Chemiluminescent substrate solution as well as preparation method and application thereof

Abstract

The invention discloses a chemiluminescence substrate solution as well as a preparation method and application thereof, and belongs to the technical field of magnetic particle chemiluminescence immunoassay. The chemiluminescent substrate solution takes a (4-chlorobenzene sulfydryl) (10-methyl-9, 10-dihydroacridine methylene) phosphate disodium salt derivative as a core luminescent substrate, the core luminescent substrate is matched with a 50-200mmol / L buffer matrix, a 0.01-10mg / L fluorescence enhancement substance containing aromatic groups and a 1-1000mg / L inorganic or organic background inhibitor and a surfactant, the pH value is controlled at 7.5-10, and 0.01-0.2% of a preservative can be selectively added. According to the substrate solution, the synergistic effect of all the components is utilized, and the advantages of short luminescence platform period, long luminescence duration and remarkably improved detection sensitivity and stability are achieved. The method can be widely applied to detection of biomarkers in chemiluminescence immunoassay, can improve the clinical detection speed, shorten the detection time and guarantee the result accuracy, and has good practical value and popularization prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of magnetic particle chemiluminescence immunoassay technology, and particularly relates to a chemiluminescence substrate liquid and a preparation method and application thereof. BACKGROUND

[0002] Chemiluminescence immunoassay technology has become an immune analysis technology widely used in the field of immune diagnosis due to high sensitivity, wide detection linear range, simple operation, short detection time, easy automation and the like. Enzymatic chemiluminescence immunoassay technology is established on the basis of enzyme-linked immune reaction and chemiluminescence, and the reaction principle is similar to enzyme-linked immune (EIA) and radioimmunoassay (RIA), except for the generation and detection of signals. After the reaction is completed, the chemiluminescence substrate forms an excited-state intermediate under the catalysis of the enzyme, and the intermediate is unstable and will radiate photons in the process of returning to the ground state. The relative light intensity can be measured by a light signal measuring instrument, and the concentration of the detected substance can be calculated.

[0003] Alkaline phosphatase (ALP) is widely used in enzymatic chemiluminescence immunoassay, and the most commonly used alkaline phosphatase luminescent substrate is 1,2-dioxane derivative, such as CDP-star, AMPPD and the like. Such luminescent substrate belongs to the glow type, and the time to reach the luminescence platform period is long, usually more than 20 minutes. In the related technology, there is an APS-5 alkaline phosphatase substrate liquid using 9,10-dihydroacridine derivative as a luminescent substrate. Although the time to reach the luminescence platform period of the substrate is shorter than that of the above-mentioned 1,2-dioxane derivative luminescent substrate, the substrate has a high background signal, poor stability, and high cost. When APS-5 is used as an alkaline phosphatase detection substrate, the storage stability is not as good as AMPPD, the use conditions are harsh, the cost is high, and the wide application and promotion of APS-5 are limited.

[0004] Therefore, it is urgent to develop a chemiluminescence substrate liquid with short time to reach the platform period, high detection sensitivity and good stability to solve the problems in the prior art. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art, and provides a chemiluminescence substrate liquid and a preparation method and application thereof. The chemiluminescence substrate liquid has short time to reach the platform period, long light duration, high detection sensitivity and good light stability, and can effectively meet the needs of clinical immune detection.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A chemiluminescent substrate solution, comprising the following components: 200-300 mg / L of (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinylmethyl)phosphonic acid disodium salt derivative, 50-200 mmol / L of buffer matrix, 0.01-10 mg / L of fluorescence enhancer, 1-1000 mg / L of background eliminator, and surfactant.

[0008] The pH of the chemiluminescent substrate solution is 7.5-10, the background eliminator is inorganic or organic reducing agent, and the fluorescence enhancer is an aromatic group-containing compound.

[0009] In the chemiluminescent substrate solution of the present application, the (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinylmethyl)phosphonic acid disodium salt derivative is an alkaline phosphatase substrate, which contains a nitrogen, oxygen or sulfur-containing heterocyclic ring system, and has an exocyclic carbon-carbon double bond and two electron-donating groups (R1 and R2). Figure One The double bond is further substituted with a phosphonic acid group and an oxygen or sulfur-containing group on the distal carbon. Specifically, the present application relates to a chemiluminescent compound containing a heterocyclic group, an enol phosphate group and an electron-donating stabilizing group (R1 and R2), which reacts with oxygen to form an enolate after the phosphatase enzyme removes the phosphonic acid group, and the enolate further reacts to produce chemiluminescence and a carbonyl compound.

[0010] Specifically, in the (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinylmethyl)phosphonic acid disodium salt derivative, R1 is an alkyl, alkenyl, alkynyl, phenyl, sulfoethyl, sulfopropyl, sulfobutyl or aralkyl group, the number of carbon atoms of which does not exceed 24, and the number of heteroatoms (nitrogen, oxygen, phosphorus and sulfur) contained therein does not exceed 20.

[0011] Specifically, in the (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinylmethyl)phosphonic acid disodium salt derivative, R2 is an alkyl, alkenyl, alkynyl, aralkyl or alkoxy group, the number of carbon atoms of which does not exceed 8 and is unbranched, and the number of carbon atoms of the side chain group exceeds 4.

[0012] The buffer matrix of the present application has the effect of maintaining pH and increasing the overall stability of the substrate solution; the fluorescence enhancer can significantly enhance the luminescence signal of the substrate solution, thereby improving the detection sensitivity; the background inhibitor with weak reducing property can reduce the background signal value and reduce interference, further improving the detection sensitivity; the non-ionic surfactant can improve the solution dispersibility, enhance the luminescence signal of the substrate solution, not only improve the detection sensitivity, but also ensure the continuous and stable luminescence signal. Through the synergistic effect and specific ratio of the above components, the luminescent efficiency of the chemiluminescent substrate solution is fast, the time to reach the luminescence plateau is short, the luminescence duration is long, the detection sensitivity is high, and the light stability is good, thereby improving the clinical immune detection speed and shortening the detection time.

[0013] As a preferred scheme of the present application, the buffer matrix is selected from one of Tris, 2-amino-2-methyl-1-propanol and 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES). The above-mentioned buffer matrix has good buffering capacity in the pH range of 7.5-10, can stably maintain the pH environment of the substrate solution, provides suitable conditions for enzyme reaction and chemiluminescence reaction, and helps to improve the storage stability of the substrate solution. Among them, Tris has better buffering effect and is the preferred choice.

[0014] As a preferred scheme of the present application, the fluorescence enhancer is an aromatic compound containing an aromatic heterocycle and at least one methyl substituent, and is selected from one of lucigen and ANS. Such a fluorescence enhancer has good adaptability with the structure of the luminescent substrate, can efficiently absorb the energy generated in the luminescence reaction process and further radiate stronger photons, significantly enhancing the luminescence signal intensity, thereby effectively improving the detection sensitivity. Among them, lucigen has better enhancement effect and is the preferred choice.

[0015] As a preferred scheme of the present application, the background luminescence inhibitor is selected from one of sodium sulfite, sodium thiosulfate and TCEP. The above-mentioned background eliminator has suitable reducing property, can effectively remove impurities or free radicals in the substrate solution that may cause background luminescence, reduce the relative luminescence value when detecting the zero concentration calibrant, thereby improving the signal-to-noise ratio and enhancing the detection sensitivity. Among them, sodium thiosulfate has stable background inhibition effect and is the preferred choice.

[0016] As a preferred scheme of the present application, the surfactant is a mixture of a non-ionic surfactant and an anionic surfactant, the non-ionic surfactant is a high molecular compound copolymerized from propylene oxide and ethylene oxide, and the anionic surfactant is sodium dodecyl sulfonate. The non-ionic surfactant and the anionic surfactant synergistically act, can further improve the dispersibility and uniformity of the solution, promote the contact reaction of the luminescent substrate and the enzyme, and at the same time enhance the stability and continuity of the luminescence signal, further improving the detection performance.

[0017] As a preferred scheme of the present application, the chemiluminescent substrate solution further comprises: a preservative with a mass percentage of 0.01-0.2%, the preservative being selected from at least one of Bovogen PC-300 and BND series. The preservative has the effect of bacteriostatic preservation, can effectively inhibit the growth and reproduction of microorganisms in the substrate solution, avoid the damage of microorganisms to the components of the substrate solution, thereby increasing the stability of the chemiluminescent substrate solution and prolonging the effective period of the chemiluminescent substrate solution.

[0018] The present application further provides a preparation method of the chemiluminescent substrate solution, comprising the following steps:

[0019] S1, measuring 800 mL of ultrapure water, adding 10-200 mmol of buffer matrix, and adjusting the pH to the target pH value;

[0020] S2, adding 200-300 mg of (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinylmethylene) phosphonic acid disodium salt derivative to the solution obtained in step S1, and mixing;

[0021] S3, adding 0.01-10 mg of a fluorescence enhancer to the solution obtained in step S2, and mixing;

[0022] S4, adding 0.1-2 g of a preservative to the solution obtained in step S3, and mixing;

[0023] S5, adding 10-2000 mg of a background inhibitor to the solution obtained in step S4, and mixing;

[0024] S6, adding 0.01-5 g of a surfactant to the solution obtained in step S5, and mixing;

[0025] S7, adjusting the pH of the solution obtained in step S6 to the target pH value, adding 0.1-10 mg of an antifoaming agent, and diluting to 1 L, to obtain the chemiluminescent substrate solution.

[0026] The preparation method is simple to operate, each component is added in a specific order, which can ensure that each component is fully dissolved and uniformly mixed, and avoid performance degradation caused by improper mixing of components. By precisely controlling the amount of each component and the pH value, the chemiluminescent substrate solution prepared finally has stable and excellent performance.

[0027] As a preferred scheme of the above preparation method, the reagent used for adjusting the pH value in steps S1 and S7 is dilute hydrochloric acid, and the target pH value is 8.8-9.0. Dilute hydrochloric acid can precisely adjust the pH value of the solution, and does not introduce other impurities, which will not adversely affect the performance of the substrate solution; when the pH value is controlled at 8.8-9.0, the efficiency of enzyme-catalyzed reaction and chemiluminescence reaction is the highest, and the stability of the substrate solution is also the best.

[0028] The application also provides application of the chemical luminescence substrate liquid in a chemiluminescence immunoassay. The chemical luminescence substrate liquid can be widely applied to various chemiluminescence immunoassays based on alkaline phosphatase, such as detection of various biomarkers such as cardiac troponin I (cTnI), thyroid stimulating hormone (TSH), tumor markers, etc., can effectively improve the speed of clinical immunoassay, shorten the detection time, and at the same time improve the detection sensitivity and result accuracy, and has a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the modified APS-5 chemical structure. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the related drawings. The preferred embodiments of the present application are shown in the drawings. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0032] Example 1

[0033] The chemiluminescence substrate liquid of the present embodiment comprises the following components: 200 mg / L of (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinemethyl) phosphonic acid disodium salt derivative, 50 mmol / L of tris-hydroxymethyl aminomethane, 1 mg / L of lusterine, 0.5 g / L of sodium dodecyl sulfonate, 100 mg / L of sodium thiosulfate, 0.2 g / L of propylene oxide and ethylene oxide copolymer, 0.02% of proclin300 by mass percentage, and the pH of the chemiluminescence substrate liquid is 8.8.

[0034] The preparation process of the chemiluminescence substrate liquid of the present embodiment is as follows:

[0035] 1) Measure 800 mL of ultrapure water, add 6.507 g of tris-hydroxymethyl aminomethane, and adjust the pH to 8.8;

[0036] 2) Add 200 mg of (4-chlorophenylthio)(10-methyl-9,10-dihydroacridinemethyl) phosphonic acid disodium salt derivative to the solution obtained in step 1) and mix thoroughly;

[0037] 3) To the solution obtained in step 2), 1 mg of lusterine was added and mixed well;

[0038] 4) To the solution obtained in step 3), 0.2 g of proclin 300 was added and mixed well;

[0039] 5) To the solution obtained in step 4), 100 mg of sodium thiosulfate was added and mixed well;

[0040] 6) To the solution obtained in step 5), 0.2 g of propylene oxide and ethylene oxide copolymer and 0.5 g of sodium dodecyl sulfonate were added and mixed well;

[0041] 7) The solution obtained in step 6) was adjusted to pH 8.8 with dilute hydrochloric acid, diluted to 1 L, and 0.5 mg of antifoaming agent was added to obtain a chemiluminescent substrate solution, which was named AS01.

[0042] Example 2

[0043] The chemiluminescent substrate solution of this example comprises the following components:

[0044] 300 mg / L of (4-chlorophenylmercapto) (10-methyl-9, 10-dihydroacridinylmethylene) phosphonic acid disodium salt derivative, 200 mmol / L of tris-hydroxymethyl aminomethane, 5 mg / L of lusterine, 1 g / L of sodium dodecyl sulfonate, 1000 mg / L of sodium thiosulfate, 1 g / L of propylene oxide and ethylene oxide copolymer, and 0.05% by mass of BND-10, and the pH of the chemiluminescent substrate solution is 9.0.

[0045] The preparation process of the chemiluminescent substrate solution of this example is as follows:

[0046] 1) 800 mL of ultrapure water was measured, 24.228 g of tris-hydroxymethyl aminomethane was added, and the pH was adjusted to 9.0 with dilute hydrochloric acid;

[0047] 2) To the solution obtained in step 1), 300 mg of (4-chlorophenylmercapto) (10-methyl-9, 10-dihydroacridinylmethylene) phosphonic acid disodium salt derivative was added and mixed well;

[0048] 3) To the solution obtained in step 2), 5 mg of lusterine was added and mixed well;

[0049] 4) To the solution obtained in step 3), 0.5 g of BND-10 was added and mixed well;

[0050] 5) To the solution obtained in step 4), 1 g of sodium thiosulfate was added and mixed well;

[0051] 6) To the solution obtained in step 5), 1 g of propylene oxide and ethylene oxide copolymer and 1 g of sodium dodecyl sulfonate were added and mixed;

[0052] 7) The pH of the solution obtained in step 6) was adjusted to 9.0 with dilute hydrochloric acid, the volume was made up to 1 L, and 1 mg of antifoaming agent was added to obtain a chemiluminescent substrate solution, which was named AS02.

[0053] Performance test

[0054] The chemiluminescent substrate solutions AS01 and AS02 prepared in Examples 1 and 2, respectively, and a commercial APS-5 alkaline phosphatase substrate solution were subjected to sensitivity test and stability test, and the specific test process was as follows:

[0055] (I) Sensitivity test of chemiluminescent substrate solution

[0056] The sensitivity of the above three chemiluminescent substrate solutions was compared and analyzed by using a cTnI assay kit produced by Shenzhen Tiaolede Medical Technology Co., Ltd., and the specific operation steps were as follows: The cTnI calibrators and the magnetically micro-particle coated antibody and the alkaline phosphatase labeled antibody were mixed together for incubation, after 12 minutes of reaction, magnetic separation was performed, and the precipitate was washed, 120 uL of the substrate solution was added to the precipitate, mixed, and then 20 s was waited, and the relative luminescence value was measured. The test data are shown in Table 1 below:

[0057] Table 1 Sensitivity test data

[0058]

[0059] The "signal-to-noise ratio" is the ratio of the relative luminescence value when measuring the maximum calibration concentration of the calibrators to the relative luminescence value when measuring the zero concentration of the calibrators, and the higher the signal-to-noise ratio, the higher the sensitivity. From the test data in Table 1, it can be seen that the relative luminescence value of the commercial APS-5 alkaline phosphatase substrate solution when measuring the maximum calibration concentration of the calibrators is higher than that of AS01 and AS02, but the relative luminescence value when measuring the zero concentration of the calibrators is also significantly higher than that of AS01 and AS02. The signal-to-noise ratios obtained by measuring AS01 and AS02 are 2825 and 3604, respectively, which are higher than the signal-to-noise ratio 858 obtained by measuring the APS-5 alkaline phosphatase substrate solution, indicating that the chemiluminescent substrate solution in the present application has higher detection sensitivity.

[0060] (II) Stability test of chemiluminescent substrate solution

[0061] AS01, AS02 and the commercial APS-5 alkaline phosphatase substrate solution were tracked for 12 months of long-term stability on the cTnI platform, and the luminescence intensity was measured by using a VIT700 chemiluminescence analyzer produced by Shenzhen Tiaolede Medical Technology Co., Ltd., and the test data are shown in Table 2 below:

[0062] Table 2 Commercial APS-5 stability test data

[0063]

[0064] Table 3 AS01 stability test data

[0065]

[0066] Table 4 AS02 stability test data

[0067]

[0068] From the data in Table 2, it can be seen that the luminescence values of the commercial APS-5 alkaline phosphatase substrate solution of each concentration calibrator and mixed plasma decreased significantly during the 12-month storage period, with a maximum decrease of 49%; while the luminescence values of the AS01 and AS02 of the present application decreased significantly less, with a maximum decrease of 20% and 22% respectively in 12 months, indicating that the 12-month long-term stability of the chemiluminescent substrate solution of the present application is significantly better than that of the commercial APS-5 alkaline phosphatase substrate solution, which is beneficial to the preparation, transportation, storage and use of the substrate solution.

[0069] In summary, the chemiluminescent substrate solution of the above embodiments has high sensitivity and good stability, which is beneficial to improving the speed of clinical immune detection and shortening the detection time; at the same time, the chemiluminescent substrate solution of the present application has a simple formula, the components used are easy to obtain, the preparation method is simple, and it is suitable for large-scale production and popularization and application.

[0070] Each technical feature of the above-described embodiments can be combined arbitrarily, and in order to make the description concise, not all possible combinations of each technical feature in the above-described embodiments are described, however, as long as the combination of these technical features does not exist contradictory, it should be considered as the scope of the present disclosure.

[0071] The above-described embodiments only express several embodiments of the present application, which are described in more detail and in detail, but should not be construed as limiting the scope of the patent. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims.

Claims

1. A chemiluminescent substrate liquid, characterized in that, It comprises the following components: 200-300 mg / L of (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacrylamide) disodium phosphate derivative, 50-200 mmol / L of buffer matrix, 0.01-10 mg / L of fluorescence enhancer, 1-1000 mg / L of background inhibitor and surfactant; the pH of the chemiluminescent substrate solution is 7.5-10, the background inhibitor is an inorganic or organic reducing agent, and the fluorescence enhancer is a compound containing an aromatic group.

2. The chemiluminescent substrate liquid according to claim 1, characterized in that, The buffer matrix is ​​selected from one of tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1-propanol and 4-hydroxyethylpiperazine ethanesulfonic acid.

3. The chemiluminescent substrate liquid according to claim 1, characterized in that, The fluorescence enhancing material is an aromatic compound containing an aromatic heterocycle and at least one methyl substituent, and preferably one of gloss enhancer and ANS.

4. The chemiluminescent substrate liquid according to claim 1, characterized in that, The background inhibitor is preferably one of sodium sulfite, sodium thiosulfate, or TCEP.

5. The chemiluminescent substrate liquid according to claim 1, characterized in that, The surfactant is a mixture of nonionic and anionic surfactants. The nonionic surfactant is a polymer compound copolymerized from propylene oxide and ethylene oxide, and the anionic surfactant is sodium dodecyl sulfonate.

6. The chemiluminescent substrate liquid according to claim 1, characterized in that, It also includes a preservative with a mass percentage concentration of 0.01%-0.2%, wherein the preservative is selected from at least one of the NUS PC-300 and BND series.

7. A method for preparing a chemiluminescent substrate solution as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Measure 800 mL of ultrapure water, add 10-200 mmol of buffer matrix, and adjust the pH to the target pH value; S2. Add 200-300 mg of (4-chlorophenylmercapto)(10-methyl-9,10-dihydroacrylamide) disodium phosphate derivative to the solution obtained in step S1, and mix well; S3. Add 0.01-10 mg of fluorescence-enhancing material to the solution obtained in step S2 and mix well; S4. Add 0.1-2g of preservative to the solution obtained in step S3 and mix well; S5. Add 1-1000 mg of background inhibitor to the solution obtained in step S4 and mix well; S6. Add 0.01-5g of surfactant to the solution obtained in step S5 and mix well; S7. Adjust the pH of the solution obtained in step S6 to the target pH value, bring the volume to 1L, add 0.1-10mg of defoamer, and obtain the chemiluminescent substrate solution.

8. The preparation method according to claim 7, characterized in that, The reagent used to adjust the pH value in steps S1 and S7 is dilute hydrochloric acid, with a target pH value of 8.8-9.

0.

9. The application of the chemiluminescent substrate solution as described in any one of claims 1-6 in chemiluminescent immunoassay.

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