1. A chemiluminescent substrate solution suitable for an acridinium ester luminescent system is characterized by comprising a substrate A solution and a substrate B solution;

wherein the content of the first and second substances,

the substrate A liquid comprises an oxidant and an inorganic acid, and the pH of the substrate A liquid is less than 1.5;

the substrate B solution comprises inorganic base and an enhancer, and the pH value of the substrate B solution is more than 13;

wherein the enhancer is a surfactant.

2. The chemiluminescent substrate solution of claim 1 wherein in the substrate solution A,

the oxidant comprises hydrogen peroxide and/or carbamide peroxide, and preferably the oxidant is hydrogen peroxide;

the mass concentration of the oxidant is 0.6-1.5 wt%.

3. A chemiluminescent substrate solution according to claim 1 wherein the inorganic acid comprises hydrochloric acid, nitric acid and sulfuric acid, preferably the inorganic acid is sulfuric acid and the sulfuric acid is 9M sulfuric acid.

4. The chemiluminescent substrate solution of claim 1 wherein in the substrate B solution,

the inorganic base comprises sodium hydroxide and/or potassium hydroxide, preferably sodium hydroxide;

the concentration of the sodium hydroxide is 0.1-0.5M.

5. The chemiluminescent substrate solution of claim 1 wherein the surfactant comprises Triton X-100 and/or Tween-20; preferably Triton X-100, and the mass concentration of the Triton X-100 is 1-3 wt%.

6. The chemiluminescent substrate solution according to claim 1 wherein, in use, the volume ratio of the substrate solution A to the substrate solution B is controlled to be 1: 1;

preferably, the chemiluminescent substrate solution comprises a substrate solution A and a substrate solution B which are independently preserved before use;

the substrate A solution: h₂O₂Adjusting the pH value to 1.0 +/-0.05 by 9M sulfuric acid with the concentration of 1.32 wt%;

the substrate B solution: 0.35M NaOH and 2 wt% Triton X-100, pH 13.48 ± 0.1;

preferably, the volume ratio of the substrate solution A to the substrate solution B is 1: 1.

7. The method for preparing a chemiluminescent substrate solution of any one of claims 1 to 6 comprising: preparing a substrate A solution and a substrate B solution; respectively and independently packaging the prepared substrate A liquid and the substrate B liquid;

preferably, the first and second liquid crystal materials are,

the preparation method of the substrate A liquid comprises the following steps: adding inorganic acid into the oxidant solution, and adding water to a constant volume;

the preparation method of the substrate B solution comprises the following steps: adding the reinforcing agent into the inorganic alkali liquor and adding water to a constant volume.

8. Use of a chemiluminescent substrate solution of any one of claims 1 to 6 in a chemiluminescent assay and/or in the manufacture of a chemiluminescent assay kit;

preferably, the chemiluminescence detection is carried out by using an acridinium ester luminescent system.

9. A chemiluminescent detection kit comprising a chemiluminescent substrate solution according to any one of claims 1 to 6; preferably, the detection kit further comprises acridinium ester.

10. A chemiluminescent assay method comprising the use of a chemiluminescent substrate solution of any one of claims 1 to 6 and/or a chemiluminescent assay kit of claim 9.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Chemiluminescence immunoassay (CLIA) was born in 1977. According to the basic principle of radioimmunoassay, a chemiluminescence immunoassay method is established by combining a high-sensitivity chemiluminescence technology with a high-specificity immunoreaction. CLIA has the characteristics of high sensitivity, strong specificity, wide linear range, simple and convenient operation, no need of expensive instruments and equipment and the like. CLIA has wide application range, can detect antigens, haptens and antibodies with different molecular sizes, and can also be used for detecting nucleic acid probes. Compared with Radioimmunoassay (RIA), fluoroimmunoassay (IFA) and Enzyme Immunoassay (EIA), CLIA has the advantages of no radiation, long effective period of the marker, full automation realization, and the like. CLIA is widely applied in the fields of environment, clinic, food, drug detection and the like, and becomes a research hotspot and development trend of an immunoassay method.

The acridinium ester has an important function in chemiluminescence immunoassay as an important chemiluminescence reagent, is used for immunoassay as a marker, and has the advantages of simple and rapid luminescence system, no need of adding a catalyst, high labeling efficiency, low background and the like. However, the studies on the chemiluminescent substrate solution of the acridinium ester chemiluminescent system are only reported, so that the chemiluminescent substrate solution more suitable for the acridinium ester chemiluminescent system needs to be developed, so that the stability, the sensitivity and the detection range of the chemiluminescent substrate solution are improved, and the production and use costs are further reduced.

Disclosure of Invention

Based on the defects of the prior art, the invention provides chemiluminescent substrate liquid suitable for an acridinium ester luminescent system and application thereof. The invention successfully prepares the chemiluminescent substrate liquid which has the advantages of good stability, high sensitivity, wide detection range, low cost and the like and is suitable for the acridinium ester luminescent system by screening and optimizing the components, the using amount, the pH value and the like of the chemiluminescent substrate liquid which is suitable for the acridinium ester luminescent system.

In a first aspect of the invention, a chemiluminescent substrate solution suitable for an acridinium ester luminescent system is provided, wherein the chemiluminescent substrate solution comprises a substrate solution A and a substrate solution B.

Wherein the content of the first and second substances,

the substrate A liquid comprises an oxidant and an inorganic acid, and the pH of the substrate A liquid is less than 1.5;

the substrate B solution comprises inorganic base and an enhancer, and the pH of the substrate B solution is more than 13.

When the method is used, the volume ratio of the substrate A liquid to the substrate B liquid is 1: 1.

In the substrate A solution,

the oxidizing agent comprises hydrogen peroxide and/or carbamide peroxide, and the hydrogen peroxide has the advantages of enhancing the stability of the reagent, improving the linear range and the sensitivity of the project test compared with carbamide peroxide through optimization verification of the invention. Therefore, the preferred oxidizing agent is hydrogen peroxide.

The inorganic acid comprises hydrochloric acid, nitric acid and sulfuric acid, and the optimization and verification of the invention prove that the sulfuric acid has more advantages in the aspects of enhancing the stability of the reagent, improving the linear range of the project test and improving the sensitivity compared with the hydrochloric acid and the nitric acid. Thus, preferably, the mineral acid is sulfuric acid, which is 9M sulfuric acid.

In the substrate B solution,

wherein the inorganic base comprises sodium hydroxide and/or potassium hydroxide, preferably sodium hydroxide.

The stability of the substrate liquid, the linear range and the sensitivity of the project test and the like can be effectively improved by adding an enhancer, wherein the enhancer is selected from surfactants such as Triton X-100 and/or Tween-20. And the overall improvement effect is better when Triton X-100 is selected as the reinforcing agent.

In a second aspect of the present invention, there is provided a method for producing the above chemiluminescent substrate solution, the method comprising: preparing a substrate A solution and a substrate B solution;

and packaging the prepared substrate A solution and the substrate B solution respectively and independently.

Wherein the content of the first and second substances,

the preparation method of the substrate A liquid comprises the following steps: adding inorganic acid into the oxidant solution, and adding water to a constant volume;

the preparation method of the substrate B solution comprises the following steps: adding the reinforcing agent into the inorganic alkali liquor and adding water to a constant volume.

In a third aspect of the present invention, there is provided an application of the above chemiluminescent substrate solution in chemiluminescent detection and/or preparation of a chemiluminescent detection kit.

Wherein, the chemiluminescence detection is carried out by adopting an acridinium ester luminescent system.

In a fourth aspect of the present invention, there is provided a chemiluminescent assay kit comprising the above chemiluminescent substrate solution; further, the detection kit also comprises acridinium ester.

In a fifth aspect of the present invention, there is provided a chemiluminescent detection method comprising the use of the above chemiluminescent substrate solution and/or the above chemiluminescent detection kit.

The beneficial technical effects of one or more technical schemes are as follows:

the technical scheme provides the chemiluminescent substrate solution suitable for the acridinium ester luminescent system, and the used acid, alkali, oxidant, reinforcing agent and the like are screened and optimized, so that the stability of the reagent can be effectively enhanced, and the linear range and the sensitivity of the project test are improved; meanwhile, the chemiluminescence substrate liquid has the advantages of cheap and easily-obtained raw materials, simple preparation method, no need of complex and expensive equipment, and suitability for industrial production, thereby having good practical application value.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described above, there are few reports on the studies of the chemiluminescent substrate solution of the acridinium ester chemiluminescent system, and therefore, it is necessary to develop a chemiluminescent substrate solution more suitable for the acridinium ester chemiluminescent system, so as to improve the stability, sensitivity and detection range thereof and further reduce the production and use costs.

In view of the above, in an exemplary embodiment of the present invention, a chemiluminescent substrate solution suitable for an acridinium ester luminescent system is provided, wherein the chemiluminescent substrate solution comprises a substrate solution a and a substrate solution B.

Wherein the content of the first and second substances,

the substrate A liquid comprises an oxidant and an inorganic acid, and the pH of the substrate A liquid is less than 1.5;

the substrate B solution comprises inorganic base and an enhancer, and the pH of the substrate B solution is more than 13.

When the method is used, the volume ratio of the substrate A liquid to the substrate B liquid is 1: 1.

In still another embodiment of the present invention, in the substrate A solution,

the oxidizing agent comprises hydrogen peroxide and/or carbamide peroxide, and the hydrogen peroxide has the advantages of enhancing the stability of the reagent, improving the linear range and the sensitivity of the project test compared with carbamide peroxide through optimization verification of the invention. Therefore, the preferred oxidizing agent is hydrogen peroxide. Meanwhile, the present invention further optimizes the concentration of the oxidizing agent, preferably, the oxidizing agent has a mass concentration of 0.6 to 1.5 wt%, such as 0.6 wt%, 0.65 wt%, 0.8 wt%, 1.0 wt%, 1.2 wt%, 1.32 wt%, 1.4 wt%, 1.5 wt%, and when the hydrogen peroxide has a mass concentration of 1.32 wt%, the linear range and sensitivity of the test item are improved optimally.

The inorganic acid comprises hydrochloric acid, nitric acid and sulfuric acid, and the optimization and verification of the invention prove that the sulfuric acid has more advantages in the aspects of enhancing the stability of the reagent, improving the linear range of the project test and improving the sensitivity compared with the hydrochloric acid and the nitric acid. Thus, preferably, the mineral acid is sulfuric acid, which is 9M sulfuric acid.

In still another embodiment of the present invention, in the substrate B solution,

wherein the inorganic base comprises sodium hydroxide and/or potassium hydroxide, preferably sodium hydroxide. Meanwhile, the stability of the reagent, the linear range of the test and the sensitivity of the concentration of the sodium hydroxide are researched, and the influence of the concentration on the detection result of the reagent is found to be small, so that the low-concentration sodium hydroxide is selected for the test in comprehensive consideration, and therefore, in one embodiment of the invention, the concentration of the sodium hydroxide is 0.1-0.5M, including 0.1M, 0.2M, 0.3M, 0.35M, 0.4M and 0.5M, and is preferably 0.35M.

The linear range, the sensitivity and the like of the project test can be effectively improved by adding an enhancer, wherein the enhancer is selected from surfactants such as Triton X-100 and/or Tween-20. The overall improvement effect of the Triton X-100 is better when the Triton X-100 is selected as an enhancer, and meanwhile, through research on the mass concentration of the Triton X-100, when the concentration of the Triton X-100 is less than 2 wt%, the improvement effects on the stability, the linear range and the sensitivity of the reagent are gradually improved along with the increase of the concentration, but when the concentration of the Triton X-100 reaches 3 wt%, the repeatability and the sensitivity of a test item are remarkably reduced, so that the mass concentration of the Triton X-100 is 1-3 wt%, including 1 wt%, 2 wt% and 3 wt%, and preferably 2 wt%.

In another embodiment of the present invention, there is provided a chemiluminescent substrate solution suitable for acridinium ester luminescent systems, comprising a substrate solution a and a substrate solution B, which are independently preserved before use;

wherein the content of the first and second substances,

the substrate A solution: h₂O₂Concentration 1.32 wt%, 9M sulfuric acid, pH to 1.0 + -0.05;

the substrate B solution: 0.35M NaOH and 2 wt% Triton X-100, pH 13.48 ± 0.1;

wherein the volume ratio of the substrate solution A to the substrate solution B is 1:1 when the substrate solution A and the substrate solution B are used.

In another embodiment of the present invention, there is provided a method for producing the chemiluminescent substrate solution, the method comprising: preparing a substrate A solution and a substrate B solution; and packaging the prepared substrate A solution and the substrate B solution respectively and independently.

Wherein the content of the first and second substances,

the preparation method of the substrate A liquid comprises the following steps: adding inorganic acid into the oxidant solution, and adding water to a constant volume;

the preparation method of the substrate B solution comprises the following steps: adding the reinforcing agent into the inorganic alkali liquor and adding water to a constant volume.

In another embodiment of the present invention, there is provided the use of the above chemiluminescent substrate solution in chemiluminescent detection and/or in the preparation of a chemiluminescent detection kit.

Wherein, the chemiluminescence detection is carried out by adopting an acridinium ester luminescent system.

In another embodiment of the present invention, there is provided a chemiluminescent assay kit comprising the above chemiluminescent substrate solution; further, the detection kit also comprises acridinium ester.

In another embodiment of the present invention, there is provided a chemiluminescent detection method comprising the use of the above chemiluminescent substrate solution and/or the above chemiluminescent detection kit.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

Substrate a solution to be verified:

formulation a 1: h₂O₂The concentration is 0.65 wt%, and the pH is adjusted to 1.0 +/-0.05 by using 4M hydrochloric acid;

formulation a 2: h₂O₂The concentration is 0.65 wt%, and the pH is adjusted to 1.0 +/-0.05 by using 8M nitric acid;

formulation a 3: h₂O₂The concentration is 0.65 wt%, and the pH is adjusted to 1.0 plus or minus 0.05 by 9M sulfuric acid;

formulation a 4: h₂O₂The concentration is 1.32 wt%, and the pH is adjusted to 1.0 +/-0.05 by using 9M sulfuric acid;

formulation a 5: urea peroxide concentration 1.32 wt%, adjusted pH to 1.0 + -0.05 with 9M sulfuric acid;

taking the formula A1 as an example, the preparation method comprises the following steps: 10.8g of 30% H are weighed out₂O₂The solution, 12.5mL of 4M hydrochloric acid, then made up to 500mL with purified water. After stirring and mixing, buffer A1 was obtained. The buffer prepared by the above method has pH of 1.0 + -0.05 and H₂O₂The concentration was 0.65 wt%, and the pH was 1.0. + -. 0.05.

Taking the formula A2 as an example, the preparation method comprises the following steps: 10.8g of 30% H are weighed out₂O₂The solution, 6.25mL of 8M nitric acid, was then made up to 500mL with purified water. After stirring and mixing, buffer A2 was obtained. The buffer prepared by the above method has pH of 1.0 + -0.05 and H₂O₂The concentration was 0.65 wt%, and the pH was 1.0. + -. 0.05.

Taking the formula A3 as an example, the preparation method comprises the following steps: 10.8g of 30% H are weighed out₂O₂The solution, 5.56mL of 9M sulfuric acid, then made up to 500mL with purified water. After stirring and mixing, buffer A3 was obtained. The buffer prepared by the above method has pH of 1.0 + -0.05 and H₂O₂The concentration was 0.65 wt%, and the pH was 1.0. + -. 0.05.

Taking the formula A4 as an example, the preparation method comprises the following steps: 22g of 30% H are weighed out₂O₂The solution, 5.56mL of 9M sulfuric acid, then made up to 500mL with purified water. After stirring and mixing, buffer A4 was obtained. The buffer prepared by the above method has pH of 1.0 + -0.05 and H₂O₂The concentration was 0.65 wt%, and the pH was 1.0. + -. 0.05.

Taking the formula A5 as an example, the preparation method comprises the following steps: 6.6g of carbamide peroxide and 5.56mL of 9M sulfuric acid were weighed out and made up to 500mL with purified water. After stirring and mixing, buffer A5 was obtained. The buffer prepared by the above method has pH of 1.0 + -0.05 and H₂O₂The concentration was 0.65 wt%, and the pH was 1.0. + -. 0.05.

And (3) substrate B solution to be verified:

formulation B1: 2.5M NaOH +0.02M EDTA-Na₂，pH＝13.32；

Formulation B2: 0.35M NaOH, pH 13.48;

formulation B3: 0.35M NaOH +1 wt% Tween-20, pH 13.48;

formulation B4: 0.35M NaOH +1 wt% Triton X-100, pH 13.48;

formulation B5: 0.35M NaOH +2 wt% Triton X-100, pH 13.48;

formulation B6: 0.35M NaOH +3 wt% Triton X-100, pH 13.48;

control substrate a solution: C-A contains 1.32% by weight of H₂O₂An acid solution (substrate A liquid of a certain imported brand, the component is a component disclosed in the specification, and the rest components are not detailed)

Control substrate B solution: C-B an alkaline solution containing 0.35M NaOH (substrate solution B used in combination with substrate solution A, the components being disclosed in the specification, the remaining components being not specified)

Taking the formula B3 as an example, the preparation method comprises the following steps: weighing 7g of NaOH solid, dissolving the NaOH solid in 400mL of purified water, then weighing 10g of Triton X-100, then using the purified water to fix the volume to 500mL, and measuring the pH value after fixing the volume, wherein the pH value is required to be within the range of 13.48 +/-0.1.

	Substrate solution A	Substrate solution B
			Comparative example 1	C-A	C-B
Example 1	A1	C-B
			Example 2	A2	C-B
Example 3	A3	C-B
			Example 4	A4	C-B
Example 5	A5	C-B
			Example 6	C-A	B1
Example 7	C-A	B2
			Example 8	C-A	B3
Example 9	C-A	B4
			Example 10	C-A	B5
Example 11	C-A	B6
			Example 12	A4	B5

(1) Testing background luminescence value of substrate solution

The background of the substrate A liquid is higher than that of the substrate A liquid in the comparative example 1 in the example 1 in which hydrochloric acid is used as inorganic acid, and the background of the substrate A liquid in the examples 2 and 3 in which nitric acid and sulfuric acid are used as inorganic acid is basically consistent with that of the substrate A liquid in the comparative example 1; examples 4, 5, in which the hydrogen peroxide concentration was increased or urea peroxide was selected as the oxidizing agent, had significantly lower background than comparative example 1. Among the three inorganic acids, nitric acid or sulfuric acid is selected, so that the background of a substrate is lower; urea peroxide or higher concentrations of hydrogen peroxide have a significant effect on reducing substrate background.

Several formulas of the substrate B liquid show a lower background compared with the substrate 1 in a background test, and the improvement of the sodium hydroxide concentration has no obvious effect on the reduction of the substrate background.

(2) Test AE sample adding repeatability

In examples 1, 2 and 3 in which the hydrogen peroxide concentration of the substrate A solution was 0.65 wt%, the luminescence value of the AFP item AE in the direct substrate test was significantly lower than that of comparative example 1 no matter what inorganic acid was used; example 4, in which the substrate a solution had a hydrogen peroxide concentration of 1.32 wt%, the luminescent value of the substrate direct test AFP item AE was substantially the same as that of comparative example 1; in example 5, after replacing the hydrogen peroxide of substrate a liquid with urea peroxide, the substrate direct test AFP item AE still had significantly lower luminescence values than comparative example 1. The increase of the hydrogen peroxide concentration has obvious improvement on the increase of the substrate luminescence value.

The luminous value of the AFP item AE directly tested by the substrate in the example 7 with the concentration of 0.35M of sodium hydroxide in the substrate B solution is higher than that of the AFP item AE directly tested by the substrate in the example 6 with the concentration of 2.5M of sodium hydroxide, and the improvement of the concentration of sodium hydroxide has no obvious improvement on the improvement of the luminous value of the AFP item AE directly tested; example 8 with 1% Tween-20 added as an enhancer showed a significant increase in luminescence value for the AFP item AE tested directly compared to example 7 without the enhancer; compared with example 8, example 9 in which the enhancing agent is replaced by 1% of Triton X-100 has the advantages that the luminescence value of the AFP item AE directly tested is further improved and can basically reach the level of comparative example 1, and examples 10 and 11 in which the concentration of the Triton X-100 is further improved to 2% and 3% have the advantages that the luminescence value of the AFP item AE directly tested is continuously improved and can reach the condition which is obviously better than that of comparative example 1. The addition of the reinforcing agent can obviously improve the luminous value, wherein the action of the Triton X-100 is more obvious than that of Tween-20, and the reinforcing effect is more obvious along with the increase of the concentration.

(3) Test items

Antigen concentration (ng/ml)	Comparative example 1	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6	Example 7	Example 8	Example 9	Example 10	Example 11	Example 12
														0	8169	9320	11582	8951	6670	8936	3701	5179	8654	10029	9987	13843	7301
7.8	33570	39009	32391	37012	34086	34092	9388	13497	25703	33620	43652	48214	37968
														31.25	87588	101354	72327	92785	87291	83029	23184	34739	64694	91447	145761	156351	128751
125	307906	328513	206541	309963	348270	319047	73733	109755	227603	313002	558721	549811	487123
														500	988085	969720	465738	934571	1120456	920017	241494	362750	740539	926299	1937863	1876324	1698635
2000	2688232	2406172	1208791	2683901	3326437	2735091	656896	946647	1647814	2236233	5390875	4805412	4702898
														Gradient of pull-apart	Comparative example 1	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6	Example 7	Example 8	Example 9	Example 10	Example 11	Example 12
B/A	4.1	4.2	2.8	4.1	5.1	3.8	2.5	2.6	3.0	3.4	4.4	3.5	5.2
														C/B	2.6	2.6	2.2	2.5	2.6	2.4	2.5	2.6	2.5	2.7	3.3	3.2	3.4
D/C	3.5	3.2	2.9	3.3	4.0	3.8	3.2	3.2	3.5	3.4	3.8	3.5	3.8
														E/D	3.2	3.0	2.3	3.0	3.2	2.9	3.3	3.3	3.3	3.0	3.5	3.4	3.5
F/E	2.7	2.5	2.6	2.9	3.0	3.0	2.7	2.6	2.2	2.4	2.8	2.6	2.8

The signal range of the test item of example 2 in which nitric acid is selected as the inorganic acid of the substrate a liquid is narrower, and is significantly lower than that of comparative example 1 and examples 1 and 3 in which hydrochloric acid and sulfuric acid are selected as the inorganic acids; example 4, which increased the hydrogen peroxide concentration to 1.32 wt%, the test AFP project had a wider overall signal range, higher sensitivity, and was significantly better than comparative example 1 and examples 1, 2, and 3; example 4 containing hydrogen peroxide, the test item is more sensitive than example 5 containing carbamide peroxide. Of the three inorganic acids, hydrochloric acid and sulfuric acid are significantly superior to nitric acid in the test items; the improvement of the sensitivity of the test for the enhancement item by hydrogen peroxide is more obvious than that by urea peroxide, and the improvement effect of the sensitivity by hydrogen peroxide with the concentration of 1.32 wt% is more obvious than that by hydrogen peroxide with the concentration of 0.65 wt%.

The overall bias ratio and the signal value of the test items of examples 6 and 7 in which sodium hydroxide with different concentrations is selected as the substrate B liquid are both significantly lower than those of the test item of the comparative example 1; in the example 8 in which 1% Tween-20 was added as an enhancer, the signal values of the test items were improved, but the overall signal values and the sensitivity of low-value parts were still significantly lower than those of the comparative example 1; example 9, in which the enhancer was replaced with 1% Triton X-100, showed a significant improvement in both the signal value and sensitivity of the test item compared to example 8, but was still significantly lower than comparative example 1; in example 10, in which the Triton X-100 concentration was further increased to 2%, the overall signal value and the bias ratio were superior to those of comparative example 1; when the concentration of Triton X-100 was again increased to 3% in example 11, the luminescence value was not further increased and the sensitivity was deteriorated. The addition of the reinforcing agent can obviously improve the luminous value and the sensitivity of a test item, wherein the action of the Triton X-100 is more obvious than that of Tween-20, and the reinforcing effect is more obvious along with the increase of the concentration, but when the concentration of the Triton X-100 reaches more than 2 percent, the improvement effect is not more obvious, and the sensitivity of the test item tends to be poor.

(4) Testing item repeatability

The three inorganic acids of the substrate A liquid, examples 1, 2 and 3, have no significant difference in repeatability tests compared with the comparative example 1, the examples 3 and 4 with different concentrations of hydrogen peroxide also have no significant difference in repeatability, and the carbamide peroxide has slightly poorer CV in test item repeatability compared with the hydrogen peroxide with the same concentration, but the overall result is still acceptable. On the repeatability test, the several formulas of the substrate A liquid have no significant difference.

The examples 6 and 7 with different concentrations of sodium hydroxide selected from the substrate B solution and the examples 8 and 9 with different types of enhancers added have no significant difference in the repeatability of the test items, but when the concentration of Triton X-100 is increased to 3%, the repeatability of the test items is significantly deteriorated. In the substrate liquid B, when the concentration of the enhancer is too high, the reproducibility of the test is remarkably deteriorated.

(5) Accelerated stability test at 37 deg.C

Among three inorganic acids of the substrate A liquid, the reduction degree of the luminescence value is more obvious in the accelerated stability test at 37 ℃ in the examples 1 and 2 which select hydrochloric acid and nitric acid than in the example 3 which selects sulfuric acid and the comparative example 1; in the accelerated stability test at 37 ℃, the reduction degrees of the luminous values of the examples 3 and 4 with different concentrations of hydrogen peroxide are basically not obviously different; the luminescence value of example 5 containing carbamide peroxide was reduced more significantly than that of example 4 containing hydrogen peroxide in the accelerated stability test at 37 ℃ compared to hydrogen peroxide at the same concentration of carbamide peroxide. Among the three inorganic acids, the stability of the substrate A liquid is obviously improved by selecting sulfuric acid; hydrogen peroxide is more stable than urea peroxide in substrate solution a.

Examples 6 and 7 in which no enhancer was added to the substrate solution B and example 8 in which Tween-20 was selected as the enhancer were slightly poor in stability and the luminescence value decreased rapidly, while example 9 in which Triton X-100 was selected as the enhancer was improved in substrate stability, examples 10 and 11 in which the concentration of Triton X-100 was continuously increased were not improved significantly in stability. The stability of the substrate can be improved by adding the enhancer into the substrate solution B, wherein the stability improvement effect of Triton X-100 is more obvious than that of Tween-20, but the stability improvement effect of increasing the concentration of Triton X-100 is not obvious.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.