BIOLOGICAL REACTION APPARATUS AND METHOD FOR PERFORMING BIOLOGICAL DETECTION ON BASIS OF APPARATUS

A biological reaction apparatus used for biological macromolecules includes a power supply module (1), a control module (2), a liquid processing module (3), a reactor module (4) and a sensor (5); the power supply module (1) includes a direct current power supply (6) and a switch (7); the control module (2) includes a system controller (8), an input device (9) and an output device (10); the liquid processing module (3) includes a valve (11) or a combination of valves (11), a pump (12) or a combination of pumps (12) and sample cells (13); the reactor module (4) includes a reactor (14); the reactor (14) includes a reactor frame (15) and a reactor cavity (16) formed by the reactor frame (15). The apparatus has a simple and practical structure and is easy to operate. In the reactor (14), a very small amount of a reagent is used to achieve a uniform and highly-sensitive reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of a Chinese patent application with the application number of 201910439559.7 and the disclosure title of “Biological Reaction Apparatus and Method for Performing Biological Detection on Basis of Apparatus”, which was submitted to the Chinese Patent Office on May 24, 2019, and all contents of the patent application is incorporated by reference in this application.

TECHNICAL FIELD

The present disclosure relates to the technical field of biology, in particular to a biological reaction apparatus and a method for performing biological detection on basis of the apparatus.

BACKGROUND

Common detection methods for biological macromolecules include forming feedback signals on basis of structures of biological macromolecules and detection reagents.

Common experimental methods are as follows:1. Protein detectionA) Gel or other vectors with proteins are put into a staining solution;B) stains are bound to the proteins by vibration and heating;C) the stained gel and the like are put into a destaining solution; andD) stains not bound to the proteins are removed from the gel and the like by vibration and heating.2. Nucleic acid detectionA) Gel or other vectors with nucleic acid are put into a staining solution;B) stains are bound to the nucleic acid by vibration and heating;C) the stained gel and the like are put into a destaining solution; andD) stains not bound to the nucleic acid are removed from the gel and the like by vibration and heating.3. Probe detectionA) Vectors with samples are put into a blocking solution;B) an unbound sample area is rapidly bound to a blocking substance by vibration and heating;C) the blocked vectors are put into a probe solution;D) probes are rapidly bound to the samples by vibration and heating;E) the vectors are put into a cleaning solution; andF) non-specifically bound probes are washed away by vibration and heating.

A general detection method for biological macromolecules can be described as a soaking method. Soaking is performed in a large amount of a solution, and specific binding and non-specific removal are performed by thermal motion of molecules. Although the thermal motion of molecules can be accelerated by vibration, heating, ultrasound, microwave and other ways, existing defects, such as high consumption of time and labors, waste of reagents, low repeatability and low uniformity, are still unavoidable.

Therefore, it is of great practical significance to provide an automatic biological reaction apparatus for detecting biological macromolecules.

SUMMARY

In view of this situation, the present disclosure provides a biological reaction apparatus and a method for performing biological detection on basis of the apparatus. The apparatus has a simple and practical structure and is easy to operate. In a reactor, a very small amount of a reagent is used to achieve a uniform and highly-sensitive reaction.

To achieve the foregoing inventive objectives, the present disclosure provides the following technical solutions:

The present disclosure provides a biological reaction apparatus, including a power supply module1, a control module2, a liquid processing module3, a reactor module4and a sensor5; the power supply module1includes a direct current power supply6and a switch7; the control module2includes a system controller8, an input device9and an output device10; the liquid processing module3includes a valve11or a combination of valves11, a pump12or a combination of pumps12and sample cells13; the reactor module4includes a reactor14; and the reactor14includes a reactor frame15and a reactor cavity16formed by the reactor frame15; the power supply module1is separately connected to the control module2, the liquid processing module3and the reactor module4through cables25; the control module2is separately connected to the liquid processing module3and the reactor module4through the cables25; the liquid processing module3is connected to the reactor module4through a pipeline26; and the control module2is connected to the reactor module4through the sensor5.

In some specific embodiments of the present disclosure, the reactor frame15is provided with at least one opening17; and the opening17is connected to the pump12or the combination of pumps12.

In some specific embodiments of the present disclosure, the reactor frame15is further provided with a pressing part18.

In some specific embodiments of the present disclosure, the reactor frame15is further provided with a sealing part19.

In some specific embodiments of the present disclosure, the reactor frame15includes a reactor front plate20and a reactor rear plate21, and a limiting part22is also arranged between the reactor front plate20and the reactor rear plate21. The limiting part is used to prevent the situation that since the stroke of96and15is too large during pressing, the volume of16is too small.

In some specific embodiments of the present disclosure, a mother liquid pool23is also arranged between the liquid processing module3and the reactor module4.

In some specific embodiments of the present disclosure, at least one diversion block24is also arranged in the reactor cavity16.

On basis of the researches above, the present disclosure also provides an application of the biological reaction apparatus in detection of biological samples.

The present disclosure also provides a biological sample detection method, including on basis of the biological reaction apparatus provided in the present disclosure, putting a vector carrying a reactant into the reactor cavity16, putting a reaction liquid into the sample cells13of the liquid processing module3and turning on the control module2by the power supply module1to control the pump12or the combination of pumps12of the liquid processing module3to deliver the reaction liquid in the sample cells (13) into the reactor cavity16, so as to make the reaction liquid mixed with the vector carrying the reactant for reaction and detection.

According to the automatic biological reaction apparatus provided in the present disclosure, the whole process can be completed automatically by only placing a sample and a reaction solution to specific positions and setting a program.

It is shown through experiments that:

In a Coomassie brilliant blue staining test, on IgG, all bands in a traditional method and the present disclosure are visible, but the color in the present disclosure is darker. On BSA, the 10thsample in the traditional method is visible, and all 12 samples in the present disclosure are visible, so that the sensitivity of the present disclosure is better. On lysozyme, the 9thsample in the traditional method is visible, and the 11thsample in the present disclosure is visible, so that the sensitivity of the present disclosure is better. It can be seen fromFIG. 9(C),FIG. 9(D),FIG. 9(E)and Tables 2-4 that compared with the traditional method, the present disclosure has the advantages that the staining degree is increased by 4.49%-22.90% based on IgG detection results; the staining degree is increased by 14.87%-60.00% based on BSA detection results; and the staining degree is increased by 9.72%-28.37% based on lysozyme detection results. Compared with the traditional method, only 50% of reagents need to be used in the present disclosure, and the sensitivity and the staining degree of the present disclosure are better than those of the traditional method.

In a silver staining test, when half of the reagents are used, the sensitivity of the present disclosure is better than that of the traditional method, and the background is better than that of the traditional method. On IgG, all bands in the traditional method and the present disclosure are visible. On BSA, the 10thsample in the traditional method is visible, and all 12 samples in the present disclosure are visible, so that the sensitivity of the present disclosure is better. It can be seen fromFIG. 10(C),FIG. 10(D)and Tables 6-7 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 114.91%-139.27% based on IgG detection results; and the staining degree is increased by 100.88%-186.47% based on BSA detection results. Compared with the traditional method, only 50% of reagents need to be used in the present disclosure, and the sensitivity and the staining degree are better than those of the traditional method.

In a WB1 test, on 93 kd, the 4thlane in the traditional method is visible, and the 5thlane in the present disclosure is visible, so that the sensitivity of the present disclosure is better. On 7 kd, the 8thlane in the traditional method is visible, and the 9th lane in the present disclosure is visible, so that the sensitivity of the present disclosure is better. It can be seen fromFIG. 11(E),FIG. 11(F)and Tables 9-10 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 102.09%-507.93% based on 93 kd detection results; and the staining degree is increased by 20.61%-432.91% based on 7 kd detection results. Compared with the traditional method, only 74% of reagents and 80% of antibodies need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

In a WB2 test, the 6thlane in the traditional method is visible, and the 8thlane in the present disclosure is visible, so that the sensitivity of the present disclosure is better. It can be seen fromFIG. 12(C)and Table 12 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 3.05%-305.33% based on β-actin detection results. Compared with the traditional method, only 65% of reagents, 40% of antibodies and a shorter processing time need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

In a WB5 test, the color ofFIG. 13(B) andFIG. 13(D) is darker than that ofFIG. 13(A) andFIG. 13(C), and it can be seen fromFIG. 13(E)and Table 13 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 50.38%-108.84% based on GAPDH detection results. That is to say, the signal intensity is higher, and it is further shown that the effect of the present disclosure is better than that of the traditional method. Compared with the traditional method, only66% of reagents need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

In a WB3 test, it can be seen fromFIG. 14that the 4thlane in the traditional method is visible, and the5th lane in the present disclosure is visible, so that the sensitivity of the present disclosure is better. It can be seen fromFIG. 14(C)and Table 15 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 27.52%-603.57% based on β-actin detection results. Compared with the traditional method, only 30% of reagents and a shorter processing time need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

In a WB4 test, it can be seen fromFIG. 15(D)and Table 16 that bands in the present disclosure are darker than those in the traditional method, and the staining degree is increased by 59.26%-111.13%, showing that the effect of the present disclosure is better. Compared with the traditional method, only 20% of reagents and a shorter processing time need to be used in the present disclosure, and the signal intensity of the present disclosure is better than that of the traditional method.

In summary, compared with the traditional method, the automatic biological reaction apparatus has the following advantages:1. Time is saved. The system is fully automatic, a lot of labors are reduced, and the efficiency is greatly improved.2. The sensitivity is high. Compared with the traditional method, the reaction efficiency is improved by liquid flow control, and thus the sensitivity is higher under conditions same as those in the traditional method.3. Reactor reagents are reduced. In the automatic biological reaction apparatus, only a small amount of a liquid needs to be used in the reactor to complete a reaction requiring a large amount of reagents in the traditional method.

DETAILED DESCRIPTION

The present disclosure provides a biological reaction apparatus and a method for performing biological detection on basis of the apparatus. Modifications can be made by those skilled in the art by appropriately changing process parameters on basis of contents of the disclosure. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art and are all deemed to be included in the present disclosure. The method and application of the present disclosure are described by using exemplary embodiments. Apparently, a person skilled in the art may make changes, appropriate modifications, and combinations to the method and application described in this specification without departing from the content, spirit and scope of the present disclosure, to implement and apply the technologies of the present disclosure.

In view of shortcomings of the prior art, an objective of the present disclosure is to provide an automatic system used for reactions of biological macromolecules.

Another objective of the present disclosure is to provide a method for detecting biological macromolecules by using the automatic reaction system for biological macromolecules.

The objective of the present disclosure may be achieved by using the following technical solutions:

The present disclosure protects an automatic reaction system for biological macromolecules (can also be called a biological reaction apparatus in the present disclosure), including a power supply module1, a control module2, a liquid processing module3, a reactor module4and a sensor5; the power supply module1provides driving energy and energy required for reactions to the liquid processing module3and the reactor module4; the liquid processing module3is controlled by the control module2, and feeds back an operating state to the control module, and the liquid processing module3inputs or outputs a reactant to the reactor module4; and the reactor module4feeds back an operating state to the control module2and the liquid processing module3, the reactor module4can carry the reactant, and the reactant is suspended in the reactor module4.

In some embodiments, the power supply module1includes a direct current power supply6and a switch7; the control module2includes a system controller8, an input device9and an output device10; the liquid processing module3includes a valve11or a combination of valves11, a pump12or a combination of pumps12and sample cells13; the reactor module4includes a reactor14; and the reactor14includes a reactor frame15and a reactor cavity16formed by the reactor frame15; the power supply module1is separately connected to the control module2, the liquid processing module3and the reactor module4through cables25; the control module2is separately connected to the liquid processing module3and the reactor module4through the cables25; the liquid processing module3is connected to the reactor module4through a pipeline26; and the control module2is connected to the reactor module4through the sensor5.

In some embodiments, the reactor module4utilizes liquid flow or gas flow to make the reactant suspended in the reactor14. A liquid film or a gas film may be formed between the reactant and the wall of the reactor14to prevent a wall attachment effect of the reactant.

In some embodiments, there are one or more reactors14, and the reactors can withstand a pressure.

In some embodiments, when there are a plurality of reactors14, the reactors may be connected in series or in parallel.

In some embodiments, the reactor14may be a disposable reactor or a reusable reactor.

In some embodiments, at least one liquid inlet and outlet is formed in the reactor14and used for adding and discharging a reaction liquid.

In some embodiments, the inner wall of the reactor14is smooth or is provided with a certain structure, such as a diversion block24, which is used to perform liquid diversion to make the liquid uniformly distributed in the reactor.

In some embodiments, the inner wall of the reactor14may be subjected to hydrophobization or hydrophilization treatment.

In some embodiments, hydrophobization may be one or more of siliconization and alkylation.

In some embodiments, hydrophilization may be one or more of hydroxylation, carboxylation and amination.

In some embodiments, the reactor frame15is further provided with a pressing part18to ensure the sealing performance after the reactor is closed.

In some embodiments, the reactor14is made from a metal material, such as stainless steel and aluminum alloy, or a polymer material, such as polypropylene and an acrylonitrile-styrene-butadiene copolymer.

In some embodiments, the reactor frame15is further provided with a sealing part19, and the sealing part19is made from an elastic material or a material repellent to a reaction solution.

In some embodiments, the reactor module4further includes an identification module; and according to the biological reaction apparatus provided in the present disclosure, the type of the reactor and an existing program are fed back through identification results.

In some embodiments, RFID is used as the identification module. The reactor module4is provided with an identifier, and the reactor is provided with a serial number chip. After the reactor14is transferred into the reactor module4, the serial number of the chip is identified by the identifier and has a corresponding program or no corresponding program in the reactor module4. When there is an existing program, the program is called by the reactor module4. When there is no corresponding program, a program may be set through the reactor module4and saved.

In some embodiments, a stopped reaction is continued by using an identification method. For example, after a reaction liquid is subjected to a reaction, the reactor14is removed from the reactor module4to stop the process. After the reactor14is put into the reactor module4again, an unfinished program is identified by the reactor module4and displayed on an interface, and a prompt about whether or not to continue the program is given to an operator.

In some embodiments, a mother liquid pool23is further included before the reactor14, the reaction solution is diluted and removed by flowing through the mother liquid pool23, and a final reaction solution is formed in the reactor14.

In some embodiments, the reactant is a biological macromolecule vector, such as a thin film, polyacrylamide gel and agarose gel.

In some embodiments, when the reactant is a thin film, a micro-cavity is formed in the reactor14. When the reactant is gel, the micro-cavity is the reactor cavity16, and can withstand a positive pressure or a negative pressure.

In some embodiments, the liquid processing module3includes one or more sample storage containers, namely, the sample cells13.

In some embodiments, the liquid processing module3includes at least one one-way and/or two-way pump12.

In some embodiments, the pump12is one or more of a peristaltic pump, a diaphragm pump, a gear pump and a plunger pump.

In some embodiments, the reaction solution is pumped into or out of the reactor14by the pump12.

In some embodiments, the liquid processing module3includes a valve11or a combination of valves11.

In some embodiments, the valve11is one or more of a diaphragm valve, a pinch valve and a column valve.

In some embodiments, two or more different solutions in the reactor14may be used and automatically exchanged freely by the liquid processing module3.

In some embodiments, the reactor14is connected to the liquid processing module3through a pipeline26or a one-way valve or a two-way fast connector. A two-way fast card is used to connect a pipeline and the reactor. When the reactor contains a liquid, the reactor is removed from the system (Example 17) and disconnected from the pipeline, and the reactor and the pipeline are blocked to prevent liquid leakage or contamination.

In some embodiments, the reaction liquid is cycled in the reactor14with the biological reaction apparatus using the pump12.

In some embodiments, the biological macromolecule is one or more of nucleic acid, protein and polypeptide.

In some embodiments, the biological reaction apparatus includes a liquid sensing module.

In some embodiments, at least one reactor14is included. The number of reactors14depends on the number of samples in the reactor14and requirements of a reaction solution. For example,1sample and1solution require1reactor14;2samples and1solution require1reactor14. The sample is put into the reactor14, and the system is operated according to settings after the solution quantity, sequence and time are set. In the biological reaction apparatus, the solution is pumped into or out of the reactor14through the pump12, and a circulation may be formed according to requirements and settings to prolong the contact time of the sample and the reaction solution.

In some embodiments, the solution may be pumped by setting pulses to increase the contact chance.

Specifically, the structure of the automatic reaction system for biological macromolecules provided in the present disclosure is as follows:

FIG. 1shows basic compositions of the present disclosure.

1refers to a power supply module, including a direct current power supply6and a switch7, and is used to convert alternative current input into direct current output and provide energy required for operation of other parts of the present disclosure.

2refers to a control module, including a system controller8, an input device9and an output device10, and is operated with energy input from the power supply module1; the energy provided by the power supply module1is transmitted to the liquid processing module3and the reactor module4through cables25; and the working state of the liquid processing module3and the reactor module4is detected through the sensor5and controlled.

3refers to a liquid processing module and includes a valve11or a combination of valves, a pump12or a combination of pumps and sample cells13, and the valve11or the combination of valves, the pump12or the combination of pumps and the reactor14are connected through a pipeline (26). The valve11or the combination of valves performs sample selection according to signals of the control module2, and the pump12or the combination of pumps moves the sample into or out of the reactor14. The sensor5is used to detect the operating state of the liquid processing module3and give feedback to the control module2. The operating energy of the liquid processing module3is transmitted from the power supply module1by the control module2, or may be directly provided by the power supply module1.

4refers to a reactor module, where14refers to a reactor,17refers to an opening formed in the reactor frame15and is a liquid inlet17-1or a liquid outlet17-2, and a liquid is pumped into or out of the reactor14by the pump12or the combination of pumps connected to the opening17through a pipeline. The sensor5is used to detect the operating state of the reactor14and give feedback to the control module2. The operating energy of the reactor module4is transmitted from the power supply module1by the control module2, or may be directly provided by the power supply module1.

FIG. 2shows an application example of the present disclosure, which includes a reactor, four reaction liquids,1pump and6two-position three-way valves.13refers to four sample cells, and each sample cell13can be filled with a reaction liquid.12refers to a pump, specifically a peristaltic pump.14refers to a reactor.17-1refers to a liquid inlet.17-2refers to a liquid outlet.23refers to a mother liquid pool.27refers to a waste liquid collector.28refers to a liquid level indicator.29refers to a reaction liquid inlet and outlet pipeline.30refers to a waste liquid discharge or air inlet pipeline.50,51,52,53,54and55each refers to a two-position three-way valve.32,33and34refer to three liquid receiving ports of a two-position three-way valve,33is a common end, and32and33are in normally open connection.38,39and40refer to three liquid receiving ports of a two-position three-way valve,39is a common end, and38and39are in normally open connection.35,36and37refer to three liquid receiving ports of a two-position three-way valve,36is a common end, and35and36are in normally open connection.41,42and43refer to three liquid receiving ports of a two-position three-way valve,43is a common end, and42and43are in normally open connection.44,45and46refer to three liquid receiving ports of a two-position three-way valve,44is a common end, and46and44are in normally open connection.47,48and49refer to three liquid receiving ports of a two-position three-way valve,49is a common end, and47and49are in normally open connection.28needs to be maintained below the liquid level of a container to ensure that enough liquid may enter during operation.30needs to be maintained above the liquid level of a container to ensure that waste liquid is not sucked into a pipeline when gas is introduced. Furthermore, a gas-liquid selection valve may be added to the tail end of30to ensure that gas intake and liquid discharge are completely isolated.

FIG. 3shows an application example of the present disclosure, which includes three reactors14, six reaction liquids, three pumps12and two valves11or combinations of valves.62refers to a gas buffer bottle.27refers to a waste liquid collector.13refers to a sample cell.11refers to a valve, specifically an eight-position selection valve.66,67,68,69,70,71,72and73each refers to a selection connector of an eight-position valve.74refers to a common connector.65refers to a selection path, which is used to make the selection connectors66,67,68,69,70,71,72and73communicated with the common connector74according to signals of the control module2.64refers to a four-position selection valve group.12refers to a pump, specifically a plunger pump.14refers to a reactor.

FIG. 4shows a flow direction of a liquid in the reactor14on a time axis. Furthermore, the liquid stationary time may be prolonged to improve the effect.

FIG. 5(A)is a schematic diagram of the reactor.19refers to a sealing part,20refers to a reactor front plate,21refers to a reactor rear plate,18-1refers to a front pressing structure of the reactor, and18-2refers to a rear pressing structure of the reactor.18-1and20may be one part, and21and18-2may be one part. After the reactor frame15of the reactor14is closed, a reactor cavity16can be formed. The size of the reactor cavity16may be determined by the sealing part19and a pressing part18. When the reactant is a thin film, a micro-cavity is formed in the reactor cavity16. Furthermore, a limiting part22may be added into20and21to maintain a certain cavity. Furthermore, the sealing part19may be omitted, and a sealing cavity is formed due to the tightness of20and21. At least one opening17is formed in the reactor14,17-1refers to a liquid inlet, and17-2refers to a liquid outlet; and the opening17is connected to the pump12, the valve11or the reactor14and used for sample input or output.

FIG. 5(B)shows a reactor design scheme,FIG. 5(C)is a cross-sectional view, andFIG. 5(D)is a rear view.18refers to a pressing part;91refers to a rotating shaft of the pressing part, and the pressing part18is fixed to the reactor frame15through the rotating shaft and can rotate around the rotating shaft;20refers to a reactor front plate;21refers to a reactor rear plate;15refers to a reactor frame;17refers to an opening, which is connected to the reactor rear plate21through the reactor frame15;19refers to a sealing part; and16refers to a formed reactor cavity.

FIG. 5(E)shows a reactor design scheme,FIG. 5(F)is a cross-sectional view, andFIG. 5(G)is a rear view.96refers to a reactor upper cover;15refers to a reactor frame;20refers to a reactor front plate;21refers to a reactor rear plate;99refers to a connecting shaft of the reactor upper cover96and the reactor frame15;18refers to a pressing part;19refers to a sealing part;22refers to a limiting part;16refers to a reactor cavity; and17refers to an opening.

FIG. 6(A),FIG. 6(B)andFIG. 6(C)are shown, and106refers to an inner wall of the reactor14.17refers to an opening.107refers to a reactant.19refers to a sealing part.109refers to a liquid flow indicator.

FIG. 6(A)shows the state of the reactant107unsuspended in the reactor14, and the reactant is in an irregular shape and may be partially attached to the inner wall106of the reactor14.

FIG. 6(B)andFIG. 6(C)are schematic diagrams of the reactant suspended in a liquid. After the liquid enters the reactor14, the liquid flows through a gap between the reactant and the reactor14to make the reactant suspended in the reactor14. The liquid is introduced through liquid inlets on both sides, and the reactant is suspended in the reactor14through the liquid flow. Furthermore, as shown inFIG. 6(B), the reactant is suspended by introducing a liquid on one side.

FIG. 7shows that the liquid flow is subjected to diversion by adding an opening17or a diversion block24to make the liquid entering the reactor14uniformly distributed.17refers to an opening and is specifically a liquid inlet;19refers to a sealing part and is specifically a sealing ring;16refers to a reactor cavity; and24refers to a diversion block in the reactor14.

FIG. 8(A)is a schematic diagram of a two-way fast card set consisting of a fast card A112and a fast card B113;FIG. 8(B)is a schematic diagram of the fast card B113;FIG. 8(C)is a cross-sectional view of the fast card B113;FIG. 8(D)is a schematic diagram of the fast card A112;FIG. 8(E)is a cross-sectional view of the fast card A112;114refers to a pagoda connector of the fast card B113, is a derivative part of125, and is used to connect pipelines or is directly connected to a container wall;125refers to a closure member of the fast card B113, and forms a containing cavity with116;118refers to an internal flow channel of114;115refers to an inner pagoda pattern of the fast card B113and is used to prevent internal leakage and falling after the fast card A and the fast card B are assembled;116refers to a main body of the fast card B113;117refers to a conductor;119refers to an internal flow channel of the conductor;120refers to a guide structure of the fast card B113and is combined with the fast card A for internal guide fixation;121refers to a limiting structure of the conductor and is used to prevent the conductor117from falling off the fast card B113;122refers to an inner limiting structure of the fast card B113and is used to limit the backward distance of the conductor117;124refers to an outer limiting structure of the fast card B113and is used to limit the outward distance of the conductor117;123refers to a structural part with one-way valve functions and is used to control that when the fast card B113is separated from the fast card A112, the fast card B is blocked in a direction from118to119and communicated in a direction from119to118;129refers to an upper blocking structure of the fast card A112and is used to fix a leakproof ring130;130refers to the leakproof ring of the fast card A112;128refers to a main body of the fast card A112;127refers to a lower blocking structure of the fast card A112;126refers to a pagoda connector of the fast card A112;131refers to a guide structure of the fast card A112;132refers to an internal flow channel of126; and133refers to a structural part with one-way valve functions and is used to control that when the fast card B113is separated from the fast card A112, the fast card A is blocked in a direction from132to129and communicated in a direction from129to132. When the fast card A and the fast card B are combined, the conductor119moves backward to122to open123of113, so as to make the structural part lose the one-way valve functions, and119is inserted into123of112, so as to make the structural part lose the one-way valve functions.

In another aspect, the present disclosure protects a method for detecting biological macromolecules by using the automatic reaction system for biological macromolecules.

The present disclosure has the following beneficial effects. Time is saved. The system is fully automatic, a lot of labors are reduced, and the efficiency is greatly improved. The sensitivity is high. Compared with the traditional method, the reaction efficiency is improved by liquid flow control, and thus the sensitivity is higher under conditions same as those in the traditional method. Reactor reagents are reduced. In the system, only a small amount of a liquid needs to be used in the reactor to complete a reaction requiring a large amount of reagents in the traditional method.

Raw materials and reagents used in the biological reaction apparatus and the method for performing biological detection on basis of the apparatus provided in the present disclosure can be purchased from the market.

The present disclosure is further described below with reference to the embodiments.

Example 1 Biological Reaction Apparatus Provided in the Present Disclosure

The present disclosure provides a biological reaction apparatus, as shown inFIG. 1, including a power supply module1, a control module2, a liquid processing module3, a reactor module4and a sensor5.

Where,1refers to a power supply module, including a direct current power supply6and a switch7, and is used to convert alternative current input into direct current output and provide energy required for operation of other parts of the present disclosure.

2refers to a control module, including a system controller8, an input device9and an output device10, and is operated with energy input from the power supply module1; the energy provided by the power supply module1is transmitted to the liquid processing module3and the reactor module4through cables (25); and the working state of the liquid processing module3and the reactor module4is detected through the sensor5and controlled.

3refers to a liquid processing module and includes a valve11or a combination of valves, a pump12or a combination of pumps and sample cells13, and the valve11or the combination of valves, the pump12or the combination of pumps and a reactor14are connected through a pipeline (26). The valve11or the combination of valves performs sample selection according to signals of the control module2, and the pump12or the combination of pumps moves a sample into or out of the reactor14. The sensor5is used to detect the operating state of the liquid processing module3and give feedback to the control module2. The operating energy of the liquid processing module3is transmitted from the power supply module1by the control module2, or may be directly provided by the power supply module1.

4refers to a reactor module, where14refers to a reactor and includes a reactor frame15and a reactor cavity16formed by the reactor frame15,17refers to an opening formed in the reactor frame15and is a liquid inlet17-1or a liquid outlet17-2, and a liquid is pumped into or out of the reactor14by the pump12or the combination of pumps connected to the opening17through a pipeline. The sensor5is used to detect the operating state of the reactor14and give feedback to the control module2. The operating energy of the reactor module4is transmitted from the power supply module1by the control module2, or may be directly provided by the power supply module1.

The power supply module1is separately connected to the control module2, the liquid processing module3and the reactor module4through cables25; the control module2is separately connected to the liquid processing module3and the reactor module4through the cables25; the liquid processing module3is connected to the reactor module4through a pipeline26; and the control module2is connected to the reactor module4through the sensor5, and the opening17is connected to the pump12or the combination of pumps12.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIG. 2, including a reactor, four reaction liquids,1pump and6two-position three-way valves.

13refers to four sample cells, and each sample cell13can be filled with a reaction liquid.12refers to a pump, specifically a peristaltic pump.14refers to a reactor.17-1refers to a liquid inlet.17-2refers to a liquid outlet.23refers to a mother liquid pool.27refers to a waste liquid collector.28refers to a liquid level indicator.29refers to a reaction liquid inlet and outlet pipeline.30refers to a waste liquid discharge or air inlet pipeline.50,51,52,53,54and55each refers to a two-position three-way valve.32,33and34refer to three liquid receiving ports of a two-position three-way valve,33is a common end, and32and33are in normally open connection.38,39and40refer to three liquid receiving ports of a two-position three-way valve,39is a common end, and38and39are in normally open connection.35,36and37refer to three liquid receiving ports of a two-position three-way valve,36is a common end, and35and36are in normally open connection.41,42and43refer to three liquid receiving ports of a two-position three-way valve,43is a common end, and42and43are in normally open connection.44,45and46refer to three liquid receiving ports of a two-position three-way valve,44is a common end, and46and44are in normally open connection.47,48and49refer to three liquid receiving ports of a two-position three-way valve,49is a common end, and47and49are in normally open connection.

28needs to be maintained below the liquid level of a container to ensure that enough liquid may enter during operation.30needs to be maintained above the liquid level of a container to ensure that waste liquid is not sucked into a pipeline when gas is introduced. Furthermore, a gas-liquid selection valve may be added to the tail end of30to ensure that gas intake and liquid discharge are completely isolated.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIG. 3, including three reactors14, six reaction liquids, three pumps12and two valves11or combinations of valves.

62refers to a gas buffer bottle.27refers to a waste liquid collector.13refers to a sample cell.11refers to a valve, specifically an eight-position selection valve.66,67,68,69,70,71,72and73each refers to a selection connector of an eight-position valve.74refers to a common connector.65refers to a selection path, which is used to make the selection connectors66,67,68,69,70,71,72and73communicated with the common connector74according to signals of the control module2.64refers to a four-position selection valve group.12refers to a pump, specifically a plunger pump.14refers to a reactor. The reactors may be connected in series or in parallel. For example, the reactor14-1and the reactor14-2are connected in series.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIG. 5(A).19refers to a sealing part,20refers to a reactor front plate,21refers to a reactor rear plate,18-1refers to a front pressing structure of a reactor, and18-2refers to a rear pressing structure of the reactor.18-1and20may be one part, and21and18-2may be one part. After a reactor frame15of the reactor14is closed, a reactor cavity16can be formed. The size of the reactor cavity16may be determined by the sealing part19and a pressing part18. When a reactant is a thin film, a micro-cavity is formed in the reactor cavity16. Furthermore, a limiting part22may be added into20and21to maintain a certain cavity. Furthermore, the sealing part19may be omitted, and a sealing cavity is formed due to the tightness of20and21. At least one opening17is formed in the reactor14,17-1refers to a liquid inlet, and17-2refers to a liquid outlet; and the opening17is connected to a pump12, a valve11or the reactor14and used for sample input or output.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIG. 5(B)toFIG. 5(D).18refers to a pressing part;91refers to a rotating shaft of the pressing part, and the pressing part18is fixed to a reactor frame15through the rotating shaft and can rotate around the rotating shaft;20refers to a reactor front plate;21refers to a reactor rear plate;15refers to the reactor frame;17refers to an opening, which is connected to the reactor rear plate21through the reactor frame15;19refers to a sealing part; and16refers to a formed reactor cavity.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIG. 5(E)toFIG. 5(G).96refers to a reactor upper cover;15refers to a reactor frame;20refers to a reactor front plate;21refers to a reactor rear plate;99refers to a connecting shaft of the upper cover and a frame body;18refers to a pressing part;19refers to a sealing part;22refers to a limiting part;16refers to a reactor cavity; and17refers to an opening.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIGS. 6(A)-6(C).106refers to an inner wall of a reactor14.17refers to an opening.107refers to a reactant.19refers to a sealing part.109refers to a liquid flow indicator.

FIG. 6(A)shows the state of the reactant107unsuspended in the reactor14, and the reactant is in an irregular shape and may be partially attached to the inner wall106of the reactor14.

FIG. 6(B)andFIG. 6(C)are schematic diagrams of the reactant suspended in a liquid.

After the liquid enters the reactor14, the liquid flows through a gap between the reactant and the reactor14to make the reactant suspended in the reactor14. The liquid is introduced through liquid inlets on both sides, and the reactant is suspended in the reactor14through the liquid flow. Furthermore, as shown inFIG. 6(B), the reactant is suspended by introducing a liquid on one side.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIG. 7.17refers to an opening and is specifically a liquid inlet;19refers to a sealing part and is specifically a sealing ring;16refers to a reactor cavity; and24refers to a diversion block in a reactor14.

On basis of the structure of the biological reaction apparatus provided in Example 1, the present disclosure further provides a biological reaction apparatus, as shown inFIGS. 8(A)-8(E).FIG. 8(A)is a schematic diagram of a two-way fast card set consisting of a fast card A112and a fast card B113;FIG. 8(B)is a schematic diagram of the fast card B113;FIG. 8(C)is a cross-sectional view of the fast card B113;FIG. 8(D)is a schematic diagram of the fast card A112; andFIG. 8(E)is a cross-sectional view of the fast card A112; and114refers to a pagoda connector of the fast card B113, is a derivative part of125, and is used to connect pipelines or is directly connected to a container wall;125refers to a closure member of the fast card B113, and forms a containing cavity with116;118refers to an internal flow channel of114;115refers to an inner pagoda pattern of the fast card B113and is used to prevent internal leakage and falling after the fast card A and the fast card B are assembled;116refers to a main body of the fast card B113;117refers to a conductor;119refers to an internal flow channel of the conductor;120refers to a guide structure of the fast card B113and is combined with the fast card A for internal guide fixation;121refers to a limiting structure of the conductor and is used to prevent the conductor117from falling off the fast card B113;122refers to an inner limiting structure of the fast card B113and is used to limit the backward distance of the conductor117;124refers to an outer limiting structure of the fast card B113and is used to limit the outward distance of the conductor117;123refers to a structural part with one-way valve functions and is used to control that when the fast card B113is separated from the fast card A112, the fast card B is blocked in a direction from118to119and communicated in a direction from119to118;129refers to an upper blocking structure of the fast card A112and is used to fix a leakproof ring130;130refers to the leakproof ring of the fast card A112;128refers to a main body of the fast card A112;127refers to a lower blocking structure of the fast card A112;126refers to a pagoda connector of the fast card A112;131refers to a guide structure of the fast card A112;132refers to an internal flow channel of126; and133refers to a structural part with one-way valve functions and is used to control that when the fast card B113is separated from the fast card A112, the fast card A is blocked in a direction from132to129and communicated in a direction from129to132. When the fast card A and the fast card B are combined, the conductor119moves backward to122to open123of113, so as to make the structural part lose the one-way valve functions, and119is inserted into123of112, so as to make the structural part lose the one-way valve functions.

Example 10 Coomassie Brilliant Blue Staining

On basis of the biological reaction apparatus provided in Example 1, the present disclosure includes the following staining steps:1: adding corresponding working solutions into reservoirs of a staining solution and a destaining solution;2: putting to-be-stained gel or a film into a reactor;3: setting a working procedure of an instrument:setting the temperature to be 20-30° C., the area of a to-be-stained object to be 7.5*8 CM and the flow rate to be 40 ml/min;A: introducing the staining solution (100 ml);B: circulating the staining solution for 1 h;C: discharging the staining solution;D: introducing the destaining solution (100 ml);E: circulating the destaining solution for 1 h;F: discharging the destaining solution;G: introducing the destaining solution (100 ml);H: circulating the destaining solution for 1 h;I: discharging the destaining solution; andJ: completing work of the instrument;4: taking out the gel and recording results.A traditional method includes the following staining process:1: putting gel or a film into a box;2: pouring 200 ml of a staining solution;3: performing vibration at room temperature for 1 h;4: removing the staining solution, and pouring 200 ml of a destaining solution;5: performing vibration at room temperature for 1 h;6: removing the destaining solution in step 4, and pouring 200 ml of a destaining solution;7: performing vibration at room temperature for 1 h; and8: repeating steps 6-7 until bands are clear and the background is clean.

According to a staining formulation, the staining solution includes 0.1% of R250, 30% of ethanol, 10% of acetic acid and 60% of water; and the destaining solution includes 10% of ethanol, 10% of acetic acid and 80% of water.

FIG. 9(A)shows staining results of the traditional method,FIG. 9(B)shows staining results of the present disclosure, andFIG. 9(C)shows comparative analysis of the staining degree of IgG in the staining results of the traditional method and the staining results of the present disclosure;FIG. 9(D)shows comparative analysis of the staining degree of lysozyme in the staining results of the traditional method and the staining results of the present disclosure;FIG. 9(E)shows comparative analysis of the staining degree of BSA in the staining results of the traditional method and the staining results of the present disclosure; and the results are shown on the same plane by using a bio-rad photosensitivity scanner.

When the traditional method is used for staining, a piece of gel requires at least 600 ml of a liquid for reaction. When the present disclosure is used, only 300 ml of a liquid is used to complete a reaction.

On IgG, all bands in the traditional method and the present disclosure are visible, but the color in the present disclosure is darker.

On BSA, the 10thsample in the traditional method is visible, and all 12 samples in the present disclosure are visible, so that the sensitivity of the present disclosure is better.

On lysozyme, the 9thsample in the traditional method is visible, and the 11thsample in the present disclosure is visible, so that the sensitivity of the present disclosure is better.

It can be seen fromFIG. 9(C),FIG. 9(D),FIG. 9(E)and Tables 2-4 that compared with the traditional method, the present disclosure has the advantages that the staining degree is increased by 4.49%-22.90% based on IgG detection results; the staining degree is increased by 14.87%-60.00% based on BSA detection results; and the staining degree is increased by 9.72%-28.37% based on lysozyme detection results.

In summary, compared with the traditional method, only 50% of reagents need to be used in the present disclosure, and the sensitivity and the staining degree of the present disclosure are better than those of the traditional method.

Example 11 Silver Staining

On basis of the biological reaction apparatus provided in Example 2, a reaction is introduced as follows:a stationary liquid includes 40% of methanol, and each 200 ml of the stationary liquid includes 100 ul of formaldehyde;a sensitizing agent includes 0.2 g/L of sodium thiosulfate;a staining agent includes 20% of AgNO3and 250 ul/50 ml of H2O; anda color developing solution includes 3% of sodium carbonate and 0.0004%L of sodium thiosulfate, and 25 ul of a formaldehyde solution/50 ml is added before use.

The present disclosure includes the following steps:1: putting gel into a reactor;2: setting a program as follows:setting the temperature to be 20-30° C. and the gel area to be 7.5*8 CM, where compared with Example 10, the reaction rate of silver staining reagents is lower than that of Coomassie brilliant blue, so that the flow rate needs to be reduced and is set to be 20 ml/min;1) pumping in 100 ml of the stationary liquid, circulating the stationary liquid for 10 min and pumping out the stationary liquid after a reaction is completed;2) pumping in 100 ml of ddH2O, circulating the water for 5 min, pumping out the water and repeating to complete the step 2 times;3) pumping in 100 ml of the sensitizing agent, circulating the sensitizing agent for 1 min and pumping out the sensitizing agent;4) pumping in 100 ml of ddH2O, circulating the water for 1 min, pumping out the water and repeating to complete the step 2 times;5) pumping in 100 ml of the staining agent, circulating the staining agent for 10 min and pumping out the staining agent;6) pumping in 100 ml of ddH2O, circulating the water for 1 min, pumping out the water and repeating to complete the step 2 times;7) pumping in 100 ml of the color developing solution, circulating the color developing solution for 10 min and pumping out the color developing solution; and8) pumping in 100 ml of ddH2O, circulating the water for 5 min and pumping out the water;3: taking out the gel and recording results.

A traditional method includes the following steps:1: putting gel into a container;2: pouring 200 ml of a stationary liquid, performing vibration at room temperature for 10 min and discharging waste liquid;3: pouring 200 ml of ddH2O, performing vibration at room temperature for 5 min and discharging waste liquid;4: repeating step 3;5: pouring 200 ml of a sensitizing agent, performing vibration at room temperature for 1 min and discharging waste liquid;6: pouring 200 ml of ddH2O, performing vibration at room temperature for 1 min and discharging waste liquid;7: repeating step 6;8: pouring 200 ml of a staining agent, performing vibration at room temperature for 10 min and discharging waste liquid;9: pouring 200 ml of ddH2O, performing vibration at room temperature for 1 min and discharging waste liquid;10: repeating step;11: pouring 200 ml of a color developing solution, performing vibration at room temperature for 10 min and discharging waste liquid;12: pouring 200 ml of ddH2O, performing vibration at room temperature for 5 min and discharging waste liquid; and13: taking out the gel and recording results.

FIG. 10(A)shows staining results of the present disclosure;FIG. 10(B)shows staining results of the traditional method;FIG. 10(C)shows comparative analysis of the staining degree of IgG in the staining results of the traditional method and the staining results of the present disclosure; andFIG. 10(D)shows comparative analysis of the staining degree of BSA in the staining results of the traditional method and the staining results of the present disclosure.

It can be seen from the figures that when half of reagents are used, the sensitivity of the present disclosure is better than that of the traditional method, and the background is better than that of the traditional method. On IgG, all bands in the traditional method and the present disclosure are visible. On BSA, the 10thsample in the traditional method is visible, and all 12 samples in the present disclosure are visible, so that the sensitivity of the present disclosure is better.

It can be seen fromFIG. 10(C),FIG. 10(D)and Tables 6-7 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 114.91%-139.27% based on IgG detection results; and the staining degree is increased by 100.88%-186.47% based on BSA detection results.

In summary, compared with the traditional method, only 50% of reagents need to be used in the present disclosure, and the sensitivity and the staining degree are better than those of the traditional method.

On basis of the biological reaction apparatus provided in Example 3, a reaction is introduced as follows:proteins with His tags and the molecular weight of 93 kd and 7 kd are used as samples.

The sample loading quantity is:1: 1 ul of WB Marker;2: 150 ng of 93 kd protein and 10 ng of 7 kd protein;3: 75 ng of 93 kd protein and 5 ng of 7 kd protein;4: 37.5 ng of 93 kd protein and 2.5 ng of 7 kd protein;5: 18.75 ng of 93 kd protein and 1.25 ng of 7 kd protein;6: 9 ng of 93 kd protein and 0.6 ng of 7 kd protein;7: 4.5 ng of 93 kd protein and 0.3 ng of 7 kd protein;8: 2.25 ng of 93 kd protein and 0.15 ng of 7 kd protein;9: 1 ng of 93 kd protein and 0.075 ng of 7 kd protein; and10: 0.5 ng of 93 kd protein and 0.0375 ng of 7 kd protein.

A blocking solution includes 5% of skimmed milk powder and a PBS solution.

PBST is obtained by adding one thousandth of Tween 20 into PBS.

A primary antibody is mouse anti-His with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:3000.

A secondary antibody is goat anti-mouse HRP with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:10000.

The present disclosure includes the following steps:1: putting two films back-to-back into a reactor and setting the temperature to be 20-30° C., the film area to be 7.5*8 CM*2 and the flow rate to be 40 ml/min;2: pumping in 8 ml of the blocking solution, circulating the blocking solution for 1 h and pumping out the blocking solution;3: pumping in 8 ml of PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;4: pumping in 8 ml of a diluted solution of the primary antibody, circulating the diluted solution of the primary antibody for 1 h and pumping out the diluted solution of the primary antibody;5: pumping in 8 ml of PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;6: pumping in 8 ml of a diluted solution of the secondary antibody, circulating the diluted solution of the secondary antibody for 1 h and pumping out the diluted solution of the secondary antibody;7: pumping in 8 ml of PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times; and8: taking out the films and exposing the films.

A traditional method includes the following steps:1: putting two films back-to-back into a container;2: adding 10 ml of a blocking solution, performing vibration for 1 h and discharging the blocking solution;3: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;4: repeating step 3 twice;5: adding 10 ml of a diluted solution of a primary antibody, performing vibration for 1 h and discharging the diluted solution of the primary antibody;6: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;7: repeating step 3 three times;8: adding 10 ml of a diluted solution of a secondary antibody, performing vibration for 1 h and discharging the diluted solution of the secondary antibody;9: repeating step 3 three times; and10: taking out the films and exposing the films.

FIG. 11(A)shows results of the traditional method;FIG. 11(B)shows results of the method provided in the present disclosure;FIG. 11(C)toFIG. 11(D)show results of the method provided in the present disclosure;FIG. 11(E)shows comparative analysis of the staining degree of a 93 kd protein in the results of the traditional method and the results of the present disclosure; andFIG. 11(F)shows comparative analysis of the staining degree of a 7 kd protein in the results of the traditional method and the results of the present disclosure.

It can be seen fromFIGS. 11(A)-11(F) that on 93 kd, the 4thlane in the traditional method is visible, and the 5thlane in the present disclosure is visible, so that the sensitivity of the present disclosure is better.

On 7 kd, the 8thlane in the traditional method is visible, and the 9thlane in the present disclosure is visible, so that the sensitivity of the present disclosure is better.

It can be seen fromFIG. 11(E),FIG. 11(F)and Tables 9-10 that compared with the traditional method, the method provided in the present disclosure has the advantages that the staining degree is increased by 102.09%-507.93% based on 93 kd detection results; and the staining degree is increased by 20.61%-432.91% based on 7 kd detection results.

In summary, compared with the traditional method, only 74% of reagents and 80% of antibodies need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

On basis of the biological reaction apparatus provided in Example 4, a reaction is introduced as follows:a 293 T cell lysis buffer is used as a sample.

The sample loading quantity is:1: 1 ul of WB marker;2: 20 ug of the cell lysis buffer;3: 10 ug of the cell lysis buffer;4: 5 ug of the cell lysis buffer;5: 2.5 ug of the cell lysis buffer;6: 1.25 ug of the cell lysis buffer;7: 0.625 ug of the cell lysis buffer; and8: 0.3125 ug of the cell lysis buffer.

A blocking solution includes 5% of skimmed milk powder and a PBS solution.

PBST is obtained by adding one thousandth of Tween 20 into PBS.

A primary antibody is rabbit anti-β-actin with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:3000.

A secondary antibody is goat anti-rabbit HRP with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:10000.

The present disclosure includes the following steps:setting the temperature to be 20-30° C., the film area to be 7.5*8 CM and the flow rate to be 20 ml/min;1: putting a film into a reactor;2: pumping in the blocking solution;3: pumping in PBST when the reactor is filled with the blocking solution;4: pumping in a diluted solution of the primary antibody when the reactor is filled with PBST;5: pumping in PBST when the reactor is filled with the diluted solution of the primary antibody;6: pumping in a diluted solution of the secondary antibody when the reactor is filled with PBST;7: pumping in PBST when the reactor is filled with the diluted solution of the secondary antibody;8: continuously pumping in 2 times volume of PBST when the reactor is filled with PBST; and9: taking out the film and performing developing.

The blocking solution, the diluted solution of the primary antibody and the diluted solution of the secondary antibody require 4 ml each, and PBST requires 16 ml.

The process of the present disclosure is completed within 2 h.

A traditional method includes the following steps:1: putting a film into a container;2: adding 10 ml of a blocking solution, performing vibration for 1 h and discharging the blocking solution;3: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;4: repeating step 3 twice;5: adding 10 ml of a diluted solution of a primary antibody, performing vibration for 1 h and discharging the diluted solution of the primary antibody;6: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;7: repeating step 3 three times;8: adding 10 ml of a diluted solution of a secondary antibody, performing vibration for 1 h and discharging the diluted solution of the secondary antibody;9: repeating step 3 three times; and10: taking out the film and exposing the film.

FIG. 12(A)shows results of the traditional method;FIG. 12(B)shows results of the present disclosure; andFIG. 12(C)shows comparative analysis of the staining degree of a β-actin protein in the results of the traditional method and the results of the present disclosure.

It can be seen from the figures that the 6thlane in the traditional method is visible, and the 8thlane in the present disclosure is visible, so that the sensitivity of the present disclosure is better.

It can be seen fromFIG. 12(C)and Table 12 that compared with the traditional method, the method provided in the present disclosure has the advantage that the staining degree is increased by 3.05%-305.33% based on β-actin detection results.

In summary, compared with the traditional method, only 65% of reagents, 40% of antibodies and a shorter processing time need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

On basis of the biological reaction apparatus provided in Example 3, a reaction is introduced as follows:a 293 T cell lysis buffer is used as a sample.

The sample loading quantity is:1: 1 ul of WB marker;2: 20 ug of the cell lysis buffer;3: 10 ug of the cell lysis buffer;4: 5 ug of the cell lysis buffer;5: 2.5 ug of the cell lysis buffer; and6: 1.25 ug of the cell lysis buffer.

A primary antibody is mouse anti-GAPDH.

A secondary antibody is goat anti-mouse.

The present disclosure includes the following steps:1: putting a film B into a reactor14-1and a film D into a reactor14-2;2: connecting the reactor14-1and the reactor14-2in series and setting the temperature to be 20-30° C., the area of the film B and the film D to be 7.5*4 CM and the flow rate to be 10 ml/min;3: pumping in a blocking solution, circulating the blocking solution for 1 h after the reactor14-1and the reactor14-2are filled with the blocking solution and pumping out the blocking solution;4: pumping in PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;5: pumping in a diluted solution of the primary antibody, circulating the diluted solution of the primary antibody for 1 h and pumping out the diluted solution of the primary antibody;6: pumping in PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;7: pumping in a diluted solution of the secondary antibody, circulating the diluted solution of the secondary antibody for 1 h and pumping out the diluted solution of the secondary antibody;8: pumping in PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times; and9: taking out the films and exposing the films.

The blocking solution, the diluted solution of the primary antibody and the diluted solution of the secondary antibody require 8 ml each, and PBST requires 7 ml.

A traditional method includes the following steps:1: putting two films (A and C) into containers respectively;2: separately adding 10 ml of a blocking solution, performing vibration for 1 h and discharging the blocking solution;3: separately adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;4: repeating step 3 twice;5: separately adding 10 ml of a diluted solution of a primary antibody, performing vibration for 1 h and discharging the diluted solution of the primary antibody;6: separately adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;7: repeating step 3 three times;8: separately adding 10 ml of a diluted solution of a secondary antibody, performing vibration for 1 h and discharging the diluted solution of the secondary antibody;9: repeating step 3 three times; and10: taking out the films and exposing the films.

FIG. 13(A)shows results of the film A;FIG. 13(B)shows results of the film B;FIG. 13(C)shows results of the film C;FIG. 13(D)shows results of the film D; andFIG. 13(E)shows comparative analysis of the staining degree of a GAPDH protein in the results of the traditional method and the results of the present disclosure.

TABLE 13GAPDHLane 2Lane 3Lane 4Lane 5Film A79.760.329.99.4Film B154.3124.443.112.3Film C84.55420.63.9Film D143.6114.344.67.7Average value of the82.157.1525.256.65film A and the film CAverage value of the148.95119.3543.8510film B and the film DImprovement of the81.43%108.84%73.66%50.38%present disclosure incomparison with thetraditional method

It can be seen from the figures that the color ofFIG. 13(B)andFIG. 13(D)is darker than that ofFIG. 13(A)andFIG. 13(C), and it can be seen fromFIG. 13(E)and Table 13 that compared with the traditional method, the method provided in the present disclosure has the advantage that the staining degree is increased by 50.38%-108.84% based on GAPDH detection results. That is to say, the signal intensity is higher, and it is further shown that the effect of the present disclosure is better than that of the traditional method. In summary, compared with the traditional method, only 66% of reagents need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

On basis of the biological reaction apparatus provided in Example 5, a reaction is introduced as follows:a 293 T cell lysis buffer is used as a sample.

The sample loading quantity is:1: 20 ug of the cell lysis buffer;2: 10 ug of the cell lysis buffer;3: 5 ug of the cell lysis buffer;4: 2.5 ug of the cell lysis buffer;5: 1.25 ug of the cell lysis buffer; and6: 0.625 ug of the cell lysis buffer.

A blocking solution includes 5% of skimmed milk powder and a PBS solution.

PBST is obtained by adding one thousandth of Tween 20 into PBS.

A primary antibody is rabbit anti-β-actin with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:3000.

A secondary antibody is goat anti-rabbit HRP with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:10000.

The present disclosure includes the following steps:1: setting the temperature to be 20-30° C., the film area to be 7.5*4 CM and the flow rate to be 10 ml/min;2: pumping in 5 ml of the blocking solution, circulating the blocking solution for 1 h and pumping out the blocking solution;3: pumping in 5 ml of PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;4: pumping in 5 ml of a mixed solution of the primary antibody and the secondary antibody, circulating the mixed solution for 1 h and pumping out the mixed solution for recovery;5: pumping in 5 ml of PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;6: pumping in a developing solution and pumping out the developing solution after a reactor is filled with the developing solution; and

7: taking out a film and performing developing.

A traditional method includes the following steps:1: putting a film back-to-back into a container;2: adding 10 ml of a blocking solution, performing vibration for 1 h and discharging the blocking solution;3: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;4: repeating step 3 twice;5: adding 10 ml of a diluted solution of a primary antibody, performing vibration for 1 h and discharging the diluted solution of the primary antibody;6: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;7: repeating step 3 three times;8: adding 10 ml of a diluted solution of a secondary antibody, performing vibration for 1 h and discharging the diluted solution of the secondary antibody;9: repeating step 3 three times; and10: taking out the film and exposing the film.

FIG. 14(A)shows results of the traditional method;FIG. 14(B)shows results of the present disclosure; andFIG. 14(C)shows comparative analysis of the staining degree of a β-actin protein in the results of the traditional method and the results of the present disclosure.

It can be seen fromFIGS. 14(A)-14(C)that the 4thlane in the traditional method is visible, and the 5thlane in the present disclosure is visible, so that the sensitivity of the present disclosure is better.

It can be seen fromFIG. 14(C)and Table 15 that compared with the traditional method, the method provided in the present disclosure has the advantage that the staining degree is increased by 27.52%-603.57% based on β-actin detection results.

In summary, compared with the traditional method, only 30% of reagents and a shorter processing time need to be used in the present disclosure, and the sensitivity and the signal intensity of the present disclosure are better than those of the traditional method.

On basis of the biological reaction apparatus provided in Example 6, a reaction is introduced as follows:a protein with an His tag and the molecular weight of 7 kd is used as a sample.

The sample loading quantity is:1:10 ng of His protein;2: 5 ng of His protein;3: 2.5 ng of His protein;4: 1.25 ng of His protein;5: 0.6 ng of His protein; and6: 0.3 ng of His protein.

A blocking solution includes 5% of skimmed milk powder and a PBS solution. PBST is obtained by adding one thousandth of Tween 20 into PBS.

A primary antibody is mouse anti-His with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:3000.

A secondary antibody is goat anti-mouse HRP with a concentration of 1 mg/ml and is diluted with the blocking solution according to a ratio of 1:10000.

The present disclosure includes the following steps:1: setting the temperature to be 20-30° C., the film area to be 7.5*8 CM and the flow rate to be 20 ml/min;2: adding a primary antibody mother liquid and a secondary antibody mother liquid into corresponding reservoirs (as shown inFIG. 15(C),23-1refers to a secondary antibody mother liquid pool,23-2refers to a primary antibody mother liquid pool, and143refers to a liquid flow direction);3: pumping in 5 ml of the blocking solution, where antibodies are diluted and introduced into a reactor when the blocking solution flows through the reservoirs; and circulating the blocking solution for 1 h and pumping out the blocking solution;4: pumping in 5 ml of PBST, circulating PBST for 5 min, pumping out PBST and repeating to complete the step 3 times;5: pumping in a TMB color developing solution and pumping out the TMB color developing solution; and6: observing conditions from an observation window and recording results.

A traditional method includes the following steps:1: putting a film back-to-back into a container;2: adding 10 ml of a blocking solution, performing vibration for 1 h and discharging the blocking solution;3: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;4: repeating step 3 twice;5: adding 10 ml of a diluted solution of a primary antibody, performing vibration for 1 h and discharging the diluted solution of the primary antibody;6: adding 10 ml of PBST, performing vibration for 5 min and discharging PBST;7: repeating step 3 three times;8: adding 10 ml of a diluted solution of a secondary antibody, performing vibration for 1 h and discharging the diluted solution of the secondary antibody;9: repeating step 3 three times; and10: adding a TMB color developing solution into the container and recording results.

FIG. 15(B)shows results of the traditional method;FIG. 15(A)shows results of the present disclosure;FIG. 15(C)shows the liquid flow direction of the primary antibody mother liquid and the secondary antibody mother liquid added into corresponding reservoirs; andFIG. 15(D)shows comparative analysis of the staining degree of a 7 kd protein in the results of the traditional method and the results of the present disclosure.

It can be seen fromFIG. 15(D)and Table 16 that bands in the present disclosure are darker than those in the traditional method, and the staining degree is increased by 59.26%-111.13%, showing that the effect of the present disclosure is better.

In summary, compared with the traditional method, only 20% of reagents and a shorter processing time need to be used in the present disclosure, and the signal intensity of the present disclosure is better than that of the traditional method.

Descriptions above are only preferred embodiments of the present disclosure. It should be pointed out that many improvements and modifications may be made by those of ordinary skill in the art without departing from the principle of the present disclosure, and those improvements and modifications also fall within the protection scope of the present disclosure.