CN112067601A - Alkaline phosphate enzymatic chemiluminescence substrate reinforcing agent and application thereof - Google Patents
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Abstract
The invention discloses a basic phosphate enzymatic chemiluminescence substrate reinforcing agent and application thereof, wherein the basic phosphate enzymatic chemiluminescence substrate reinforcing agent comprises a fluorescent compound and a surfactant, wherein the fluorescent compound is a water-soluble quantum dot, the surfactant is a Gemini surfactant, and chemiluminescence substrate liquid containing the reinforcing agent comprises the following components: 50-500 mM of 2-amino-2-methyl-1-propanol, 0.1-1 mM of AMPPD, and MgCl21-10 mM, 10-30 mg/L of reinforcing agent and 100-1000 mg/L of preservative. The invention creatively selects a Gemini surfactant to be matched with water-soluble quantum dots to obtain the alkaline phosphatase-catalyzed chemiluminescence substrate reinforcing agent, and the water-soluble quantum dots and the AMPPD substrate are enabled to be in contact with the water-soluble quantum dots by utilizing the internal hydrophobic microenvironment of micelles formed by the Gemini surfactantThe distance of the substance is shortened, and light energy transfer is generated along with the distance, and the quantum yield of the water-soluble quantum dots is far higher than that of chemiluminescent molecules, so that the luminous value of chemiluminescent substrate liquid containing the reinforcing agent is obviously enhanced, and the performances of precision, sensitivity, luminous rate stability and the like of luminous value measurement are further enhanced.
Description
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to an alkaline phosphate enzymatic chemiluminescence substrate enhancer and application thereof.
Background
Chemiluminescence is a light radiation phenomenon accompanying substances in the process of carrying out chemical reactions and can be divided into direct luminescence and indirect luminescence. Direct emission is the simplest chemiluminescent reaction and consists of two key steps: namely excitation and radiation. For example, A, B, two substances react chemically to form substance C, and the energy released by the reaction is absorbed by the molecules of substance C and transits to excited state C, which generates optical radiation during the return to the ground state. Where C is a luminophore, in this process C is called direct chemiluminescence because it is directly involved in the reaction. Indirect luminescence, also known as energy transfer chemiluminescence, is mainly composed of three steps: firstly, reacting reactants A and B to generate excited state intermediate C (energy donor); when C decomposes, energy is released and transferred to F (energy acceptor), so that F is excited and transits to an excited state F; finally, when F transitions back to the ground state, light emission occurs. Due to the transfer of the cleavage energy of the chemical bond, if all of the energy is converted into light energy, the generated signal is strong and stable.
1, 2-dioxetane compound (AMPPD) is used as an ultra-sensitive alkaline phosphatase substrate, phosphate radical is hydrolyzed under the catalytic action of Alkaline Phosphatase (AP) to form an unstable intermediate, the intermediate is decomposed automatically, and a large amount of energy is released by breaking of-O-O-four-membered ring, so that chemiluminescence reaction is excited, and photon is emitted, although the signal can last for more than 20 hours, the luminous efficiency is low, and commercially available chemiluminescence substrate liquid is substrate liquid prepared by compounding an enhancer and AMPPD, so that the luminous intensity is improved. The prior art finds that the enhancers for luminescent substrates mainly comprise globular proteins such as bovine serum albumin and cationic surfactant enhancers such as polyethylene polymers with quaternary phosphonium side groups, but the enhancers are expensive and have large required amount, and excessive addition of surfactant is easy to generate excessive foam, thereby affecting the accuracy of luminescent value detection; meanwhile, the enhancement factor of the reinforcing agent is low, so that the problems of low precision of light-emitting value measurement, uneven light-emitting rate and the like are caused.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the alkaline phosphoric acid enzymatic chemiluminescence substrate reinforcing agent, a Gemini surfactant is specially selected to be matched with water-soluble quantum dots for use, wherein a small amount of the Gemini surfactant can quickly form more micelles without generating foam, the water-soluble quantum dots are close to the AMPPD substrate, and light energy transfer is generated to remarkably reinforce a luminescence signal, so that the alkaline phosphoric acid enzymatic chemiluminescence substrate liquid with remarkably enhanced precision, sensitivity and stability and stable luminescence rate is obtained.
In order to achieve the purpose, the invention adopts the technical scheme that:
a basic phosphate enzymatic chemiluminescent substrate enhancer comprises a fluorescent compound and a surfactant, wherein the fluorescent compound is a water-soluble quantum dot, and the surfactant is a Gemini surfactant.
The technical principle of the invention is as follows: the Gemini surfactant solution has the advantages of more compact arrangement on the surface, lower surface energy and higher surface activity, thereby increasing the solubility of AMPPD in an aqueous solution, promoting the chemiluminescence reaction rate, and having lower critical micelle concentration (CMC value), so a large amount of micelles can be quickly generated in the aqueous solution by using a small amount of Gemini surfactant, and the generation of foams can be avoided due to the low use amount. The Gemini surfactant and the water-soluble quantum dots are matched for use, the hydrophobic microenvironment in the micelle is utilized to enable the distance between the water-soluble quantum dots and the AMPPD substrate to be shortened, light energy transfer is generated, the quantum yield of the water-soluble quantum dots is far higher than that of chemiluminescent molecules, and the reaction efficiency and the quantum yield of chemiluminescence can be improved under the hydrophobic microenvironment, so that luminescence signals are obviously enhanced, and further the precision, the sensitivity and the stability of luminescence rate of luminescence value measurement are obviously enhanced.
Preferably, the mass ratio of the water-soluble quantum dots to the Gemini surfactant is 1: 15-25.
Preferably, the concentration ratio of the water-soluble quantum dots to the Gemini surfactant is 1: 20.
Preferably, the structural formula of the Gemini surfactant is as follows:
wherein the R group is selected from C8-20 alkyl.
Preferably, the water-soluble quantum dots are selected from one or more of water-soluble quantum dots CdTe, water-soluble quantum dots CdSe, water-soluble quantum dots ZnS and water-soluble quantum dots ZnCdS.
The invention also provides alkaline phosphate enzymatic chemiluminescent substrate liquid, which comprises the enhancer.
Preferably, the chemiluminescent substrate solution comprises:
the pH was 9.5.
Preferably, the chemiluminescent substrate solution comprises:
the pH was 9.5.
Preferably, the preservative in the chemiluminescent substrate solution is at least one of sodium azide, Proclin series, potassium sorbate and sodium benzoate.
Compared with the prior art, the invention has the beneficial effects that: the invention creatively selects the Gemini surfactant, which not only can enhance the water solubility of AMPPD to enhance the chemiluminescence reaction rate, but also can reduce the usage amount of the surfactant so as to reduce the influence of foam on the luminous value. The Gemini surfactant and the water-soluble quantum dots are matched to obtain the alkaline phosphate enzymatic chemiluminescence substrate reinforcing agent, the hydrophobic microenvironment in the micelle can enable the distance between the water-soluble quantum dots and the AMPPD substrate to be shortened, light energy transfer is generated, and the quantum yield of the water-soluble quantum dots is far higher than that of chemiluminescence molecules, so that the luminous value of the alkaline phosphate enzymatic chemiluminescence substrate containing the reinforcing agent is remarkably enhanced, and the performances of precision, sensitivity, luminous rate stability and the like of luminous value measurement in chemiluminescence detection are remarkably enhanced.
Drawings
FIG. 1 is a graph showing changes in luminescence rates of example 1 and comparative examples 1 to 5 measured in application example 3 of the present invention;
FIG. 2 is a graph showing the luminescence kinetics of example 1 and comparative example 1 catalyzed by alkaline phosphatase at a high concentration (0.1. mu.g/mL) in application example 4 of the present invention;
FIG. 3 is a graph showing the luminescence kinetics of example 1 and comparative example 1 catalyzed by low concentration of alkaline phosphatase (0.025. mu.g/mL) in application example 4 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The embodiment provides a basic phosphate enzymatic chemiluminescent substrate enhancer, which specifically comprises: the water-soluble quantum dot and the Gemini surfactant are in a mass ratio of 1:20, wherein the Gemini surfactant is Gemini C12, and the structural formula is as follows:
wherein the R group is C12H25An alkyl group; the water-soluble quantum dots are water-soluble quantum dots CdTe.
The Gemini surfactant is a surfactant formed by connecting ionic head groups of two traditional surfactants through chemical bonds and tightly connecting the two ionic heads; the strong interaction effectively prevents the repulsion of the charges of the ion head groups, so that the polar groups are arranged more closely, the hydrophobic bonding force between hydrocarbon chains is enhanced by the structure, and the repulsion between the ion head groups is greatly weakened due to the inhibition of chemical bonding force. Therefore, compared with the common surfactant, the Gemini surfactant solution has the advantages of more compact arrangement on the surface, lower surface energy and higher surface activity, thereby increasing the solubility of AMPPD in an aqueous solution and promoting the rate of chemiluminescence reaction. And the surfactant has a lower CMC value, so a small amount of Gemini surfactant can quickly generate a large amount of micelles in an aqueous solution, and the use amount is low, so that foam is avoided. The Gemini surfactant and the water-soluble quantum dots are matched for use, the hydrophobic microenvironment in the micelle is utilized to enable the distance between the water-soluble quantum dots and the AMPPD substrate to be shortened, and light energy transfer is generated.
The embodiment also provides alkaline phosphate enzymatic chemiluminescent substrate solution containing the enhancer, which specifically comprises:
the pH value is 9.0-9.5
Weighing the components according to the formula, dissolving the components in ultrapure water, adjusting the pH value to 9.0-9.5 by using hydrochloric acid, and performing constant volume to 1L to obtain the chemiluminescent substrate solution.
Example 2
The present embodiment is different from embodiment 1 in that: the mass ratio of the water-soluble quantum dots to the Gemini surfactant in the reinforcing agent is 1: 15.
Example 3
The present embodiment is different from embodiment 1 in that: the mass ratio of the water-soluble quantum dots to the Gemini surfactant in the reinforcing agent is 1: 30.
Example 4
The present embodiment is different from embodiment 1 in that: the Gemini surfactant in the reinforcing agent is Gemini C10, namely R group in the structural formula is C10H21The basic phosphoenzyme-catalyzed chemiluminescence substrate solution containing the enhancer comprises the following components in percentage by weight:
example 5
The present embodiment is different from embodiment 1 in that: the Gemini surfactant in the reinforcing agent is Gemini C18, namely R group in the structural formula is C18H37The basic phosphoric acid enzymatic chemiluminescence substrate solution containing the reinforcing agent comprises the following components in percentage by weight:
comparative example 1
This comparative example differs from example 1 in that: BSA (bovine serum albumin) is used as an enhancer in the alkaline phosphatase-catalyzed chemiluminescent substrate solution.
Comparative example 2
This comparative example differs from example 1 in that: the alkaline phosphatase-catalyzed chemiluminescence substrate solution takes cationic polymer polyacrylamide as an enhancer.
Comparative example 3
This comparative example differs from example 1 in that: the reinforcing agent is a conventional fluorescent compound fluorescein isothiocyanate and a Gemini surfactant in a mass ratio of 1: 20.
Comparative example 4
This comparative example differs from example 1 in that: the reinforcing agent is water-soluble quantum dot ZnS and a conventional surfactant TritonX-114 in a mass ratio of 1: 20.
Comparative example 5
This comparative example differs from example 1 in that: the reinforcing agent is a conventional fluorescent compound fluorescein and a conventional surfactant sodium fatty alcohol polyoxyethylene ether carboxylate in a weight ratio of 1: 20.
Application example 1 enhancement factor of luminescence signal of substrate solution
The concentration of alkaline phosphatase was adjusted to 0.0025. mu.g/mL using Tris buffer, 2.0. mu.L of the diluted alkaline phosphatase was added to each well of a 96-well microplate, 200. mu.L of each of the chemiluminescent substrate solutions prepared in examples 1-5 and comparative examples 1-5 was added thereto, and the chemiluminescent signal value of each substrate solution was determined by the MD microplate reader Spectra MAX i3X end point method. The enhancement factor of each example and comparative example compared to that without the reinforcing agent was calculated by using no reinforcing agent (the reinforcing agent was replaced with ultrapure water of the same volume), no water-soluble quantum dot (containing only 20mg/mL of Gemini C12), and no Gemini surfactant (containing only 1mg/mL of water-soluble quantum dot CdTe) as controls, and the enhancement factor without the reinforcing agent as 1.0, and the results are shown in Table 1.
TABLE 1 fold enhancement of chemiluminescence of substrate fluids
| Group of | Luminous signal (RLU) | Multiple of enhancement |
| No reinforcing agent | 3026 | 1.0 |
| Water-insoluble quantum dots | 6852 | 2.3 |
| Gemini-free surfactant | 5360 | 1.8 |
| Example 1 | 89653 | 29.6 |
| Example 2 | 86524 | 28.6 |
| Example 3 | 86532 | 29.0 |
| Example 4 | 82475 | 27.3 |
| Example 5 | 88543 | 29.3 |
| Comparative example 1 | 5823 | 1.9 |
| Comparative example 2 | 4986 | 1.6 |
| Comparative example 3 | 67325 | 23.5 |
| Comparative example 4 | 73498 | 24.7 |
| Comparative example 5 | 75453 | 22.2 |
Comparing the luminescence signal values of the substrate solutions in the examples and comparative examples and the fold signal enhancement compared to that without the enhancing agent, it can be found from the results in table 1. The enhancement factor of the luminescent signal of the enhancer used in the prior art, such as BSA or a common surfactant (comparative examples 1-2), is lower and is only increased by about 2 times, compared with the situation that a water-soluble quantum dot is added alone or a Gemini surfactant is added alone, the two are compounded according to the mass ratio of 1: 15-25 to be used as the enhancer (examples 1-5), the luminescent signal is obviously enhanced, the enhancement factor is more than 26, and when any one of the two is changed (comparative examples 3-4) or the two are replaced by other conventional substances (comparative example 5), the enhancement factor of the luminescent signal is obviously reduced.
Application example 2 substrate liquid self-luminescence and sensitivity evaluation
Detecting the self-luminescence value of each substrate solution when no alkaline phosphatase is added for catalysis, and specifically: the substrate of examples 1 to 5 and comparative examples 1 to 5 was applied in an amount of 200. mu.L per well in a 96-well plate, and the luminescence values of each group were measured by a MD plate reader Spectra MAX i3X, and the results of measuring the mean luminescence values within 10min of each group are shown in Table 2.
And (3) measuring the sensitivity: the substrate is used for detecting an alpha-fetoprotein (AFP) project, and the detection method specifically comprises the following steps: 50 muL of AFP antibody coated by magnetic beads is taken, 10 muL of AFP samples with the concentration of 0 mug/L, 0.04 mug/L, 1 mug/L and 50 mug/L are respectively added, 50 muL of AFP antibody of marked alkaline phosphatase with the concentration of 0.05 mug/mL is added, after uniform mixing, the mixture is combined for 10 minutes at 37 ℃, washed, 200 muL of chemiluminescent substrate liquid prepared in each example and comparative example is added, after incubation, the luminescence value is detected by an MD microplate reader Spectra MAX i3X, the ratio of the luminescence value of each AFP sample concentration to that of the AFP sample with the concentration of 0 mug/L, namely the signal to noise ratio is calculated, and the results are shown in Table 3, and the signal to noise ratio is used as the basis of sensitivity.
TABLE 2 spontaneous luminescence values of substrate solutions
| Group of | Mean value of luminescence signal (RLU) |
| Blank hole | 0.2 |
| Example 1 | 49 |
| Example 2 | 256 |
| Example 3 | 358 |
| Example 4 | 275 |
| Example 5 | 362 |
| Comparative example 1 | 1340 |
| Comparative example 2 | 844 |
| Comparative example 3 | 507 |
| Comparative example 4 | 717 |
| Comparative example 5 | 923 |
TABLE 3 sensitivity measurement data
| 0.04μg/L | 1μg/L | 50μg/L | |
| Example 1 | 4.03 | 82.74 | 3716.35 |
| Example 2 | 3.98 | 80.51 | 3689.85 |
| Example 3 | 3.71 | 73.34 | 3652.33 |
| Example 4 | 3.88 | 79.35 | 3675.63 |
| Example 5 | 3.59 | 69.52 | 3628.00 |
| Comparative example 1 | 2.35 | 52.48 | 2755.48 |
| Comparative example 2 | 2.78 | 55.79 | 2943.95 |
| Comparative example 3 | 3.32 | 65.33 | 3568.34 |
| Comparative example 4 | 3.07 | 64.78 | 3513.75 |
| Comparative example 5 | 3.15 | 64.23 | 3557.41 |
According to the results in Table 2, the self-luminescence values of the substrate solutions obtained in examples 1 to 5 are all significantly lower than those of comparative examples 1 to 5, wherein the self-luminescence value of the substrate is the background value in the detection process, and the lower the background value is, the higher the signal to noise ratio is, according to the results in Table 3, the signal to noise ratios of examples 1 to 5 are all significantly higher than those of comparative examples 1 to 5 for samples with different concentrations, which indicates that the self-luminescence value of the substrate solution has less interference with the detection result, i.e., the detection sensitivity of the substrate solution is high.
Application example 3 evaluation of light emission Rate
Whether the luminescence rates of the examples and the comparative examples are uniform and stable is detected, and the specific method comprises the following steps: alkaline phosphoric acid was diluted to 0.05. mu.g/mL with Tris buffer, 2.0. mu.L of diluted alkaline phosphatase was added to each well of a 96-well microplate, 200. mu.L of each of the chemiluminescent substrate solutions prepared in examples 1-5 and comparative examples 1-5 was added, the chemiluminescence signal value of each substrate solution was measured with an MD microplate reader Spectra MAX i3X, the luminescence signal values at 3 minutes to 8 minutes after the start of the reaction were counted, the measurement was performed every half minute, the change in the luminescence signal value every half minute and the coefficient of variation in the luminescence signal change value were calculated, wherein the smaller the coefficient of variation, the more uniform the change in the luminescence signal was, and the results are shown in Table 4. And the luminescence rate graphs of example 1 and comparative examples 1 to 5 are shown in fig. 1.
TABLE 4 coefficient of variation of luminescence signal
| Group of | Coefficient of variation |
| Example 1 | 3.13％ |
| Example 2 | 3.53％ |
| Example 3 | 3.49％ |
| Example 4 | 4.03％ |
| Example 5 | 3.87％ |
| Comparative example 1 | 13.63％ |
| Comparative example 2 | 11.80％ |
| Comparative example 3 | 8.22％ |
| Comparative example 4 | 8.89％ |
| Comparative example 5 | 9.56％ |
As can be seen from the results in Table 4, the variation coefficient of the variation values of the luminescence signal values of examples 1-5 is significantly lower than that of comparative examples 1-4, i.e., the luminescence rates of the substrate solutions prepared in examples 1-5 are more uniform, so that the detection deviation of the luminescence values due to the non-uniform luminescence rate of the substrate is avoided during the detection process, and the interference caused by the time difference of the test can be corrected during the instrument test. Meanwhile, as can be seen from fig. 1, the light emission value and the light emission rate of example 1 are significantly higher than those of comparative examples 1 to 5.
Application example 4 evaluation of luminescence kinetics
Alkaline phosphate was diluted with Tris buffer to 0.1. mu.g/mL (high concentration) and 0.025. mu.g/mL (low concentration), 2.0. mu.L of the above-mentioned alkaline phosphatase at high and low concentrations was added to each well of a 96-well microplate, and 200. mu.L of the chemiluminescent substrate solutions prepared in example 1 and comparative example 1 was added thereto, respectively, and the luminescence kinetics curves at different alkaline phosphatase concentrations for 0 to 30 minutes were measured with a MD microplate reader Spectra MAX i3X, and the results are shown in FIG. 2 and FIG. 3.
Wherein, fig. 2 is a graph showing the luminescence power curve of the added high concentration alkaline phosphatase, fig. 3 is a graph showing the luminescence power curve of the added low concentration alkaline phosphatase, and according to the results, it can be found that the luminescence value and the stability of the substrate solution prepared in example 1 are higher than those of comparative example 1, no matter under the catalysis of the high concentration alkaline phosphatase or the low concentration alkaline phosphatase, that is, compared with the prior art that BSA is used as the reinforcing agent, the luminescence signal value and the luminescence stability of the reinforcing agent prepared in the invention are both significantly improved.
Application example 5 precision evaluation
Alkaline phosphate was diluted to 0.025. mu.g/mL with Tris buffer, 2.0. mu.L of the diluted alkaline phosphatase was added to each well of a 96-well microplate, 200. mu.L of each of the chemiluminescent substrate solutions prepared in examples 1-5 and comparative examples 1-5 was added, the luminescence value was measured with a MD microplate reader Spectra MAX i3X, the measurement was repeated 10 times, and the average value, standard deviation (S) and Coefficient of Variation (CV) were calculated, and the results are shown in Table 5.
TABLE 5 repeated measurement of precision data
| Group of | Mean value | Standard deviation of | Coefficient of variation |
| Example 1 | 8.45×106 | 1.15×104 | 0.14％ |
| Example 2 | 6.48×106 | 3.76×104 | 0.58％ |
| Example 3 | 7.33×106 | 2.57×104 | 0.35％ |
| Example 4 | 7.25×106 | 7.51×104 | 1.09％ |
| Example 5 | 7.84×106 | 3.55×104 | 0.45％ |
| Comparative example 1 | 5.44×105 | 4.45×104 | 8.19％ |
| Comparative example 2 | 4.88×105 | 3.84×104 | 7.88％ |
| Comparative example 3 | 3.03×106 | 6.90×104 | 2.28％ |
| Comparative example 4 | 4.55×106 | 8.74×104 | 1.92％ |
| Comparative example 5 | 2.85×106 | 7.20×104 | 2.53％ |
As can be seen from the results in table 5, the variation coefficients of the repeated tests in examples 1 to 5 are significantly lower than those in comparative examples 1 to 5, i.e., the precision of the reagent is significantly lower than that of the substrate solution prepared by the present invention, compared to the reinforcing agent used in the prior art, or any one of the water-soluble quantum dots or the Gemini surfactant in the reinforcing agent is changed.
Application example 6 stability evaluation
The signal retention rates of the substrate solutions prepared in examples 1 to 5 and comparative examples 1 to 5 after being placed at 4 ℃ for 16 days or at 37 ℃ for 16 days were measured as stability evaluations, and the specific measurement methods were as follows: alkaline phosphate was diluted to 0.025. mu.g/mL with Tris buffer, 2.0. mu.L of diluted alkaline phosphatase was added to each well of a 96-well microplate, 200. mu.L of each of the chemiluminescent substrate solutions prepared in examples 1-5 and comparative examples 1-5 was added, the change in luminescence before and after the standing was measured with a MD microplate reader Spectra MAX i3X, and the luminescence signal retention was calculated, the results of which are shown in Table 6.
TABLE 6 stability data
| Group of | Retention of 16d signal at 4 ℃ | Retention of 16d signal at 37 ℃ |
| Example 1 | 154.2％ | 94.2％ |
| Example 2 | 109.6％ | 87.5％ |
| Example 3 | 105.9％ | 88.3％ |
| Example 4 | 102.5％ | 86.7％ |
| Example 5 | 107.4％ | 87.9％ |
| Comparative example 1 | 86.4％ | 75.3％ |
| Comparative example 2 | 85.7％ | 72.6％ |
| Comparative example 3 | 96.3％ | 84.7％ |
| Comparative example 4 | 95.7％ | 85.1％ |
| Comparative example 5 | 92.8％ | 83.7％ |
According to the determination results in table 6, when a conventional enhancer is selected or any one of the water-soluble quantum dots and the Gemini surfactant in the enhancer is replaced by the conventional material, the signal retention rate of the substrate liquid is remarkably reduced compared with that of the substrate liquid in examples 1-5 after the substrate liquid is placed at 4 ℃ for 16 days or at 37 ℃ for 16 days, namely the chemiluminescent substrate liquid prepared by the method has good stability.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. The basic phosphate enzymatic chemiluminescence substrate enhancer comprises a fluorescent compound and a surfactant, and is characterized in that the fluorescent compound is a water-soluble quantum dot, and the surfactant is a Gemini surfactant.
2. The alkaline phosphatase-catalyzed chemiluminescent substrate enhancer as claimed in claim 1, wherein the mass ratio of the water-soluble quantum dots to the Gemini surfactant is 1: 15-25.
3. The alkaline phosphatase-based chemiluminescent substrate enhancer according to claim 2, wherein the mass ratio of the water-soluble quantum dots to the Gemini surfactant is 1: 20.
4. The alkaline phosphatase-based chemiluminescent substrate enhancer according to claim 1, wherein the Gemini surfactant has the structural formula:
wherein the R group is selected from C8-20 alkyl.
5. The alkaline phosphatase enzymatic chemiluminescent substrate enhancer as claimed in claim 1, wherein the water soluble quantum dots are selected from one or more of water soluble quantum dots CdTe, water soluble quantum dots CdSe, water soluble quantum dots ZnS, water soluble quantum dots ZnCdS.
6. A basic phosphoenzymatic chemiluminescent substrate solution comprising the enhancer of any one of claims 1-5.
7. The alkaline phosphatase-based chemiluminescent substrate solution according to claim 6, comprising:
the pH value is 9.0-9.5.
8. The alkaline phosphatase-based chemiluminescent substrate solution according to claim 7, comprising:
the pH value is 9.0-9.5.
9. The alkaline phosphatase-based chemiluminescent substrate solution according to claim 7 or 8, wherein the preservative in the chemiluminescent substrate solution is at least one of sodium azide, Proclin series, potassium sorbate and sodium benzoate.
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