A visual positioning solid-phase protein chip

Abstract

The present invention provides the preparation and application of a visual positioning solid-phase protein chip. By using a protein fixing solution containing water-soluble dyes, surfactants, water-soluble high molecular polymers and other components, the protein is fixed on a specific solid-phase carrier to obtain a visual positioning solid-phase protein chip that does not require sealing or cleaning; and in the process of using the solid-phase protein chip for measurement, the water-soluble dye will be completely removed, and will not affect the signal reading of the analyte detection process at all. The visual positioning solid-phase protein chip provided by the present invention is convenient for quality control of the solid-phase protein chip and simultaneous analysis of a large number of indicators due to visual positioning, which reduces the manufacturing difficulty of the solid-phase protein chip and improves the quality, and is very worthy of promotion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro clinical testing and biochips, and in particular, relates to the preparation and application of a visualized positioning solid-phase protein chip. Background Art

[0002] Protein chip is a high-throughput protein function analysis technology. The essence of solid-phase protein chip is a protein dot array on a two-dimensional plane. Use appropriate methods to distribute tiny droplets containing protein molecules to different positions of the solid-phase carrier and fix them in situ by appropriate methods. The dot positions of different indicators are the basis for distinguishing different indicators. In the actual fixation process, maintaining the activity of biological macromolecules mainly protein molecules and ensuring the precise control of tiny droplets are often a contradiction. In order to maintain the biological activity of biological macromolecules, it is necessary to avoid the use of high temperature, high pressure, electrolysis and other technical means to improve the accuracy of mechanical control.

[0003] At present, there are very mature commercial machines for the manufacture of solid-phase chips. There are roughly two types, one is contact type and the other is non-contact type. The so-called contact type refers to using a solid needle with a special structure on the head to dip the spotting reagent, and then controlling the solid needle tip to contact the hard chip substrate, such as glass, to transfer the liquid at the solid needle head to the solid phase plane. This method can control the transferred liquid to the pL level, and the position is relatively accurate. The so-called non-contact type refers to the use of piezoelectric principles to manufacture nozzles that can spray pL levels, without contacting the substrate, to spray tiny droplets within a certain distance range, such as 4 mm on a substrate. However, both methods have the problem of low efficiency. The main reasons are: 1. Different spotting needles or nozzles cannot be integrated on a chip area, so that different points cannot be spotted on a chip at the same time. At present, most commercial spotting equipment adopts the method of spotting one reagent first, then cleaning the needle or pipeline before spotting the next reagent. 2. Even if the preparation of high-density biochips can be achieved through mechanical control and the volume of sample points, this method has low production efficiency and is difficult to form large-scale preparation for situations where the number of samples is limited, such as scientific research and drug screening, or areas where more than dozens of sample points are not actually required in daily testing, which limits its application in these fields.

[0004] Commercial machines do not fully consider the relationship between the volume of the spotting liquid and the formation of droplets, as well as the relationship between the chip substrate and the spotting liquid, and only emphasize the accuracy of moving droplets or spraying droplets. In fact, the volume of the droplet is in a cubic relationship with the droplet diameter. When the volume of the droplet reaches 5nL, the spherical diameter can reach 0.212mm, which is larger than the accuracy that can be controlled by ordinary mechanical movement. In the early experiments, the inventors of the present application invented a new spotting method. Droplets are formed at the spotting needle after liquid injection. When the droplets contact the substrate, the interaction between the biochip substrate and the spotting liquid breaks the balance between the adsorption of the needle top and the droplets and the gravity of the droplets themselves. When the spotting needle moves vertically upward, the droplets will remain on the chip substrate. This spotting method is more convenient to operate and can easily realize large-scale manufacturing of solid-phase chips.

[0005] Although the above-mentioned spotting methods can realize large-scale manufacturing of solid-phase chips, due to the limited area of ​​the chip and the inevitable small displacement of each point during the manufacturing process, there will inevitably be spotting errors during the manufacturing process, thus affecting the quality of the solid-phase protein chip. Since the fixed protein sites in the existing solid-phase protein chips cannot be distinguished by the naked eye (such as CN202010108061.5 and CN201810188202.1), it is difficult to achieve visual and convenient quality control, and it is difficult to ensure the quality of the solid-phase protein chip, which increases the difficulty of manufacturing; especially when it is necessary to realize the simultaneous analysis of a large number of indicators on a protein chip, it is very easy to make mistakes.

[0006] Therefore, it is necessary to visualize the fixed protein sites to facilitate quality control and improve the user experience of solid-phase protein chips. However, it is difficult to achieve visualization by adding dyes to the protein sites fixed on the solid-phase protein chip. Some dyes may affect the signal reading of the analyte detection process, thereby affecting the test results; and currently, solid-phase protein chips generally need to go through blocking, washing and other steps before sample testing. The dye is likely to be removed during the blocking and washing process, resulting in positioning failure. It can be seen that there is an urgent need to find a new method that can achieve visual positioning of solid-phase protein chips. Summary of the invention

[0007] In order to solve the above problems, the present invention provides the preparation and application of a visual positioning solid-phase protein chip. By using a protein fixing solution containing water-soluble dyes, surfactants, water-soluble high molecular polymers and other ingredients, the protein is fixed on a specific solid-phase carrier to obtain a visual positioning solid-phase protein chip that does not require sealing or cleaning; and in the process of using the solid-phase protein chip for measurement, the water-soluble dye will be completely removed, and will not affect the signal reading of the analyte detection process at all. The visual positioning solid-phase protein chip provided by the present invention is convenient for quality control of the solid-phase protein chip and simultaneous analysis of a large number of indicators due to visual positioning, which reduces the difficulty of manufacturing the solid-phase protein chip and improves the quality, and is very worthy of promotion and application.

[0008] In traditional solid-phase protein chips, the proteins fixed on the chip cannot be directly observed visually. The present invention provides a novel visual positioning protein chip, which does not require additional pre-operations such as blocking, and does not affect the signal reading of the test results, providing more convenience for the preparation and use of solid-phase protein chips.

[0009] In one aspect, the present invention provides a protein fixing solution, which comprises a water-soluble dye and a protein capable of binding to a substance to be detected.

[0010] The present invention creatively adds a water-soluble dye in the preparation process of the protein fixative. The addition of the water-soluble dye mainly assists the image recognition of the protein array position and the single fixed area morphology of the finished solid-phase protein chip. In the process of manufacturing the solid-phase protein chip, especially in the process of fixing the protein molecules, the water-soluble dye does not affect the interaction between the protein and the surface of the solid-phase carrier. After the entire measurement process is completed, the water-soluble dye will be completely removed and will not affect the reading of the signal related to the analyte in the fixed area.

[0011] According to the classic ELISA, the fixed protein sites on the chip need to go through the steps of coating, washing, and blocking before they can be used. The role of blocking and washing is to help block the non-specific sites on the solid surface, so that the molecules of the enzyme conjugate will not be non-specifically adsorbed on them during use, which can improve the signal-to-noise ratio. This method has been used to this day, so the solid-phase protein chips currently on the market also need to go through the above steps.

[0012] However, after a large number of experiments, the inventors found that the main reason for the need for blocking and washing in the past was that the specific titer of the enzyme conjugate was not high and the concentration used was high, so blocking was useful. However, for the current enzyme conjugates with high titers and small dosages, blocking is not necessary. For example, when measuring alpha-fetoprotein AFP, a serum sample must be added. In fact, the serum sample contains 60 mg / mL of human serum albumin and about 10 mg / mL of immunoglobulin G, which can play a blocking role and does not affect the detection effect. In contrast, the measured concentration of AFP is only about 10 ng / mL, and the amount of anti-AFP antibodies fixed on the chip is only a few ug / mL. After fixation, the surface density is only 100 ng / cm 2 On the other hand, during the reagent application process, the solution in contact with the solid phase chip also contains a large amount of impurities such as proteins that play a blocking role. Therefore, the visual positioning solid phase protein chip prepared by the protein fixative provided by the present invention does not require additional pretreatment steps such as blocking and cleaning.

[0013] Since the steps of blocking and washing are omitted, the water-soluble dye added to the protein fixative in the present invention will not be removed due to the steps of blocking and washing before detection, making it difficult to play the role of visual positioning and providing analysis area information.

[0014] Furthermore, the water-soluble dye is one or more of a water-soluble azo dye, a non-azo dye, and a dye having the property of stimulating fluorescence.

[0015] Furthermore, the water-soluble azo dye is 3-carboxy-5-hydroxy-(p-benzenesulfonic acid)-4-(p-benzenesulfonic acid azo) pyrazole trisodium salt and / or 1-(4'-sulfonic acid-1'-naphthazo)-2-naphthol-3,6-disulfonic acid trisodium salt; the non-azo dye is indigo and / or its derivatives; the dye having the characteristic of exciting fluorescence is rhodamine and / or fluorescein.

[0016] Furthermore, the protein fixative solution also includes a surfactant and a balanced salt; the surfactant is one or more of hydroxyethylated sorbitol fatty acid ester (Tween), polyoxyethylene and aromatic hydrocarbon polymer (Triton), or 3-[3-(cholamidopropyl)dimethylammonium]-1-propanesulfonic acid inner salt (CHAPS); the balanced salt is one or more of phosphate buffer, citric acid, glycine, tris, and carbonate buffer.

[0017] The protein fixative contains a certain amount of surfactant, such as a nonionic surfactant or a zwitterionic surfactant, etc. The surfactant can adjust the surface contact angle between the protein site and the solid phase carrier of the chip, so that the protein site can have a certain infiltration on the plane of the solid phase carrier, maintain the uniformity of the protein site, and will not shift on the solid phase carrier.

[0018] The balanced salt described in the present invention is a salt that can form an acid-base pair and can maintain a certain acid-base buffering capacity, such as a phosphate buffer solution, in which the phosphate and hydrogen phosphate can relatively stabilize the hydrogen ion concentration in a certain solution, and the amount of the substance is relatively large between pH 6-8, and has a buffering effect. A pair with such ability also includes other salts, such as citric acid, glycine, tris, carbonate buffer, etc. The protein fixative is an aqueous solution, which contains a certain amount of balanced salt according to the characteristics of protein biomacromolecules to control the hydrogen ion concentration and salt ion concentration in the solution, so that the surface charge of the protein has consistency, which can improve the contact direction and binding efficiency between the protein and the solid phase carrier surface of the chip to a certain extent.

[0019] Furthermore, the protein fixative also includes a water-soluble high molecular polymer, and the water-soluble high molecular polymer is one or more of oxyethylene glycol, polyvinyl pyrrolidone, dextran, and pullulan.

[0020] Furthermore, the protein fixative also includes other proteins, and the other proteins are one or more of animal serum albumin, casein, and gelatin.

[0021] The role of water-soluble polymers and other proteins is relatively complex, and they have different roles for different target fixed proteins, including blocking, regulating the fixation of target fixed proteins, and protecting target fixed proteins. From a microscopic perspective, the surface of macromolecular polymers and other proteins have interactions with the chip surface and the surface of the fixed "capture" protein. These interactions will affect the surface properties of the "capture" protein itself fixed on the chip point, and will also affect the interaction between the detection protein labeled with the enzyme conjugate and the chip point.

[0022] Therefore, for different proteins to be tested and fixed "captured" proteins, a large number of experiments must be carried out to find the most suitable protein fixative formula to achieve the best detection effect.

[0023] On the other hand, the present invention provides a solid phase protein chip for visual positioning, wherein the solid phase protein chip contains fixed protein formed by fixing and drying the protein fixing solution as described above, and the water-soluble dye in the protein fixing solution can be completely removed during the cleaning process of the solid phase protein chip.

[0024] Furthermore, the solid phase protein chip also includes a solid phase carrier, and the solid phase carrier is prepared by using an organic high molecular polymer with thermoplasticity and surface polarity, or is prepared by coating with the organic high molecular polymer.

[0025] Since the surface of protein macromolecules is charged and not evenly distributed, the exposed amino acid residues have a certain degree of chemical activity, and the protein amino acid chain is folded and flexible, which can give the protein a variety of types of interactions with the outside world. It mainly manifests itself in a large number of electrostatic adsorption, hydrophobic interactions, and covalent bonds. These interactions form the basis for protein macromolecules to be adsorbed and finally fixed on the surface of a certain material. There are many materials with these surface characteristics, including hard materials based on silicon oxide, metal oxide thin film sheets, organic polymers, etc.

[0026] Considering the convenience of manufacturing and use, the present invention adopts thermoplastic organic high molecular polymer or coating film with certain surface polarity.

[0027] Furthermore, the organic high molecular polymer is one or more of polyvinyl chloride, polystyrene, polyphenylene propylene, polyethylene terephthalate, and polyvinylidene fluoride; titanium dioxide is also added to the organic high molecular polymer.

[0028] Suitable organic high molecular polymer materials include, but are not limited to, polyvinyl chloride, polystyrene, polypropylene, polyethylene terephthalate, polyvinylidene fluoride, etc., or a mixture of two or more polymers.

[0029] The addition of titanium dioxide and mixing with organic high molecular polymer materials can produce a solid phase carrier with a white background, which is more convenient for the identification of protein sites with water-soluble dyes and makes the visual positioning effect better.

[0030] In yet another aspect, the present invention provides a method for preparing a solid phase protein chip.

[0031] The present invention adopts a spotting unit to spot the protein planar array on a solid phase carrier, thereby completing the efficient preparation of a solid phase protein chip, and is suitable for large-scale production of biochips.

[0032] The spotting unit is composed of a capillary clamping sheet and a spotting capillary.

[0033] Furthermore, a capillary clamping groove is provided on the capillary clamping sheet, the capillary clamping groove is semicircular, and the outer diameter of the spotting capillary is equal to the semicircular diameter of the capillary clamping groove.

[0034] Furthermore, the capillary clamping sheets can be stacked layer by layer, and the semicircular diameter of the capillary clamping groove on each layer of the capillary clamping sheets is consistent with the diameter of the sample spotting capillary clamped.Figure 1 and Figure 2 As shown, taking a spotting unit with 16 dots as an example, firstly, four spotting capillaries are arranged in parallel in the corresponding capillary clamping grooves of the capillary clamping sheet 1, and then the capillary clamping sheet 2 is superimposed, and the clamping groove interface diameter and position of the capillary clamping sheet 2 are the same and consistent with those of the first layer of spotting capillaries; according to the above method, the superposition of 5 capillary clamps and corresponding spotting capillaries is completed in sequence.

[0035] Furthermore, the spotting capillary has certain rigidity and uniformity, and the material includes but is not limited to different types of stainless steel.

[0036] Furthermore, the spotting capillary clamping sheet is made of a material with a certain rigidity, including but not limited to stainless steel, aluminum, copper machined parts; it can also be a polymer plastic material by injection molding or 3D printing.

[0037] Furthermore, the method for spotting the protein planar array on the solid phase carrier by the spotting unit is: fixing the capillary and the capillary clamping sheet, and keeping the capillary spotting head horizontal for spotting.

[0038] Furthermore, there are two ways to keep the plane horizontal. One way is to level the plane with appropriate looseness and fixture cooperation, such as using a height measuring instrument, and fix the spotting capillary and the spotting capillary support sheet when all the spotting capillary heads are in a plane 3. The other way is to ignore the problem of keeping the plane horizontal, and after fixing it, use a wire cutting process to cut the spotting needle flat.

[0039] Furthermore, the bottom of the capillary array of the spotting unit is a spotting needle array, and the upper part is sheathed with a capillary tube; the capillary tube can be made of chemically inert plastic material, including but not limited to polytetrafluoroethylene, polyethylene, polypropylene, PEEK, silicone, etc.

[0040] Furthermore, the capillary tube is connected to a syringe pump controlled by a stepper motor or a servo motor, and the motor is controlled to control the injection of micro-droplets from the sampling capillary sampling head. Taking a common 100uL syringe pump with a piston diameter of 1.5mm as an example, when the piston is moved 1μm, about 2nL of liquid can be injected. Using a conjoined syringe pump, tiny liquid transfers of multiple sampling capillaries can be completed by controlling one motor.

[0041] Furthermore, the spotting unit is fixed in the xy plane, the solid phase carrier is placed below the spotting unit, and the spotting unit is moved downward along the z-axis direction and stops above the solid phase carrier.

[0042] Furthermore, the vertical distance between the plane formed by the spotting capillary array and the solid phase carrier is smaller than the diameter of the droplet ejected from the spotting capillary.

[0043] Furthermore, there are two ways of spotting: one way is to eject droplets while moving downward, and when the movement stops, the bottom of the droplet contacts the solid phase carrier, and when the spotting unit moves upward, the droplet remains on the solid phase carrier; the other way is to start ejecting droplets after the spotting unit stops moving downward, and when sufficient droplets are ejected, move the spotting unit upward, and the droplet will remain on the solid phase carrier.

[0044] Furthermore, after one spotting is completed, the solid phase carrier is removed, and another one or a batch of solid phase carriers are placed at the same position, and the above steps are repeated.

[0045] On the other hand, the present invention provides a method for preparing a solid-phase protein chip with visual positioning, which mainly adopts the method for preparing a solid-phase protein chip as described above, wherein the spotting capillary is filled with the protein fixing solution as described above, and the water-soluble dye in the protein fixing solution can be completely removed during the cleaning process of the solid-phase protein chip.

[0046] Furthermore, in a clean environment, such as a 100,000-class clean room, at a temperature of 20-25°C and a relative humidity of 40-60%, the protein is diluted with an appropriate fixative to a fixative solution of a certain concentration, loaded into an industrial syringe pump, and the syringe pump is driven at the same time to squeeze out trace amounts of different fixative solutions from the capillary at the same time, revealing droplets at the capillary head.

[0047] Due to the atmospheric pressure and the surface tension of the droplets, the liquid on each capillary takes the shape of a spherical crown, and the protruding droplet spherical crown is dipped onto the plane of the solid phase carrier in the vertical direction.

[0048] Furthermore, the solid phase carrier dipped with the array protein solution is naturally dried in an environment with a temperature of 20-25°C and a relative humidity of 40-60%, and then moved to an environment with a relative humidity below 30% and a temperature controlled at 20-70°C for full drying.

[0049] In another aspect, the present invention provides a method for determining a protein, the method comprising the following steps:

[0050] 1) Take the solid phase protein chip as described above, place it at a specific position, and use a camera to photograph the chip to visually locate the array of fixed proteins on the chip. The software stores the position information and analysis area of ​​each fixed protein.

[0051] 2) Take the sample dilution solution to be tested and mix it evenly with the chip, let it stand, wash the chip, add the specific reagent containing the marker enzyme, let it stand, wash the chip, add the substrate solution, let it stand, wash the chip, and absorb the residual liquid on the chip;

[0052] 3) The chip obtained in step 2) is placed at the specific position in step 1) and the camera takes a picture of the chip;

[0053] 4) Use the information stored in the software to calculate the grayscale value of each fixed protein in the corresponding area.

[0054] Furthermore, the visual positioning of the array of fixed proteins on the chip in step 1) includes collecting visual array images on the chip by naked eyes and machine vision.

[0055] The naked eye observation is mainly suitable for situations where there are fewer types of analytes and for judging the distribution of proteins fixed at various array points on the chip; the machine vision can greatly expand the number of analytes and store the generated data.

[0056] For a solid-phase chip that can obtain the results of multiple indicators at one time, when the number of detected indicators is small, for example, no more than 4 items, it can be used to obtain the test results by direct observation with the naked eye. When the number of detection indicators reaches ten or even dozens, it is obviously very difficult to judge by the naked eye alone. Therefore, it is necessary to use visual sensors, such as taking pictures with smart phones, scanners, etc. to assist in acquiring images, and synchronously obtain the test results of various indicators with the help of image analysis software. The present invention uses a camera to shoot the chip, which is very suitable for situations where the number of detection indicators is large.

[0057] Furthermore, the determination step described in step 2) is mainly based on immunological principles.

[0058] The use of protein chips depends on the ability of antigens and antibodies to form immune complexes. Liquid reagents involved in immune reactions need to be modified to improve the observability of immune complexes. So far, a variety of modification methods have been developed, such as the use of isotope labels, substances that can be excited by external light sources to produce fluorescence, substances that can emit light under certain chemical conditions, chromophores that can be observed through accumulation, and enzyme labels that amplify signals with the assistance of enzymes.

[0059] Furthermore, the present invention preferably uses an enzymatic method to initiate signal amplification of immune complexes formed with the participation of immobilized proteins.

[0060] This is mainly because this method has sufficient sensitivity and the signal formed can be observed in the visible light range. It can not only be observed by the naked eye, but also recorded by ordinary visual sensors and further realize qualitative and even quantitative analysis of the analytes in the tested samples.

[0061] Furthermore, the enzyme includes but is not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase and the like.

[0062] The common use of these enzymes is to use chemical methods to label liquid reagent components involved in the immune complex reaction. When the component participates in the formation of the immune complex, the enzyme is also added to the immune complex. Under appropriate conditions, the enzyme uses its high efficiency and specificity to catalyze the substrate and form visible spots around the immune complex. Usually, the presence or depth of the spots reflects the presence and content of the analyte in the sample.

[0063] Furthermore, the immunological principles mainly include indirect method or sandwich method, competition method, blocking method and the like.

[0064] For the indirect method or sandwich method, a solid phase carrier with protein macromolecules fixed thereon is contacted with a liquid sample or sample diluent (hereinafter collectively referred to as a sample). The analyte in the liquid sample reacts immunologically with the protein macromolecules fixed on the carrier. After a period of time, the chip is separated from the sample, and after sufficient cleaning, the chip is reacted with another reagent, such as for the sandwich method, the liquid contains a specific antigen or specific antibody of the marker enzyme; for the indirect method, the liquid contains a secondary antibody of the marker enzyme. After a period of time, an antigen chip-antibody sample-antigen enzyme conjugate liquid reagent (double antigen sandwich method), an antibody chip-antigen sample-antibody enzyme conjugate liquid reagent (double antibody sandwich method), and an antigen chip-antibody sample-secondary antibody enzyme conjugate liquid reagent (indirect method) will be formed. In particular, for the sandwich method, the chip and the sample-liquid reagent mixture can also be reacted simultaneously. After a period of time, an antigen chip-antibody sample-antigen enzyme conjugate liquid reagent (double antigen sandwich method) and an antibody chip-antigen sample-antibody enzyme conjugate liquid reagent (double antibody sandwich method) will be formed. At this time, the presence or depth of the complex is determined by the presence or content of the analyte in the sample. Afterwards, the highly efficient and specific catalytic activity of the enzyme in the complex is used to produce visual spots under appropriate conditions. The presence or depth of the spots reflects the presence or content of the enzyme in the complex. Since the enzyme in the structure of the complex corresponds to the analyte in the sample one by one, the spots reflect the presence and content of the analyte in the sample.

[0065] For the competitive method, the chip can react with the sample and reagent mixture. When the sample contains the analyte, an antigen chip-antibody sample (to measure the antibody in the sample) or an antibody chip-antigen sample (to measure the antibody in the sample) is formed; when the sample does not contain the analyte, an antigen chip-antibody enzyme conjugate liquid reagent (to measure the antibody in the sample) or an antibody chip-antigen enzyme conjugate liquid reagent (to measure the antibody in the sample) is formed. After that, the efficient and specific catalytic activity of the enzyme in the complex is used to produce visual spots under appropriate conditions. The presence or depth of the spots reflects the presence or content of the enzyme in the complex. Since the enzyme in the structure of the complex corresponds one-to-one with the analyte in the sample, the spots reflect the presence and content of the analyte in the sample.

[0066] For the blocking method, the chip first reacts with the sample, or after washing, or directly reacts with the liquid reagent. When the sample contains the analyte, an antigen chip-antibody sample (determine the antibody in the sample) or an antibody chip-antigen sample (determine the antibody in the sample) is formed; when the sample does not contain the analyte, an antigen chip-antibody enzyme conjugate liquid reagent (determine the antibody in the sample) or an antibody chip-antigen enzyme conjugate liquid reagent (determine the antibody in the sample) is formed. After that, the efficient and specific catalytic activity of the enzyme in the complex is used to produce visual spots under appropriate conditions. The presence or depth of the spots reflects the presence or content of the enzyme in the complex. Since the enzyme in the structure of the complex corresponds one-to-one with the analyte in the sample, the spots reflect the presence and content of the analyte in the sample.

[0067] Furthermore, step 4) is the step of data processing and result feedback during the protein determination process.

[0068] The so-called data processing and result feedback refers to locating the location of the analyte and the analyzed area at that location through the information stored before the test, and then collecting the signal amount in that area. The analyte is determined by the location, and the concentration of the analyte that causes the immune response in the area is fed back through the presence or strength of the signal.

[0069] The solid-phase protein chip with visualized positioning provided by the present invention has the following beneficial effects:

[0070] 1. By using a protein fixative containing water-soluble dyes, surfactants, water-soluble high molecular polymers and other components, the protein is fixed on a specific solid phase carrier to obtain a visual positioning solid phase protein chip that can be directly observed and positioned;

[0071] 2. The visual positioning method can be used to match the position of the spot-fixed protein on the chip with the position of the result interpretation. This not only brings more convenience for the simultaneous analysis of a large number of indicators, but also reduces the difficulty of manufacturing solid-phase chips. At the same time, visual detection can be introduced into the quality control link of solid-phase chip manufacturing to improve the quality of solid-phase chips;

[0072] 3. The obtained visualization positioning chip does not require pretreatment such as sealing and can be directly used for protein detection, which provides more convenience for the preparation and use of solid-phase protein chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Schematic diagram of 16 dot matrix integrated spotting units in Example 1

[0074] Figure 2 Schematic diagram of the equipment of 16 dot matrix integrated spotting units in Example 1

[0075] Figure 3 Schematic diagram of a single spotting process in Example 1

[0076] Figure 4 Schematic diagram of the 16-dot dot pattern effect in Example 1

[0077] Figure 5 This is the image information collection diagram of each point before the measurement in Example 2

[0078] Figure 6 The location information and the distribution map of the analyzed area obtained in Example 2

[0079] Figure 7 The detection result image acquisition diagram in Example 2

[0080] Figure 8 This is a visualization image information collection diagram of each chip before the measurement in Example 3

[0081] Fig. 9 This is a visualization result information collection diagram of each chip after the measurement in Example 3

[0082] Fig.10 The four-parameter equation simulation curve diagram between the alpha-fetoprotein AFP concentration and the grayscale average value of the sample in Example 3 DETAILED DESCRIPTION

[0083] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0084] Example 1 Preparation of the solid-phase protein chip for visual positioning provided by the present invention

[0085] The preparation of the solid-phase protein chip for visual positioning provided in this embodiment is as follows Figure 1-4 As shown, Figure 1 It is a schematic diagram of 16 dot matrix integrated spotting units; Figure 2 This is a schematic diagram of the equipment for 16 dot matrix integrated spotting units. Figure 3 It is a schematic diagram of a single spotting process; Figure 4 This is a schematic diagram of the 16-dot dot matrix patterning effect.

[0086] The solid-phase protein chip with visualized positioning prepared in this embodiment is prepared by using a spotting unit to spot a protein planar array on a solid-phase carrier, thereby completing the efficient preparation of the solid-phase protein chip, which is suitable for large-scale production of biochips. Figure 1As shown, the spotting unit 10 provided in this embodiment is a spotting unit with 16 dots, which is composed of a capillary clamping sheet 11 and a spotting capillary 12. The capillary clamping sheet 11 is provided with a capillary clamping groove 13, which is semicircular, and the outer diameter of the spotting capillary 12 is equal to the diameter of the semicircle of the capillary clamping groove 13.

[0087] Preferably, the capillary clamping sheets 11 can be stacked layer by layer, and the semicircular diameter of the capillary clamping groove 13 on each layer of the capillary clamping sheets 11 is consistent with the diameter of the sample spotting capillary clamped.

[0088] Preferably, the installation process of the spotting unit 10 is as follows: Figure 2 As shown, firstly, four spotting capillaries 12 are arranged in parallel in the corresponding capillary clamping grooves of the capillary clamping sheet 1, and then the capillary clamping sheet 2 is superimposed. The clamping groove interface diameter and position of the capillary clamping sheet 2 are the same and consistent with the first layer of spotting capillaries 12; according to the above method, the superposition of 5 capillary clamps and corresponding spotting capillaries 12 is completed in sequence.

[0089] Preferably, the spotting capillary 12 is made of 304 stainless steel; and the spotting capillary clamping sheet 11 is made of polyvinyl chloride.

[0090] like Figure 3 As shown, the method for spotting the protein planar array on the solid phase carrier by the spotting unit 10 is as follows: the spotting capillary 12 and the capillary clamping sheet 11 are fixed, and the capillary spotting head 14 is kept horizontal for spotting. There are two ways to keep the plane horizontal. One way is to level it with appropriate looseness and the cooperation of the clamp, for example, using a height measuring instrument. When all the spotting capillary spotting heads 14 are in a plane 3, the spotting capillary and the spotting capillary holding sheet are fixed. Another way is to ignore the problem of keeping the plane horizontal, and after fixing it, use a wire cutting process to cut the spotting needle flat.

[0091] The bottom of the capillary array of the spotting unit 10 in this embodiment is a spotting needle array, and the upper part is sheathed with a capillary tube (not shown in the figure); the capillary tube can be made of chemically inert plastic material, including but not limited to polytetrafluoroethylene, polyethylene, polypropylene, PEEK, silicone, etc. The capillary tube is connected to a syringe pump controlled by a stepper motor or a servo motor, and the motor is controlled to control the injection of micro droplets from the spotting capillary spotting head 14. Taking the common 100uL syringe pump with a piston diameter of 1.5mm as an example, when the piston is moved 1μm, about 2nL of liquid can be injected. Using a conjoined syringe pump, the tiny liquid transfer of multiple spotting capillaries can be completed by controlling one motor.

[0092] Preferably, during the spotting process, the spotting unit 10 is fixed in the xy plane, the solid phase carrier 15 is placed below the spotting unit 10, and the spotting unit is moved downward along the z-axis direction and stopped above the solid phase carrier 15. The vertical distance between the plane formed by the spotting array of the spotting capillary 12 and the solid phase carrier 15 is less than the diameter of the droplet injected by the spotting capillary. There are two ways of spotting: one way is to inject the droplet while moving downward, and when the movement stops, the bottom of the droplet contacts the solid phase carrier 15, and when the spotting unit 10 moves upward, the droplet remains on the solid phase carrier 15; the other way is to start injecting the droplet after the spotting unit 10 stops moving downward, and when a sufficient amount of droplets are injected, the spotting unit is moved upward, and the droplet will remain on the solid phase carrier 15. After one spotting is completed, the solid phase carrier 15 is removed, and another or a batch of solid phase carriers 15 are placed in the same position, and the above actions are repeated.

[0093] The tiny liquid is a protein fixative, which contains a water-soluble dye, a surfactant, a balanced salt, and a protein that can bind to the object to be detected. The water-soluble dye can be completely removed during the cleaning process of the solid-phase protein chip.

[0094] Preferably, the solid-phase protein chip with visualized positioning provided in this embodiment is prepared in a 100,000-class clean room at a temperature of 20-25°C and a relative humidity of 40-60%. The protein is diluted with an appropriate fixative to form a protein fixative, which is loaded into an industrial syringe pump. The syringe pump is driven at the same time to squeeze out trace amounts of different fixative solutions from the capillary at the same time, revealing droplets at the capillary head.

[0095] Due to the atmospheric pressure and the surface tension of the droplet, the liquid on each capillary is in the shape of a spherical cap, and the protruding droplet spherical cap 16 is dipped vertically onto the plane of the solid phase carrier 15, as shown in FIG. Figure 4 As shown, the solid phase carrier 15 dipped with the array protein solution is then naturally dried in an environment with a temperature of 20-25°C and a relative humidity of 40-60%, and then moved to an environment with a relative humidity below 30% and a temperature controlled at 20-70°C to fully dry, thereby obtaining a visually positioned solid phase protein chip.

[0096] Example 2 Example of location identification and result calculation of the solid-phase protein chip with visual location provided by the present invention

[0097] This example adopts the preparation method provided in Example 1 to prepare a solid-phase protein chip for visual positioning, wherein the solid-phase carrier is made of a hot-pressed polyvinyl chloride sheet with titanium dioxide added; according to the preparation method of the protein fixative, it is divided into the following five types: 4 μg / mL cytomegalovirus antigen is dissolved in a solution containing:

[0098] ①40μg / mL Tween20, 2μg / mL bovine serum albumin, 10μg / mL carmine, 0.01M phosphate buffer solution (pH7.0);

[0099] ②40μg / mL Tween20, 10μg / mL dextran (5000Da), 2μg / mL bovine serum albumin, 10μg / mL carmine, 0.01M phosphate buffer solution (pH7.0);

[0100] ③10μg / mL Tween20, 10μg / mL dextran (20000Da), 2μg / mL bovine serum albumin, 10μg / mL carmine, 0.01M phosphate buffer solution (pH7.0);

[0101] ④40μg / mL Tween20, 10μg / mL pullulan, 2μg / mL bovine serum albumin, 10μg / mL carmine, 0.01M phosphate buffer solution (pH7.0);

[0102] ⑤10μg / mL carmine, 0.01M phosphate buffer solution (pH 7.0).

[0103] The single-channel spotter is used to spot the solid phase carrier with 1.5μL / spot in a non-precise positioning manner. The chip is placed in an environment with a relative humidity of 40-50% and a temperature of 20-25°C for 3 hours, and then the chip is placed in an environment with a relative humidity of 10-20% and a temperature of 20-25°C for 12 hours, and then stored in a moisture-proof state for use. A solid phase protein chip with visual positioning containing ①, ②, ③, ④, and ⑤ 5 kinds of protein fixatives is prepared. At this time, each area of ​​the fixed protein can be observed on each chip.

[0104] Before measuring the sample, visually locate and analyze the array on the chip: use a camera to capture the position information and analysis area of ​​each fixed protein on the chip, and store at least these two basic information in the software.

[0105] Determination: Cytomegalovirus antibody negative serum samples, low-concentration positive serum samples, and high-concentration positive serum samples were reacted with different chips to be used. Specifically, the sample was diluted 1 / 40 times with a phosphate buffer solution containing 10 mg / mL bovine serum albumin, and then the sample dilution was added to the chip, mixed evenly and allowed to stand for 5 minutes, the chip was cleaned, and then the horseradish peroxidase-protein A conjugate solution was added to the chip, mixed evenly and allowed to stand for 5 minutes, the chip was cleaned, and then the substrate solution was added, allowed to stand for 5 minutes, the chip was cleaned, and the residual liquid on the chip was absorbed. The chip was placed in the same position as before the sample was measured and photographed by a camera.

[0106] Data processing and result feedback: Use the information stored in the software to calculate the average gray value in the corresponding area. The image information of each point before the measurement is collected as follows: Figure 5 As shown, the position information and distribution of the analyzed regions obtained by analysis (the software stores the position information and analysis regions of each fixed protein) are shown in Figure 6 As shown, the detection result image acquisition is as follows Figure 7 As shown. The grayscale value of each fixed protein is calculated in the corresponding area using the information stored in the software. Grayscale uses black tones to represent objects, that is, black is used as the base color, and different saturation blacks are used to display images. Each grayscale object has a brightness value from 0% (white) to 100% (black). The grayscale value calculation results are shown in Table 1.

[0107] Table 1. Grayscale calculation results of different corresponding points

[0108]

[0109] Depend on Figure 7 As can be seen from Table 1, when using ①, ②, ③, and ④, the cytomegalovirus content in the three serum samples of negative, low-concentration positive, and high-concentration positive can be measured more accurately. Among them, the gray value measured by protein fixative ③ is higher, which can further widen the gray value range of low-concentration and high-concentration serum samples, and the detection sensitivity is higher. This may be due to the two components and contents of 10μg / mL Tween20 and 10μg / mL dextran (20000Da), which can play a better blocking role, or regulate the fixation of the target fixed protein and better promote its reaction.

[0110] Although protein fixative ⑤ can also measure the cytomegalovirus content, the test result is significantly lower than that of the other four. The reason is probably because protein fixative ⑤ contains only water-soluble dyes and buffers, without other ingredients, which makes the target protein fixation effect insufficient. It may also fail to play a blocking role because it does not contain other proteins.

[0111] Therefore, protein fixative solution ③ is the best protein fixative solution formula for the visualization and localization solid phase protein chip of cytomegalovirus.

[0112] Example 3 Qualitative and quantitative application examples of the solid-phase protein chip with visual positioning provided by the present invention

[0113] This embodiment adopts the preparation method provided in Example 1 to prepare a solid-phase protein chip for visual positioning, wherein the solid-phase carrier is made of polystyrene with titanium dioxide added by hot injection molding; the preparation method of the protein fixative is as follows: 6 μg / mL anti-alpha-fetoprotein monoclonal antibody is dissolved in 20 μg / mL Tween20, 2 μg / mL bovine serum albumin, 10 μg / mL dextran (5000Da), 10 μg / mL amaranth, 0.05M carbonate buffer solution (pH9.6), and the 18-channel spotting instrument is simultaneously spotted on the solid-phase carrier at 0.5 μL / spot. Stand for 3 hours in an environment with a relative humidity of 40-50% and a temperature of 20-25°C, then stand the chip in a relative humidity of 10-20% and a temperature of 20-25°C for 12 hours, and then store it in a moisture-proof state for use. At this time, each area of ​​the fixed protein can be observed on each chip.

[0114] Before measuring the sample, visually locate and analyze the array on the chip: use a camera to capture the position information and analysis area of ​​each fixed protein on the chip, and store at least these two basic information in the software. Figure 8 shown.

[0115] Determination: Samples containing different concentrations of alpha-fetoprotein antigens are reacted with different chips to be used. Specifically, the samples are added to the chip, mixed evenly and allowed to stand for 10 minutes, the chip is cleaned, and then horseradish peroxidase-anti-alpha-fetoprotein antibody conjugate solution is added to the chip, mixed evenly and allowed to stand for 10 minutes, the chip is cleaned, and then the substrate solution is added, allowed to stand for 10 minutes, the chip is cleaned, and the residual liquid on the chip is blotted dry. The chip is placed in the same position as before the sample was measured and photographed by a camera. After the measurement, the visual result information of each chip is as follows: Fig. 9 Data processing and result feedback: The average gray value is calculated in the corresponding area using the information stored in the software. The gray value calculation results are shown in Table 2.

[0116] Table 2. Grayscale calculation results of different corresponding points

[0117]

[0118] There is a positive correlation between the grayscale average and the AFP concentration in the sample, which can be simulated using a four-parameter equation. The curve and simulation equation are as follows: Fig.10 As shown, the four-parameter equation simulates the dose-effect relationship: AFP concentration (ng / ml) = 53.97 ((gray value - 0.095) / (69.44-gray value)) （1 / 1.24） According to this relationship, the AFP concentration of the unknown sample can be calculated.

[0119] This embodiment is only a detection of one indicator, and the purpose is only to illustrate that the present invention can be used to achieve qualitative and quantitative determination through visual precise positioning. When the indicators of each point in the chip are independent of each other, that is, the formation of immune complexes is not interfered by other indicators, the present invention can be used to obtain quantitative detection results of multiple indicators in one detection.

[0120] Example 4 Comparative verification of the application of the solid-phase protein chip for visual positioning provided by the present invention

[0121] In this example, the preparation method provided in Example 1 was used to prepare a solid-phase protein chip with visualized positioning and an invisible solid-phase protein chip (Huaying Bio) available on the market. At the same time, cytomegalovirus antibody detection was performed on the same batch of samples, and the detection results were compared.

[0122] The solid phase protein chip with visualized positioning provided in Example 1 is made of polystyrene with titanium dioxide added by hot injection molding, and the preparation method of the protein fixative is as follows: 6 μg / mL anti-alpha-fetoprotein monoclonal antibody is dissolved in 20 μg / mL Tween20, 2 μg / mL bovine serum albumin, 10 μg / mL dextran (5000Da), 10 μg / mL amaranth, 0.05M carbonate buffer solution (pH9.6), and the 18-channel spotting instrument is simultaneously spotted on the solid phase carrier at 0.5 μL / spot. The chip is placed in an environment with a relative humidity of 40-50% and a temperature of 20-25°C for 3 hours, and then the chip is placed in a relative humidity of 10-20% and a temperature of 20-25°C for 12 hours, and then stored in a moisture-proof state for standby use. At this time, each area of ​​the fixed protein can be observed on each chip. Visual positioning and analysis of the array on the chip: Use a camera to capture the position information and analysis area of ​​each fixed protein on the chip, and store at least these two basic information in the software.

[0123] The invisible solid phase protein chip labels the alpha-fetoprotein serum sample to be tested with fluorescent dye, and the protein bound to the chip will emit a specific signal.

[0124] Determination: Samples containing different concentrations of alpha-fetoprotein antigen are reacted with a visually positioned solid phase protein chip and an invisible solid phase protein chip respectively.

[0125] The specific steps are:

[0126] (1) For cleaning of the invisible solid-phase protein chip, add blocking solution prepared with bovine serum albumin, block overnight, and then clean; for the visual positioning solid-phase protein chip, no operation is required.

[0127] (2) Add the sample to the visualized positioning solid phase protein chip, mix well and let it stand for 10 minutes, wash the chip, then add the horseradish peroxidase-anti-alpha-fetoprotein antibody conjugate solution to the chip, mix well and let it stand for 10 minutes, wash the chip, then add the substrate solution, let it stand for 10 minutes, wash the chip, and dry the residual liquid on the chip;

[0128] At the same time, the sample was labeled with fluorescein and added to the invisible solid phase protein chip. After being mixed evenly, it was allowed to stand for 10 minutes. The chip was cleaned and the residual liquid on the chip was aspirated.

[0129] (3) Place the visualized solid-phase protein chip in the same position as before the sample was measured and take a picture with a camera; place the invisible solid-phase protein chip in a fluorescence meter to read the fluorescence value.

[0130] Data processing and result feedback: The detection results of the visual solid phase protein chip were used to calculate the average gray value in the corresponding area using the information stored in the software, and the concentration of alpha-fetoprotein AFP was calculated using the four-parameter equation provided in Example 3 to simulate the dose-effect relationship; the invisible solid phase protein chip calculated the concentration of alpha-fetoprotein AFP based on the read fluorescence value. The results are shown in Tables 3 and 4, respectively.

[0131] Table 3. Determination results of the visual positioning solid phase protein chip provided by the present invention

[0132]

[0133] Table 4. Results of the invisible solid phase protein chip assay

[0134]

[0135] It can be seen from Table 3 and Table 4 that both the visualized positioning solid-phase protein chip provided by the present invention and the existing invisible solid-phase protein chip can be used to quantitatively detect the concentration of alpha-fetoprotein AFP in a sample, and the visualization positioning solid-phase protein chip provided by the present invention has higher detection accuracy and a more convenient detection process, without any pre-operation such as sealing and cleaning. In addition, due to its visualization positioning feature, it is convenient for quality control of the solid-phase protein chip and simultaneous analysis of a large number of indicators, which reduces the manufacturing difficulty of the solid-phase protein chip and improves the quality, and has huge market potential.

[0136] Although the present invention is disclosed as above, the present invention is not limited thereto. For example, it can be expanded according to its application range in medicine. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims

1. A method of preparing a reagent for visual positioning solid-phase protein chip for improving the sensitivity of cytomegalovirus detection by using a protein fixative, characterized in that: The protein fixative solution is composed of 10 μg / mL Tween20, 10 μg / mL dextran, 2 μg / mL bovine serum albumin, 10 μg / mL carmine, 0.01M phosphate buffer solution and cytomegalovirus antigen, wherein the molecular weight of the dextran is 20000Da; the visual positioning solid phase protein chip does not require blocking pretreatment and can be directly used for cytomegalovirus detection; the fixed protein formed by the protein fixative solution after being fixed and dried, and the water-soluble dye in the protein fixative solution can be completely removed during the cleaning process of the solid phase protein chip; the cytomegalovirus detection method comprises the following steps: 1) Take a visual positioning solid phase protein chip, place it at a specific position, and use a camera to photograph the chip to visually position the array of fixed proteins on the chip. The software stores the position information and analysis area of ​​each fixed protein. 2) Mix the sample dilution solution and the chip evenly, let it stand, wash the chip, add a specific reagent containing a marker enzyme, let it stand, wash the chip, add a substrate solution, let it stand, wash the chip, and dry the residual liquid on the chip; 3) placing the chip obtained in step 2) at the specific position in step 1) and photographing the chip with a camera; 4) Calculate the grayscale value of each fixed protein in the corresponding area using the information stored in the software.

2. The protein fixative for use according to claim 1, characterized in that: The invention is composed of 10 μg / mL Tween 20, 10 μg / mL dextran, 2 μg / mL bovine serum albumin, 10 μg / mL carmine, 0.01M phosphate buffer solution and cytomegalovirus antigen, wherein the molecular weight of the dextran is 20000 Da.

3. A solid-phase protein chip for visual positioning, characterized in that: The solid phase protein chip contains fixed protein formed by fixing and drying the protein fixing solution as claimed in claim 2, and the water-soluble dye in the protein fixing solution can be completely removed during the cleaning process of the solid phase protein chip.

4. The solid phase protein chip according to claim 3, characterized in that The solid phase protein chip also includes a solid phase carrier, which is prepared by using an organic high molecular polymer with thermoplasticity and surface polarity, or is prepared by coating with the organic high molecular polymer.

5. The solid phase protein chip according to claim 4, characterized in that The organic high molecular polymer is one or more of polyvinyl chloride, polystyrene, polyphenylene propylene, polyethylene terephthalate, and polyvinylidene fluoride; titanium dioxide is also added to the organic high molecular polymer.

6. A method for determining protein, characterized in that: The following steps are involved: 1) taking the solid phase protein chip according to any one of claims 3 to 5, placing it at a specific position, visually positioning the array of fixed proteins on the chip by photographing the chip with a camera, and storing the position information and analysis area of ​​each fixed protein in the software; 2) Mix the sample dilution solution and the chip evenly, let it stand, wash the chip, add a specific reagent containing a marker enzyme, let it stand, wash the chip, add a substrate solution, let it stand, wash the chip, and dry the residual liquid on the chip; 3) placing the chip obtained in step 2) at the specific position in step 1) and photographing the chip with a camera; 4) Calculate the grayscale value of each fixed protein in the corresponding area using the information stored in the software.

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