CN117740765A - Cleaning liquid suitable for chemiluminescent system and preparation method and application thereof - Google Patents

Abstract

The invention discloses a cleaning solution suitable for a chemiluminescent system, which comprises buffer solution in every 1000mL of cleaning solution: 300-600mM; chloride salt: 300-600mM; and (2) a surfactant: 0.1-0.3mL; preservative: 0.05-0.2g; defoaming agent: 0.2-1.0mL; the balance being water. The cleaning solution suitable for the chemiluminescent system provided by the invention has the advantages that the 2-hydroxypyridine-N-oxide is used as the preservative, the preservative has low cost, is convenient to use, has a good antiseptic and antibacterial effect, has no obvious adverse effect on animals and human bodies, has less environmental pollution, has an excellent cleaning effect, ensures the stability and accuracy of detection results, and can be popularized and used on a large scale.

Description

Cleaning liquid suitable for chemiluminescent system and preparation method and application thereof

Technical Field

The invention belongs to the technical field of chemiluminescent immunoassay, and particularly relates to a cleaning solution suitable for a chemiluminescent system, a preparation method and application thereof, and further relates to magnetic bead cleaning for an acridine ester direct chemiluminescent detection reagent and system cleaning for an acridine ester direct chemiluminescent detection instrument.

Background

The chemiluminescence technology combines magnetic particle separation, chemiluminescence and immunoreaction, has the characteristics of wide application range, high sensitivity, good specificity and the like, and is getting more attention in various fields. Depending on the light-emitting system, chemiluminescence can be classified into direct chemiluminescence immunoassay, indirect (enzymatic) chemiluminescence immunoassay, and electrochemiluminescence immunoassay techniques. The direct chemiluminescence is an immunoassay method for directly labeling antigens or antibodies through a chemiluminescent agent, and the method has the characteristics of high detection sensitivity, low background interference and the like. The more commonly used chemiluminescent agents are acridine esters compounds, the compounds can directly label antibodies or antigens, then react with the corresponding antigens or antibodies in a sample to be detected and the antibodies or antigens coated by magnetic particles, the free state and the combined state luminescent markers are separated through a magnetic field, finally strong visible light is rapidly emitted under the action of strong alkali excitation liquid containing hydrogen peroxide, and qualitative or quantitative detection can be carried out through determination of luminous relative light intensity.

In general, the steps of chemiluminescent technology mainly include sample addition, incubation, washing, excitation and reading of luminescence values. Wherein washing is one of the key steps of detection, and the purpose of the step is to put the mixture after the reaction into a magnetic separation system for treatment so as to clean impurities and excessive reagents and reduce the influence of non-specific interfering substances on detection results; meanwhile, the cleaning liquid can provide a proper buffer environment for the reaction, and plays a key role in the accuracy, sensitivity and precision of the test result. In addition, the cleaning liquid is also of great significance to the maintenance of the pipeline and the sample adding needle of the chemiluminescent immunoassay analyzer.

The existing cleaning liquid on the market is of various types, wherein the imported original cleaning liquid can ensure the better cleaning effect and the stability and consistency of the inspection result, but the cleaning liquid is often expensive, and the cleaning liquid used by most factories (such as Siemens and the like) contains dangerous chemical reagents such as sodium azide, which can block the electron transfer chain of cells, have toxicity to a plurality of organisms and are convenient to transport, use, treat waste liquid and the like. The self-prepared cleaning solution of some domestic reagent companies has low price, but most cleaning solutions have poor cleaning effect, poor result accuracy and stability and over-high luminous background value, and most domestic cleaning solutions depend on chemical compositions imported from abroad in large quantity, so that the cost is increased and the risk of raw material outage is also caused at any time.

CN113484306a discloses a magnetic particle chemiluminescence cleaning solution, a preparation method and application thereof, and the components of the magnetic particle chemiluminescence cleaning solution comprise PBS buffer solution, sodium ions, disodium ethylenediamine tetraacetate, SDS, tween-20, sodium azide and silicone oil type defoamer. The sodium azide is a high-risk chemical reagent, has high requirements on transportation, storage, use and waste liquid treatment, and has high use cost. SDS has a stimulating effect on mucous membrane and upper respiratory tract of human, has a stimulating effect on eyes and skin, can cause allergic reaction of respiratory system, also needs training personnel when in use, brings danger and increases cost.

CN107118865a discloses a cleaning solution, which uses sodium cholate as a surfactant, and this component has the disadvantages of strong washing capability, capability of dissolving cell membranes, easy washing away of antigens or antibodies which are specifically bound in the detection process, and easy occurrence of non-specific reaction during immunodetection, and sodium cholate contains nitrogen-hydrogen bonds with reactivity. In addition, the patent only uses Proclin series preservative which is safer and cheaper than sodium azide, but has far inferior preservative effect compared with sodium azide, and particularly is easy to precipitate out after long-time storage, thus influencing the cleaning effect.

In chemical cleaning solutions, a certain amount of preservative is often required to be added in order to ensure the accuracy and stability of the measured result, but in most cleaning solutions on the market at present, the used preservative either contains extremely toxic substances (such as sodium azide) or lacks stability, and is easy to lose efficacy when stored for a long time; in order to solve the problems, a plurality of preservatives are matched for use in the cleaning liquid formula so as to achieve a better anti-corrosion effect, but the method often causes the cleaning liquid to be more troublesome to prepare, and the risks of increased cost and broken raw material supply are faced. Therefore, it is very important to find a preservative which is low in price, good in preservative effect, single in component and high in localization degree.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the main purpose of the invention is to provide a cleaning solution which has low cost, simple components, good anti-corrosion effect and high localization degree and is suitable for a chemiluminescent system; the invention also provides a preparation method and application of the chemiluminescent cleaning liquid.

The invention aims at realizing the following technical scheme:

a wash solution suitable for use in a chemiluminescent system, comprising buffer solution per 1000mL of wash solution: 300-600mM; chloride salt: 300-600mM; and (2) a surfactant: 0.1-0.3mL; preservative: 0.05-0.2g; defoaming agent: 0.2-1.0mL; the balance being water.

Preferably, the buffer is one of sodium phosphate buffer or Tris-HCl buffer.

Preferably, the chloride salt is one of potassium chloride or sodium chloride.

Preferably, wherein the surfactant is one of tween-20 or tween-80.

Preferably, wherein the preservative is 2-hydroxypyridine-N-oxide.

Preferably, wherein the defoamer is a silicone defoamer.

The preparation method of the cleaning liquid suitable for the chemiluminescent system comprises the following steps:

1) Preparing a buffer solution with the pH of 6.5-7.5;

2) According to the formula requirement, adding chloride, preservative, surfactant and defoamer into buffer solution, and using water to fix the volume to 1000mL to obtain the cleaning agent.

Compared with the prior art, the invention has at least the following advantages:

the cleaning solution suitable for the chemiluminescent system provided by the invention has the advantages that the 2-hydroxypyridine-N-oxide is used as the preservative, the preservative has low cost, is convenient to use, has a good antiseptic and antibacterial effect, has no obvious adverse effect on animals and human bodies, has less environmental pollution, has an excellent cleaning effect, ensures the stability and accuracy of detection results, and can be popularized and used on a large scale.

Detailed Description

The invention will now be further described in detail with reference to the following specific examples, which are intended to be illustrative only and not limiting in any way.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as an upper range limit, or as a lower range limit, it is to be understood that any range is specifically disclosed by combining any pair of the upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise indicated, the numerical range values set forth herein are intended to include the endpoints of the range, and all integers and fractions within the range.

All percentages, parts, ratios, etc. herein are by weight unless otherwise specified.

The materials, methods, and examples herein are illustrative and, unless otherwise indicated, should not be construed as limiting. Example 1

The cleaning solution suitable for the chemiluminescent system is prepared by the following preparation method:

preparing 300mM buffer solution (sodium phosphate buffer solution) with pH of 7.0; then 600mM chloride, 0.05g preservative, 0.1mL surfactant and 0.3mL defoamer are added, and water is used for constant volume to 1000mL, thus obtaining chemiluminescent cleaning liquid;

example 2

The cleaning solution suitable for the chemiluminescent system is prepared by the following preparation method:

preparing 500mM buffer (Tris-HCl buffer) with pH of 7.0; then adding 500mM chloride, 0.1g preservative, 0.2mL surfactant and 0.5mL defoamer, and fixing the volume to 1000mL with water to obtain chemiluminescent cleaning liquid;

example 3

The cleaning solution suitable for the chemiluminescent system is prepared by the following preparation method:

600mM buffer (sodium phosphate buffer) at pH 7.0 was prepared; then adding 300mM chloride, 0.2g preservative, 0.3mL surfactant and 0.5mL defoamer, and fixing the volume to 1000mL with water to obtain chemiluminescent cleaning liquid;

comparative example 1:

this comparative example is essentially the same as example 2 except that the 2-hydroxypyridine-N-oxide is replaced with sodium azide.

Comparative example 2

This comparative example is essentially the same as example 2 except that 2-hydroxypyridine-N-oxide is replaced with Proclin 300.

Test example:

the present application takes example 2 and/or comparative examples 1-3 as examples to perform performance tests on the obtained chemiluminescent cleaning solution, and simultaneously introduces similar products with large use amount on the market at present as comparative examples, namely original cleaning solution suitable for being used by weight, celebration biological technology, inc. model number SMART 6500S.

The method comprises the following steps:

1) Accuracy verification

The present application selects 3 representative items of significant clinical significance, namely KIM-1 (kidney injury molecule 1), IGFBP7 (human insulin-like growth factor binding protein 7) and IS (indoxyl sulfate), each of which was visualized as 10 samples. The chemiluminescent washing solution of example 2 was diluted 10-fold, and after the original washing solution of the SMART 6500S chemiluminescent analyzer of Chongqing Ke-Si Biotechnology Co., ltd was diluted a specified number of times, a corresponding procedure was started for detection to verify the accuracy of the chemiluminescent washing solution of the present invention. The experimental results are shown in table 1:

TABLE 1 accuracy of chemiluminescent washing solutions in example 2 and original washing solution (control)

As is clear from Table 1, the chemiluminescent washing liquid of example 2 showed a deviation of not more than 5% from the original washing liquid, indicating that the chemiluminescent washing liquid was more accurate and reached the same level as the original washing liquid.

2) Precision verification

Two batches of cleaning solutions were prepared according to the method of example 2, namely a first batch (abbreviated as example 2-1) and a second batch (abbreviated as example 2-2), and the original cleaning solution and the prepared two batches of cleaning solutions were diluted in respective proportions and the three cleaning solutions were measured by using a SMART 6500S chemiluminescent analyzer, a company of Chongkesimai biotechnology, respectively. The test items were KIM-1 (kidney injury molecule 1), IGFBP7 (human insulin-like growth factor binding protein 7) and IS (indoxyl sulfate), respectively, each item tested one sample (repeated 20 times), and the precision within the batch of each sample and the precision between the batches of two self-assembled samples were calculated, respectively. The results are shown in Table 2:

table 2 for each batch of example 2, the chemiluminescent washing liquid of the control was tested for accuracy

As can be seen from the data in Table 2, the cleaning solution of the present invention has good detection precision and reactivity, and the precision in batch and the precision between batches can be controlled within 2%, which is even better than the original cleaning solution to some extent. And meets the requirement that the intra-batch variation coefficient of the common diagnostic reagent is less than 10 percent and the inter-batch variation coefficient is less than 15 percent.

3) Stability verification

10 bottles of 1L of each of the washing liquid concentrates of example 2, comparative example 1 and comparative example 2 were stored at room temperature together with the corresponding original washing liquid (comparative example), and were subjected to measurement after being left for 0, 1, 3, 6, 9, 12, 18, 24, 32 and 36 months, diluted according to the corresponding proportions before the measurement, and then subjected to washing liquid long-term blank stability identification and KIM-1 sample measurement with a concentration of 30pg/mL by using a SMART 6500S chemiluminescent analyzer of Chongke Kelvin biological technology Co., ltd.) for four washing liquids, and the measurement was repeated ten times to observe the Coefficient of Variation (CV) thereof, and the results are shown in tables 1, 2, 3 and 4:

TABLE 1 detection of Long-term stability of wash appearance

As is clear from the data in Table 3, except for example 2 of the present application, the other cleaning solutions were changed in physical properties at the later stage of storage, for example, the color of comparative example 2 was changed due to the inclusion of Proclin 300, and a small amount of precipitate was deposited; the original washing liquid and the comparative example 1 also have obvious turbidity in the later period, which suggests that the original washing liquid and the comparative example 1 can be polluted by miscellaneous bacteria, so that the washing liquid can be invalid; the physical properties of the cleaning solution of the present invention (example 1) were not changed when it was stored for 36 months, indicating that the cleaning solution of the present invention has longer stability than the cleaning solution of the remaining comparative examples or the conventional original cleaning solution.

32 Test results of long-term stability of cleaning solution on blank sample

TABLE 4 test results of long-term stability of cleaning fluids on blank samples

As can be seen from the data in Table 4, the blank luminescence value of comparative example 2 at 12 months was already over 500, and the blank luminescence values of the original cleaning liquid and comparative example 1 were also over 500 at 24 months, whereas the blank luminescence value of inventive example 2 was kept low after being stored at room temperature for over 36 months; meanwhile, the comparison of CV values also shows that the CV values of the other cleaning fluids except for the example 2 of the present invention are all larger and larger in the long-term storage process and are more than 10% in the later period, but the example 2 does not show the phenomenon, and the CV value at 36 months can be kept at 5%. The change in the post-period of these cleaning fluids is suspected to be caused by contamination after long-term storage in combination with the change in physical properties that occurs after long-term storage of the remaining cleaning fluids.

33 30pg/mL KIM-1 sample long-term detection results

TABLE 5 30pg/mL KIM-1 sample Long-term detection results

As can be seen from the data of the KIM-1 sample in Table 5, the CV values of the original cleaning solution (control group), the cleaning solutions in comparative example 1 and comparative example 2 are all larger and larger in the long-term storage process, while the formulation of the cleaning solution in example 2 can still maintain CV values below 5% at 36 months, which indicates that the cleaning solution has strong repeatability and high stability. In summary, the cleaning solution of the application has the advantages that after the 2-hydroxypyridine-N-oxide is adopted to replace the preservative commonly used in the existing cleaning solution, the corrosion resistance is improved, and meanwhile, the cleaning performance and the stability are better than those of the existing cleaning solution.

Compared with other preservatives, the preservative has the advantages of simple components, low cost, convenient use, good preservative effect and the like, has no obvious adverse effect on animals and human bodies, has relatively less environmental pollution, and ensures that the cleaning liquid has good cleaning effect after long-time storage.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.

Claims (7)

1. A wash solution suitable for use in a chemiluminescent system, comprising, per 1000mL of wash solution, a buffer solution: 300-600mM; chloride salt: 300-600mM; and (2) a surfactant: 0.1-0.3mL; preservative: 0.05-0.2g; defoaming agent: 0.2-1.0mL; the balance being water.

2. The wash solution for chemiluminescent system of claim 1 wherein the buffer is one of a sodium phosphate buffer or Tris-HCl buffer.

3. The cleaning solution for chemiluminescent system of claim 1 wherein the chloride salt is one of potassium chloride or sodium chloride.

4. The cleaning solution for chemiluminescent system of claim 1 wherein the surfactant is one of tween-20 or tween-80.

5. The cleaning solution for chemiluminescent system of claim 4 wherein the preservative is 2-hydroxypyridine-N-oxide.

6. The cleaning solution for chemiluminescent system of claim 5 wherein the defoamer is a silicone defoamer.

7. A method for preparing a cleaning solution suitable for use in chemiluminescent systems according to any one of claims 1-6 comprising the steps of:

1) Preparing a buffer solution with the pH of 6.5-7.5;

2) According to the formula requirement, adding chloride, preservative, surfactant and defoamer into buffer solution, and using water to fix the volume to 1000mL to obtain the cleaning agent.