Selection and Application Skills of Nitrocellulose Film for Making Colloidal Gold Test Paper
The nitrocellulose membrane, also known as the NC membrane, is used as a carrier for the C/ T line in colloidal gold test paper and is also the site of the immune reaction. Therefore, the NC membrane becomes the most important consumable in this test, and because it belongs to non- Standard devices, basically at the beginning of each project will encounter the problem of how to choose the right NC film. There is also a discussion on whether to post-process the NC film and how to deal with it.
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I. The production principle of NC film Although this seems to belong to the manufacturer, but GMP has a point of view that the control of the process will have good results. Then only understand the general production process and basic principles of the NC film can be better. Master the characteristics of this material and finally produce a satisfactory test strip. Do you understand?
I have been looking for a long time and finally found some more intuitive pictures. You can go to Debao 's official website to see The process of producing NC film is very similar to the ordinary paper making process. We can learn from papermaking. Know to understand.
First, the raw material nitrocellulose particles purchased from the homogenate ratio is a very common organic chemical that dissolves to form a mixed slurry. In the slurry, a certain proportion of reagents are added to adjust the properties of the finally formed film. Is a reagent formulation, mainly containing surfactant / polymer / salt ion / molding agent, etc. dissolved in a buffer system. Different manufacturers added solution formulations are not the same, directly leading to differences in the product.
Secondly, the roller film is equipped with a homogenate through the roller to form a film which is spread over a very smooth flat carrier. This process is very similar to the papermaking process.
Finally, the molding agent formed in the homogenate begins to volatilize, and the film is gradually dried and formed. At the same time, due to the relatively high temperature in the process, some manufacturers adopt a form in the closed cavity while replenishing the form of the formulation solution to avoid Evaporation of some active ingredients.
The film produced by the above steps is a product with a very wide width. The width is directly related to the size of the drum. The larger the drum, the more convenient the production, but the higher the cost of the equipment. The wide film must be cut. It is 25mm or 18mm (or 20mm) wide, and the length of the finished roll film and wide film are the same. In theory, the manufacturer can cut the width to the width you need, but this will cause waste of raw materials. And the increase in labor costs, and later in the process of coordination with the test paper manufacturers, the comprehensive material cost and production convenience basically determine the width mentioned above, the second is the standard. Regarding the difference in use of different widths, detailed later .
From the production process, we can know that the NC membrane itself has been added with surfactants to improve the hydrophilic capacity, and there is already a certain buffer system (although it will not affect the paper strip test). Some of the problems I get are easier to understand.
2. Suppliers and current status of NC film The models of NC film brands that are currently sold are as follows:
MILLIPORE (USA), M135 (backed / unbacked) M180 (backed / unbacked)
WHA T MAN and S&S, Immunopore RP, 100 sec/4cm, with lining, C T thread does not spread, very concentrated, clean background. Suitable for all kinds of tests . (only 25mm x 50M); PuraBind R, 140sec/4cm , without lining, suitable for small molecule competition t es t et al. (20/25mm x 50M)
SAR T ORIUS (Germany), CN140
Debao Film Industry (domestic), 8um 6um
MDI (India), 8um 6um
Speaking of these suppliers, I have to talk about the history of development. S&S is the originator of the film industry. All the film companies admit that .millipore, SAR T ORIUS and wha t man are all younger generations. My understanding is that S&S is in the market. In the process of competition, the operation mode was backward, and it was finally acquired by wha t man. S&S AE99 and AE98 are very good in terms of product quality.
MDI is a film company that has only appeared in recent years. It may also be closely related to the rapid development of the Indian colloidal gold test paper production industry. All its products are produced in India. The operators are father and son. The father is responsible for technology and the son is operating. I have tested the samples in .03 and found that there are uneven waves in the running board, and the results of the accelerated stability test are not ideal. I received some samples at the beginning of this year, and the situation still has not improved much. No longer considered for use.
Domestic film Debao has entered the market in recent years. The main feature is that it can reduce the cost. I have also tested several batches of samples. The performance has been very close to the imported film. After some targeted optimization, it can be used in HCG products. Production. But not all factories can easily complete this optimization work. The localization of membranes is a trend, but importing manufacturers to face localization still faces great problems, and the relocation of plant equipment is a very big problem. .
The domestic contacts and technical support of several suppliers are
MILLIPORE (United States), Mrs Yang, harry
WHA T MAN and S&S, Clark, kevin
SAR T ORIUS (Germany), Mr Xu, Iric
Debao (domestic), Mr Zhang
MDI (India), Ashawan t Gup t a
The sales model is direct sales. In the case of a small purchase, the agent should go through the account. The price is similar to WHA T MAN, SAR T ORIUS is cheaper, domestic film is cheaper. Indian film and SAR T ORIUS are the same price. should not be concerned. in the research market, MILLIPORE use the most. Since the research is only used to test the market, so the demand for small film, not taken seriously suppliers. production customers, MILLIPORE, WHA T MAN, SAR T ORIUS possession of quite, Germany Bao now also has some factory customers, and the minimum number of MDI complaints is also the most.
3. Choice of membranes The above talks about some of the development status of the industry. Then, after we have a general understanding, we will return to the most concerned issues and how to choose membranes. The problem that I often encounter is that I am doing ** projects, I should Which type of film is chosen?
This involves a classification criteria for a membrane. One supplier may provide this membrane for 8um, but another supplier tells you that the membrane is 135s. What is the difference and connection between them?
Um refers to the pore size of the membrane, and from the production process of the membrane above, we can see that the pore size of the membrane is virtually impossible to define. Due to the non-absolute uniformity of the process such as dry molding, the pore size of the membrane is also non-uniform. The theory of aperture is actually a long-standing image. The definition in seconds is that every 4cm film, the chromatographic time of water is ***s. The unit has been increasingly used by major manufacturers. Accepted, has become a general comparison standard. Below we will use s units for communication.
The conversion is roughly as follows: 8um=135s; 6um=180s.
What effect does the film of different seconds have on the reaction?
First, we analyze the movement of the liquid on the membrane. A film of 4 cm in length, make a mark every 1 cm, record the time point when the liquid moves past the mark. Then you will find the movement of the liquid on the film. It is in the direction of deceleration. Two different seconds of film (such as 135s, 180s) have different speeds of movement at the same time mark. This test is not easy to observe with water, and it can be considered to use an aqueous solution of pigment, which is very obvious.
From this test, it can be seen that the velocity of the gold solution passing through the same T- line is the fast film > slow film. The faster the passing speed and the shorter the reaction time of the coating on the T line. The reading is fast, then the sensitivity is lower. Conversely, the reaction time is long, the reading is slow, and the sensitivity is high. At the same time, there is a problem that the longer the reaction time, the more likely the non-specific binding occurs, so Long-term reactions don't necessarily improve sensitivity. So there is an equalization of reading time/reaction sensitivity/non-specific binding.
In fact, we don't have so many choices. After using the practice, each supplier generally only provides two models, namely 135s and 180s. Although we see some manufacturers have a wide variety of models on the catalogue, the order quantity of these two models is Accounted for more than 95%, other models are far less successful than these two models. 135s is generally used in the double antibody sandwich method, 180s is generally used in the competition method. What you have to do is to select one of the models first. And then compare the differences between the different models of different suppliers. Due to the difference between the added formula and the test conditions mentioned above, this difference may be large or small. The final choice should be determined based on the test results.
I personally do not recommend carpet testing of different specifications / different manufacturers of film, so the cost is high, the chance of success is also small.
4. Quality control method of membrane This section is suitable for users with large membrane volume. For users who can use a film for several months, the quality control standard of the manufacturer can fully meet your requirements, instead of Need to do the relevant quality control yourself.
Although the quality control of the film belongs to the QC function of the original auxiliary material, due to its strong professionalism, it is generally executed by the small sample debugging personnel. After the film is imported into the raw material warehouse, the following inspection work is required:
1. Check COA
At the time of purchase, the supplier will provide a paper quality certificate for each lo t film, which is called COA. If you have purchased a batch of film and have not obtained the corresponding certificate, If necessary, the batch number can be provided to the manufacturer for reissue. In fact, in many industries, manufacturers have the habit of attaching COA to their products.
COA is generally only available to large customers, and it does not make sense to small customers.
The content is mainly listed by the manufacturer when the product is shipped from the factory. The role can be understood as the quality certificate and test data reference.
2. Check physical properties The physical properties of the membrane are mainly 2 parameters, membrane thickness and width. The length does not need to be tested. In use, you can pay attention to whether there is cross-section reconnection in the length, and the section has more materials and more, a few occur.
Thickness, measured by spiral micrometer, select several measurement points and count after averaging. The measured parameters of membrane production. The main performance is that the thickness unevenness affects the diffusion performance of biological materials on the membrane, and the C/ T line width is not narrow. 1. Also affects the speed of climbing.
Width, ordinary ruler measurement.
Physical performance generally does not have any major problems. After all, the testing standards are objective and easy to grasp. Manufacturers without conditions can be omitted.
3. The running water performance sample uses an aqueous solution containing colored food coloring for easy observation. A simple stent is required.
Operation: Inject enough solution into the lower tank, mark each batch of membrane every 1cm (total length is more than 4cm) and put it on the inclined bracket. Place the lower end of the whole stent into the solution tank, and the membrane starts to absorb liquid. Time. Record each The passage time at the mark and compared with the control group.
When running the board, theoretically the solution is sucked up in a horizontal line to observe whether there is an abnormal phenomenon such as wave inclination or encapsulation.
4. Spot test
C/ T line exit time, when other test conditions are the same, record and compare the C/ T line appearance time is different from the control.
Sensitivity, compared with the different sample concentrations, whether the change of T line is the same as that of the control group. Generally, the front end of each batch of film is used for testing. Due to the heterogeneity of the manufacturing process, the sensitivity of different batches may be different. when used in large quantities, this effect is a very big difference, then they would behave in accordance with the sensitivity of the different batches of film classification. in S T OCK film to be able to easily tell the difference. when entering the post-production called, According to these classifications, different batches of membranes can be used according to the order requirements, or with strong sensitive materials and poor sensitivity membranes.
Regarding the intra-batch difference of the film. The intra-batch difference is definitely present, but the problem of the size of the gap. It is very difficult to obtain detailed values ​​according to the general test conditions, because you cannot make detailed details for each segment of each film. Test. The most effective way to reduce the difference in the film batch is to not buy the edge film strip. As mentioned above, the production method of the film, the semi-finished product produced is a wide film surface, which can be obtained by section cutting to obtain 25mm. Or 20mm width. Then there is the middle part, and the edge part. The closer to the middle part, the better the uniformity of the film, and the edge part is relatively poor, which causes the difference in the batch. Generally, the specific position can be obtained by COA. Information such as MILLIPORE uses the letters A, B, C... after cutting to indicate the position of the cut narrow film in the wide film.
If you are trying a sample of a certain type of membrane, you need to add a film accelerated aging test after the above test. It includes the aging test of the film alone and the aging test of the product after the film is finished. For the specific test method, please refer to the research and development ideas I wrote before. .
V. In-depth discussion and application of the film From the beginning of this section, we began to discuss the film in depth and talk about some application skills.
1. The principle of binding of protein to membrane The principle of binding of protein to membrane, the known binding force includes hydrophobic force \H bond \ electrostatic force, etc. The exact combination principle is not clear, mainly supported by hypothesis. There are two Hypothesis:
1) First, the two are combined by electrostatic force, and then the H bond and hydrophobic action are used to maintain long-term bonding.
2) First, the two are combined by hydrophobic interaction, and then rely on static electricity to maintain long-term bonding.
The two hypotheses all indicate that the combination process is divided into two steps. First, the combination is combined with the latter for a long time. Due to the ambiguity of the combination principle, the work in this area is very dependent on practical experience.
2. Effect of membrane on bonding
1) Membrane pore size Some technicians prefer to use membrane pore size to distinguish different membranes, but please note that this is only limited to the same manufacturer's products. If it is a different manufacturer's product, this comparison is meaningless. Membrane pore size and chromatographic speed The relationship has been described above.
As the pore size of the membrane decreases, the actual available surface area of ​​the membrane increases, and the amount of membrane-bound protein also increases. The parameter for estimating the surface area is the surface area ratio (the ratio of the actual available surface area to the flat area of ​​the membrane used).
In addition, the smaller the membrane pore size, the smaller the chromatographic speed, then the longer the gold standard complex passes through the T line, and the more complete the reaction.
Combining the above two points, the conclusion is that the smaller the membrane aperture is, the higher the sensitivity is. However, it also slows down the running speed and increases the chance of non-specific binding, that is, the higher the false positive. Therefore, it is necessary to select the suitable project according to the test results. Membrane, find the right balance point.
2) The difference in membranes between different manufacturers mainly comes from two points:
1> When producing membranes, the sources, types and quantities of polymers and surfactants used are different. Similarly, in membrane treatment, these two substances generally have a greater impact on performance.
2> The process is different.
3. Bio-materials, buffer solution reagents and formulations
1) Biological raw materials, as the biological raw materials of the C T line are used differently, so only a brief description will be given here.
First, the binding of monoclonal antibodies to membranes is superior to polyclonal antibodies, mainly because polyclonal antibodies have many different surface sites, and the optimal binding conditions for each spot and membrane are subtly different, and there is no doubt that it increases. The optimization difficulty.
Second, the larger the molecular weight, the harder it is to bind to the solid phase material.
2) Buffers The most important concern for everyone is to obtain a formulation with excellent performance, including buffer, blocking solution, etc.
In fact, I can't provide you with a list of universal formulas. Because different reaction systems require different formulas to support, and the reaction systems of different institutions are different. I want to learn "fish" first to learn "fishing". In order not to mislead everyone. I am concerned with the formulation of the problem below, only provide ideas, specific formula please explore.
The composition of the buffer is generally: PBS (or other buffer system) + action substance (for a specific problem) + PH adjustment. After I have consulted the previous various materials, the personal opinion is that the formula principle should be simple and not suitable. Adding substances according to their own needs, many of the original substances that need to be added, because the improvement of membrane manufacturing technology is no longer needed.
The recommended buffer system is 0.01M PBS PH7-7.2. The buffer system has good adaptability to various antigens and antibodies.
The situation of the substance is roughly as follows:
A small amount of NACL, reducing signal strength, eliminating false positives.
Organic alcohol (methanol, isopropanol, etc.), wet the film, reduce the static electricity carried by the film, which is conducive to the combination of coating. Personally not recommended, due to the improvement of the film making process.
Surfactant ( T W20, T X100), increase the hydrophilic force, can avoid the hollow phenomenon of the line, can also add color.
Sugar, protective agent, slow down the aging rate, can also increase the hydrophilic force as above.
Adjusting the PH to a certain position can eliminate the fake sun.
4. The ambient humidity is very important to the filming process. The optimum humidity is generally 45-65%.
If the humidity is too low, the film will easily accumulate static charges, and the film will be prone to scatter, resulting in hydrophobic spots on the test.
When the humidity is too high, the capillary action on the membrane is strengthened, and the film is likely to cause the C T line to widen or even spread.
In order to ensure the uniformity of the film moisture during spotting, the film is generally placed under the humidity condition for a certain period of time before spotting.
5. The relationship between the spotting instrument and the film surface condition There are currently two kinds of spotting methods, the film type and the non-contact film type. The non-contact film type is superior to the film type, and the imported film type is superior to the domestic film type. .
Because the film is a hose to the antibody to the surface of the film, and the physical properties of the film itself is soft and brittle, the pipe will leave a mark on the surface. The imported filming machine is better because of the materials and control system used. The scratches left are lighter, while the domestic instruments are poor, and the scratches left are more serious. The scratches tend to form resistance to the gold-labeled complex of the chromatogram, resulting in false positives. At the same time, it is easy to appear in the running plate at T The line position appears if there is a thin line (ghost line), and the ghost line disappears after the end of the running board.
6. The width of the film and the width of the film at the spotting position are generally 18mm (or 20mm) and 25mm, which are used for test strips and test boards, respectively.
However, different T- line spotting positions will bring different sensitivities. When the spotting position is moved up, the speed of the gold-marked complex is slowed by the T- line position, the reaction time is increased, and the sensitivity is increased. Conversely, the sensitivity is lowered. This method can be Used to change sensitivity and eliminate false positives.
7. The amount of the solution on the membrane of the diffusion solution on the membrane is generally 1 ul / cm.
The diffusion of the solution on the membrane tends to both ends, and the spray point is a uniform antibody solution, but when drying, the edge of the line is dried faster than the middle, and the intermediate antibody will continuously spread to both sides, so the antibody is oriented to the line after drying. The two ends are gathered. Under normal circumstances, it does not affect your experiment. If you find that the lines appear red at both ends and the middle is light, you should consider this problem. You can add the action substance as mentioned above to solve.
8. The sealing effect before and after the film is basically optimized for the film purchased from the supplier. The film can be used directly. However, we often encounter discussions about sealing.
In fact, the closure is not as closed as everyone thinks. Any problem that has been closed is solved. The old problem has come again, and it is more troublesome. I have to say that it is closed with caution.
I am extremely opposed to the method of sealing before the film. The reason is that when the manufacturer produces the film, the various formulas are mixed and added to the original slurry. When the film is sealed before the film is immersed in the blocking liquid, it will inevitably disturb the normal inside the film. Material distribution. This has caused many problems, and also reduced the efficiency of work. There are also problems that manufacturers can not cover the film when they are not closed. In fact, they can be solved by other methods. The closure must be the next test.
There are two kinds of closed techniques that I often use.
The flow is closed and the active substance is treated on the sample pad.
The membrane is fixedly closed, and the active substance is formulated into a solution spray point at a specific position of the membrane. This method requires the use of BIODO T AIRJE T nozzle. The unqualified semi-finished board can be revived.
As long as the closure is done on the membrane, it will inevitably affect the stability of the product. The specific degree of influence should be judged by the stability test.
9. Storage of membranes The membranes that have just been produced generally contain 5-10% of water. There is a theoretical support for the aging mechanism of the membrane, but the controversy is relatively large. The theory is that the aging of the membrane is due to the evaporation of water on the membrane. The membrane becomes hydrophobic, charged and brittle. The storage membrane is generally required to be protected from light and sealed. It is not good to be too dry or too wet. Under such storage conditions, it can generally be placed for two years. However, if the membrane is closed, It is necessary to judge according to the specific test situation.
Some membranes will change the sensitivity after use due to the production process. After encountering this problem, it needs to be placed for a period of time after the film is to be stabilized before entering the debugging production.
The above sequence is some of my experience in the use of nitrocellulose membranes in colloidal gold chromatography diagnostic tests. It took about a month from the beginning of the pen to the end, intermittently, and finally completed. Thanks to all membrane manufacturers for their technical expertise. I have done a lot of technical training in my previous work contacts, many of which have become friends who are often contacted. As the film industry is constantly evolving, new problems or new solutions may emerge. Welcome to discuss.