Selection and application skills of nitrocellulose membrane for making colloidal gold test paper

Nitrocellulose membrane, also known as NC membrane, is used as a carrier for C/T lines in colloidal gold test papers, and is also where immune reactions occur. Therefore, the NC film became the most important consumable in the test, and since it was a non-standard device, the problem of how to select a suitable NC film was encountered at the beginning of each project. At the same time, there are also discussions on whether post-processing and how to treat NC film. First, the principle of production of NC film Although this seems to be a matter of manufacturer, but GMP has a point of view is that the control of the process will have good results. Only by understanding the general production process and basic principles of NC film can we better grasp the characteristics of this material, and finally produce a satisfactory test paper. Do you understand? I have been looking for a long time, finally found some more intuitive pictures, we can go to the official website of Iraqi can see http://. The process of producing NC film is very similar to the ordinary papermaking process. We can learn from the understanding of papermaking. First, the homogenate ratio. The raw material nitrocellulose that is purchased 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 resulting film. Generally, it is a reagent formulation. Contains surfactants/polymers/salt ions/formers etc dissolved in a buffer system. Different manufacturers add solution formulations that directly lead to differences in the product. Second, the drum is filmed. The homogenized slurry passes through the drum to form a film that is spread flat on a very smooth planar carrier. This process is very similar to papermaking. Finally, molding. The forming agent in the homogenate starts to volatilize, and the film is gradually dried and shaped. At the same time, due to the relatively high temperature in this process, some manufacturers take the form of molding in sealed chambers and supplement the formulation solution to avoid the evaporation of some active components. Cut out the product. The film produced through the above steps is a product with a very large width. The width is directly related to the size of the drum. The larger the drum, the more convenient it is to produce, but the higher the cost of the equipment. The wide film has to be cut to become 25mm or 18mm (or 20mm) wide, which we purchased, and the length of the finished film and the wide film is the same. Theoretically, it can allow manufacturers to cut into any width you need, but this will lead to waste of raw materials and increase in labor costs. Later, in the process of coordination with test strip manufacturers, the manufacturer’s comprehensive use of materials costs and production facilities basically determine the above The width of the statement is followed by the standard. The difference in use of different widths will be described in detail later. From the production process, we can learn that the NC film itself has been added with surfactants to improve the hydrophilicity, and there is already a certain buffer system (although the paper strip test will not be very significant). Some of the issues discussed later are easier to understand. Second, the NC film suppliers and the status of the current sales of the NC film brand models are as follows: MILLIPORE (United States), M135 (with backing / no backing two) M180 (with backing / no backing two) WHATMAN and S&S, Immunopore RP, 100 sec/4cm, taped, CT lines do not spread, very concentrated, background clean. Suitable for all kinds of test. (only 25mm x 50M); PuraBind R, 140sec/4cm, without lining, suitable for small molecule competition test etc. (20/25mm x 50M) SARTORIUS (Germany), CN140 (China), 8um 6umMDI (India), 8um 6um Speaking of these suppliers, we have to talk about the history of development. S&S is the originator of the film industry, which is recognized by all film companies. Both millipore, SARTORIUS and whatman are descendants, and my understanding is that S&S lags behind in the process of market competition and is eventually acquired by whatman. S&S's AE99 and AE98 are very good in terms of product quality. MDI is a film company that appeared in recent years. It may also be closely related to the rapid development of India's colloidal gold test paper production industry. All of its products are produced in India. The operators are father and son. The father is responsible for the technology and the son runs the business. . I tested the samples in 2003 and found that there were uneven waves on the running board, and the accelerated stability test results were not ideal. At the beginning of this year, I received some samples again. The situation is still not much improved and I will no longer consider using them. The domestic film Ienon started to enter the market last year. Its main feature is that it can reduce costs. I also test several batches of samples. The performance is very close to that of the imported film. After some targeted optimization, it can be used in the production of HCG products. . But not all factories can easily complete this optimization work. The localization of membranes is a trend, but importing manufacturers are also faced with major problems in achieving localization. The relocation of plant and equipment is a very big problem. The domestic contacts and technical support of several suppliers are MILLIPORE (USA), Mrs Yang, harryWHATMAN and S&S, Clark, kevinSARTORIUS (Germany), Mr Xu, Iric (domestic), connie, Mr YangMDI (India), Ashawant Gupta sales model is direct sales, under a small amount of purchase to go through the agent account. MILLIPORE is equivalent to WHATMAN on price, SARTORIUS is cheaper, and domestic film is cheaper. The prices of Indian membrane and SARTORIUS are the same and basically do not need to be considered. In the scientific market, MILLIPORE is the most used. Because the scientific research market is only used for testing, the demand for membranes is small and it is not valued by suppliers. Among the production customers, MILLIPORE, WHATMAN, and SARTORIUS occupy a similar amount. Yi Neng now also has some factory customers, and the MDI volume has the smallest number of complaints. Third, the choice of membrane above talked about some of the industry's development status, then we generally understand the problem back to the most concern, how to choose the membrane? The problem often encountered is that I am doing ** project, I should Which type of membrane is used? This involves a classification problem for a membrane. One supplier may provide this membrane with 8um, but another supplier tells you that the membrane is 135s. What is the difference between this and what is the relation? Um refers to the membrane pore size, and from the production process of the membrane above, we can see that the pore size of the membrane is actually not defined. Due to the non-absolute uniformity of the drying and forming processes, the pore size of the membrane is also non-uniform. The term “membrane aperture” is actually an image name that has always been used. The definition in seconds is that for every 4cm membrane, the chromatographic time of water is ***s. This unit has been increasingly accepted by major manufacturers and has become a universal comparison standard. In the following we will use s units to communicate. The conversion is roughly as follows: 8um = 135s; 6um = 180s. What are the effects of different seconds on the reaction of the membrane? First, we analyze the movement of the liquid on the membrane. A piece of film with a length of 4 cm is marked every 1 cm, and the time is recorded when the liquid moves past the mark. Then you will find that the movement of the liquid on the membrane is slowing down. Two different seconds of film (for example, 135s, 180s) have different movement speeds at the same time point. This test uses clear water for poor observation and can be considered as an aqueous dye solution. From this test, it can be seen that the gold solution passes through the fast film and slow film at the point of spraying the same T line. The faster the passing speed and the shorter the reaction time of the material coated on the T line, the faster the reading, and the lower the sensitivity. On the contrary, the reaction time is long, the reading is slow, and the sensitivity is high. At the same time, there is also a problem that the longer the reaction time is, the greater the possibility of non-specific binding occurs. Therefore, long-term reactions may not be able to truly increase the sensitivity. So there is a reading time/response sensitivity/non-specific binding equilibrium. In fact, we do not have so many choices. After practice, each supplier generally provides only two models, namely, 135s and 180s. Although some manufacturers have seen a wide variety of models on the product catalog, the order volume of these two models accounts for more than 95%. Other models The supply is far less smooth than the two models. 135s is generally used in double antibody sandwich method, and 180s is generally used in competition method. All you have to do is select one of the models first, and then compare the differences between different suppliers for the same model. This difference may be large or small due to the difference between the previously mentioned additive recipe and your test conditions. According to the test results, the final choice should be determined. I personally do not recommend carpet-type testing of membranes of different specifications/different manufacturers. This is costly and has a low chance of success. Fourth, the film quality control method This section is suitable for users with a large amount of film reference. For users who can use a roll of film for several months, the manufacturer's quality control standards can fully meet your requirements without having to do related quality control. Although the quality control of the membrane belongs to the QC function of the raw materials, due to its strong professionalism, it is generally performed by sample debugging personnel. After the film is put into the raw material warehouse, the following inspection work is required: 1. When the COA is inspected, the supplier will supply a paper quality certificate for the product for each lot of the film. This certificate is called COA. If you have purchased A batch of film, without requesting a corresponding certificate, can be provided to the manufacturer for reissue if necessary. In fact, in many industries, manufacturers have the habit of attaching COA to their products. COA is generally provided only to large customers and is meaningless to small customers. Its content is mainly listed by the manufacturer when the product was shipped out. The role can be understood as quality certificate and test data reference. 2. Checking physical properties The physical properties of the film are mainly 2 parameters, film thickness and width. The length does not need to be detected. In use, attention may be paid to whether there is a section reconnection phenomenon in length, and more sections and more materials are consumed in the section, and a few occur. The thickness is determined by the spiral micrometer after selecting several measuring points. Membrane production must be measured parameters. The main manifestation is that the non-uniform thickness affects the diffusion properties of biological materials on the membrane, and the C/T line widths are different. It also affects the climb speed. Width, normal ruler measurement. Physical performance is generally not a big problem. After all, the testing standard is objective and easy to grasp. Manufacturers without conditions can be omitted. 3. The running performance samples were prepared using an aqueous solution containing a colored food coloring. The purpose was to facilitate observation. Need to make a simple bracket. Operation: Inject enough solution into the lower tank, mark different batches of membrane once every 1cm (greater than 4cm in total length) and place it on the inclined support. Put the lower end of the whole stent into the solution tank, and the membrane starts to absorb liquid and time. The time of passage at each marker was recorded and compared with the control group. When running the board, the solution is theoretically sucked up in the form of a horizontal line, observing whether there is an anomaly such as wavy slope or enveloping wetting. 4. When the test sample C/T cable outlet time is the same, if other test conditions are the same, record and compare whether the C/T line appearance time is different from the control. Sensitivity, compared with different sample concentrations, the T-line change is the same as the control group. The front end of each batch of membrane is usually used for testing. Due to the inhomogeneity of the manufacturing process, the sensitivity of different lot numbers will be different. When used in large batches, the impact of this difference is very large. Then the different batches of membranes should be classified according to the sensitivity performance. It should be easily discernable in the STOCK of the film. When entering a later production call, it is possible to use different batches of membranes according to the order requirements, or to use a combination of a highly sensitive raw material and a poorly sensitive membrane. Regarding the intra-assay of the film. The intra-batch difference is certainly present, but it is only the size of the gap. Based on the general test conditions, it is very difficult to obtain detailed values, because you cannot perform detailed tests on each segment of each film. The most effective way to reduce the internal tolerance of the film is to not purchase the edge film tape. As mentioned before, the semi-finished product produced by the production method of the film is a film surface with a large width, and a width of 25 mm or 20 mm can be obtained by section cutting. 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 worse the edge part, resulting in a difference within the batch. Generally, specific position information can be obtained through COA. For example, after cutting, MILLIPORE uses the letters A, B, C,... to indicate the position of the narrow film that is cut out in the wide film. If you are testing a certain type of membrane sample, you need to add a membrane accelerated aging test after the above test. Including the aging test of the film alone and the aging test of the after-film product. For detailed test methods, please refer to the article I wrote previously. V. Membrane in-depth discussion and application skills From the beginning of this section, we began to discuss the film in depth and discuss some application techniques. 1. The principle of protein-membrane binding The principle of protein-membrane binding. Known binding forces include hydrophobic forces, H-bonds, and electrostatic forces. The exact binding principle is not clear, and it is mainly supported by hypotheses. There are two main hypotheses: (1) First, the two are combined by electrostatic force, and then the H bond and hydrophobic effect are used to maintain long-term binding. (2) First, the two are combined by hydrophobic interaction, and then the electrostatic interaction is used to maintain long-term binding. Two hypotheses indicate that the process of integration is divided into two steps, first combining and later combining for a long time. Due to the ambiguity of the principle of integration, the work in this area is very dependent on practical experience. 2. Influence of membrane on binding (1) Membrane pore size Some technicians tend to use membrane pore size to distinguish different membranes, but please note that this is only limited to the same manufacturer's products. If the products are from different manufacturers, this comparison is meaningless. of. The relationship between membrane pore size and chromatographic velocity has been described above. As the membrane pore size decreases, the actual usable surface area of ​​the membrane increases and the amount of membrane-bound protein also increases. The parameter to estimate the surface area is the surface area ratio (the ratio of the actual usable surface area to the flat area of ​​the membrane used). In addition, the smaller the pore size of the membrane, the lower the chromatographic velocity, and the longer the gold-labeled complex passes through the T-ray, the more reactive the reaction will be. Based on the above two points, the conclusion is that the smaller the membrane aperture, the higher the sensitivity. But at the same time, it slows down the running speed and increases the chance of non-specific binding, that is, the higher the false positive. Therefore, according to the test results, select the appropriate film for the actual project and find the right balance point. (2) Membrane differences from different manufacturers This difference mainly comes from two points: A. When the film is produced, the source, type, and quantity of the polymer and surfactant used are different. By the same token, these two types of materials generally have a large impact on performance in membrane processing. B. The process is different. 3. Biological raw materials, reagents and recipes for buffer solutions (1) Biological raw materials, The use of biological materials as CT lines is different, so this is only outlined here. First, monoclonal antibodies and membranes bind better than polyclonal antibodies. The main reason is that polyclonal antibodies have many different surface sites, and there are subtle differences in the optimal binding conditions between the individual sites and membranes, which undoubtedly increases. The difficulty of optimization. Second, the larger the molecular weight, the harder it is for proteins to bind to solid-phase materials. (2) Buffer Most people may be interested in obtaining a formula with excellent performance, including buffers, blocking solutions, and other treatment solution formulations. In fact, I can't provide you with a list of all-purpose recipes. Because different reaction systems require different formulations to support, different organizations have different reaction systems. Want to get "fish" first learn "fishing". In order not to mislead you, I will only provide ideas on the following issues related to formulas. The composition of the buffer is generally: PBS (or other buffer system) + acting substance (for a particular problem) + PH adjustment. After I have consulted various previous materials, my opinion is that the formulation principle should not be too complicated, and the substance should be added according to my own needs. Many of the original substances that need to be added are no longer needed due to improvements in membrane manufacturing technology. The recommended buffer system is 0.01M PBS PH7-7.2. This buffer system has good adaptability to various antigens and antibodies. The situation of the active substances is roughly listed as follows: A small amount of NACL reduces signal intensity and eliminates false positives. Organic alcohols (methanol, isopropanol, etc.) wet the membrane and reduce the electrostatic charge on the membrane, which facilitates binding and coating. Personally not recommended because of the improvement of the film making process. Surfactants (TW20, TX100) increase the hydrophilicity to avoid hollow lines and enhance color. Sugar, protective agents, slowing down the aging process can also increase the hydrophilicity as above. Adjusting PH to a certain position can eliminate false positives. 4. Spotting the ambient environment humidity is very important for the spotting process. The optimum humidity is generally 45-65%. If the humidity is too low, electrostatic charges tend to accumulate on the film, and dot films tend to scatter, resulting in hydrophobic spots on the test. If the humidity is too high, the capillary action on the membrane is strengthened, and the spot film easily causes the CT line to become wider or even spread. In order to ensure the uniformity of film moisture during dispensing, the film is generally equilibrated for a period of time before being spotted under this humidity condition. 5. The relationship between the spotting instrument and the film surface There are currently two spotting methods, film-stripping and non-contact film. The non-contact film type is better than the film type, and the imported film type is superior to the domestic film type. Because the membrane is a hose, the antibody is drawn on the membrane surface, and the physical nature of the membrane is soft and brittle, and the marking tube will leave an imprint on the surface. Imported film-squeezing machines use better materials and control systems, so the scratches left are lighter, and domestic equipment is poorer, leaving more scratches. Scratches tend to create resistance to chromatographic gold-labeled complexes, leading to false positives. At the same time, it is prone to the strange phenomenon that if there is no thin line (ghost line) at the position of the T line when running the board, the ghost line disappears after the board runs. 6, the width of the film and spot location The width of the film is generally 18mm (or 20mm) and 25mm two, were used in the test strip and test board. However, different T line spotting positions will bring different sensitivities. As the spotting position moves upwards, the velocity of the gold-label composite passes through the T-line position, the reaction time increases, and the sensitivity increases. On the contrary, the sensitivity decreases. This method can be used to change the sensitivity and eliminate false positives. 7. Diffusion of the solution on the membrane The amount of spotted solution on the membrane is generally 1 ul/cm. The diffusion of the solution on the membrane is toward both ends, and the sprayed point is a uniform antibody solution, but when the lines are dry The drying speed of the edge is higher than that of the middle, and the antibody in the middle will continue to spread to both sides. Therefore, after the drying, the antibodies are gathered at both ends of the line. Under normal circumstances does not affect your test. If you find that the lines appear red at the ends and light in the middle, consider this issue. Can be added as the above-mentioned acting substance to solve. 8, before and after the point of film sealing effect to buy back from the supplier of the film are basically has been optimized to deal with a good direct film can be used. However, we often encounter discussions about closure. In fact, closure is not what everyone thinks, and what problems are closed are solved. The old problem has gone and the new problem has come again, and it is even more troublesome. What I want to say is to use caution with caution. I am extremely opposed to the practice of closing the membrane. The reason is that when manufacturers produce films, various formulations are mixed into the original slurry. However, when the membrane is closed, the membrane is immersed in the blocking solution, which inevitably disturbs the normal material distribution in the membrane. This has caused many problems and also reduced work efficiency. There are also the problems that manufacturers can not be coated on the membrane when they are not closed. In fact, they can be solved by other means. The closure must be tested. There are two types of closure techniques that I often use. The flow was closed and the affected substance was treated on the sample pad. The membrane is sealed at a fixed point and the active substance is sprayed onto the membrane at a specific location. This method requires the use of BIODOT's AIRJET spray head. Unqualified semi-finished boards can be revived. As long as it is sealed on the membrane, it will inevitably affect the stability of the product. The specific degree of influence is judged by the stability test. 9. Membrane storage The membrane just produced typically contains 5-10% moisture. There is a theoretical support for the aging mechanism of the membrane, but the controversy is relatively large. Theory holds that the aging of the membrane is due to the evaporation of water on the membrane, rendering the membrane hydrophobic, charged and brittle. Storage film is generally required to be protected from light and sealed. Being too dry or too wet is not good. In this preservation condition, it can generally be placed for two years. However, if the membrane is closed, it must be judged according to the specific test conditions. Some membranes may have changes in sensitivity over a period of time because of problems with the production process. When this type of problem is encountered, it may be necessary to place the membrane for a period of time after it has been spotted.