseradish peroxidase, the luminous value of system and fluorescent lifetime graph of a relation.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer; to technical scheme of the present invention be carried out to clear, complete description below; based on the embodiment in the present invention; all other embodiments that those of ordinary skill in the art obtain under the prerequisite of not making creative work, all belong to the scope that the present invention protects.
This chemiluminescence enhanced sensitivity liquid provided by the invention is that 0.01%-20% organic solvent, ionic strength are the damping fluid that 0.01-10mol/L, pH value are 7-11 comprising p bromophenol, hydrogen peroxide, 0.05-100mmol/L luminol or the Derivative of Luminol of 0.05-100mmol/L, the alkali of 0.01-10mol/L, the percent by volume of 0.1-100mmol/L.In addition, in order to realize better enhancing effect, preferably, in enhanced sensitivity liquid, the content range of each component is lock pin suitably, as: its p bromophenol, hydrogen peroxide, 0.05-50mmol/L luminol or the Derivative of Luminol of 0.05-50mmol/L, the alkali of 0.01-5mol/L, percent by volume that can comprise 0.1-50mmol/L is the damping fluid that 0.01%-10% organic solvent, ionic strength are 0.01-5mol/L; In addition, preferred, for each component, described Derivative of Luminol is luminol sodium salt, different luminol or 7-amino-6-sulfydryl phthalylhydrazine; Described alkali is NaOH, potassium hydroxide or calcium hydroxide; Described organic solvent is dimethyl formamide, acetone, methyl alcohol or acetonitrile; And/or described damping fluid is phosphate buffer or Tris-HCl damping fluid.Specifically, in the process of preparation, enhanced sensitivity liquid of the present invention can possess above-mentioned condition simultaneously, or only meets one or more in above-mentioned qualifications.
Next,, by the content in conjunction with above-mentioned, the present invention provides following specific embodiment to the chemiluminescence enhanced sensitivity liquid based on using p bromophenol as reinforcing agent, please refer to following content:
Embodiment mono-
This chemiluminescence enhanced sensitivity liquid that the present embodiment provides, it comprises: the NaOH of the p bromophenol of 0.1mmol/L, the hydrogen peroxide of 0.05mmol/L, 0.05mmol/L luminol, 0.01mol/L, percent by volume are that 0.01% dimethyl formamide, ionic strength are the phosphate buffer that 0.01mol/L, pH value are 7-11.
Embodiment bis-
This chemiluminescence enhanced sensitivity liquid that the present embodiment provides, it comprises: 7-amino-6-sulfydryl phthalylhydrazine of the p bromophenol of 10mmol/L, the hydrogen peroxide of 10mmol/L, 8mmol/L, the potassium hydroxide of 5mol/L, percent by volume are that 10% dimethyl formamide, ionic strength are the Tris-HCl damping fluid that 5mol/L, pH value are 7-11.
Embodiment tri-
This chemiluminescence enhanced sensitivity liquid that the present embodiment provides, it comprises: the calcium hydroxide of the different luminol of the p bromophenol of 50mmol/L, the hydrogen peroxide of 50mmol/L, 50mmol/L or luminol sodium salt, 5mol/L, percent by volume are that 10% acetone, methyl alcohol or acetonitrile, ionic strength are the Tris-HCl damping fluid that 5mol/L, pH value are 7-11.
Embodiment tetra-
This chemiluminescence enhanced sensitivity liquid that the present embodiment provides, it comprises: the luminol of the p bromophenol of 100mmol/L, the hydrogen peroxide of 100mmol/L, 100mmol/L, the NaOH of 10mol/L, percent by volume are that 20% acetone, methyl alcohol or acetonitrile, ionic strength are the Tris-HCl damping fluid that 10mol/L, pH value are 7-11.
For the chemiluminescence enhanced sensitivity liquid that makes the above embodiment of the present invention is better applied, more effectively be applied in the field of chemiluminescence immune assay, the present invention also provides embodiment five on the basis of above-described embodiment, embodiment five has provided the preparation method of the chemiluminescence enhanced sensitivity liquid of above-described embodiment, is now described in detail and explains.
Embodiment five
The preparation method of the chemiluminescence enhanced sensitivity liquid that the embodiment of the present invention provides specifically comprises the following steps, and please refer to Fig. 1:
Step 101: add luminol or Derivative of Luminol in aqueous slkali, obtain the luminol of set concentration or the mother liquor of Derivative of Luminol;
Concrete, in this step, take 111mg luminol, join in 0.1mol/LNaOH aqueous solution, and be settled to 100mL, the luminol mother liquor that acquisition concentration is 6.25mmol/L, for subsequent use.
Step 102: p bromophenol is joined in organic solvent, obtain the p bromophenol mother liquor of set concentration;
Concrete, take 86.5mg p bromophenol, join in 20%DMF aqueous solution, and be settled to 100mL, the p bromophenol mother liquor that acquisition concentration is 5mmol/L, for subsequent use.
Step 103: after the mother liquor of described luminol or Derivative of Luminol being mixed with described p bromophenol mother liquor according to set volume ratio, add successively therein damping fluid and superoxol, mix, obtain chemiluminescence enhanced sensitivity liquid.
In this step, the volume ratio of the mother liquor of described luminol or Derivative of Luminol and described p bromophenol mother liquor is 1:1.In the process of operation, draw respectively 2mL luminol mother liquor and 2mL p bromophenol mother liquor, with 16mL0.1M, the Tris-HCl damping fluid that pH value is 8.5 dilutes, and adds the superoxol of 10 μ L30%, mixes and get final product.
Wherein, the collocation method of damping fluid comprises: by 1M hydrochloric acid solution adjustment pH value to 9.0, be settled to 1000mL with deionized water afterwards, acquisition concentration is the Tris-HCl damping fluid that 0.05mol/L, pH value are 7.2, in concrete use, is adjusted to set pH and ion concentration according to specific demand.
In addition, concrete, in the present embodiment, whole layoutprocedure can be carried out according to following operation: take 111mg luminol, be settled to 100mL by 0.1mol/LNaOH aqueous solution, the luminol mother liquor that to obtain concentration be 6.25mmol/L; Take 86.5mg p bromophenol, be settled to 100mL by 20%DMF aqueous solution, the p bromophenol mother liquor that to obtain concentration be 5mmol/L; Take trishydroxymethylaminomethane 6.05g, first use after 900mL deionized water dissolving, adjust pH value to 9.0 with 1M hydrochloric acid solution, be settled to 1000mL with deionized water afterwards, acquisition concentration is the Tris-HCl damping fluid that 0.05mol/L, pH value are 7.2; Draw respectively 2mL luminol mother liquor and 2mL p bromophenol mother liquor, with 16mL0.05M, the Tris-HCl damping fluid that pH value is 9.0 dilutes, and adds the superoxol of 10 μ L30%, obtains chemiluminescence enhanced sensitivity liquid after mixing.
Please refer to Fig. 2, the chemiluminescence enhanced sensitivity liquid of making by said method, it is added in the immune response system that contains horseradish peroxidase, can occur chemiluminescence reaction, detects luminous value and fluorescent lifetime by chemiluminescence detector.As shown in Figure 2, as can be seen from Figure 2, the chemiluminescence enhanced sensitivity liquid based on reinforcing agent p bromophenol has effectively strengthened the luminous intensity of luminol-horseradish peroxidase-hydrogen peroxide chemical luminous system, and (maximum emission intensity can reach 2.5 × 10 to testing result
7), extend its lighting time interval (can reach 25 minutes); Its there is obvious luminous enhancing effect and improve fluorescent lifetime with and the effect of stability of photoluminescence.In addition, the present invention has also investigated under the condition of the horseradish peroxidase (HRP) at variable concentrations, measures its luminous power in 20 minutes.Concrete outcome please refer to table 1:
Table 1 horseradish peroxidase concentration and p bromophenol enhanced sensitivity liquid are on luminous impact
Wherein, in table 1, RLU (max): maximum luminous value; Time (RLU max): the time that reaches maximum luminous value; Constant Time (s): lasting time when luminous value rate of change is less than 1%; " instability ": the time lasting when luminous value rate of change is less than 1% is less than 10s.
Lasting time when Constant Time (CT) is defined as values of chemiluminescence rate of change and is less than 1%, the larger explanation chemiluminescence intensity of CT value is longer stabilization time, more favourable to detection method stability, for example CLEIA.Experimental result shows (in table 1), along with the increase of HRP concentration, the maximum chemical luminous value RLU (max) of reinforcing agent (p bromophenol) increases gradually, and arriving the peaked time is that Time (RLU max) also shortens gradually; Particularly in this system, horseradish peroxidase concentration, in the time of 15-25ng/mL, has better enhancing effect and lighting time interval.
Therefore, it can be applied to this enhanced sensitivity liquid that the embodiment of the present invention provides in the chemiluminescence enzyme immunoassay taking horseradish peroxidase as marker enzyme widely, the illumination effect of enhancing system, prolongation light duration, and then improve and detect sensitivity and the accuracy analyzed.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. chemiluminescence enhanced sensitivity liquid, is characterized in that, described chemiluminescence enhanced sensitivity liquid comprises:
The alkali of the p bromophenol of 0.1-100mmol/L, the hydrogen peroxide of 0.05-100mmol/L, 0.05-100mmol/L luminol or Derivative of Luminol, 0.01-10mol/L, percent by volume are that 0.01%-20% organic solvent, ionic strength are the damping fluid that 0.01-10mol/L, pH value are 7-11.
2. chemiluminescence enhanced sensitivity liquid according to claim 1, is characterized in that, described chemiluminescence enhanced sensitivity liquid comprises:
The alkali of the p bromophenol of 0.1-50mmol/L, the hydrogen peroxide of 0.05-50mmol/L, 0.05-50mmol/L luminol or Derivative of Luminol, 0.01-5mol/L, percent by volume are the damping fluid that 0.01%-10% organic solvent, ionic strength are 0.01-5mol/L.
3. chemiluminescence enhanced sensitivity liquid according to claim 1, is characterized in that: described Derivative of Luminol is luminol sodium salt, different luminol or 7-amino-6-sulfydryl phthalylhydrazine.
4. chemiluminescence enhanced sensitivity liquid according to claim 3, is characterized in that, described alkali is NaOH, potassium hydroxide or calcium hydroxide.
5. chemiluminescence enhanced sensitivity liquid according to claim 1, is characterized in that, described organic solvent is dimethyl formamide, acetone, methyl alcohol or acetonitrile;
And/or;
Described damping fluid is phosphate buffer or Tris-HCl damping fluid.
6. a preparation method for chemiluminescence enhanced sensitivity liquid according to claim 1, is characterized in that, comprises the following steps:
(1), in aqueous slkali, add luminol or Derivative of Luminol, obtain the luminol of set concentration or the mother liquor of Derivative of Luminol;
(2), p bromophenol is joined in organic solvent, obtain the p bromophenol mother liquor of set concentration;
(3), according to set volume ratio, the mother liquor of described luminol or Derivative of Luminol is mixed with described p bromophenol mother liquor after, add successively therein damping fluid and superoxol, mix, obtain chemiluminescence enhanced sensitivity liquid.
7. preparation method according to claim 6, is characterized in that, in step (1), specifically comprises:
Take 111mg luminol, join in 0.1mol/LNaOH aqueous solution, and be settled to 100mL, the luminol mother liquor that acquisition concentration is 6.25mmol/L.
8. preparation method according to claim 7, is characterized in that, in step (2), specifically comprises:
Take 86.5mg p bromophenol, join in 20%DMF aqueous solution, and be settled to 100mL, the p bromophenol mother liquor that acquisition concentration is 5mmol/L.
9. preparation method according to claim 8, is characterized in that, in described step (3), the volume ratio of described luminol mother liquor and described p bromophenol mother liquor is 1:1.
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| CN115128062A (en) * | 2022-08-29 | 2022-09-30 | 中储粮成都储藏研究院有限公司 | Method for detecting freshness of grains and application |
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