阅读说明：本技术 用于提高免疫荧光检测灵敏度的过滤膜前处理液及其制备方法与应用 (Filtering membrane pretreatment liquid for improving immunofluorescence detection sensitivity and preparation method and application thereof ) 是由 唐海燕 钟越 杨燕斌 于梦露 赵朝辉 于 2021-07-21 设计创作，主要内容包括：本发明公开了一种用于提高免疫荧光检测灵敏度的过滤膜前处理液及其制备方法与应用,用于提高免疫荧光检测灵敏度的过滤膜前处理液包括溶剂和溶质,所述溶剂为缓冲液Tris-HCL,所述Tris-HCL的浓度为50Mm/L-100Mm/L,所述溶质包括2g/L-6g/L的表面活性剂,1g/L-5g/L的PVP,3g/L-6g/L的PEG6000,20g/L-60g/L的糖类,30g/L-60g/L的惰性蛋白,1mg/L-5mg/L的异噬性抗体,1mg/L-5mg/L抗红细胞抗体,0.5g/L-1g/L的防腐剂。过滤膜前处理液具有提高微流控芯片检测的灵敏度的优点。(The invention discloses a filter membrane pretreatment liquid for improving immunofluorescence detection sensitivity and a preparation method and application thereof, the filter membrane pretreatment liquid for improving immunofluorescence detection sensitivity comprises a solvent and a solute, the solvent is buffer solution Tris-HCL, the concentration of the Tris-HCL is 50-100 Mm/L, the solute comprises 2-6 g/L of surfactant, 1-5 g/L of PVP, 3-6 g/L of PEG6000, 20-60 g/L of sugar, 30-60 g/L of inert protein, 1-5 mg/L of heterophagy antibody and 1-5 mg/L of anti-erythrocyte antibody, 0.5g/L-1g/L preservative. The pretreatment liquid for the filter membrane has the advantage of improving the detection sensitivity of the microfluidic chip.)

1. A pretreatment liquid for a filtration membrane for improving the immunofluorescence detection sensitivity, which is characterized in that: the solvent is buffer Tris-HCL, the concentration of the Tris-HCL is 50-100 Mm/L, and the solute comprises 2-6 g/L of surfactant, 1-5 g/L of PVP, 3-6 g/L of PEG6000, 20-60 g/L of carbohydrate, 30-60 g/L of inert protein, 1-5 mg/L of autophagy antibody, 1-5 mg/L of anti-erythrocyte antibody and 0.5-1 g/L of preservative.

2. The pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to claim 1, wherein: the surfactant is one or more of Triton X-100, Tween20, S10 and S11.

3. The pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to claim 2, wherein: the saccharide is one or more of glucose, sucrose, fructose and trehalose.

4. The pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to claim 3, wherein: the inert protein is one or more of BSA, casein and skimmed milk powder.

5. The pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to claim 4, wherein: the preservative is one or more of PC300 and sodium azide.

6. The pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to claim 4, wherein: the saccharide is glucose, the inert protein is BSA, and the ratio of glucose: PEG 6000: BSA ratio 10: 1: 10.

7. the pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to claim 6, wherein: the solute comprises: 6g/L Triton X-100, 5g/L PVP,6g/L PEG6000, 60g/L glucose, 60g/L BSA, 5mg/L heterophagic antibodies, 5mg/L anti-erythrocyte antibodies, 1g/L PC 300.

8. A method for preparing a pretreatment liquid for filtration membrane for use in enhancing immunofluorescence detection sensitivity according to any one of claims 1 to 7, comprising the steps of: adjusting the pH of the buffer Tris-HCL to 7.8-8.2, mixing and stirring surfactants PVP, PEG6000, saccharides, inert protein, heterophagic antibody and anti-erythrocyte antibody with the buffer Tris-HCL until dissolving.

9. Use of a pretreatment liquid for filtration membrane for improving immunofluorescence detection sensitivity according to any one of claims 1 to 7, wherein: and (3) putting the pre-treatment liquid into a filtering membrane, soaking for 5min-10min, taking out, horizontally placing, drying at 20-40 ℃ for 12h-24h, taking out, sealing and storing for later use.

Technical Field

The invention belongs to the technical field of microfluidic immunofluorescence detection, and particularly relates to a filtering membrane pretreatment liquid for improving immunofluorescence detection sensitivity, and a preparation method and application thereof.

Background

The microfluidic technology is a scientific technology which is mainly characterized by controlling fluid in a micron-scale space, and the technical core of the technology is a microfluidic chip. The microfluidic chip has the advantages of high detection speed, high flux, high sensitivity, low reagent consumption and the like, and is rapidly developed in recent years. In the 90 s of the 20 th century, a.mans et al put forward the concept of a micro total analysis system (microfluidic chip) using a chip, in which the functions of sample introduction, pretreatment, chemical reaction, separation, detection and the like of an analysis sample realize the miniaturization, automation and integration of equipment.

The detection principle of the microfluidic immunofluorescence detection chip is as follows: the whole blood passes through a filter membrane on the detection card, blood cells and plasma are separated, the plasma flows into a mixing area in the detection card, wherein the antigen or antibody in the plasma generates immunoreaction with the fluorescence labeling antibody in the mixing area, then the fluorescence antibody and the antigen or antibody in the plasma flow into a flow channel of the microstructure along with the reaction liquid to be captured by another antibody fixed in the flow channel, and the concentration of a substance to be detected is calculated by measuring the fluorescence intensity. The sample introduction and the pretreatment to the filtering membrane are the first two links of the micro-fluidic chip.

High sensitivity is one of the main advantages of microfluidic chips, but is also one of the biggest challenges. The detection of many projects in the market has extremely high sensitivity requirements, for example, the lower limit of BNP detection is 5pg, the lower limit of cTnI detection is 50pg, and the like, which has extremely high requirements on each link of the operation of the microfluidic chip. In order to improve the sensitivity of detection of microfluidic detection, researchers mainly start with changing an antibody crosslinking system, fluorescent materials and the like. The filter membrane plays an important part in the pretreatment of a sample, and the currently commonly used filter membrane only plays two roles of filtering red blood cells and promoting the flow of plasma. Early-stage researches in a laboratory show that a formula for pretreatment of a filter membrane has a key influence on the detection sensitivity of projects, and for the detection of projects with higher detection threshold values, such as MYO, the requirements can only be met by using a common filter membrane by methods such as optimizing a crosslinking system, but the common filter membrane is slightly poor for the projects with higher detection sensitivity, so that the development of a treatment formula of the filter membrane is very important.

Disclosure of Invention

In order to improve the detection sensitivity of the microfluidic chip, the invention provides a filtering membrane pretreatment liquid for improving the immunofluorescence detection sensitivity.

The filtering membrane pretreatment liquid for improving the immunofluorescence detection sensitivity comprises a solvent and a solute, wherein the solvent is buffer Tris-HCL, the concentration of the Tris-HCL is 50-100 Mm/L, the solute comprises 2-6 g/L of a surfactant, 1-5 g/L of PVP, 3-6 g/L of PEG6000, 20-60 g/L of a sugar, 30-60 g/L of an inert protein, 1-5 mg/L of an autophagic antibody, 1-5 mg/L of an anti-erythrocyte antibody and 0.5-1 g/L of a preservative.

By adopting the technical scheme, the buffer Tris-HCL provides the optimal PH and the optimal ionic strength of the immunoreaction of the antigen antibody in the plasma and the antibody antigen antibody coated on the chip bottom plate; the surfactant and PVP promote the flow of blood plasma, which is beneficial to the dispersion of fluorescent substances; the PEG6000 and the carbohydrate can enhance the reaction sensitivity of the antigen and the antibody; the inert protein can play a role in sealing the filtering membrane, reduce non-specific adsorption and enhance the reaction sensitivity of the antigen and the antibody; the antibody reduces the response to non-specific antigen; the anti-erythrocyte antibody improves the filtering efficiency of the erythrocyte, and reduces the interference of substances generated after the erythrocyte is broken to the detection; the preservative can prolong the storage time of various components of the pretreatment liquid.

Preferably, the surfactant is one or more of Triton X-100, Tween20, S10 and S11.

By adopting the technical scheme, one or more of Triton X-100, Tween20, S10 and S11 can control the plasma flow rate, but the effect of the Triton X-100 is optimal.

Preferably, the saccharide substance is one or more of glucose, sucrose, fructose and trehalose.

By adopting the technical scheme, one or more of glucose, sucrose, fructose and trehalose can enhance the antigen-antibody reaction sensitivity, but the use effect of the glucose is optimal.

Preferably, the inert protein is one or more of BSA, casein, skimmed milk powder and the like.

By adopting the technical scheme, one or more of BSA, casein, skimmed milk powder and the like can play a role in blocking the filtering membrane, reduce non-specific adsorption and enhance the reaction sensitivity of antigen and antibody, but the using effect of the BSA is optimal.

Preferably, the preservative is one or more of PC300 and sodium azide.

By adopting the technical scheme, one or more of PC300 and sodium azide can play a role of a preservative, but the using effect of the PC300 is optimal.

Preferably, the saccharide is glucose, the inert protein is BSA, and the ratio of glucose: PEG 6000: BSA ratio 10: 1: 10.

by adopting the technical scheme, the combination of three substances, namely BSA, PEG6000 and glucose can obviously improve the detection sensitivity to antigen and antibody in blood plasma, and when the trehalose: PEG 6000: BSA ratio 10: 1: the effect is best when the dosage is 10 days.

Preferably, the solutes comprise: 6g/L Triton X-100, 5g/L PVP,6g/L PEG6000, 60g/L glucose, 60g/L BSA, 5mg/L heterophagy antibodies, 5mg/L anti-erythrocyte antibodies, 1g/L PC 300.

By adopting the above technical scheme, when the concentrations of various solutes are the above concentrations, the detection result of the antigen-antibody in the plasma is optimal at this time.

A preparation method of a filter membrane pretreatment liquid for improving the immunofluorescence detection sensitivity comprises the following steps: adjusting the pH of Tris to a pH of 7.8-8.2, mixing and stirring solute surfactants PVP, PEG6000, saccharides, inert proteins, heterophagy antibodies, and anti-erythrocyte antibodies with Tris until dissolved.

By adopting the technical scheme, the pH value of Tris is firstly adjusted, detection signals are reduced when the pH value is too high or too low, and then the pretreatment liquid is prepared by mixing and stirring.

The application of the filter membrane pretreatment liquid for improving the immunofluorescence detection sensitivity comprises the following steps: and (3) putting the pre-treatment liquid into a filtering membrane, soaking for 5min-10min, taking out, horizontally placing, drying at 20-40 ℃ for 12h-24h, taking out, sealing and storing for later use.

By adopting the technical scheme, the filtering membrane is soaked in the pretreatment liquid for less than 5min, and the filtering membrane does not completely and uniformly absorb the solution, so that the difference between the filtering membranes in the same batch is large, and the detection repeatability is influenced; the filtering membrane excessively absorbs the solution to swell for more than 10min, and is partially dissolved in the solution to influence the shape of the filtering membrane, so that the shape of the filtering membrane is not suitable for the bottom plate; after drying, the dried blood plasma is stored, and the detection sensitivity of the microfluidic chip is enhanced through the filtering membrane of the pretreatment liquid, so that the detection of the antigen antibody in the blood plasma is more accurate.

In conclusion, the invention has the following beneficial effects:

1. the invention adopts Tris-HCL as solvent, and the solute comprises: surfactant, PVP, PEG6000, saccharides, inert protein, heterophagic antibody and anti-erythrocyte antibody preservative; the buffer solution Tris-HCL provides the optimal PH and the optimal ionic strength for the reaction of the antigen antibody to be detected in the anti-plasma and the antigen antibody coated on the chip bottom plate; the surfactant and PVP promote the flow of blood plasma, which is beneficial to the dispersion of fluorescent substances; the PEG6000 and the carbohydrate can enhance the reaction sensitivity of the antigen and the antibody; the inert protein can play a role in sealing the filtering membrane, reduce non-specific adsorption and enhance the reaction sensitivity of the antigen and the antibody; the antibody reduces the response to non-specific antigen; the anti-erythrocyte antibody improves the filtering efficiency of the erythrocyte, and reduces the interference of substances generated after the erythrocyte is broken to the detection; the preservative can prolong the storage time of various components of the pretreatment liquid.

2. The glucose in the invention: PEG 6000: BSA ratio 10: 1: 10, the three substances of BSA, PEG6000 and glucose have synergistic effect, so that the detection sensitivity can be obviously improved, and when the content of glucose: PEG 6000: BSA ratio 10: 1: when 10, the effect is best;

3. when the pretreatment liquid is applied, the filtering membrane is placed into the pretreatment liquid, the soaking is carried out for 5min-10min, the filtering membrane is taken out and horizontally placed for drying, the filtering membrane is taken out after being dried for 12h-24h at the temperature of 20-40 ℃, the filtering membrane is sealed and stored for later use, and the stability of the detection of the filtering membrane is improved through the soaked filtering membrane.

Detailed Description

The existing filtering membrane commonly used for immunofluorescence detection only plays a role in promoting the flow of a sample, and the potential value of the pretreatment link of the filtering membrane to the detection is ignored. The invention aims to develop a formula of a filter membrane pretreatment liquid for a filter membrane which is used for an immunofluorescence detection product on the market and takes glass fiber as a raw material, and the detection sensitivity of a target object can be improved when the filter membrane treated by the treatment liquid is used on a microfluidic chip; the adsorption of the glass fiber to a target object is reduced, the non-specific adsorption is reduced, and the detection CV is reduced; the blood plasma flowing speed is promoted, and the detection time length is shortened; efficient filtration of red blood cells reduces its impact on the assay.

The present invention will be described in further detail with reference to examples.

The raw materials of the invention are all commercially available.

Examples

Examples 1 to 7

The following example 1 illustrates a method for preparing a pretreatment liquid for a filtration membrane for improving the immunofluorescence detection sensitivity, which comprises the following steps:

(1) preparing a buffer solution Tris-HCL with the concentration of 50Mm/L, and adjusting the pH value by using hydrochloric acid with the concentration of 4M/L;

(2) weighing surface active agents PVP, PEG6000, saccharides, inert proteins, heterophagy antibodies and anti-erythrocyte antibodies in a clean beaker;

(3) then adding the buffer Tris-HCL into the beaker in the step (2), stirring for 20min on a magnetic stirrer at the stirring speed of 300-500rpm until the substances are completely dissolved, and continuing stirring for 10min after the volume is up to 100 ml.

The values of the solvent concentration, the solvent pH and the respective solute concentrations of examples 1 to 6 are shown in tables 1, 2 and 3.

Table 1 is a table of solvent concentration, solvent pH and solute concentrations for examples 1-5

	Example 1	Example 2	Example 3	Example 4	Example 5
						Buffer pH	7.8	7.9	8	8.1	8.2
Tris-HCL(mM/L)	50	60	70	80	100
						Triton X-100(g/L)	2	3	4	5	6
PVP(g/L)	1	2	3	4	5
						PEG6000(g/L)	3	3.5	4	5	6
Glucose (g/L)	20	30	40	50	60
						BSA(g/L)	30	40	45	50	60
Heterophagy antibody (g/L)	1	2	3	4	5
						Anti-erythrocyte antibody (g/L)	1	2	3	4	5
PC300(g/L)	0.5	0.6	0.8	0.9	1

Table 2 shows the solvent concentration, solvent pH and solute concentration of example 6

Table 3 shows the solvent concentration, solvent pH and solute concentration in example 7

	Example 6
		Buffer pH	8.2
Tris-HCL(mM/L)	100
		Triton X-100(g/L)	6
PVP(g/L)	5
		PEG6000(g/L)	6
Glucose (g/L)	60
		Skimmed milk powder (g/L)	60
Isophagocytic antibody (mg/L)	5
		Anti-erythrocyte antibody (mg/L)	5
PC300(g/L)	1

The surfactants of examples 1-6 may be replaced with one or more of Triton X-100, Tween20, S10, S11; the saccharide can be replaced by one or more of glucose, sucrose, fructose, and trehalose; the inert protein can be replaced by one or more of BSA, casein, skimmed milk powder, etc.

Comparative example

Comparative examples 1 to 2

The difference from example 1 is that: the solvent concentration, solvent pH and solute concentration are different. The values of solvent concentration, solvent PH and solute concentration for comparative examples 1-2 are shown in table 4.

Table 4 is a table of solvent concentration, solvent pH and solute concentration for comparative examples 1-2

	Comparative example 1	Comparative example 2
			Buffer pH	7	9
Tris-HCL(mM/L)	20	200
			Triton X-100(g/L)	1	10
PVP(g/L)	0.5	10
			PEG6000(g/L)	1	10
Glucose (g/L)	10	100
			BSA(g/L)	10	100
Isophagocytic antibody (mg/L)	0.5	10
			Anti-erythrocyte antibody (mg/L)	0.5	10
PC300(g/L)	0.1	5

Comparative example 3

The formula of the conventional pad fluid is 0.1-2% of surfactant, and the proportion of the surfactant adopted in the comparative example is 1%.

The application of the filter membrane pretreatment liquid for improving the immunofluorescence detection sensitivity comprises the following steps: putting the pretreatment liquid prepared in the examples 1-7 and the comparative examples 1-3 into a filtering membrane, and soaking for 5-10 min; then taking out the filtering membrane, flatly placing and drying, drying at 20-40 ℃ for 12-24 h, taking out, sealing and storing for later use. The soaking time, the drying temperature and the soaking time of examples 1 to 7 and comparative examples 1 to 3 are shown in Table 5.

TABLE 5 soaking duration, drying temperature and duration tables for examples 1-7 and comparative examples 1-3

Performance test

The pre-prepared solutions of examples 1-7 and comparative examples 1-3 were immersed in a filtration membrane for 5min-10min, dried at 20-40 ℃ for 12h-24h, and then placed on a microfluidic chip for testing, the results of which are shown in Table 6.

TABLE 6 inspection structure table for multiple filtration membranes

From Table 6, it can be seen that the results of the detection of the filtration membranes treated with the pretreatment liquids of examples 1 to 7 and comparative example 3 are: the fluorescence signal is enhanced, the background signal is weakened, the fluorescent substance is not agglomerated, the flowing time is relatively short, and the filtering membrane treated by the method can improve the flowing speed of the blood plasma and the detection sensitivity of the micro-fluidic chip.

According to Table 6, when the filter membrane treated with the pretreatment liquid of comparative examples 1 to 2 and examples 1 to 7 showed the results of detection, the pH of the treatment liquid was more than 8.2 or less than 7.8, the concentration of the buffer solution was less than 50mM/L or more than 100mM/L, the concentration of PVP was less than 1g/L or more than 5g/L, the concentration of PEG6000 was less than 3g/L or more than 6g/L, the concentration of saccharide was less than 20g/L or 60g/L, the concentration of inert protein was less than 30g/L or more than 60g/L, the concentration of the heterophagy antibody was less than 1ng/L or more than 5ng/L, the concentration of the anti-erythrocyte antibody was less than 1ng/L or more than 5ng/L, and the concentration of the preservative was less than 0.5g/L or more than 1g/L, the detection result shows that the fluorescence signal is weakened, the background signal is enhanced, the fluorescent substance is agglomerated, and the flowing time is prolonged.

As is apparent from tables 1 and 2, in example 5, the conditions were the same as in example 6 except that glucose was replaced with trehalose, and from table 6, the fluorescence signal was decreased and the background signal was increased as compared with the detection results of the filtration membrane treated with the pretreatment liquid in example 6 and example 5.

As is apparent from tables 1 and 3, in example 5, the conditions were the same as in example 7 except that the skim milk powder was used instead of BSA, and from table 6, the fluorescence signal was decreased and the background signal was increased as compared with the detection results of the filtration membrane treated with the pretreatment liquid in example 7 and example 5.

Therefore, when the saccharide in the current treatment solution is glucose and the inert protein is BSA, the detection result is optimal, and the main principle is that the detection effect of the filter membrane is improved together due to the synergistic effect of the glucose, the BSA and the PEG 6000.

As is clear from Table 6, in examples 1 to 3, compared with the results of the tests in examples 4 to 5, the ratio of glucose: PEG 6000: the concentration ratio of BSA was 10: 1: at 10, the fluorescence signal is optimal.

As can be seen from Table 6, the solutes of examples 1 to 5 are the same in raw material, but when the solutes are in the following proportions: 6g/L Triton X-100, 5g/L PVP,6g/L PEG6000, 60g/L glucose, 60g/L BSA, 5mg/L heterophagic antibody, 5mg/L anti-erythrocyte antibody and 1g/L PC300, the detection result shows that the fluorescence signal is strongest, the background signal is lowest, the fluorescent substance is not agglomerated, the flowing time is low, and the mixture ratio is the optimal mixture ratio of the invention.

The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.