Continuous flow reaction control device and control method for antibody nucleic acid coupling

Abstract

The invention discloses a continuous flow reaction control device for antibody nucleic acid coupling and a control method, and relates to the field of biology and medicine, the control device comprises a device main body, and a fluid input part and a reaction control part which are sequentially connected and arranged on the device main body according to a reaction path; the fluid input component comprises a flow channel assembly and a step converging flow channel connected to the bottom of the flow channel assembly. The reaction control component comprises a snakelike reaction flow channel and a reaction control assembly; the top end of the snakelike reaction flow channel is communicated with the bottom of the stepped converging flow channel, and a fluid output port is formed in the bottom end of the snakelike reaction flow channel. According to the present invention, the coupling process of the antibody and the metal isotope is optimized by precisely regulating the chemical reaction environment, including the micron-sized channel design, such that the binding efficiency of the antibody or oligonucleotide and the metal isotope is improved, and the non-specific binding is significantly reduced so as to improve the stability and the reliability of the whole antibody nucleic acid coupling process.

Description

Technical Field

[0001] The present invention relates to the field of biology and medicine technology, and in particular to a continuous flow reaction control device and a control method for antibody nucleic acid coupling. Background Art

[0002] As a cutting-edge technology, antibody-nucleic acid coupling technology (such as antibody-oligonucleotide coupling, antibody-metal isotope labeling, etc.) is of great significance for drug development, disease diagnosis and precision medicine. This technology achieves targeted recognition and labeling of specific cells or tissues by coupling antibodies with nucleic acid molecules, thereby improving the accuracy and effectiveness of treatment. With the development of precision medicine and spatial omics technology, higher requirements are placed on the efficiency and specificity of coupling products.

[0003] Traditional antibody-nucleic acid coupling methods usually use intermittent reactions, that is, all reactants are added to the reaction vessel at once, and then stirred and reacted. Although this method is simple to operate, it has problems such as difficult to accurately control reaction conditions, poor product uniformity, and many by-products, which seriously affect the quality and application effect of the coupling product. Especially when dealing with trace reactants or reaction systems that require high sensitivity and high specificity, the limitations of traditional methods are more obvious.

[0004] Therefore, it is necessary to improve the deficiencies in the prior art to solve the above problems. Summary of the invention

[0005] The present invention overcomes the shortcomings of the prior art and provides a continuous flow reaction control device and control method that can accurately control reaction conditions, improve coupling efficiency and reduce non-specific binding, so as to meet the urgent needs of the biotechnology and pharmaceutical research and development fields for antibody nucleic acid coupling technology.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a continuous flow reaction control device for antibody nucleic acid coupling, comprising: a device body, a fluid input component and a reaction control component arranged on the device body are sequentially connected according to a reaction path;

[0007] The fluid input component comprises: a flow channel component for providing dispersed phase liquid and continuous phase liquid input respectively, and a stepped converging flow channel connected to the bottom of the flow channel component for converging the dispersed phase liquid and the continuous phase liquid and reducing the formation of bubbles;

[0008] The reaction control component includes: a serpentine reaction channel, and a reaction control assembly for gradient-controlling the internal temperature of the serpentine reaction channel and synchronously controlling the internal size to manipulate the reaction time; the top end of the serpentine reaction channel is communicated with the bottom of the stepped converging channel, and the bottom end is provided with a fluid outlet; at least four parallel reaction segments are included in the serpentine reaction channel, and at least two groups of the reaction control assemblies are included and are respectively located between every two segments of the serpentine reaction channel.

[0009] In a preferred embodiment of the present invention, the reaction control assembly includes: a fluid input pipeline for inputting a fluid with a required temperature or pressure, a plurality of control channels opened on the inner wall of the serpentine reaction channel, and a fluid permeable membrane fixed on the inner side of the control channels;

[0010] One end of the fluid input pipeline is respectively communicated with the interiors of the plurality of control channels, and the fluid permeable membrane is used for permeating the input fluid from the high-pressure side to the low-pressure side to change the internal temperature or the internal size of the serpentine reaction channel.

[0011] In a preferred embodiment of the present invention, the flow channel assembly includes: a dispersed-phase input channel, and at least one continuous-phase input channel located on the side of the dispersed-phase input channel;

[0012] Fluid inlets are provided at the top ends of the dispersed-phase input channel and the continuous-phase input channel, and the flow path of the fluid inlet to the continuous-phase input channel is two segments with widths gradually decreasing in sequence, which is used to increase the flow velocity of the continuous-phase liquid; the top end of the stepped converging channel is communicated with the bottom end of the dispersed-phase input channel, and the side is communicated with the bottom end of the continuous-phase input channel.

[0013] In a preferred embodiment of the present invention, at least three stepped portions are provided inside the stepped converging channel; the plurality of stepped portions are alternately and symmetrically distributed from top to bottom inside the stepped converging channel.

[0014] The present invention provides a control method for a continuous flow reaction control device for antibody nucleic acid conjugation, including the following steps:

[0015] S1. Input the dispersed-phase liquid and the continuous-phase liquid respectively through the flow channel assembly, and enter the stepped converging channel from the bottom of the flow channel assembly in a state where the flow velocity of the continuous-phase liquid is greater than that of the dispersed-phase liquid for convergence;

[0016] S2. The converged liquid enters the serpentine reaction channel and flows and reacts along at least four parallel reaction segments, and the reaction control assembly controls the temperature gradient and the reaction time within each gradient in the interior of every two parallel reaction segments in the serpentine reaction channel;

[0017] S3. The reacted liquid is discharged through the fluid outlet, and the product concentration and by-product content are monitored in real time.

[0018] S4. Repeat steps S1 - S3 to achieve continuous flow reaction control.

[0019] In a preferred embodiment of the present invention, in the step of S1, the flow rate of the continuous phase liquid is 2 - 4 times that of the dispersed phase liquid.

[0020] In a preferred embodiment of the present invention, in the step of S1, the flow rate gradient in the stepped converging flow channel is 0.5 - 1.5 m / s.

[0021] In a preferred embodiment of the present invention, in the step of S2, the temperature gradient is specifically: initially heating up 40 - 45 °C to accelerate the reaction, and later cooling down 20 - 25 °C to reduce non - specific binding.

[0022] In a preferred embodiment of the present invention, the initial reaction time is 10 - 20 s, and the later reaction time is 15 - 30 s.

[0023] In a preferred embodiment of the present invention, in the step of S3, the real - time monitoring indicators include: coupling efficiency and non - specific binding rate.

[0024] The present invention solves the defects existing in the background technology and has the following beneficial effects:

[0025] (1) The present invention provides a continuous flow reaction control device and control method for antibody - nucleic acid conjugation. By precisely regulating the chemical reaction environment, including the precise control of fluid flow rate, pressure, and temperature through the design of micro - scale channels, the conjugation process of antibodies and metal isotopes is optimized. It not only improves the binding efficiency of antibodies or oligonucleotides with metal isotopes, but also significantly reduces non - specific binding. The efficient conjugation reaction ensures that more metal isotopes are labeled onto the target antibody, improving the detection signal intensity. At the same time, reducing non - specific binding avoids the random binding of non - target molecules with isotopes, reduces background interference, improves the signal - to - noise ratio, makes weak signals easier to be recognized, and thus improves the stability and reliability of the entire antibody - nucleic acid conjugation process.

[0026] (2) In the present invention, by utilizing the continuous-phase liquid flowing at high speed to exert a high-shear effect on the dispersed phase, the dispersed-phase droplets are forced to break into smaller units within the microchannel. The reduction in droplet size significantly increases the reaction surface area, promotes the contact efficiency between the antibody or nucleic acid and metal ions, shortens the diffusion distance of the reactants in the reaction channel, further accelerates the reaction process, and the smaller droplets are more likely to achieve uniform mixing in the serpentine channel, avoiding the formation of local concentration gradients, ensuring the full interaction of the reactants at the microscale, and reducing the residue of uncoupled free molecules.

[0027] (3) In the present invention, the stepped converging flow channel gradually reduces the flow velocity gradient of the mixed liquid through the multi-stage stepped structure, converting the turbulent flow into a laminar flow state. Under laminar flow conditions, the viscous force of the fluid dominates the flow, making it difficult for bubbles to form due to the lack of turbulent disturbance, avoiding the influence of the presence of a large number of bubbles on the mixing effect and reaction rate of the reactants, and the structure with staggered and symmetric distribution of the stepped parts further disperses the fluid impact, reducing the shear effect at the gas-liquid interface. Furthermore, the reduction of bubbles avoids the adsorption loss of antibody or nucleic acid molecules at the gas-liquid interface in the reaction system, while ensuring the continuity and stability of the reaction in the channel, thereby improving the product yield and reducing the batch-to-batch variation.

[0028] (4) In the present invention, through the fine regulation of the reaction process by segmental temperature control during the reaction, the high temperature in the initial stage enhances the flexibility of the active groups in the antibody or nucleic acid molecules, promoting their reversible binding with metal ions. The low temperature in the later stage locks the formed specific binding conformation by reducing the molecular kinetic energy, reduces the bond breakage or mismatch caused by thermal motion, and inhibits side reactions, ensuring the integrity of the product structure, thereby optimizing the reaction path and improving the purity and yield of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings;

[0030] Figure 1 It is a schematic structural diagram of the reaction control device of the preferred embodiment of the present invention;

[0031] Figure 2 It is a schematic diagram of the state of the fluid permeable membrane when there is no fluid input in the control channel of the preferred embodiment of the present invention;

[0032] Figure 3 It is a schematic diagram of the state of the fluid permeable membrane when the control channel has fluid input of the preferred embodiment of the present invention;

[0033] Figure 4 It is a schematic diagram of the distribution structure of the stepped part in the preferred embodiment of the present invention;

[0034] In the figure: 1, device main body; 21, flow channel assembly; 211, dispersed phase input flow channel; 212, continuous phase input flow channel; 213, fluid input port; 22, stepped converging flow channel; 221, stepped part; 31, serpentine reaction flow channel; 311, fluid output port; 32, reaction control assembly; 321, fluid input pipeline; 322, control flow channel; 323, fluid permeable membrane. Specific embodiments

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0036] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention may also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0037] In the description of the present application, it should be understood that the terms "center", "longitudinal", "transverse", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be understood as limiting the protection scope of the present application. In the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0038] In the description of the present application, it should be noted that, unless otherwise clearly defined and limited, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection, or an integral connection; it may be a mechanical connection or an electrical connection; it may be directly connected, or indirectly connected through an intermediate medium, and it may be the internal communication of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood through specific situations.

[0039] Such as Figure 1As shown in the figure, a continuous flow reaction control device for antibody-nucleic acid conjugation includes: a device main body 1, a fluid input component and a reaction control component that are sequentially connected and arranged on the device main body 1 according to the reaction path; the fluid input component includes: a flow channel assembly 21 for respectively providing the input of the dispersed phase liquid and the continuous phase liquid, and a stepped converging flow channel 22 connected to the bottom of the flow channel assembly 21 for the convergence of the dispersed phase liquid and the continuous phase liquid and reducing the formation of bubbles; the reaction control component includes: a serpentine reaction flow channel 31, and a reaction control assembly 32 for gradient control of the temperature inside the serpentine reaction flow channel 31 and synchronously controlling the internal size to achieve the manipulation of the reaction time; the top end of the serpentine reaction flow channel 31 is communicated with the bottom of the stepped converging flow channel 22, and the bottom end is provided with a fluid outlet 311; at least four parallel reaction segments of the serpentine reaction flow channel 31 are included, and at least two groups of reaction control assemblies 32 are included, and are respectively located between every two segments of the serpentine reaction flow channel 31.

[0040] It should be noted that the width of the connection channel between the stepped converging flow channel 22 and the serpentine reaction flow channel 31 is reduced by at least 30% relative to the width of the stepped converging flow channel 22, which is used to introduce the mixed liquid into the serpentine reaction flow channel 31, reduce the fluid swirl by using the inertial effect, avoid the droplet breakage, and a tapered transition section is provided at the connection to reduce the fluid resistance and maintain the laminar flow state; by precisely controlling the input of the dispersed phase and continuous phase liquids, the stepped converging flow channel 22 is used to reduce the formation of bubbles, and then the double control of the temperature gradient and reaction time is carried out in the serpentine reaction flow channel 31, which helps to optimize the conditions of the antibody-nucleic acid conjugation reaction and improve the reaction efficiency and product quality.

[0041] As Figure 2 shown, in some embodiments, the reaction control assembly 32 includes: a fluid input pipeline 321 for inputting the fluid of the required temperature or pressure, a plurality of control flow channels 322 opened on the inner wall of the serpentine reaction flow channel 31, and a fluid permeable membrane 323 fixed on the inner side of the control flow channels 322; one end of the fluid input pipeline 321 is respectively communicated with the inside of the plurality of control flow channels 322, and the fluid permeable membrane 323 is used to permeate the input fluid from the high-pressure side to the low-pressure side to achieve the change of the temperature or the internal size inside the serpentine reaction flow channel 31.

[0042] It should be noted that the material of the fluid permeable membrane 323 is one of polydimethylsiloxane (PDMS) or perfluoropolyether (PFPE). Its high gas permeability (N2 permeability coefficient ≥ 500 Barrer) and chemical inertness ensure the efficient permeation of the temperature control fluid without contaminating the reaction system. In the serpentine reaction flow channel 31, a number of control flow channels 322 on each parallel reaction section are evenly distributed in a linear array, which is used to evenly distribute the fluid permeable membrane 323 and improve the environmental uniformity inside the serpentine reaction flow channel 31. The fluid is preferably nitrogen. Utilizing the characteristics of the fluid permeable membrane 323, when the external temperature or pressure of nitrogen is injected into the control flow channel 322 through the fluid input pipeline 321, a pressure difference is formed on both sides of the fluid permeable membrane 323, driving nitrogen to permeate to the inner wall of the serpentine reaction flow channel 31, and the pressure difference can prevent the internal reaction mixture from passing through the fluid permeable membrane 323. Furthermore, by adjusting the temperature of the input nitrogen, the temperature of the flow channel wall surface can be changed, achieving a temperature control accuracy of ±0.5°C, enabling dynamic adjustment of the temperature and size inside the serpentine reaction flow channel 31. When nitrogen permeates, it can drive the fluid permeable membrane 323 to deform towards the inside of the serpentine reaction flow channel 31( Figure 3 as shown), so as to change the internal size of the channel, extend the required reaction time, meet the requirements of different reaction stages, and thus optimize the reaction process.

[0043] As Figure 1 and Figure 4 shown, in some embodiments, the flow channel assembly 21 includes: a dispersed phase input flow channel 211, and at least one continuous phase input flow channel 212 located on the side of the dispersed phase input flow channel 211; fluid inlets 213 are provided at the tops of both the dispersed phase input flow channel 211 and the continuous phase input flow channel 212. The flow path of the fluid inlet 213 leading to the continuous phase input flow channel 212 is two segments with gradually decreasing widths, which is used to increase the flow velocity of the continuous phase liquid. The top of the stepped converging flow channel 22 is connected to the bottom of the dispersed phase input flow channel 211, and the side is connected to the bottom of the continuous phase input flow channel 212.

[0044] It should be noted that the number of the continuous phase input flow channels 212 is preferably two and they are symmetrically distributed on both sides of the dispersed phase input flow channel 211. Through the dispersed phase input flow channel 211 and the continuous phase input flow channel 212, the addition of the dispersed phase liquid and the continuous phase liquid can be respectively carried out. Since the continuous phase input flow channel 212 has two segments with gradually decreasing widths, at a constant flow rate, according to Bernoulli's principle, compared with the dispersed phase input flow channel 211, the cross-sectional area decreases, resulting in a significant increase in the flow velocity. When entering the stepped converging flow channel 22, the high-speed continuous phase exerts a high shear force on the dispersed phase at the intersection to break the dispersed phase droplets, which helps to improve the uniformity and stability of the reaction system and increase the efficiency and product purity of the antibody-nucleic acid conjugation reaction.

[0045] As Figure 4As shown, in some embodiments, at least three stepped portions 221 are provided inside the stepped converging flow channel 22; a number of stepped portions 221 are staggeredly and symmetrically distributed in the stepped converging flow channel 22 from top to bottom in sequence.

[0046] It should be noted that by geometric symmetry to disperse the fluid impact force and avoid the formation of eddy currents in the local high-pressure area, each stepped portion 221 converts the turbulent kinetic energy into heat energy dissipation through the sudden change of the flow channel cross-section, gradually reduces the Reynolds number, thereby eliminating the turbulent conditions required for bubble generation. The staggered distribution of the stepped portions 221 forms a periodic flow velocity perturbation, breaks the axisymmetry of the fluid, enhances the radial mixing, and avoids the aggregation caused by the too high local concentration of the antibody / nucleic acid.

[0047] The present invention provides a control method for a continuous flow reaction control device for antibody-nucleic acid conjugation, including the following steps:

[0048] S1. Input the dispersed phase liquid and the continuous phase liquid respectively through the flow channel assembly 21, and enter the stepped converging flow channel 22 for convergence from the bottom of the flow channel assembly 21 in a state where the flow velocity of the continuous phase liquid is greater than that of the dispersed phase liquid.

[0049] S2. The converged liquid enters the serpentine reaction flow channel 31 and flows and reacts along at least four parallel reaction segments. The reaction control assembly 32 controls the temperature gradient and the reaction time within each gradient inside every two parallel reaction segments in the serpentine reaction flow channel 31.

[0050] S3. The reacted liquid is discharged through the fluid outlet 311, and the product concentration and the by-product content are monitored in real time.

[0051] S4. Repeat the steps of S1-S3 to achieve continuous flow reaction control.

[0052] In some specific embodiments, in the step of S1, the flow velocity of the continuous phase liquid is 2-4 times that of the dispersed phase liquid.

[0053] In some specific embodiments, in the step of S1, the flow velocity gradient in the stepped converging flow channel 22 is 0.5-1.5 m / s.

[0054] In some specific embodiments, in the step of S2, the temperature gradient is specifically: initially heating up by 40-45 °C to accelerate the reaction, and later cooling down by 20-25 °C to reduce non-specific binding.

[0055] In some specific embodiments, the initial reaction time is 10-20 s, and the later reaction time is 15-30 s.

[0056] In some specific embodiments, in the step of S3, the real-time monitoring indicators include: coupling efficiency and non-specific binding rate.

[0057] To further make the objectives and effects of the present invention simple and easy to understand, the present invention is further described in combination with examples and comparative examples. Among them, the parallel reaction section of the serpentine reaction channel 31 includes four sections, and each two sections are respectively a set of reaction control components 32; the material of the fluid permeable membrane 323 is PDMS; the number of continuous phase input channels 212 is two, symmetrically distributed on both sides of the dispersed phase input channel 211; the number of internal stepped parts of the stepped converging channel 22 is three; the dispersed phase liquid is an antibody solution: humanized IgG antibody (1 mg / mL, dissolved in pH 7.4 PBS buffer), and the continuous phase liquid is a complex of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and a chelating agent: 10 mM DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) + 5 mM EDC, dissolved in pH 6.0 MES buffer.

[0058] More specifically, the preparation of the PDMS membrane includes the following steps:

[0059] a. Mix the PDMS prepolymer (Sylgard 184) with the platinum catalyst at a ratio of 10:1, and remove air bubbles by vacuum degassing;

[0060] b. Spin-coat on the silicon template at a speed of 2000 rpm to form a uniform film layer, and bake at 80 °C for 2 h to complete cross-linking;

[0061] c. Permanently bond both sides of the PDMS membrane to the inner wall of the serpentine reaction channel by oxygen plasma treatment (power 50 W, time 30 s).

[0062] It should be noted that the thickness of the PDMS membrane is 50 μm, the N2 permeability coefficient ≥ 500 Barrer, the working temperature is -50 to 200 °C, and the elastic modulus is 0.5 - 2 MPa (reversibly deformed under the pressure regulation of 50 - 150 kPa, accurately compressing the channel width by 50 - 100 μm).

[0063] Example 1

[0064] A control method for a continuous flow reaction control device for antibody-nucleic acid coupling, comprising the following steps:

[0065] S1. Inject the antibody solution (1 mg / mL) into the dispersed phase input channel 211 at a flow rate of 0.4 mL / min. The width of the dispersed phase input channel 211 is 500 μm. Inject the EDC-DOTA complex solution into the continuous phase input channel 212 at a flow rate of 1.2 mL / min. The width of the first section of the continuous phase input channel 212: 500 μm, the width of the second section: 125 μm. The two-phase liquids converge in the stepped convergence channel 22. The flow rate gradient in the stepped convergence channel 22: the inlet flow rate is 1.5 m / s, and the flow rate at the outlet of the stepped part is 0.5 m / s;

[0066] S2. Input nitrogen at 43 °C into the fluid input pipeline 321. Through the fluid permeable membrane 323, raise the temperature of the first two parallel reaction section channels of the serpentine reaction channel 31 to 43 °C. Pressure control: 150 kPa. Compress the channel to a width of 50 μm and extend the residence time to 20 s. Input nitrogen at 25 °C into the fluid input pipeline 321. Through the fluid permeable membrane 323, lower the temperature of the last two parallel reaction section channels of the serpentine reaction channel 31 to 25 °C. Pressure control: 100 kPa. Restore the channel width to 75 μm and the residence time is 15 s;

[0067] S3. Collect the product through the fluid outlet 311; Real-time detection:

[0068] Coupling efficiency: Detect the DOTA-antibody binding rate by HPLC;

[0069] Non-specific binding rate: Detect the DOTA residue in the BSA interference sample by ELISA.

[0070] Example 2

[0071] This example is basically the same as Example 1, except that the EDC-DOTA complex solution is injected at a flow rate of 0.8 mL / min.

[0072] Example 3

[0073] This example is basically the same as Example 1, except that the EDC-DOTA complex solution is injected at a flow rate of 1.6 mL / min.

[0074] Comparative Example 1

[0075] This comparative example is basically the same as Example 1, with the difference that: The steps of S2 are specifically as follows: Input nitrogen gas at 43°C into the fluid input pipeline 321. Through the fluid permeable membrane 323, the temperature of the first two parallel reaction section channels of the serpentine reaction channel 31 is raised to 43°C, pressure control: 150 kPa, the channel is compressed to a width of 50 μm, and the residence time is extended to 20 s; Input nitrogen gas at 43°C into the fluid input pipeline 321. Through the fluid permeable membrane 323, the temperature of the last two parallel reaction section channels of the serpentine reaction channel 31 is maintained at 43°C, pressure control: 100 kPa, the channel is restored to a width of 75 μm, and the residence time is 15 s.

[0076] Comparative Example 2

[0077] This comparative example is basically the same as Example 1, with the difference that: The steps of S2 are specifically as follows: Input nitrogen gas at 25°C into the fluid input pipeline 321. Through the fluid permeable membrane 323, the temperature of the first two parallel reaction section channels of the serpentine reaction channel 31 is raised to 25°C, pressure control: 150 kPa, the channel is compressed to a width of 50 μm, and the residence time is extended to 20 s; Input nitrogen gas at 43°C into the fluid input pipeline 321. Through the fluid permeable membrane 323, the temperature of the last two parallel reaction section channels of the serpentine reaction channel 31 is raised to 43°C, pressure control: 100 kPa, the channel is restored to a width of 75 μm, and the residence time is 15 s.

[0078] Comparative Example 3

[0079] This comparative example is basically the same as Example 1, with the difference that: The EDC-DOTA complex solution is injected at a flow rate of 0.4 mL / min.

[0080] Comparative Example 4

[0081] This comparative example is basically the same as Example 1, with the difference that: The EDC-DOTA complex solution is injected at a flow rate of 2.0 mL / min.

[0082] Comparative Example 5

[0083] This comparative example is basically the same as Example 1, with the difference that: The flow velocity gradient in the stepped converging flow channel 22: The inlet flow velocity is 1.5 m / s, and the flow velocity at the stepped part outlet is 1.5 m / s.

[0084] Performance detection record: In Examples 1-3 and Comparative Examples 1-5, the results of the coupling efficiency and non-specific binding rate detected in real time are shown in Table 1.

[0085] Coupling efficiency: Detected by HPLC (high performance liquid chromatography), separating the unreacted free metal isotope and the coupling product, and calculating the coupling efficiency through the peak area:

[0086]

[0087] Non-specific binding rate: The residual DOTA in the BSA interference sample was detected by ELISA method.

[0088] Table 1: Performance test record results of Examples 1-3 and Comparative Examples 1-5

[0089] Performance detection Coupling efficiency (%) Non-specific binding rate (%) Example 1 87.3 4.1 Example 2 83.2 5.3 Example 3 85.6 4.8 Comparative example 1 72.1 12.3 Comparative example 2 68.9 15.7 Comparative example 3 79.5 9.5 Comparative example 4 81.7 8.6 Comparative example 5 76.3 10.4

[0090] As shown in Table 1:

[0091] From the comparison between Example 1 and Comparative Example 1, it can be known that continuous high temperature will lead to intensified molecular thermal motion, resulting in over-activation of the active groups of antibody molecules, increasing the random collision probability with metal chelators, and the non-specific binding rate increases. Among them, although high temperature accelerates the reaction start, it does not lock the conformation by cooling in the later stage, that is, the formed conjugate undergoes bond breakage or mismatch due to thermal motion, and side reactions (such as metal self-aggregation) are intensified, so that the non-specific binding rate rises from 4.1% to 12.3%, and the coupling efficiency drops from 87.3% to 72.1%.

[0092] From the comparison between Example 1 and Comparative Example 2, it can be known that low temperature in the initial stage inhibits the activation of the carboxylic acid group of DOTA by EDC, resulting in a delay in the start of the coupling reaction and a reduction in the overall efficiency. Although heating up in the later stage accelerates the reaction, insufficient time is reserved to stabilize the product, accelerating the entropy-driven binding of metal ions to heteroproteins, the non-specific binding rate increases significantly, the coupling efficiency drops sharply, and the non-specific binding rate is as high as 15.7%.

[0093] From the comparison between Examples 1-3 and Comparative Example 3, it can be known that the continuous phase flow rate is not higher than the dispersed phase, and the insufficient flow rate ratio results in insufficient fragmentation of the dispersed phase droplets, reduced reaction surface area, reduced contact efficiency, and insufficient internal eddy current intensity of the droplets, limited diffusion of antibodies and metal ions, and increased uncoupled residues.

[0094] From the comparison between Examples 1-3 and Comparative Example 4, it can be known that too high continuous phase flow rate leads to too large shear force, and the droplets break into nanoscale. Although the surface area is increased, the droplet stability decreases, and partial droplet fragmentation leads to antibody inactivation. At the same time, high-speed flow shortens the reaction time, the coupling reaction is not fully completed, and some intermediates are not converted into stable products, resulting in a reduction in the coupling efficiency and an increase in the non-specific binding rate to 8.6%.

[0095] From the comparison between Example 1 and Comparative Example 5, it can be known that when there is no flow rate gradient in the stepped converging flow channel 22, that is, there is no buffer in the stepped part, the fluid maintains a high Reynolds number, the proportion of turbulent bubbles increases, adsorbing the active sites on the antibody surface, and the droplets coalesce due to turbulence, and local concentration gradients cause uncoupled residues.

[0096] Based on the inspiration of the ideal embodiments of the present invention, through the above description, for those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims involved.

[0097] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A continuous flow reaction control device for antibody-nucleic acid conjugation, characterized in that, Comprising: A device main body, successively connecting a fluid input component and a reaction control component provided on the device main body according to a reaction path; The fluid input component includes: a flow channel assembly for respectively providing the input of a dispersed phase liquid and a continuous phase liquid, and a stepped confluence flow channel connected to the bottom of the flow channel assembly for the confluence of the dispersed phase liquid and the continuous phase liquid and reducing the formation of bubbles; The reaction control component includes: a serpentine reaction flow channel, and a reaction control assembly for gradiently controlling the temperature inside the serpentine reaction flow channel and synchronously controlling the internal size to achieve the manipulation of the reaction time; the top end of the serpentine reaction flow channel is communicated with the bottom of the stepped confluence flow channel, and the bottom end is provided with a fluid outlet; at least four parallel reaction segments are included in the parallel reaction segment of the serpentine reaction flow channel, and at least two groups are included in the reaction control assembly, and are respectively located between every two segments of the serpentine reaction flow channel.

2. The continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 1, wherein: The reaction control assembly includes: a fluid input pipeline for inputting a fluid with a required temperature or pressure, a plurality of control flow channels opened on the inner wall of the serpentine reaction flow channel, and a fluid permeable membrane fixed on the inner side of the control flow channel; One end of the fluid input pipeline is respectively communicated with the inside of a plurality of the control flow channels, and the fluid permeable membrane is used for permeating the input fluid from the high-pressure side to the low-pressure side to change the temperature or the internal size inside the serpentine reaction flow channel.

3. The continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 1, wherein: The flow channel assembly includes: a dispersed phase input flow channel, and at least one continuous phase input flow channel located on the side of the dispersed phase input flow channel; Fluid inlets are provided at the top ends of the dispersed phase input flow channel and the continuous phase input flow channel, and the flow path of the fluid inlet to the continuous phase input flow channel is two sections with gradually decreasing widths for increasing the flow velocity of the continuous phase liquid; the top end of the stepped confluence flow channel is communicated with the bottom end of the dispersed phase input flow channel, and the side is communicated with the bottom end of the continuous phase input flow channel.

4. A continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 1, characterized in that: At least three stepped portions are provided inside the stepped confluence flow channel; a plurality of the stepped portions are alternately and symmetrically distributed from top to bottom inside the stepped confluence flow channel.

5. A control method for a continuous flow reaction control device for antibody-nucleic acid conjugation according to any one of claims 1-4, characterized in that Including the following steps: S1. Respectively input the dispersed phase liquid and the continuous phase liquid through the flow channel assembly, and enter the stepped confluence flow channel from the bottom of the flow channel assembly in a state where the flow velocity of the continuous phase liquid is greater than that of the dispersed phase liquid for confluence; S2. The confluent liquid enters the serpentine reaction flow channel, flows and reacts along at least four parallel reaction segments, and the reaction control assembly controls the temperature gradient and the reaction time within each gradient inside every two parallel reaction segments in the serpentine reaction flow channel; S3. The reacted liquid is discharged through the fluid outlet, and the product concentration and the by-product content are monitored in real time; S4. Repeat steps S1-S3 to achieve continuous flow reaction control.

6. The control method of a continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 5, characterized in that: In the step of S1, the flow velocity of the continuous phase liquid is 2-4 times that of the dispersed phase liquid.

7. A control method for a continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 5, characterized in that: In the step of S1, the flow velocity gradient inside the stepped confluence flow channel is 0.5-1.5 m / s.

8. The control method of a continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 5, characterized in that: In the step of S2, the temperature gradient is specifically: initially heating up 40-45 °C to accelerate the reaction, and later cooling down 20-25 °C to reduce non-specific binding.

9. The control method of a continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 8, characterized in that: The initial reaction time is 10 - 20 s, and the later reaction time is 15 - 30 s.

10. The control method of a continuous flow reaction control device for antibody-nucleic acid conjugation according to claim 5, characterized in that: In the step of S3, the real-time monitoring indicators include: coupling efficiency and non-specific binding rate.

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