CN115541893B

CN115541893B - Saccharification detection card film treatment method

Info

Publication number: CN115541893B
Application number: CN202110734283.2A
Authority: CN
Inventors: 谭中; 华一民; 李俊; 邹丽洁; 林滔; 彭伟
Original assignee: WUXI BOHUISI BIOLOGICAL MEDICINES TECHNOLOGY CO LTD
Current assignee: WUXI BOHUISI BIOLOGICAL MEDICINES TECHNOLOGY CO LTD
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2023-10-24
Anticipated expiration: 2041-06-30
Also published as: CN115541893A

Abstract

The invention relates to the technical field of blood glucose monitoring equipment, and provides a saccharification detection card film treatment method, which comprises the following steps: step one, membrane activation reaction; step two, pretreatment reaction; step three, membrane treatment one; fourthly, membrane treatment II, namely putting the membrane subjected to the first membrane treatment into a second membrane treatment reaction solution; and fifthly, drying. The invention overcomes the defects of the prior art, has reasonable design and compact structure, utilizes the porous PE film treated by water as a base material, and has the advantages of simple whole processing process, no pollution, high processing efficiency, thorough reaction and strong practicability through film activation reaction, pretreatment reaction, film treatment I, film treatment II and drying, the five-step physicochemical reaction is used for functionalizing the porous PE film, and the functionalized porous PE film is assembled into a detection card.

Description

Saccharification detection card film treatment method

Technical Field

The invention relates to the technical field of blood glucose monitoring equipment, in particular to a saccharification detection card film treatment method.

Background

Blood sugar monitoring is an important link in daily diagnosis and treatment of diabetes, and good blood sugar control can effectively delay occurrence and development of acute and chronic complications of diabetes.

Currently, there are a wide variety of HbA1c (glycosylated hemoglobin) measurement methods used clinically and in the laboratory, and they can be classified into two types based on the principle: (1) Based on the difference in charge between HbA1c and non-glycosylated hemoglobin, the main methods are ion exchange and electrophoresis; (2) Based on the structural differences between HbA1c and non-glycosylated hemoglobin, the structural differences are mainly derived from glycosylation sites, and the main methods are immunological methods and affinity chromatography methods.

Liquid chromatography is a currently internationally recognized method for measuring HbA1c, and is also used in a large amount clinically. Although the method is stable, has reference value and good linearity, the method has the defects of expensive equipment, huge instrument, long detection time and inconvenience for wide use, and needs professional operators to operate.

The electrophoresis method uses the principle that hemoglobin samples with different charges migrate at different speeds on a detection card (electrophoresis gel) to measure HbA1c. Because of the complex operation of electrophoresis, the time for obtaining results is long, and the method is rarely applied to clinic. The immune method is based on the principle of specific binding of antibodies and antigens, wherein the lateral immune chromatography test paper takes fluorescent quantum dots/fluorescent microspheres/gold nanoparticles as signal carriers, and takes signals of a detection line and a quality control line as detection results, so that the method is suitable for departments and families with rapid diagnosis requirements, but has small linear range, is easily interfered by hemoglobin variants and HbA1c precursors, and is greatly limited in application.

The affinity chromatography is based on the adsorption capacity of boric acid groups on polyhydroxy substances, the boric acid groups in the affinity chromatography column are firstly combined with cis-diol of HbA1c glycosylation sites, then the part flowing out of the column is non-glycosylated hemoglobin (the concentration is judged by ultraviolet-visible absorption spectrum), then the polyhydroxy substances such as Tris, mannitol, sorbitol and the like are used for competing and eluting HbA1c on the column, and finally the part flowing out of the column is HbA1c. The measurement of HbA1c by the affinity chromatography is also popular in clinic, and the affinity chromatography is not affected by many hemoglobin variants. The presence of HbA1c precursor has no significant effect on the results of the measurement, and the blood sample does not need to be pretreated to remove HbA1c precursor.

However, the detection card membrane of the current affinity chromatography column method is expensive, complex to operate and requires a long time to obtain the result.

In both the ion exchange method and the affinity chromatography method, hbA1c is detected by a saccharification detection card, and the saccharification detection card reacts with HbA1c through a membrane on the saccharification detection card to realize detection, but the traditional saccharification detection card membrane treatment method has higher cost, complex process flow and longer time for obtaining a result.

For this reason, we propose a saccharification detection card membrane treatment method combining two methodologies of ion exchange and affinity chromatography.

Disclosure of Invention

(one) solving the technical problems

Aiming at the defects of the prior art, the invention provides a saccharification detection card film treatment method, which overcomes the defects of the prior art, has reasonable design and compact structure, utilizes a porous PE film treated by water as a base material, and has the advantages of simple whole processing process, no pollution, high processing efficiency, complete reaction and strong practicability through film activation reaction, pretreatment reaction, film treatment I, film treatment II and drying.

(II) technical scheme

In order to achieve the above purpose, the invention is realized by the following technical scheme:

the saccharification detection card film treatment method comprises the following steps:

step one, membrane activation reaction

Plasma activation treatment is adopted;

preparing a porous PE film, fixing the porous PE film on a treatment rack, and operating a low-temperature plasma treatment system to perform film activation reaction;

step two, pretreatment reaction

Step two (1) PEI reaction

Placing the membrane subjected to the membrane activation reaction into a shaking table containing PEI reaction liquid, reacting for 3 minutes, and then cleaning and drying for multiple times;

step two (2) polyacrylic acid reaction

Placing the PEI reacted film obtained in the step two (1) into a shaking table containing polyacrylic acid reaction liquid, reacting for 8 minutes, then cleaning for multiple times, and drying for 25 minutes at 95 ℃;

step three, film treatment one

Soaking the membrane subjected to the pretreatment reaction in a membrane treatment reaction liquid, performing sealing treatment, and simultaneously heating at 60 ℃ for reaction for 2 hours;

then cleaning for multiple times, and drying for 25 minutes by blowing at 95 ℃;

step four, film treatment two

Placing the membrane subjected to the first membrane treatment into a second membrane treatment reaction solution, and simultaneously starting a shaking table with the speed of 150r/min for reaction for 10min;

then putting the membrane into MES buffer solution for cleaning for a plurality of times;

step five, drying treatment

And (3) drying the film treated in the step four, wherein the drying time is not less than 12 and h.

Further, the plasma treatment conditions in the first step are as follows: background air pressure 20Pa, oxygen flow 100sccm, discharge power 500W, and discharge time 300s.

Further, in the first step, a water climbing test needs to be performed on the porous PE film, and a specific operation flow is as follows: before the first step, marking small strips with a certain area on the porous PE film, marking the corresponding positions at the two ends of the small strips by scribing respectively, cutting the marked small strips along the marking lines after the first step, putting the marked ends at the two ends of the small strips into purified water, and recording the time of the purified water penetrating from the lower end of the film to the marking height.

Further, the PEI reaction solution in the second step (1) comprises the following components:

35-50 parts of PEI stock solution, 3800-4200 parts of purified water and 3000-3200 parts of ethanol.

Further, the polyacrylic acid reaction solution in the step two (2) comprises the following components:

15-25 parts of polyacrylic acid stock solution, 6500-7000 parts of purified water and 900-920 parts of ethanol.

Further, the membrane treatment one reaction liquid in the third step comprises the following components:

25-30 parts of phthalic anhydride, 0.5-1 part of triethanolamine hydrochloride and 800-850 parts of DMSO;

and the preparation process of the membrane treatment reaction liquid comprises the following steps: DMSO was added to the solid mixture of phthalic anhydride and triethanolamine hydrochloride and the container was sealed with a preservative film and stirred with a magnetic stirrer to completely dissolve the solids.

Further, the fourth step and the second film treatment further include cutting:

the membrane after the first reaction of membrane treatment is cut into a plurality of membrane strips with equal width according to the set size, and the membrane strips are sequentially and orderly put into the second reaction liquid of membrane treatment, so that continuous observation is needed in the reaction process, and the membrane strips are required to be stirred from time to time.

Further, the second reaction solution for membrane treatment in the fourth step comprises the following components:

1-1.5 parts of m-aminophenylboronic acid, 8-10 parts of methanol and 4600-4900 parts of MES buffer solution;

and the preparation process of the membrane treatment two reaction liquid comprises the following steps: 1 to 1.5 parts of m-aminophenylboronic acid are weighed into a mortar, 8 to 10 parts of methanol is added, grinding and dissolving are carried out, then 4600 to 4900 parts of MES buffer solution is weighed, the methanol solution of m-aminophenylboronic acid is diluted by the MES buffer solution, and the diluted methanol solution is poured into a special bottle for preparing a membrane treatment two-reaction solution.

Furthermore, in the fourth step, 1.2-1.5 parts of EDC needs to be added into the second membrane treatment reaction solution before the second membrane treatment reaction is performed, and the EDC is quickly and uniformly mixed, so that the second membrane treatment reaction is started within 3 minutes after the EDC is added into the second membrane treatment reaction solution.

Further, the MES buffer comprises the following components:

200-220 parts of MES, 9800-10000 parts of purified water and 1000-1500 parts of sodium hydroxide solution, and the pH value of the MES buffer solution is controlled between 6.3 and 6.7.

(III) beneficial effects

The embodiment of the invention provides a saccharification detection card film processing method. The beneficial effects are as follows:

1. the invention uses the porous PE film treated by water as the base material, and the porous PE film is functionalized by the five-step physicochemical reaction through the film activation reaction, the pretreatment reaction, the first film treatment, the second film treatment and the drying, and the functionalized porous PE film is assembled into the detection card, so that the quick measurement of glycosylated hemoglobin can be realized, and the whole processing process is simple.

2. PEI is uniformly dissolved into a solution by PEI reaction, and the PEI film is modified by utilizing the inherent property of PEI and the reaction of the activated film, so that PE has chemical activity.

3. The polyacrylic acid reaction is adopted, and the PE film of the grafted PEI has insufficient hydrophilicity, so that the polyacrylic acid is grafted on the PE film by the reaction (amide condensation reaction) of the polyacrylic acid and the amino group on the PEI, a large amount of carboxyl groups are introduced, the PE film has very good hydrophilicity, and meanwhile, the large amount of carboxyl groups are introduced into the PE film to be in actual use (1. The hydrophilicity is increased, 2. The effect of combining hemoglobin is exerted in the actual use process).

4. By carrying out a water climbing test on the original film, the specific operation flow is as follows: before the first step, marking a small strip with a certain area on the original film, marking the corresponding positions at the two ends of the small strip by scribing, cutting the marked small strip along a marking line after the first step, putting the marked ends at the two ends of the small strip into purified water, and recording the time of the purified water penetrating from the lower end of the film to the marking height.

5. Through cutting the membrane after a reaction of membrane treatment into a plurality of membrane strips with equal width according to the size that sets for, a plurality of membrane strips are put into membrane treatment two reaction liquid in proper order, and in the reaction process, need continuously observe, and need stir the membrane strip from time to time, can improve the efficiency of reaction, also can not cause extravagant simultaneously, pollution abatement, environmental protection more.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

step one, membrane activation reaction

Climbing water sample mark

A porous PE film was sampled, the strips were marked along the edges with a pencil and a graduated scale, and scored at 1cm and 0.5cm each of the ends of the strips.

Plasma activation treatment

Checking plasma processing conditions, wherein the plasma processing conditions are as follows: the air pressure is 20Pa, the oxygen flow is 100sccm, the discharge power is 500W, and the discharge time is 300s.

And (3) operating a low-temperature plasma treatment system to perform membrane activation reaction, and activating the inert PE membrane by adopting a physical and chemical method through plasma treatment to enable the inert PE membrane to have reaction sites and to start to participate in grafting reaction.

Climbing test

Cutting off the marked strips along a marking line, putting the marked ends of the strips into purified water, and recording the time for the purified water to permeate from the lower end of the membrane to the marked height.

Step two, pretreatment reaction

Step two (1) PEI reaction

Placing the membrane into a shaking table containing PEI reaction liquid, reacting for 3 minutes, then washing twice with an ultrasonic instrument for 3 minutes each time, sucking water on the membrane by filter paper, adopting PEI reaction to uniformly dissolve PEI into the solution, and utilizing the inherent property of PEI and the activated membrane reaction to modify the PE membrane so that the PE has chemical activity.

Step two (2) PEI solution preparation

40.0 parts of PEI stock solution, 4000 parts of purified water and 3156 parts of ethanol are weighed, the weighed PEI stock solution is dissolved and diluted by the purified water and the ethanol, and the mixture is fully and uniformly mixed by magnetic stirring for later use.

Polyacrylic acid reaction

The membrane is put into a shaking table containing polyacrylic acid reaction liquid to react for 8 minutes, then the membrane is washed twice by an ultrasonic instrument for 3 minutes each time, dried for 25 minutes at 95 ℃, cooled in a drying room, polyacrylic acid reaction is adopted, and the PE membrane of the grafted PEI has insufficient hydrophilicity, so that polyacrylic acid is grafted on the PE membrane by the reaction of polyacrylic acid and amino groups on the PEI (amide condensation reaction), a large amount of carboxyl groups are introduced, the PE membrane has very good hydrophilicity, and meanwhile, a large amount of carboxyl groups are introduced into the membrane to meet the actual use requirement (1. The hydrophilicity is increased, 2. The effect of combining hemoglobin is exerted in the actual use process).

The polyacrylic acid solution is prepared and weighed into 20.0 parts of polyacrylic acid stock solution, 6800 parts of purified water and 915 parts of ethanol, the purified water and the ethanol are used for dissolving and diluting the weighed polyacrylic acid stock solution, and the polyacrylic acid stock solution is fully and uniformly mixed by magnetic stirring for later use.

Step three, film treatment one

Pouring the film treatment reaction liquid into a rectangular glass basin, stacking and immersing the film layers in the reaction liquid, sealing the reaction container by using a preservative film, and heating at 60 ℃ for reaction for 2 hours.

The mixture is washed by shaking with a small amount of water for 5-10 minutes, then washed twice by an ultrasonic instrument, dried by blowing at 95 ℃ for 25 minutes, and cooled in a drying room.

The membrane which is treated by plasma and reacted by PEI and acrylic acid has good hydrophilicity, but the number of carboxyl (-COOH) grafted on the membrane is seriously insufficient, and the first membrane treatment process is adopted, dianhydride is used for reacting with amino (mainly secondary amino, namely-NH-) on the membrane, so that the amino is blocked and more carboxyl is introduced, thereby improving the capacity of membrane to combine with hemoglobin under weak acid condition (ion exchange method), and achieving the functionalization effect.

Preparing a reaction solution for membrane treatment

30.0 parts phthalic anhydride, 0.75 parts triethanolamine hydrochloride, and 825 parts DMSO were weighed, DMSO was added to the solid mixture, the container was sealed with a preservative film, and the solid was completely dissolved (if not completely dissolved, dissolution was accelerated by ultrasound or heating) with stirring by a magnetic stirrer.

Step four, film treatment two

since the first film treatment is an excessive reaction, the surface of the film on which the first film treatment is completed is substantially all carboxyl groups. The glycosylated hemoglobin can be coordinately combined with the boric acid groups under alkaline condition (affinity chromatography), so that partial boric acid groups are grafted on the membrane, so that the boric acid groups are uniformly grafted by amide condensation reaction of boric acid reagent with amino groups and carboxyl groups on the membrane, the quantity of the boric acid groups and the carboxyl groups reaches a proper proportion, and the functionalization effect is achieved.

the strip cutting device is used before the second reaction of the membrane treatment, the membrane after the first membrane treatment is cut into 24 membrane strips with the same width according to the set size, the contact area of the membrane and the second reaction liquid of the membrane treatment can be increased, the reaction efficiency is improved, the consumption of the reaction liquid can be reduced, and the method is more environment-friendly.

After finishing the second reaction of membrane treatment, the reaction liquid is sequentially emptied, 600mL of MES buffer solution is sequentially added into each reactor immediately and sequentially, the first time of cleaning is performed by counting down for 5min after the buffer solution is poured into the first reactor.

After the first cleaning is completed for 5min, the reaction liquid is sequentially emptied, 600mL of MES buffer liquid is sequentially added into each reactor immediately, the time is counted down for 15min from the time of pouring the buffer liquid into the first reactor, and the second cleaning is performed.

After the second washing is completed for 15min, the shaking table is closed, and the washing buffer solution is recovered as the first washing liquid of the next batch of membranes.

The membrane strips were removed and arranged in a single layer of filter paper (or on a filter pad) to avoid overlapping placement of the membrane strips.

MES buffer preparation

213.3 parts of MES and 9850 parts of purified water were weighed, the weighed MES and purified water were transferred to a glass bottle, stirred with a magnetic stirrer, the pH was measured with a pH meter, and the pH of the buffer was adjusted to 6.50 with sodium hydroxide solution. Filtering with microporous membrane, and storing.

Preparation of membrane treatment two-reaction liquid

1.484 parts of m-aminophenylboronic acid are weighed into a mortar, 8.5 parts of methanol are added, and the mixture is ground and dissolved. 4800 parts of MES buffer solution is weighed, a proper amount of MES buffer solution is used for diluting a methanol solution of m-aminophenylboronic acid, and the methanol solution is poured into a bottle special for preparing a membrane treatment two-reaction solution. And (3) rinsing the inner wall of the mortar for three times by using a proper amount of MES buffer solution, and pouring the rinsing solutions into a special bottle prepared by membrane treatment two solutions to ensure that no m-aminophenylboric acid remains in the mortar. Pouring the rest MES buffer solution into a special bottle for preparing the membrane treatment two solutions, and fully and uniformly mixing.

Step five, drying treatment

And (5) drying the membrane strip in a drying room for at least 12 h.

It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

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 technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The saccharification detection card film treatment method is characterized by comprising the following steps of:

step one, membrane activation reaction;

plasma activation treatment is adopted;

step two, pretreatment reaction;

step two (1) PEI reaction;

placing the membrane subjected to membrane activation reaction into a shaking table containing PEI reaction liquid, reacting for 3-5 minutes, and then cleaning and drying for multiple times;

step two (2) polyacrylic acid reaction;

placing the PEI reacted film obtained in the step two (1) into a shaking table containing polyacrylic acid reaction liquid, reacting for 8-10 minutes, then cleaning for multiple times, and drying for 20-30 minutes at 95 ℃;

step three, membrane treatment one;

soaking the membrane subjected to the pretreatment reaction in a membrane treatment reaction liquid, sealing, and heating at 50-70 ℃ for 2-3 hours;

then cleaning for multiple times, and drying for 20-30 minutes by blasting at 9 0-100 ℃;

fourthly, membrane treatment II;

placing the membrane subjected to the first membrane treatment into a second membrane treatment reaction solution, and simultaneously starting a shaking table with the speed of 140-160r/min for reaction for 8-10min;

step five, drying treatment;

drying the film treated in the step four for at least 12 h;

the plasma treatment conditions in the first step are as follows: background air pressure 15-25Pa, oxygen flow 80-120sccm, discharge power 450-500W, and discharge time 300-360s;

the PEI reaction liquid in the second step (1) comprises the following components:

35-50 parts of PEI stock solution, 3800-4200 parts of purified water and 3000-3200 parts of ethanol;

the polyacrylic acid reaction solution in the step two (2) comprises the following components:

15-25 parts of polyacrylic acid stock solution, 6500-7000 parts of purified water and 900-920 parts of ethanol;

and in the third step, the membrane treatment reaction liquid comprises the following components:

and the preparation process of the membrane treatment reaction liquid comprises the following steps: adding 800-850 parts of DMSO into a solid mixture of 25-30 parts of phthalic anhydride and 0.5-1 part of triethanolamine hydrochloride, sealing a container by using a preservative film, and stirring by a magnetic stirrer to completely dissolve the solid;

and in the fourth step, the second reaction liquid for membrane treatment comprises the following components:

2. The saccharification detection card film treatment method of claim 1, wherein: in the first step, a water climbing test is required to be carried out on the porous PE film, and the specific operation flow is as follows: before the first step, marking small strips with a certain area on the porous PE film, marking the corresponding positions at the two ends of the small strips by scribing respectively, cutting the marked small strips along the marking lines after the first step, putting the marked ends at the two ends of the small strips into purified water, and recording the time of the purified water penetrating from the lower end of the film to the marking height.

3. The saccharification detection card film treatment method of claim 1, wherein: and step four, the film treatment II further comprises cutting:

4. The saccharification detection card film treatment method of claim 1, wherein: in the fourth step, 1.2-1.5 parts of EDC is required to be added into the second reaction solution for membrane treatment before the second reaction is carried out, and the EDC is quickly and uniformly mixed, and meanwhile, the second reaction for membrane treatment is ensured to be started within 3 minutes after the EDC is added into the second reaction solution for membrane treatment.

5. The saccharification detection card film treatment method of claim 1, wherein: the MES buffer comprises the following components: