A high-throughput screening method for glycosylase module based on digital microfluidic platform and application thereof

Abstract

The application discloses a glycosylation enzyme module high-throughput screening method based on a digital microfluidic (DMF) platform and application thereof, wherein the DMF platform comprises a DMF chip, a detection module and a software control module; wherein the DMF chip comprises: (a) a storage unit for pre-storing reaction reagents; (b) a reaction unit for fusing sample droplets with reaction reagents; (c) a magnetic bead purification unit for binding target products in the sample droplets with magnetic beads and purifying the target products; and (d) a waste liquid recovery unit. The application develops a high-throughput screening technology of high-efficiency alpha2,6-sialylation, beta1,4-N-acetylglucosamine glycosylation, alpha2,8-sialylation, alpha2,8-poly-sialylation and other enzyme modules based on DMF, and applies the high-throughput screening technology to efficient and low-cost synthesis of functional sugar chains such as disialyl lactose-N-tetrasaccharide (DSLNT) and ganglioside GM2 sugar chains.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a high-throughput screening method for glycosylation enzyme modules based on a digital microfluidic platform and its application. Background Technology

[0002] Glycans are the third set of codes for life after DNA and proteins. They are core modifications of cell surface and intracellular proteins, determining cell recognition, signaling, immunity, protein homeostasis, disease development, and drug efficacy. They are a core target for life regulation and biomedicine. How to efficiently obtain glycans is a key technology that needs to be solved.

[0003] Glycan structures are highly complex (multiple isomers, numerous branches, and diverse glycosidic bond linkages), and their chemical synthesis suffers from limitations such as cumbersome steps, poor stereoselectivity, complex handling of protecting groups, high pollution, and high costs. Enzymatic synthesis of glycans, however, is gaining increasing attention due to its advantages, including high selectivity, mild reaction conditions, and environmental friendliness. Glycosylating enzyme modules (including monosaccharide kinases, nucleotide pyrophosphorylases or nucleoside transferases, pyrophosphatases, glycosyltransferases, or their mutants) have been successfully used in the synthesis of various glycans. Developing efficient glycosylating enzyme modules is the cornerstone of controllable, efficient, and targeted glycan synthesis, and a crucial prerequisite for fundamental research in glycobiology, the preparation of functional oligosaccharides, and the development of glycopharmaceuticals.

[0004] Necrotizing enterocolitis (NEC) of the newborn is a common gastrointestinal critical illness in premature infants, with an incidence of 3%–10% in extremely low birth weight infants (born weight less than 1500 grams) at gestational age less than 32 weeks, and a related mortality rate as high as 80%. Reports indicate that the annual cost of treating NEC in the United States reaches as high as $1 billion. The pathogenesis of NEC remains unclear, and effective pharmacological prevention and treatment strategies have not yet emerged. Research shows that a specific oligosaccharide in human milk oligosaccharides (HMOs) (i.e., disialial lactalbumin-N-tetrasaccharide (DSLNT)) is a core component in the prevention and treatment of NEC with human milk (Jantscher-Krenn et al, Gut, 2012, 61, 1417-1425). Currently, there is a lack of efficient methods for synthesizing DSLNT. Chemical synthesis of DSLNT has not been reported. Due to the advantages of enzymatic synthesis of sugar chains, such as good selectivity, mild reaction conditions, and environmental friendliness, enzymatic synthesis of DSLNT has attracted attention. However, all reported literature uses cell-expressed human α2,6-sialyltransferase ST6GalNAc5 and cytidine (CMP)-sialic acid as the sialic acid donor (Prudden et al, Proc Nat Acad Sci USA, 2017, 114, 6954-6959). Due to the high cost of cell-expressed enzymes and CMP-sialic acid, they are unsuitable for large-scale production of DSLNT. Microbial-derived α2,6-sialyltransferases are more suitable for DSLNT synthesis due to their broad substrate spectrum, ease of expression, and good stability. However, studies have found that when microbial-derived α2,6-sialyltransferases catalyze sialyl-lactose-N-tetrasaccharide a (LSTa, a precursor for DSLNT synthesis, with the structural formula shown below), the synthesis of DSLNT is affected. Figure 1 (as shown) is converted to DSLNT (structure as shown) Figure 2 When (as shown), the desired product DSLNT was not obtained; instead, an isomer of DSLNT was generated.

[0005] Ganglioside glycans play crucial roles in intercellular recognition, neural development, signaling pathway regulation, neurotransmitter regulation, immune recognition and regulation, pathogen toxin binding, and cell fate regulation. Therefore, ganglioside glycans can be used in neuroprotection and the development of new drugs for neurodegenerative diseases, tumor immunology, tumor vaccines, targeted therapies, anti-infection, and antitoxin applications. However, currently, efficient methods for synthesizing ganglioside glycans such as GM2 and GD3 are lacking.

[0006] Oligosialyllactose (OSAL) plays important roles in nervous system development and plasticity, immune regulation, cell migration, and immune escape, and therefore has applications in glycan vaccines, anti-inflammatory and anti-tumor immunotherapy, nerve injury repair and neuroprotection, drug delivery, tissue engineering and biomaterials, and cosmetics. However, efficient methods for synthesizing OSAL are currently lacking.

[0007] Currently, enzyme discovery mainly relies on high-throughput screening technologies. However, existing enzyme screening methods primarily depend on microplates, flow cytometry, and traditional continuous flow microfluidics. These technologies have the following drawbacks: (1) Microplates have limited throughput, high reagent consumption, and complicated product purification steps after reaction, requiring manual transfer operations, resulting in low automation and large errors. (2) Flow cytometry technology makes it difficult to achieve long-term in situ incubation of glycosylation enzyme module-catalyzed reactions and cannot complete in situ purification of products, which is prone to detection interference. (3) Continuous flow microfluidic channels are fixed and have poor flexibility, making it difficult to expand multi-condition combination screening and difficult to achieve magnetic bead-mediated in-situ purification. Based on continuous flow microfluidic channels, the isolation of single clones or single mutants is not thorough, and cross-interference is easy. At the same time, the product purification efficiency is low, resulting in poor detection accuracy and poor data repeatability.

[0008] The high-throughput screening method for glycosylation enzyme modules (e.g., 2,6-sialylation modules) based on a digital microfluidic platform, as claimed in this invention, and its application, are not found in the prior art. Summary of the Invention

[0009] Based on this, the present invention provides a high-throughput screening method for glycosylation enzyme modules based on a digital microfluidic platform, the method comprising the following steps: (1) Using sugar as a substrate, the tagged derivative substrate sugar is obtained by reductive amination reaction between the primary amino group of the derivatizing reagent and the aldehyde group of the sugar, or by nucleophilic substitution reaction between the N-methylhydroxylamine moiety of the derivatizing reagent and the reducing end of the sugar. (2) Different types of droplets are dispensed on a digital microfluidic chip, including glycosylation enzyme module sample droplets, labeled derivatized substrate sugar droplets, buffer droplets, functionalized magnetic bead droplets, elution buffer droplets and ultrapure water droplets; (3) Drive the glycosylation enzyme module sample droplet, the derivatized substrate sugar droplet and the buffer droplet to fuse and carry out an enzymatic reaction to obtain the reaction droplet; (4) The reaction-driven droplet fuses with the functionalized magnetic bead droplet to react, remove impurities, wash away impurities, and elute to obtain a purified sample; and (5) The purified sample was tested and the results were judged to screen for highly efficient glycosylation enzyme modules; The digital microfluidic chip drives discrete droplets to flow on its surface through an array of electrodes.

[0010] Furthermore, the digital microfluidic platform includes a digital microfluidic chip, a detection module, and a software control module.

[0011] Furthermore, the digital microfluidic chip includes: (a) a reagent storage unit for pre-storing reaction reagents; (b) a reaction unit for fusing sample droplets with reaction reagents; (c) a magnetic bead purification unit for binding the target product in the sample droplet with magnetic beads and eluting the target product from the magnetic beads to obtain a purified product; and (d) a waste liquid recovery unit.

[0012] Furthermore, the digital microfluidic chip uses glass, ITO, or a hydrophobic dielectric layer as a substrate.

[0013] Furthermore, a hydrophobic functional layer is fabricated on the surface of the digital microfluidic chip.

[0014] Furthermore, the digital microfluidic chip includes an upper electrode and a lower electrode.

[0015] Furthermore, the upper electrode plate is equipped with sampling through holes through an ITO glass drilling process.

[0016] Furthermore, when the droplet volume is approximately 5 μL and the gap between the upper and lower electrode chambers is approximately 1.25 mm, the thickness of the glass is 0.3 mm and the diameter of the sampling orifice is approximately 2 mm.

[0017] Furthermore, the droplet manipulation of this digital microfluidic platform is performed in an air phase medium environment or an oil phase medium environment, for example, in an oil phase medium environment.

[0018] Furthermore, the oil phase medium is silicone oil.

[0019] Furthermore, the glycosylation enzyme module sample droplet contains a surfactant.

[0020] Furthermore, the surfactant includes L64, F68, Pluronic F127 and / or T90R4, such as Pluronic F127.

[0021] Further, in step (1), the sugar is sialyllactose-N-tetrasaccharide a.

[0022] Further, in step (1), the derivatizing agent includes a sulfonate-containing compound or biotin, such as 7-amino-1,3-naphthalene disulfonate or 7-(2-(2-(N-methylaminooxy)ethoxy)ethoxy)naphthalene-1,3-disulfonic acid.

[0023] Furthermore, in step (1), the labeling includes sulfonate modification or biotin modification.

[0024] Furthermore, when the derivatizing reagent is a compound containing sulfonate, the functionalized magnetic bead is a DEAE magnetic bead.

[0025] Furthermore, when the derivatizing agent is biotin, the functionalized magnetic bead is avidin or streptavidin-modified magnetic bead.

[0026] Furthermore, in step (1), the reductive amination reaction further includes a reducing agent.

[0027] Furthermore, the reducing agent is sodium cyanoborohydride.

[0028] Further, in step (1), the molar ratio of the derivatizing reagent, substrate and reducing agent in the reductive amination reaction is (8~12):(0.5~2):(3~5).

[0029] Furthermore, the molar ratio of the derivatizing reagent, substrate, and reducing agent in this reductive amination reaction is approximately 10:1:4.

[0030] Further, in step (1), the reaction conditions for the reductive amination reaction include: the reaction is carried out in a shaker, the reaction solvent is an approximately 15% acetic acid solution, the reaction temperature is approximately 37°C, the shaker speed is approximately 200 r / min, and the reaction time is approximately 15 h.

[0031] Furthermore, step (1) further includes a gel filtration chromatography separation and purification step after the reductive amination reaction.

[0032] Furthermore, the gel filtration chromatography separation and purification method includes: using Bio-Gel P-2 Gel molecular sieve as the stationary phase, rinsing the molecular sieve column with pure water, adding the reducing amination reaction product, and performing isocratic elution with eluent at a flow rate of 0.5~2 ml / min, collecting the eluent fraction; detecting the eluent fraction using thin-layer chromatography with ethyl acetate-methanol-water-acetic acid as the developing solvent, and collecting the fraction containing the labeled derivatized substrate sugar after development and color development.

[0033] Furthermore, the gel particles of Bio-Gel P-2 Gel molecular sieve have a particle size of 100~1800 mesh.

[0034] Furthermore, the volume of the pure water is 100~300 ml, for example, about 200 ml.

[0035] Furthermore, the volume of the eluent is 150-350 ml, for example, about 250 ml.

[0036] Furthermore, the flow rate is approximately 1.0 ml / min.

[0037] Furthermore, the volume ratio of the ethyl acetate, methanol, water, and acetic acid in the developing solvent is approximately 4: approximately 6: approximately 1: approximately 0.2.

[0038] Further, in step (2), the glycosylation enzyme module is an α2,6-sialylase module.

[0039] Furthermore, the α2,6-sialylase module contains CMP-sialic acid synthase and / or its mutants, α2,6-sialyl transferase and / or its mutants.

[0040] Furthermore, the concentration of the CMP-sialic acid synthase and / or its mutant is 30–40 μM or 0.8–1.0 mg / ml.

[0041] Furthermore, the concentration of the α2,6-sialyltransferase and / or its mutant is 10–20 μM or 0.75–0.95 mg / ml.

[0042] Furthermore, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes.

[0043] Further, in step (2), the glycosylation enzyme module is a β1,4-N-acetylgalactosylase module.

[0044] Furthermore, the β1,4-N-acetaminogalactosyltransferase module contains N-acetaminohexokinase and / or its mutants, N-acetaminoglucosyltransferase and / or its mutants, and β1,4-N-acetaminogalactosyltransferase and / or its mutants.

[0045] Furthermore, the concentration of the N-acetylglucuronyltransferase and / or its mutant is 5–20 μM or 0.4–0.6 mg / ml.

[0046] Furthermore, the concentration of the N-acetaminohexokinase and / or its mutant is 5–20 μM or 0.3–0.5 mg / ml.

[0047] Furthermore, the concentration of the β1,4-N-acetylgalactosyltransferase and / or its mutant is 10–30 μM or 0.6–0.8 mg / ml.

[0048] Furthermore, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes.

[0049] Further, in step (2), the glycosylation enzyme module is an α2,8-sialylase module.

[0050] Furthermore, the α2,8-sialylase module contains CMP-sialic acid synthase and / or its mutants, α2,8-sialyl transferase and / or its mutants.

[0051] Furthermore, the concentration of the CMP-sialic acid synthase and / or its mutant is 30–40 μM or 0.8–1.0 mg / ml.

[0052] Furthermore, the concentration of the α2,8-sialyltransferase and / or its mutant is 10–20 μM or 0.3–0.5 mg / ml.

[0053] Furthermore, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes.

[0054] Further, in step (2), the glycosylation enzyme module is an α2,8-polysialidase module; Furthermore, the α2,8-polysialyltransferase module contains CMP-sialic acid synthase and / or its mutants, α2,8-polysialyltransferase and / or its mutants.

[0055] Furthermore, the concentration of the CMP-sialic acid synthase and / or its mutant is 30–40 μM or 0.8–1.0 mg / ml.

[0056] Furthermore, the concentration of the α2,8-polysialyltransferase and / or its mutant is 10–20 μM or 0.9–1.1 mg / ml.

[0057] Furthermore, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes.

[0058] Further, in step (4), the reaction includes the following steps: driving the reaction droplet to fuse with the functionalized magnetic bead droplet, oscillating and incubating for 2 to 10 minutes, for example, about 5 minutes, to adsorb and fix the functionalized magnetic beads that can specifically bind to the target product.

[0059] Furthermore, in step (4), the impurity removal is a process of driving the removal of unadsorbed and fixed droplets.

[0060] Furthermore, in step (4), the impurity washing is performed by driving ultrapure water droplets to remove impurities adsorbed on the surface of the magnetic beads.

[0061] Furthermore, the washing process is performed 2 to 4 times, for example, 3 times.

[0062] Furthermore, in step (4), the elution is carried out by driving the eluent droplets or by heating.

[0063] Furthermore, the eluent is a salt solution, such as ammonium bicarbonate solution, sodium chloride solution, and / or sodium hydroxide solution.

[0064] Furthermore, in step (5), the detection includes capillary electrophoresis detection and / or mass spectrometry detection.

[0065] According to another aspect of the present invention, the use of the above method in a screening enzyme module is provided.

[0066] Furthermore, this enzyme is an enzyme that synthesizes functional sugar chains.

[0067] Furthermore, the enzyme is selected from one or more of the following: α2,6-sialylase, β1,4-N-acetylgalactosylase, α2,8-sialylase, and α2,8-polysialylase.

[0068] According to another aspect of the present invention, the use of an enzyme module obtained by the above method in the synthesis of functional sugar chains is provided.

[0069] Furthermore, this enzyme is an enzyme that synthesizes functional sugar chains.

[0070] Furthermore, the enzyme is selected from one or more of the following: α2,6-sialylase, β1,4-N-acetylgalactosylase, α2,8-sialylase, and α2,8-polysialylase.

[0071] Furthermore, the functional sugar chain is selected from one or more of the following: disialyllactose-N-tetrasaccharide, ganglioside GM2 sugar chain, ganglioside GD3 sugar chain, and oligosialyllactose (OSAL).

[0072] The beneficial effects of this invention are: The digital microfluidics of this invention uses discrete droplets as operating units and manipulates droplets through the electric field of an electrode array. It is flexibly programmable, highly integrated, and volume controllable, enabling in-situ integration of reaction and purification. Combined with substrate sugar derivatization and specific adsorption of functionalized magnetic beads, it can effectively simplify the purification process and is very suitable for the high-throughput screening needs of ultra-high-throughput, low-consumption, and automated glycosyltransferases and mutants.

[0073] Discovering efficient glycosylation enzyme modules is the core foundation for the controllable, efficient, and targeted synthesis of glycans, and a key prerequisite for basic research in glycobiology, preparation of functional oligosaccharides, and development of glycopharmaceuticals. Based on this, the present invention develops a high-throughput screening technology based on digital microfluidics for highly efficient 2,6-sialylase modules (including CMP-sialic acid synthase, 2,6-sialic acid transferase or their mutants), β1,4-N-acetaminogalactosyltransferase modules (including N-acetaminohexokinase, N-acetaminoglucuryltransferase, β1,4-N-acetaminogalactosyltransferase or their mutants), α2,8-sialylase modules (including CMP-sialic acid synthase, α2,8-sialic acid transferase or their mutants), and α2,8-polysialylase modules (including CMP-sialic acid synthase, α2,8-polysialic acid transferase or their mutants), and applies it to the efficient and low-cost synthesis of disialyllactose-N-tetrasaccharide (DSLNT), ganglioside GM2 glycan, ganglioside GD3 glycan, and oligosialyllactose (OSAL). Attached Figure Description

[0074] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0075] Figure 1 This is a structural schematic diagram of LSTA.

[0076] Figure 2 This is a structural diagram of DSLNT.

[0077] Figure 3 This is a schematic diagram of the screening process based on a digital microfluidic platform.

[0078] Figure 4 The diagrams show the detection results of the tag-glycan chain. (a) shows the tag-LSTa mass spectrometry analysis results, and (b) shows the tag-DSLNT mass spectrometry analysis results. The 2 in parentheses represents [(M-2H) / 2]. - .

[0079] Figure 5 This is a schematic diagram of the DSLNT mass spectrometry analysis results. The '1' in parentheses represents [M-1H]. - 2 represents [(M-2H) / 2] - .

[0080] Figure 6This is a schematic diagram of tag-Lac mass spectrometry analysis results. The '1' in parentheses represents [M-1H]. - .

[0081] Figure 7 This is a schematic diagram of the GM2 sugar chain.

[0082] Figure 8 This is a schematic diagram of the tag-GM2 mass spectrometry analysis results. The '1' in parentheses represents [M-1H]. - 2 represents [(M-2H) / 2] - .

[0083] Figure 9 This is a schematic diagram of the mass spectrometry analysis results for GM2 glycans. The "1" in parentheses represents [M-1H]. - 2 represents [(M-2H) / 2] - .

[0084] Figure 10 This is a schematic diagram of the structure of the GD3 sugar chain.

[0085] Figure 11 This is a schematic diagram of the tag-GD3 mass spectrometry analysis results. The '1' in parentheses represents [M-1H]. - 2 represents [(M-2H) / 2] - .

[0086] Figure 12 This is a schematic diagram of the GD3 glycan mass spectrometry analysis results. The '1' in parentheses represents [M-1H]. - .

[0087] Figure 13 This is a schematic diagram of the structure of oligosialyllactose (OSAL) (n = 3-8).

[0088] Figure 14 This is a schematic diagram of the tag-OSAL mass spectrometry analysis results. The horizontal axis represents elution time, and the vertical axis represents the detector response signal. The chromatographic peaks correspond to oligosaccharides with polymerization degrees of DP5, DP6, DP7, DP8, DP9, DP10, and DP11, respectively. DP=5 corresponds to an m / z of 750.19213 and an ionic form of [(M-2H) / 2]. - DP=6, DP=7, DP=8, DP=9, DP=10, and DP=11 correspond to m / z values ​​of 596.82468, 693.85663, 790.88861, 887.91944, 985.28512, and 1082.31723, respectively, and their ionic form is [(M-3H) / 3]. - .

[0089] Figure 15 This is a schematic diagram of the mass spectrometry analysis results of oligosialic acid lactose (OSAL). Detailed Implementation

[0090] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0091] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0092] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0093] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0094] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0095] As described in the background section, existing methods for synthesizing DSLNTs are not suitable for large-scale production. To address this problem, this invention provides a high-throughput screening method for glycosylation enzyme modules based on a digital microfluidic platform, comprising the following steps: (1) Using sugar as a substrate, the tagged derivative substrate sugar is obtained by reductive amination reaction between the primary amino group of the derivatizing reagent and the aldehyde group of the sugar, or by nucleophilic substitution reaction between the N-methylhydroxylamine moiety of the derivatizing reagent and the reducing end of the sugar. (2) Different types of droplets are dispensed on a digital microfluidic chip, including glycosylation enzyme module sample droplets, labeled derivatized substrate sugar droplets, buffer droplets, functionalized magnetic bead droplets, elution buffer droplets and ultrapure water droplets; (3) Drive the glycosylation enzyme module sample droplet, the derivatized substrate sugar droplet and the buffer droplet to fuse and carry out an enzymatic reaction to obtain the reaction droplet; (4) The reaction-driven droplet fuses with the functionalized magnetic bead droplet to react, remove impurities, wash away impurities, and elute to obtain a purified sample; and (5) The purified sample was tested and the results were judged to screen for highly efficient glycosylation enzyme modules; The digital microfluidic chip drives discrete droplets to flow on its surface through an array of electrodes.

[0096] In a preferred embodiment, the digital microfluidic platform includes a digital microfluidic chip, a detection module, and a software control module.

[0097] Compared with existing technologies, the digital microfluidic platform of the present invention has the following advantages: (1) High efficiency screening capability: It breaks through the throughput limitation of traditional enzyme efficacy verification and can realize large-scale single screening (testing enzyme mutants one by one) and combination screening (testing multiple enzyme / substrate combinations at the same time) of enzyme-catalyzed reactions, greatly improving the efficiency of data acquisition.

[0098] (2) Multi-enzyme system optimization capability: It can accurately assess and optimize the compatibility and stability between components in a multi-enzyme system, providing key technical support for constructing efficient and stable artificial multi-enzyme cascade reaction pathways.

[0099] (3) Complex product synthesis and prediction capabilities: Targeting cutting-edge fields such as glycobiology, it can realize the automated synthesis of glycan structures and establish models based on high-throughput data to achieve accurate prediction of synthesis results.

[0100] (4) In-situ separation and purification capability: By utilizing the specific binding of functionalized magnetic beads with sugar derivatives, the product can be purified in situ efficiently, reducing the interference of impurities on the detection results and improving the detection accuracy. (5) Advantages of technology integration: By integrating the above functions into the miniaturized platform, the reaction system is miniaturized (reducing reagent consumption), accelerated (shortening reaction time) and automated (reducing human intervention), which greatly reduces costs and provides a new technological paradigm for synthetic biology research.

[0101] In a preferred embodiment, the digital microfluidic chip includes: (a) a reagent storage unit for pre-storing reaction reagents; (b) a reaction unit for fusing sample droplets with reaction reagents; (c) a magnetic bead purification unit for binding a target product in the sample droplet with a magnetic bead and eluting the target product from the magnetic bead to obtain a purified product; and (d) a waste liquid recovery unit.

[0102] In a preferred embodiment, the digital microfluidic chip uses glass, ITO, or a hydrophobic dielectric layer as a substrate.

[0103] In a preferred embodiment, a hydrophobic functional layer is prepared on the surface of the digital microfluidic chip.

[0104] In a preferred embodiment, the digital microfluidic chip includes an upper electrode and a lower electrode.

[0105] In a preferred embodiment, the upper electrode plate is provided with a sampling through hole by ITO glass drilling process.

[0106] In a preferred embodiment, when the droplet volume is about 5 μL and the gap between the upper and lower electrode chambers is about 1.25 mm, the thickness of the glass is 0.3 mm and the diameter of the sampling through-hole is about 2 mm.

[0107] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25; "about 1.25" includes 1.25 ± 5%, or from 1.1875 to 1.3125; and "about 2" includes 2 ± 5%, or from 1.9 to 2.1.

[0108] In a preferred embodiment, the droplet manipulation of the digital microfluidic platform is performed in an air phase medium environment or an oil phase medium environment, for example, in an oil phase medium environment.

[0109] In a preferred embodiment, the oil phase medium is silicone oil.

[0110] In a preferred embodiment, the glycosylation enzyme module sample droplet contains a surfactant.

[0111] In a preferred embodiment, the surfactant includes L64, F68, Pluronic F127 and / or T90R4, such as Pluronic F127.

[0112] In a preferred embodiment, in step (1), the sugar is sialyl lactose-N-tetrasaccharide a.

[0113] In a preferred embodiment, in step (1), the derivatizing agent comprises a sulfonate-containing compound or biotin, such as 7-amino-1,3-naphthalene disulfonate or 7-(2-(2-(N-methylaminooxy)ethoxy)ethoxy)naphthalene-1,3-disulfonic acid.

[0114] In a preferred embodiment, in step (1), the labeling includes sulfonate modification or biotin modification.

[0115] In a preferred embodiment, when the derivatizing agent is a sulfonate-containing compound, the functionalized magnetic bead is a DEAE magnetic bead.

[0116] In a preferred embodiment, when the derivatizing agent is biotin, the functionalized magnetic bead is an avidin or streptavidin-modified magnetic bead.

[0117] In a preferred embodiment, in step (1), the reductive amination reaction further includes a reducing agent.

[0118] In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

[0119] In a preferred embodiment, in step (1), the molar ratio of the derivatizing agent, substrate and reducing agent in the reductive amination reaction is (8~12):(0.5~2):(3~5).

[0120] In this invention, when molar ratio, flow rate, particle size, volume, concentration, time, number of times, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “8~12” is disclosed, the described range should be interpreted as including ranges “8~12”, “8~11”, “8~10”, “8~9”, “9~12”, “9~11”, “9~10”, “10~12”, “10~11”, “11~12”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0121] In a preferred embodiment, the molar ratio of the derivatizing agent, substrate, and reducing agent in the reductive amination reaction is about 10: about 1: about 4.

[0122] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5; "about 1" includes ±5% of 1, or from 0.95 to 1.05; and "about 4" includes ±5% of 4, or from 3.8 to 4.2.

[0123] In a preferred embodiment, in step (1), the reaction conditions for the reductive amination reaction include: the reaction is carried out in a shaker, the reaction solvent is an approximately 15% acetic acid solution, the reaction temperature is approximately 37°C, the shaker speed is approximately 200 r / min, and the reaction time is approximately 15 h.

[0124] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15%" includes 15% ±5%, or from 14.25% to 15.75%; "about 37" includes 37 ±5%, or from 35.15% to 38.85%; "about 200" includes 200 ±5%, or from 190% to 210%; and "about 15" includes 15 ±5%, or from 14.25% to 15.75%.

[0125] In a preferred embodiment, step (1) further includes a gel filtration chromatography separation and purification step after the reductive amination reaction.

[0126] In a preferred embodiment, the gel filtration chromatography separation and purification method includes: using Bio-GelP-2 Gel molecular sieve as the stationary phase, rinsing the molecular sieve column with pure water, adding the reducing amination reaction product, eluting isocratically with eluent at a flow rate of 0.5~2 ml / min, and collecting the eluent fraction; detecting the eluent fraction using thin-layer chromatography with ethyl acetate-methanol-water-acetic acid as the developing solvent, and collecting the fraction containing the labeled derivatized substrate sugar after development and color development.

[0127] In a preferred embodiment, the gel particles of the Bio-Gel P-2 Gel molecular sieve have a particle size of 100~1800 mesh.

[0128] In a preferred embodiment, the volume of the pure water is 100-300 ml, for example, about 200 ml.

[0129] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 200" includes ±5% of 200, or from 190 to 210.

[0130] In a preferred embodiment, the volume of the eluent is 150-350 ml, for example, about 250 ml.

[0131] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 250" includes ±5% of 250, or from 237.5 to 262.5.

[0132] In a preferred embodiment, the flow rate is approximately 1.0 ml / min.

[0133] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0134] In a preferred embodiment, the volume ratio of the ethyl acetate, methanol, water and acetic acid in the developing solvent is about 4: about 6: about 1: about 0.2.

[0135] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 4" includes ±5% of 4, or from 3.8 to 4.2; "about 6" includes ±5% of 6, or from 5.7 to 6.3; "about 1" includes ±5% of 1, or from 0.95 to 1.05; and "about 0.2" includes ±5% of 0.2, or from 0.19 to 0.21.

[0136] In a preferred embodiment, in step (2), the glycosylation enzyme module is an α2,6-sialylase module.

[0137] In a preferred embodiment, the α2,6-sialylase module contains CMP-sialic acid synthase and / or its mutants, α2,6-sialyl transferase and / or its mutants.

[0138] In a preferred embodiment, the concentration of the CMP-sialic acid synthase and / or its mutant is 30-40 μM or 0.8-1.0 mg / ml.

[0139] In a preferred embodiment, the concentration of the α2,6-sialyltransferase and / or its mutant is 10-20 μM or 0.75-0.95 mg / ml.

[0140] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 15" includes ±5% of 15, or from 14.25 to 15.75; "about 0.5" includes ±5% of 0.5, or from 0.475 to 0.525.

[0141] In a preferred embodiment, in step (3), the enzymatic reaction takes 20 to 40 minutes, for example, about 30 minutes.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0143] In a preferred embodiment, in step (2), the glycosylation enzyme module is a β1,4-N-acetylgalactosylase module.

[0144] In a preferred embodiment, the β1,4-N-acetaminogalactosyltransferase module contains N-acetaminohexokinase and / or its mutants, N-acetaminoglucosyltransferase and / or its mutants, and β1,4-N-acetaminogalactosyltransferase and / or its mutants.

[0145] In a preferred embodiment, the concentration of the N-acetylglucuronidase and / or its mutant is 5-20 μM or 0.4-0.6 mg / ml.

[0146] In a preferred embodiment, the concentration of the N-acetaminohexokinase and / or its mutant is 5-20 μM or 0.3-0.5 mg / ml.

[0147] In a preferred embodiment, the concentration of the β1,4-N-acetylgalactosyltransferase and / or its mutant is 10-30 μM or 0.6-0.8 mg / ml.

[0148] In a preferred embodiment, in step (3), the enzymatic reaction takes 20 to 40 minutes, for example, about 30 minutes.

[0149] In a preferred embodiment, in step (2), the glycosylation enzyme module is an α2,8-sialylase module.

[0150] In a preferred embodiment, the α2,8-sialylase module contains CMP-sialic acid synthase and / or its mutants, α2,8-sialyl transferase and / or its mutants.

[0151] In a preferred embodiment, the concentration of the CMP-sialic acid synthase and / or its mutant is 30-40 μM or 0.8-1.0 mg / ml.

[0152] In a preferred embodiment, the concentration of the α2,8-sialyltransferase and / or its mutant is 10-20 μM or 0.3-0.5 mg / ml.

[0153] In a preferred embodiment, in step (3), the enzymatic reaction takes 20 to 40 minutes, for example, about 30 minutes.

[0154] In a preferred embodiment, in step (2), the glycosylation enzyme module is an α2,8-polysialidase module; In a preferred embodiment, the α2,8-polysialylase module contains CMP-sialic acid synthase and / or its mutants, α2,8-polysialyltransferase and / or its mutants.

[0155] In a preferred embodiment, the concentration of the CMP-sialic acid synthase and / or its mutant is 30-40 μM or 0.8-1.0 mg / ml.

[0156] In a preferred embodiment, the concentration of the α2,8-polysialyltransferase and / or its mutant is 10-20 μM or 0.9-1.1 mg / ml.

[0157] In a preferred embodiment, in step (3), the enzymatic reaction takes 20 to 40 minutes, for example, about 30 minutes.

[0158] In a preferred embodiment, in step (4), the reaction includes the following steps: driving the reaction droplet to fuse with the functionalized magnetic bead droplet, oscillating and incubating for 2 to 10 minutes, for example, about 5 minutes, to adsorb and fix the functionalized magnetic beads that can specifically bind to the target product.

[0159] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 5" includes 5 ± 5%, or from 4.75 to 5.25.

[0160] In a preferred embodiment, in step (4), the impurity removal is a drive to remove unadsorbed and fixed droplets.

[0161] In a preferred embodiment, in step (4), the impurity washing is performed by driving ultrapure water droplets to remove impurities adsorbed on the surface of the magnetic beads.

[0162] In a preferred embodiment, the washing process is performed 2 to 4 times, for example, 3 times.

[0163] In a preferred embodiment, in step (4), the elution is performed by driving the eluent droplets or by heating.

[0164] In a preferred embodiment, the eluent is a salt solution, such as ammonium bicarbonate solution, sodium chloride solution, and / or sodium hydroxide solution.

[0165] In a preferred embodiment, in step (5), the detection includes capillary electrophoresis detection and / or mass spectrometry detection.

[0166] According to another aspect of the invention, the use of the above method in screening α2,6-sialylase modules is provided.

[0167] According to another aspect of the present invention, the use of the α2,6-sialylase module obtained by the above method in the synthesis of disialyllactose-N-tetrasaccharide is provided.

[0168] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0169] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0170] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0171] Example Example 1: Digital Microfluidic Platform (e.g.) Figure 3 (As shown) Preparation (1) Composition of the microfluidic chip: The core functional component of the instrument, adopting an electrowetting digital microfluidic (EWOD) architecture, using a glass / ITO / hydrophobic dielectric layer as the substrate to replace the traditional continuous flow channel. It independently drives discrete droplets through an array of electrodes, achieving programmable control of droplet (sample, substrate, magnetic beads, eluent, etc.) distribution, movement, merging, splitting, and incubation. The chip integrates a reaction unit, a magnetic bead purification unit, a reagent storage unit, and a waste liquid recovery unit. It no longer relies on a continuous microfluidic channel, but instead achieves precise control of droplets on the chip surface through a programmable electrode array. Among them: Reagent storage unit: Used to pre-store reaction reagents such as enzymes, substrates, buffer solutions, and elution solutions. They are sealed in the designated electrode area of ​​the chip in the form of independent discrete droplets and can be precisely retrieved and driven as needed.

[0172] Reaction unit: Under the control of electrode timing, sample droplets and reagent droplets are precisely fused, and efficient mixing is achieved through droplet reciprocating motion and rotational oscillation. Compared with traditional continuous flow channels, the mixing is more uniform, reagent consumption is lower, and reaction efficiency is higher.

[0173] Magnetic bead purification unit: After the reaction is completed, the droplets are fused with the magnetic bead droplets under the drive of the electrode and fully oscillated and incubated, so that the target product specifically binds to the functionalized magnetic beads; under the action of an external fixed magnetic field, the magnetic beads are captured and fixed in situ, and the impurity droplets that are not bound are removed by the electrode in a directional manner; then the elution droplets are introduced and fully interact with the magnetic beads to elute and release the high-purity target product, providing purified samples for subsequent detection.

[0174] (2) Detection module (the detection module is independent of the microfluidic chip or the microfluidic chip is integrated with the detection module): used to detect signals in the purified sample after elution, for example, by liquid chromatography-mass spectrometry, capillary electrophoresis, etc.

[0175] (3) Software control: realizes screening process programming, droplet trajectory control, reaction parameter adjustment, signal acquisition and analysis, and screening result determination.

[0176] Example 2: In-situ sampling process and parameter matching for drilling holes in the ITO glass of the upper electrode of a digital microfluidic chip. The upper layer of the chip uses ITO conductive glass, with sampling vias pre-processed at designated locations. After the droplet reacts in the chip cavity, it is driven to move directly below the via, allowing the sample to be extracted in situ through the pre-drilled hole. Once the target droplet is manipulated, it is driven to the corresponding chamber position of the via, and the droplet can naturally overflow through the via, completing the non-destructive sampling operation.

[0177] ITO glass should not be too thick (e.g., >0.8 mm), otherwise it will hinder droplet overflow and increase sampling resistance. The glass size and perforation diameter need to be matched with the droplet volume and the spacing between the upper and lower electrode chambers. As a preferred implementation example: when the droplet volume is 5 μL and the spacing between the upper and lower electrode chambers is 1.25 mm, the glass thickness is 0.3 mm and the perforation diameter is set to 2 mm, which can achieve smooth droplet overflow and efficient and accurate sampling, while not affecting the normal driving and reaction process of surrounding droplets.

[0178] Example 3: Selection of Media System and Application of Silicon Oil Phase Droplet manipulation in digital microfluidic platforms can be performed in either an air phase or an oil phase medium. For short-duration reaction processes, the air phase can be used directly as the reaction environment, which is simple to operate and has a simple medium system. For experimental systems that require long-term incubation and continuous reaction, an oil phase is usually preferred as a closed medium environment to effectively avoid the problem of evaporation of reagent components inside the droplets.

[0179] The oil phase medium used in this system is selected from silicone oil reagents. Silicone oil itself is a non-polar inert medium. Under the digital microfluidic electric field driving mechanism, it does not produce any electrophoretic, dielectric response or displacement driving behavior. It can be stably used as a filling and isolation medium for microfluidic chambers to achieve the separation of adjacent droplets and suppress reagent volatilization.

[0180] A hydrophobic functional layer is fabricated on the surface of a digital microfluidic chip, which is the core structure for achieving independent droplet dispersion and low-loss transport. When some polar organic reagents (such as acetonitrile) come into direct contact with the hydrophobic functional layer, they can erode and damage the material structure and surface hydrophobic properties of the hydrophobic layer, leading to problems such as droplet adhesion and drive failure. When a silicone oil phase is used as the coating medium, the oil phase can form an isolation and protective layer between the droplet and the chip's hydrophobic layer, preventing highly polar reagents from directly contacting the hydrophobic functional layer. This reduces corrosion, modification, and performance degradation of the hydrophobic layer, effectively extending the lifespan of the digital microfluidic chip and ensuring the repeatability and stability of multiple batches of experiments.

[0181] Example 4: Screening of magnetic bead elution solutions under different phase environments In an open air-phase reaction environment, the reagents used for elution and desorption of magnetic beads only need to meet the requirement of not damaging the hydrophobic functional layer structure and surface hydrophobic properties of the chip. The reagent selection constraints are relatively relaxed, and various acid-base buffer solutions, salt solutions and elution buffer systems can be routinely adapted.

[0182] In an oil-phase closed chamber environment, the elution and desorption reactions of magnetic beads must meet the following conditions: Since bubbles can easily cause droplet disturbance and morphological distortion, it is strictly forbidden to generate bubbles during the interaction between the elution reagent and the magnetic beads.

[0183] Taking the DEAE functionalized magnetic bead desorption process as an example, the ammonium bicarbonate (NH4HCO3) buffer system is suitable for magnetic bead elution in an air phase environment, but cannot be applied to an oil phase system. The reason is that ammonium bicarbonate is prone to decompose and generate gas to form bubbles during the reaction process. The oil phase is a closed chamber structure, and the generated bubbles cannot be discharged on their own or eliminated by manipulation. They will occupy the chamber space, interfere with the droplet driving path, and thus interfere with the subsequent experimental process.

[0184] Example 5: Selection of Surfactants and Optimization of Enzyme Concentration Because surfactants can encapsulate enzymes in droplets and reduce protein adhesion to the electrode surface, making enzyme droplets easier to drive on digital microfluidic chips, surfactants such as Pluronic F127, Pluronic F68, and Tetronic 90R4 are added to the droplets to enable enzyme droplet actuation on digital microfluidic chips. The concentration of surfactants is optimized, and it is finally determined that 0.1% of Tetronic 90R4 is added in the enzyme reaction experiment. If the surfactant concentration is too low (e.g., less than 0.05%), droplet actuation is hindered, and if the concentration is too high (e.g., greater than 0.5%), enzyme activity will be affected.

[0185] In addition, in order to make enzyme droplets easy to drive on digital microfluidic chips, the enzyme concentration needs to be optimized. For example, enzyme droplets of 1 mg / ml will increase the risk of chip biocontamination and greatly affect droplet driving, while enzyme droplets of 0.5 mg / ml can be driven normally.

[0186] Example 6: Screening process of DSLNT synthesis-related enzymes (1) Derivatization of substrate sugar LSTa LSTA is derivatized using reagents that can specifically bind to it, such as compounds containing sulfonate groups (which can bind to DEAE (diethylaminoethyl) via charge interaction) or biotin (which can specifically bind to avidin or streptavidin). These compounds are then efficiently and specifically bound to functionalized magnetic beads (magnetic microspheres with chemically modified surfaces containing specific functional groups or affinity ligands, enabling specific binding to the derivatized substrate sugar products). This efficient and specific binding allows for the separation of reaction products from non-target components such as enzymes, buffer solutions, and metal ions, providing a foundation for subsequent in-situ purification and detection on the chip (mass spectrometry, liquid chromatography-mass spectrometry, or capillary electrophoresis). Under mild conditions, the derivatizing reagent undergoes a covalent reaction with the aldehyde and / or 1-hydroxyl group of the reducing end monosaccharide of the substrate sugar, yielding sulfonic acid or biotin-labeled derivatives. The sulfonate group imparts a strong negative charge to the originally uncharged or weakly charged substrate sugar molecule. Because DEAE magnetic beads have diethylaminoethyl groups bonded to their surface, they are positively charged under working pH conditions. Sulfonate-modified substrate sugars and their corresponding 2,6-sialylase modules (including CMP-sialic acid synthase, pyrophosphatase, 2,6-sialyl transferase, or their mutants) can bind to DEAE magnetic beads efficiently and specifically through electrostatic interactions. Biotin has a very strong affinity for avidin or streptavidin, therefore biotinylated substrate sugars and their corresponding 2,6-sialylase module reaction products can bind to avidin or streptavidin-modified magnetic beads efficiently and specifically.

[0187] ① Derivatization (using 7-amino-1,3-naphthalenedisulfonate, LSTa as an example): The sugar (LSTa, sialylated lacto-N-tetrasaccharide a) was reductively amination in the presence of 7-amino-1,3-naphthalenedisulfonate and sodium cyanoborohydride (NaCNBH3). The substrate molar ratio was 7-amino-1,3-naphthalenedisulfonate: sugar: NaCNBH3 = 10: 1: 4; the reaction solvent was 15% acetic acid (v / v). The reaction was carried out at 37 °C and 200 rpm in a shaker for 15 h. This completed the derivatization of the substrate sugar.

[0188]

[0189] ② Purification: After the reaction, the crude product was separated and purified by gel filtration chromatography. Bio-Gel P-2 Gel molecular sieve (100–1800 mesh) was used as the stationary phase. Isocratic elution was performed at a flow rate of 1 mL / min, and the eluent was collected stepwise. First, the molecular sieve column was rinsed with 200 mL of pure water. Then, the reaction product of the sugar and tag reaction was added, and isocratic elution was performed with 250 mL of water. The eluent was collected using a collector. The components of the eluent were detected by TLC. The developing solvent was ethyl acetate: methanol: water: acetic acid = 4:6:1:0.2 (v / v / v / v). Color development was performed after development. The fraction containing the tag-sugar was collected, lyophilized, and stored. This yielded the purified derivatized product, tag-LSTa. Mass spectrometry analysis of the derivatized product tag-LSTa was performed to predict... m / z ([M-2H / 2]) - 641.67, actual m / z ([M-2H / 2]) - )641.67072 (e.g.) Figure 4 (as shown in (a)).

[0190] (2) Digital microfluidic chip allocation Different types of droplets are dispensed on a digital microfluidic chip, including: α2,6-sialylase module (including CMP-sialic acid synthase, α2,6-sialyl transferase or their mutants) sample droplets, derivatized substrate sugar droplets, buffer droplets, functionalized magnetic bead (DEAE or (streptomycin) avidin magnetic beads) droplets, elution buffer droplets, and ultrapure water.

[0191] (3) Enzyme-catalyzed reaction Using electrode-driven induction, the sample droplets from the α2,6-sialyltransferase module, the derivatized substrate sugar droplets, and the buffer droplets were precisely combined and vortexed to mix thoroughly. The reaction system contained 100 mM Tris buffer (pH 7.5), magnesium chloride (30 mM), 3.0 mM CTP (cytidine triphosphate), 3.0 mM sialic acid (Neu5Ac), and 3 mM LSTa. CMP-sialic acid synthase or mutant (35 μM) or (0.9 mg / ml) and α2,6-sialyltransferase or mutant (15 μM) or (0.85 mg / ml) were added to the system. The reaction was incubated in the chip reaction area for 30 min to complete the α2,6-sialyltransferase reaction, and the results were detected by mass spectrometry.

[0192] (4) In-situ magnetic bead purification ① After the reaction is completed, the droplets are fused with droplets containing functionalized magnetic beads under the drive of the electrode and fully oscillated and incubated for 5 min to allow the derived substrate sugar and reaction products to bind to the DEAE magnetic beads (specifically). ②Using the magnet integrated in the chip magnetic bead purification area, the DEAE magnetic beads bound to the product are adsorbed and fixed, and the unadsorbed reaction droplets (containing impurities) are removed and discarded by electrode driving. ③ Drive the water washing liquid droplets to move to the magnetic bead purification zone, contact the fixed DEAE magnetic beads, clean them, use a magnet to continue fixing the magnetic beads, and remove the water washing liquid droplets; repeat this water washing operation three times to completely remove the impurities adsorbed on the surface of the magnetic beads; ④ Drive the eluent droplets (ammonium bicarbonate, high-concentration sodium chloride or sodium hydroxide, or other salt solutions) to move to the magnetic bead purification zone, contact them with the fixed DEAE magnetic beads, and desorb the reaction products bound to the DEAE magnetic beads to obtain the purified product sample.

[0193] (5) Product testing The purified product sample after elution is sent to the detection unit through the chip sample export terminal for detection such as capillary electrophoresis or mass spectrometry.

[0194] (6) Result determination and recovery verification Based on the detection signal, the screening results are judged, the corresponding positive sample droplets are recovered, and a second screening is performed for verification in order to obtain the desired high-efficiency α2,6-sialylase module.

[0195] Example 7: Synthesis of DSLNT DSLNT was synthesized using a screened α2,6-sialyltransferase module. The reaction system contained 100 mM Tris buffer (pH 7.0-9.0), magnesium chloride (20-100 mM), 1.0-200.0 mM CTP (cytidine triphosphate), 1.0-200.0 mM sialic acid (Neu5Ac), and 1.0-200.0 mM LSTa. CMP-sialic acid synthase or mutant (5-80 μM) and α2,6-sialyltransferase or mutant (5-40 μM) were added to the system. The reaction was carried out at 37°C in a shaker for 12 hours. Yield >90%, and the reaction product was analyzed by mass spectrometry. The DSLNT mass spectrometry analysis results are shown in the tag below. Figure 4 As shown in (b), the DSLNT mass spectrometry analysis results are as follows: Figure 5 As shown.

[0196] Example 8: Screening process of GM2 glycan synthesis-related enzymes (1) Derivatization of the substrate lactose Except for replacing LSTA in the above process with lactose (Lac) to synthesize tag-Lac, the remaining processing steps are the same as in Example 6 (1). The derivatized and purified product tag-Lac was obtained. Mass spectrometry analysis was performed on the derivatized tag-Lac to predict... m / z ([M-1H])- 628.101, actual m / z ([M-1H]) - )628.10225 (e.g.) Figure 6 (As shown).

[0197] (2) Digital microfluidic chip allocation Except that the α2,6-sialyltransferase module (including N-acetaminohexokinase, N-acetaminoglucosyltransferase, β1,4-N-acetaminogalactosyltransferase or mutants thereof) in the above process is replaced with a β1,4-N-acetaminogalactosyltransferase module (including CMP-sialic acid synthase, α2,6-sialic acid transferase or mutants thereof) in the above process, the rest of the process is the same as (2) in Example 6.

[0198] (3) Enzyme-catalyzed reaction Using electrode-driven induction, sample droplets from the β1,4-N-acetaminogalactosyltransferase module, derivatized substrate sugar droplets, and buffer droplets were precisely combined and thoroughly mixed by vortexing. The reaction system contained 100 mM Tris buffer (pH 7.5), magnesium chloride (30 mM), 3.0 mM ATP (adenosine triphosphate), 3.0 mM UTP (uridine triphosphate), 3.0 mM N-acetaminogalactosamine, and 3 mM tag-Lac. N-acetaminohexokinase or mutant (10 μM or 0.41 mg / ml), N-acetaminoglucosamine uridine acyltransferase or mutant (10 μM or 0.5 mg / ml), and β1,4-N-acetaminogalactosyltransferase or mutant (20 μM or 0.72 mg / ml) were added to the system. The reaction was incubated in the chip reaction area for 30 minutes. The β1,4-N-acetaminogalactosylation reaction was completed within min, and the result was detected by mass spectrometry.

[0199] (4) In-situ magnetic bead purification The process is exactly the same as (4) in Example 6.

[0200] (5) Product testing The process is exactly the same as (5) in Example 6.

[0201] (6) Result determination and recovery verification Based on the detection signal, the screening results are judged, the corresponding positive sample droplets are recovered, and a second screening is performed for verification in order to obtain the desired high-efficiency β1,4-N-acetylgalactosylase module.

[0202] Example 9: Synthesis of GM2 sugar chains GM2 glycans were synthesized using a screened β1,4-N-acetylgalactosyltransferase module. The reaction system contained 100 mM Tris buffer (pH 7.0-9.0), magnesium chloride (20-100 mM), 1.0-200.0 mM ATP (adenosine triphosphate), 1.0-200.0 mM UTP (uridine triphosphate), 1.0-200.0 mM 3'-sialyllactose, and 1.0-200.0 mM N-acetylgalactosyltransferase. N-acetylaminohexokinase or its mutant (5-50 μM), N-acetylglucosamine uridine acyltransferase (5-40 μM), and β1,4-N-acetylgalactosyltransferase (5-60 μM) were added to the system. The reaction was carried out at 37°C in a shaker for 12 hours. The yield was >90%, and the reaction products were analyzed by mass spectrometry.

[0203] The structure of the GM2 sugar chain is as follows: Figure 7 As shown, the tag-GM2 mass spectrometry analysis results are as follows: Figure 8 As shown, the mass spectrometry analysis results of GM2 glycans are as follows: Figure 9 As shown.

[0204] Example 10: Screening process for enzymes related to GD3 glycan synthesis (1) Digital microfluidic chip allocation Different types of droplets are dispensed on a digital microfluidic chip, including: α2,8-sialylase module (including CMP-sialic acid synthase, α2,8-sialyl transferase or their mutants) sample droplets, derivatized substrate sugar droplets, buffer droplets, functionalized magnetic bead (DEAE or (streptomycin) avidin magnetic bead) droplets, elution buffer droplets, and ultrapure water.

[0205] (2) Enzyme-catalyzed reaction Using electrode-driven induction, the sample droplets from the α2,8-sialyltransferase module, the derivatized substrate sugar droplets, and the buffer droplets were precisely combined and vortexed to mix thoroughly. The reaction system contained 100 mM Tris buffer (pH 7.5), magnesium chloride (30 mM), 3.0 mM CTP (cytidine triphosphate), 3.0 mM sialic acid (Neu5Ac), and 3 mM tag-Lac. CMP-sialic acid synthase or mutant (35 μM) or (0.9 mg / ml) and α2,8-sialyltransferase or mutant (15 μM) or (0.38 mg / ml) were added to the system. The reaction was incubated in the chip reaction area for 30 min to complete the α2,8-sialyltransferase reaction, and the results were detected by mass spectrometry.

[0206] (3) In-situ magnetic bead purification The process is exactly the same as (4) in Example 6.

[0207] (4) Product testing The process is exactly the same as (5) in Example 6.

[0208] (5) Result determination and recovery verification Based on the detection signal, the screening results are judged, the corresponding positive sample droplets are recovered, and a second screening is performed for verification in order to obtain the desired high-efficiency α2,8-sialylase module.

[0209] Example 11: Synthesis of GD3 Glycan Chains GD3 glycans were synthesized using a screened α2,8-sialyltransferase module. The reaction system contained 100 mM Tris buffer (pH 7.0-9.0), magnesium chloride (20-100 mM), 1.0-200.0 mM CTP (cytidine triphosphate), 1.0-200.0 mM sialic acid (Neu5Ac), and 1.0-200.0 mM 3'-sialyl lactose. CMP-sialic acid synthase or mutant (5-80 μM) and α2,8-sialyltransferase or mutant (5-80 μM) were added to the system. The reaction was carried out at 37°C in a shaker for 12 hours. The yield was >90%, and the reaction products were analyzed by mass spectrometry.

[0210] The structure of the GD3 sugar chain is as follows: Figure 10 As shown, the tag-GD3 mass spectrometry analysis results are as follows: Figure 11 As shown, the mass spectrometry analysis results of GD3 glycans are as follows: Figure 12 As shown.

[0211] Example 12: Screening process for enzymes related to the synthesis of oligosialic acid lactose (OSAL) (1) Digital microfluidic chip allocation Different types of droplets are dispensed on a digital microfluidic chip, including: α2,8-sialylase module (including CMP-sialic acid synthase, α2,8-sialyl transferase or their mutants) sample droplets, derivatized substrate sugar droplets, buffer droplets, functionalized magnetic bead (DEAE or (streptomycin) avidin magnetic bead) droplets, elution buffer droplets, and ultrapure water.

[0212] (2) Enzyme-catalyzed reaction Using electrode-driven induction, the sample droplets from the α2,8-polysialidase module, the derivatized substrate sugar droplets, and the buffer droplets were precisely combined and vortexed to mix thoroughly. The reaction system contained 100 mM Tris buffer (pH 7.5), magnesium chloride (30 mM), 30.0 mM CTP (cytidine triphosphate), 30.0 mM sialic acid (Neu5Ac), and 3 mM tag-Lac. CMP-sialic acid synthase or mutant (35 μM) or (0.9 mg / ml) and α2,8-polysialidase or mutant (15 μM) or (1.0 mg / ml) were added to the system. The reaction was incubated in the chip reaction area for 30 min to complete the α2,8-polysialidization reaction, and the results were detected by mass spectrometry.

[0213] (3) In-situ magnetic bead purification The process is exactly the same as (4) in Example 6.

[0214] (4) Product testing The process is exactly the same as (5) in Example 6.

[0215] (5) Result determination and recovery verification Based on the detection signal, the screening results are judged, the corresponding positive sample droplets are recovered, and a second screening is performed for verification in order to obtain the desired high-efficiency α2,8-polysialidase module.

[0216] Example 13: Synthesis of Oligosialyllactose (OSAL) Oligosialyllactose was synthesized using a screened α2,8-polysialylase module. The reaction system contained 100 mM Tris buffer (pH 7.0-9.0), magnesium chloride (20-100 mM), 5-2000 mM CTP (cytidine triphosphate), 5-2000 mM sialic acid (Neu5Ac), and 1.0-200.0 mM GD3 glycans. CMP-sialic acid synthase or a mutant (5-80 μM) and α2,8-sialyltransferase or a mutant (5-30 μM) were added to the system. The reaction was carried out at 37°C in a shaker for 12 hours. The conversion rate was >90%, and the reaction product was analyzed by mass spectrometry.

[0217] The structure of OSAL sugar chains is as follows: Figure 13 As shown, the tag-OSAL mass spectrometry analysis results are as follows: Figure 14 As shown, the mass spectrometry analysis results of oligosialyl lactose (OSAL) are as follows: Figure 15As shown. The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A high-throughput screening method for glycosylation enzyme modules based on a digital microfluidic platform, characterized in that, The method includes the following steps: (1) Using sugar as a substrate, the tagged derivative substrate sugar is obtained by reductive amination reaction between the primary amino group of the derivatizing reagent and the aldehyde group of the sugar, or by nucleophilic substitution reaction between the N-methylhydroxylamine moiety of the derivatizing reagent and the reducing end of the sugar. (2) Distributing different types of droplets on a digital microfluidic chip, the droplets including glycosylation enzyme module sample droplets, tagged derivatized substrate sugar droplets, buffer droplets, functionalized magnetic bead droplets, elution buffer droplets and ultrapure water droplets; (3) Drive the glycosylation enzyme module sample droplet, the derivatized substrate sugar droplet and the buffer droplet to fuse and carry out an enzymatic reaction to obtain the reaction droplet; (4) Drive the reaction droplets to fuse with the functionalized magnetic bead droplets to react, remove impurities, wash and elute, and obtain a purified sample; as well as (5) The purified sample is tested and the results are judged to screen for a high-efficiency glycosylation enzyme module; The digital microfluidic chip drives discrete droplets to flow on its surface via array electrodes.

2. The method according to claim 1, characterized in that, The digital microfluidic platform includes a digital microfluidic chip, a detection module, and a software control module; Preferably, the digital microfluidic chip includes: (a) a reagent storage unit for pre-storing reaction reagents; (b) a reaction unit for fusing sample droplets with reaction reagents; (c) a magnetic bead purification unit for binding the target product in the sample droplets with magnetic beads and eluting the target product from the magnetic beads to obtain a purified product; and (d) a waste liquid recovery unit.

3. The method according to claim 2, characterized in that, The digital microfluidic chip uses glass, ITO, or a hydrophobic dielectric layer as a substrate. Preferably, a hydrophobic functional layer is prepared on the surface of the digital microfluidic chip; Preferably, the digital microfluidic chip includes an upper electrode plate and a lower electrode plate; More preferably, the upper electrode plate is provided with a sampling through hole by ITO glass drilling process; Preferably, when the droplet volume is about 5 μL and the gap between the upper and lower electrode chambers is about 1.25 mm, the thickness of the glass is 0.3 mm and the diameter of the sampling through hole is about 2 mm.

4. The method according to claim 1, characterized in that, The droplet manipulation of the digital microfluidic platform is performed in an air phase medium environment or an oil phase medium environment, for example, in an oil phase medium environment; Preferably, the oil phase medium is silicone oil; More preferably, the glycosylation enzyme module sample droplet contains a surfactant; Preferably, the surfactant includes L64, F68, Pluronic F127 and / or T90R4, such as Pluronic F127.

5. The method according to claim 1, characterized in that, In step (1), the sugar is sialyllactose-N-tetrasaccharide a; Preferably, in step (1), the derivatizing agent comprises a sulfonate-containing compound or biotin, such as 7-amino-1,3-naphthalene disulfonate or 7-(2-(2-(N-methylaminooxy)ethoxy)ethoxy)naphthalene-1,3-disulfonic acid; Preferably, in step (1), the labeling includes sulfonate modification or biotin modification; Preferably, when the derivatizing reagent is a compound containing sulfonate, the functionalized magnetic bead is a DEAE magnetic bead; Preferably, when the derivatizing agent is biotin, the functionalized magnetic beads are magnetic beads modified with avidin or streptavidin; Preferably, in step (1), the reductive amination reaction further includes a reducing agent; More preferably, the reducing agent is sodium cyanoborohydride; More preferably, in step (1), the molar ratio of the derivatizing reagent, substrate and reducing agent in the reductive amination reaction is (8~12):(0.5~2):(3~5); Preferably, the molar ratio of the derivatizing reagent, substrate, and reducing agent in the reductive amination reaction is approximately 10: approximately 1: approximately 4. Preferably, in step (1), the reaction conditions for the reductive amination reaction include: the reaction is carried out in a shaker, the reaction solvent is about 15% acetic acid solution, the reaction temperature is about 37°C, the shaker speed is about 200 r / min, and the reaction time is about 15 h; Preferably, step (1) further includes a gel filtration chromatography separation and purification step after the reductive amination reaction; More preferably, the gel filtration chromatography separation and purification method includes: using Bio-Gel P-2 Gel molecular sieve as the stationary phase, rinsing the molecular sieve column with pure water, adding the reducing amination reaction product, eluting isocratically with eluent at a flow rate of 0.5~2 ml / min, and collecting the eluent fraction; detecting the eluent fraction using thin-layer chromatography with ethyl acetate-methanol-water-acetic acid as the developing solvent, and collecting the fraction containing the labeled derivatized substrate sugar after development and color development; Particularly preferred is that the gel particles of the Bio-Gel P-2 Gel molecular sieve have a particle size of 100~1800 mesh; Particularly preferably, the volume of the pure water is 100-300 ml, for example, about 200 ml; Particularly preferably, the volume of the eluent is 150-350 ml, for example, about 250 ml; Particularly preferably, the flow rate is about 1.0 ml / min; Particularly preferably, the volume ratio of the ethyl acetate, methanol, water and acetic acid in the developing solvent is about 4: about 6: about 1: about 0.

2.

6. The method according to claim 1, characterized in that, In step (2), the glycosylation enzyme module is an α2,6-sialylase module; Preferably, the α2,6-sialylase module contains CMP-sialic acid synthase and / or its mutants, α2,6-sialyl transferase and / or its mutants; More preferably, the concentration of the CMP-sialic acid synthase and / or its mutant is 30-40 μM or 0.8-1.0 mg / ml; More preferably, the concentration of the α2,6-sialyltransferase and / or its mutant is 10-20 μM or 0.75-0.95 mg / ml; More preferably, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes; Alternatively, in step (2), the glycosylation enzyme module is a β1,4-N-acetylgalactosylase module; Preferably, the β1,4-N-acetaminogalactosyltransferase module contains N-acetaminohexokinase and / or its mutant, N-acetaminoglucosyltransferase and / or its mutant, and β1,4-N-acetaminogalactosyltransferase and / or its mutant. More preferably, the concentration of the N-acetylglucuronidase and / or its mutant is 5-20 μM or 0.4-0.6 mg / ml; More preferably, the concentration of the N-acetaminohexokinase and / or its mutant is 5-20 μM or 0.3-0.5 mg / ml; More preferably, the concentration of the β1,4-N-acetylgalactosyltransferase and / or its mutant is 10-30 μM or 0.6-0.8 mg / ml; More preferably, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes; Alternatively, in step (2), the glycosylation enzyme module is an α2,8-sialylase module; Preferably, the α2,8-sialylase module contains CMP-sialic acid synthase and / or its mutant, α2,8-sialyl transferase and / or its mutant; More preferably, the concentration of the CMP-sialic acid synthase and / or its mutant is 30-40 μM or 0.8-1.0 mg / ml; More preferably, the concentration of the α2,8-sialyltransferase and / or its mutant is 10-20 μM or 0.3-0.5 mg / ml; More preferably, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes; Alternatively, in step (2), the glycosylation enzyme module is an α2,8-polysialidase module; Preferably, the α2,8-polysialylase module contains CMP-sialic acid synthase and / or its mutant, α2,8-polysialyltransferase and / or its mutant; More preferably, the concentration of the CMP-sialic acid synthase and / or its mutant is 30-40 μM or 0.8-1.0 mg / ml; More preferably, the concentration of the α2,8-polysialyltransferase and / or its mutant is 10-20 μM or 0.9-1.1 mg / ml; More preferably, in step (3), the time for the enzymatic reaction is 20 to 40 minutes, for example, about 30 minutes.

7. The method according to claim 1, characterized in that, In step (4), the reaction includes the following steps: driving the post-reaction droplet to fuse with the functionalized magnetic bead droplet, oscillating and incubating for 2 to 10 minutes, for example, about 5 minutes, to adsorb and fix the functionalized magnetic beads that can specifically bind to the target product; Preferably, in step (4), the impurity removal is a process of driving the removal of unadsorbed and fixed droplets; Preferably, in step (4), the washing process involves driving ultrapure water droplets to remove impurities adsorbed on the surface of the magnetic beads; More preferably, the washing process is performed 2 to 4 times, for example, 3 times; More preferably, in step (4), the elution is performed by driving the eluent droplets or by heating; Preferably, the eluent is a salt solution, such as ammonium bicarbonate solution, sodium chloride solution and / or sodium hydroxide solution.

8. The method according to claim 1, characterized in that, In step (5), the detection includes capillary electrophoresis detection and / or mass spectrometry detection.

9. Use of the method according to any one of claims 1 to 8 in a screening enzyme module; Preferably, the enzyme is an enzyme that synthesizes functional sugar chains; Preferably, the enzyme is selected from one or more of the following: α2,6-sialylase, β1,4-N-acetylgalactosylase, α2,8-sialylase, and α2,8-polysialylase.

10. Use of the enzyme module obtained by the method of any one of claims 1 to 8 in the synthesis of functional sugar chains; Preferably, the enzyme is an enzyme that synthesizes functional sugar chains; Preferably, the enzyme is selected from one or more of the following: α2,6-sialidase, β1,4-N-acetylgalactosylase, α2,8-sialidase and α2,8-polysialidase; Preferably, the functional sugar chain is selected from one or more of the following: disialyllactose-N-tetrasaccharide, ganglioside GM2 sugar chain, ganglioside GD3 sugar chain, and oligosialyllactose (OSAL).

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