Digital micro-fluidic chip, preparation method and sugar sequencing method

Abstract

The digital micro-fluidic chip comprises a plurality of control units, each control unit comprises an upper polar plate and a lower polar plate which are oppositely arranged, hydrophobic layers are arranged on the upper and lower surfaces of the upper polar plate and the lower polar plate, an electrode control structure is arranged on the upper polar plate, and an electrode driving structure, a dielectric layer and a hydrophobic layer are arranged on the lower polar plate; an electrode control structure layer and an electrochemical sensing layer are sequentially arranged on the upper polar plate in the direction towards the lower polar plate, the electrochemical sensing electrode is arranged on the electrochemical sensing layer, the electrode control structure is arranged on the electrode control structure layer, and at least one electrochemical sensor is communicated with the electrode control structure. The invention provides a special digital micro-fluidic chip, an electrochemical sensor is integrated on the digital micro-fluidic chip, a matrix of the electrochemical sensor can be formed, automatic treatment of a sample is realized through a digital micro-fluidic technology, and in-situ rapid high-sensitivity electrochemical detection of sample liquid drops is realized through an electrochemical electrode array.

Description

Technical Field

[0001] The present invention relates to the field of chips, and in particular to a digital microfluidic chip, a preparation method and a sugar sequencing method. Background Art

[0002] The application of micro total analysis system (mTAS) in the field of chemistry and biology has aroused great interest. These integrated, miniaturized and automated analysis systems are also called lab-on-a-chip (LOC). Microfluidics technology, as one of its important branches, has received widespread attention and a large amount of research. The main advantages of this technology are to improve the automation of traditional biochemical experimental processes, reduce the consumption of samples and reagents, and improve the operability of samples. Digital microfluidics technology based on the principle of droplet manipulation based on dielectric wetting (EWOD) can achieve precise manipulation of discrete droplets with a volume of microliters to nanoliters through a series of electrode arrays protected by insulating layers and hydrophobic layers. It has the advantages of traditional microfluidic chips such as low reagent consumption, small overall size and easy integration, while not requiring micropumps, microvalves and other structures or power sources, and the chip has a higher degree of generalization and automation. However, in practical applications, the most important step after the sample undergoes a series of reactions and treatments is to quickly and accurately detect the product, which is difficult to achieve with conventional digital microfluidic chips. The most commonly used detection method for digital microfluidics is optical method, such as visual observation, fluorescence, chemiluminescence, etc. At present, there are related studies combining electrochemical sensing with digital microfluidics, but these studies are all focused on the combination of a single electrochemical sensor and digital microfluidics technology. However, complex applications involve a large number and variety of droplets, and different applications have different electrochemical detection requirements. Different droplets in the same application may also need to face their own specific electrochemical detection requirements. These unique requirements require electrochemical sensors to have their own specific size, shape, structure and modification. A single electrochemical electrode cannot meet these requirements. However, optical detection methods often rely on the luminescence or absorption behavior of the reagent itself, and the system for accurately detecting and collecting optical signals is usually large, which is not conducive to integration and miniaturization.

[0003] In summary, the prior art lacks an electrochemical electrode structure based on a transistor switch matrix, which can meet multiple electrochemical detection requirements and improve the efficiency of electrochemical sensing by specifically designing each electrochemical electrode in the electrochemical electrode array. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide an electrochemical electrode control structure based on a transistor switch matrix, which satisfies multiple electrochemical detection requirements at the same time by specifically designing each electrochemical electrode in the electrochemical electrode array.

[0005] Based on the above technical problems, the present invention provides a digital microfluidic chip, including several control units, the control unit including an upper electrode plate and a lower electrode plate which are arranged relatively to each other and have hydrophobic layers on the upper and lower surfaces, the upper electrode plate is provided with an electrode control structure, a dielectric layer and a hydrophobic layer, the lower electrode plate is provided with an electrode driving structure, the upper electrode plate is provided with an electrode control structure layer and an electrochemical sensing layer in sequence in the direction toward the lower electrode plate, the electrochemical sensing electrode is provided in the electrochemical sensing layer, the electrode control structure is provided in the electrode control structure layer, and at least one electrochemical sensor is connected to the electrode control structure.

[0006] The present invention also provides a method for preparing a digital microfluidic chip, and the method for preparing the digital microfluidic chip as described above comprises the following steps:

[0007] (1) After the electrode control structure on the upper electrode plate is prepared, a first dielectric layer is prepared on the electrode control structure, and a connection point between the electrode control structure and the electrochemical sensor is exposed on the first dielectric layer;

[0008] (2) forming the electrochemical sensor on the first dielectric layer;

[0009] (3) forming a second dielectric layer on the electrochemical sensor and exposing three electrodes of the electrochemical sensor;

[0010] (4) preparing a hydrophobic layer and exposing the three electrodes of the chemical sensor;

[0011] (5) Modification of the working electrode surface.

[0012] Preferably, the method for preparing a digital microfluidic chip with integrated electrochemical sensor is characterized in that: in the step (1),

[0013] The first dielectric layer is prepared by a thin film deposition process.

[0014] The connection points between the electrode control structure and the electrochemical sensor are exposed by photoresist covering and stripping.

[0015] Preferably, the step (2) comprises the following steps:

[0016] Prepare the electrode photoresist pattern by photolithography, expose the position where the electrode needs to be prepared according to the pattern, and cover the other positions with photoresist;

[0017] depositing a metal layer;

[0018] The photoresist is removed from the glass using a stripping process to form the three electrodes of the electrochemical sensor.

[0019] Preferably, the step (2) comprises the following steps:

[0020] A ground electrode of the electrode driving structure is formed while the electrochemical sensor is formed on the first dielectric layer.

[0021] Preferably, in step (2), the following steps are included:

[0022] After creating the electrochemical sensor and the ground electrode, metal is deposited over the electrodes.

[0023] Preferably, the method further comprises the following steps:

[0024] (6) Prepare the electrode driving structure on the lower electrode plate.

[0025] The present invention also provides a sugar sequencing method for a digital microfluidic chip, wherein a digital microfluidic chip prepared by any of the above methods is used to perform sugar sequencing. Preferably, the sugar sequencing method may include the following steps:

[0026] 1) using the electrode driving structure to drive the oligosaccharide droplet to mix with the first glycosidase droplet to perform enzymatic cleavage of the oligosaccharide; wherein the enzymatic cleavage can be performed by mixing the oligosaccharide droplet with magnetic beads and then mixing with the first glycosidase for enzymatic cleavage;

[0027] 2) Using a magnet to separate the oligosaccharide chains and monosaccharide products after enzyme cleavage into droplets;

[0028] 3) using the electrode driving structure to move the droplets of the enzyme cleavage product to the electrochemical sensor for concentration detection;

[0029] 4) Repeating the above steps 1) to 3), sequentially using the electrode driving structure to drive the oligosaccharide droplet to mix with the second glycosidase droplet, the third glycosidase droplet, etc. until the oligosaccharide chain is completely hydrolyzed.

[0030] Preferably, the step 1) comprises the following steps:

[0031] mixing the oligosaccharide solution with magnetic beads to form oligosaccharide droplets;

[0032] The oligosaccharide droplet is mixed with the first glycosidase droplet to complete the enzymatic cleavage of the oligosaccharide.

[0033] The beneficial effects of the present invention are:

[0034] (1) The present invention provides a special digital microfluidic chip, and integrates an electrochemical sensor into the digital microfluidic chip to form a matrix of electrochemical sensors, realizes automated sample processing through digital microfluidic technology, and realizes in-situ rapid and highly sensitive electrochemical detection of sample droplets through an electrochemical electrode array. Each individual electrochemical electrode in the electrochemical electrode array can be specifically modified to realize the detection of specific samples, and has a wide range of applicability.

[0035] (2) The present invention provides a large-scale electrochemical sensor matrix based on a transistor switch matrix. The transistor switch matrix can be a thin film transistor (TFT), a field effect transistor (FET), a triode or other switching transistors, which can realize large-scale complex droplet manipulation and concentration detection, and can gradually decompose large-scale complex liquid experimental operations to form a simple and easy-to-operate automatic operation system.

[0036] (3) Each electrochemical sensor structure of the present invention is independently controlled, which is very efficient and easy to maintain. The electrochemical sensing electrode array based on the transistor switch matrix designed by the present invention connects the reference electrode (RE) of each column of electrochemical electrodes in series, and can realize the control of the signals of n×n reference electrodes through n channels. The counter electrode (CE) in the electrochemical electrode adopts the same control scheme as the reference electrode. The switch of each electrochemical working electrode in the array is a transistor switch. Taking the TFT thin film transistor as an example, when a positive voltage is applied to the gate, an upward electric field is generated. The electrons in the active layer move toward the insulating layer under the action of the electric field, and an electron accumulation layer is formed at the interface between the active layer and the insulating layer (front channel). At this time, a positive voltage is applied between the source and drain electrodes, and the electrons in the electron accumulation layer move toward the drain electrode to form a conduction current. Therefore, only when the gate and the drain electrode are simultaneously applied with an effective voltage, the source and the drain electrode will form a conduction current. By connecting the source electrode of the TFT to the working electrode in the electrochemical electrode, the current on and off of the electrochemical working electrode can be controlled by the TFT. The gates of each row of TFTs in the TFT array are connected in series, and the drains of each column of TFTs are connected in series, so that m×n TFT switches can be controlled by m+n electrical signals, thereby controlling m×n electrochemical sensors. Each individual electrochemical electrode in the electrochemical sensor array can be controlled and used individually.

[0037] (4) Based on the present invention, the sensitivity of electrochemical sensing can be further increased through specific modifications. In order to enhance the electrochemical signal, the surface of the working electrode of the present invention can be modified with nanoparticles through electrochemical deposition, slurry printing, thermal evaporation and other processes to enhance the electrochemical signal. In addition, the working electrode of each electrochemical electrode can be specially modified to achieve signal acquisition of specific samples. For example, the working electrode is modified with glucose oxidase to detect the glucose concentration in the droplet. The working electrode is modified with a specific antibody marker to detect specific antigens in the droplet. Certain electrochemical electrodes are designed as interdigitated electrodes, which can be used in applications such as impedance detection.

[0038] (5) The present invention also provides a specific method for preparing a digital droplet microfluidic chip, through which the digital droplet microfluidic chip of the present invention can be quickly formed, thereby realizing the rapid preparation of the structure of the present invention.

[0039] (6) The present invention also constructs an automated sugar sequencing system through the digital microfluidic chip of this patent, and realizes full automation of sugar sequencing through the digital microfluidic chip, thereby completely improving the efficiency of sugar sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the cross-sectional structure of a digital microfluidic chip control unit according to Embodiment 1 of the present invention.

[0041] Figure 2 It is a connection diagram of the electrochemical detection electrode array based on the transistor switch array of the present invention in the first embodiment.

[0042] Figure 3 yes Figure 2 A magnified schematic diagram of part A in the figure.

[0043] Figure 4 This is one of the flow charts for preparing the upper electrode plate of the digital microfluidic chip according to the second embodiment of the present invention.

[0044] Figure 5 This is the second flow chart of preparing the upper electrode plate of the digital microfluidic chip according to the second embodiment of the present invention.

[0045] Figure 6 This is the third flow chart of preparing the upper electrode plate of the digital microfluidic chip of the second embodiment of the present invention.

[0046] Figure 7 This is the fourth flow chart of preparing the upper electrode plate of the digital microfluidic chip of the second embodiment of the present invention.

[0047] Figure 8 This is the fifth flow chart of preparing the upper electrode plate of the digital microfluidic chip according to the second embodiment of the present invention.

[0048] Fig. 9It is a flowchart of the third embodiment of the present invention.

[0049] Fig.10 It is a schematic diagram of process simulation of the third embodiment of the present invention. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0051] Embodiment 1:

[0052] The present invention provides an embodiment 1. Specifically, a digital microfluidic chip is provided in this embodiment, including several control units, and the control unit includes at least one electrode control structure. In this embodiment, this electrode control structure is particularly a transistor, and in particular can be set as a TFT structure. An electrochemical sensing electrode is connected in each TFT transistor. Such an electrode control structure, i.e., a TFT transistor switch, can control an electrochemical sensing electrode, and an array arrangement of electrochemical sensing electrodes can be realized to realize electrochemical sensing of droplets, and each electrochemical sensing electrode can be controlled separately. The core component of electrochemical detection is the electrochemical sensing electrode. Integrating the electrochemical sensing electrode on the digital microfluidic chip is the premise and important basis for the chip to have electrochemical detection. Electrochemical sensing electrode detection often uses a three-electrode system, which includes a working electrode, a counter electrode, and a reference electrode. In the three-electrode system, the current between the working electrode and the auxiliary electrode is regulated by an external power supply. The relative electrode potential of the working electrode is tested by testing the voltage between the working electrode and the reference electrode, thereby obtaining a functional relationship curve between the current and the potential.

[0053] Specifically, in this embodiment, the specific structure of the control unit may include an upper plate and a lower plate that are relatively arranged, such as Figure 1 As shown, the electrode control structure is a TFT structure. The electrode control structure here mainly controls electrochemical sensing. In this embodiment, a TFT structure can be used. In other embodiments, other types of transistors can also be used to achieve the same technical effect. It is arranged on the upper plate, and a droplet electrode driving structure 13 is arranged on the lower plate. A hydrophobic layer 11 is arranged on the lower surface of the upper plate and the upper surface of the lower plate.

[0054] An upper plate substrate 1 is provided on the upper plate, a lower plate substrate 14 is provided on the lower plate, an electrode control structure layer is provided in sequence from the upper plate 1 toward the lower plate, the electrode control structure layer includes a TFT gate 2 and a TFT drain 4, a TFT active layer 3 in this embodiment, an electrochemical sensing layer is provided with an electrochemical reference electrode 5, an electrochemical working electrode (WE) 8, an electrochemical counter electrode (CE) 9, and a ground electrode layer 6, the electrochemical sensing electrode is provided in the electrochemical sensing layer, the electrode control structure is provided in the electrode control structure layer, and is separated from the electrochemical sensing layer by a dielectric layer 10, the electrode control structure is connected to one end of the working electrode 8 of the electrochemical sensing electrode, and the other end of the working electrode 8 is connected to the ground electrode layer 6 and the lower surface of the upper plate. A hydrophobic layer 11 is provided on the lower surface of the upper plate except for the working electrode, and the ground electrode layer 6 and the hydrophobic layer 11 are also separated by a dielectric layer.

[0055] The working electrode has a working electrode surface modifier 12 on its lower surface, and the working electrode surface modifier 12 can expand the detection range of electrochemical sensing. The electrode control structure is set as a transistor switch, and the digital microfluidic chip with an integrated electrochemical electrode array is composed of upper and lower plates, wherein an electrochemical electrode array based on a transistor switch array is prepared on the upper plate. Taking a TFT thin film transistor switch as an example, the upper plate substrate 1 is made of glass or other materials, and a TFT thin film transistor is prepared on the substrate 1. The structure of the TFT includes a gate 2, an active layer 3, a drain 4, and a source 7. There is a dielectric layer 10 (usually silicon oxide, silicon nitride, etc.) between the gate 2 and the active layer. Each electrochemical electrode consists of a working electrode (WE) 8, a counter electrode ( The upper plate is composed of a ground electrode 6 and a reference electrode (RE) 5. The function of the ground electrode 6 of the upper plate is to form an electric field with the droplet electrode driving structure array of the lower plate for droplet control. The ground electrode 6 is covered with a dielectric layer 10 and a hydrophobic layer 11. The hydrophobic layer 11 is the same as the lower plate substrate 14, but the surface of the electrochemical three electrodes needs to be exposed. The dielectric layer 6 and the hydrophobic layer 10 on the surface of the electrochemical electrode are removed by dry etching or wet etching, so that the droplets can fully contact the electrochemical electrodes to realize the collection of electrochemical sensing signals. The electrode driving structure, namely the droplet driving electrode 13, is arranged on the lower plate.

[0056] like Figure 2 When multiple control units are integrated, the reference electrodes 5 of the electrochemical sensing electrodes of each control unit are connected in series. The working electrode 8 of the electrochemical sensing electrode is connected to the source electrode 7 of the TFT. The gate electrodes 2 of the TFTs in several control units are connected in series. The drain electrodes 4 of the TFTs in several control units are connected in series.

[0057] The electrochemical sensing electrode array based on the transistor switch matrix designed by the present invention connects the reference electrode (RE) 5 of each column of electrochemical electrodes in series, and can realize the control of the signals of n×n reference electrodes through n channels. The counter electrode (CE) 9 in the electrochemical electrode adopts the same control scheme as the reference electrode 5. The switch of each electrochemical working electrode in the array is a transistor switch. Taking a TFT thin film transistor as an example, the structure of the TFT is as follows: Figure 3 As shown, when a positive voltage is applied to the gate 2, an upward electric field is generated. The electrons in the active layer move toward the insulating layer under the action of the electric field, and an electron accumulation layer is formed at the interface between the active layer and the insulating layer (front channel). At this time, a positive voltage is applied between the source and drain electrodes, and the electrons in the electron accumulation layer move toward the drain electrode to form a conduction current. Therefore, only when the gate 2 and the drain 4 are simultaneously applied with an effective voltage, the source 7 and the drain electrode will form a conduction current. Therefore, in this embodiment, the source electrode of the TFT is connected to the working electrode in the electrochemical electrode, and the current on and off of the electrochemical working electrode can be controlled by the TFT. The gates of each row of TFTs in the TFT array are connected in series, and the drains of each column of TFTs are connected in series, so that m×n TFT switches can be controlled by m+n electrical signals, thereby realizing the control of m×n electrochemical sensors. Each individual electrochemical electrode in the electrochemical sensor array can be controlled and used individually, and the active matrix is ​​used to control the electrochemical array. Large-scale arrays are controlled by scanning, and only one electrode is tested at a specific moment, so the pressure on the electrochemical workstation is not great.

[0058] Embodiment 2:

[0059] In this embodiment, a specific preparation method of the digital microfluidic chip of embodiment 1 is provided, in particular, a specific preparation method of a digital microfluidic chip with an integrated electrochemical electrode array. The preparation process of the TFT thin film transistor array of the upper plate is the same as the conventional TFT preparation process, and the present invention focuses on the preparation of the electrochemical sensing electrode array based on TFT. The specific process flow is as follows: Figure 4 As shown:

[0060] like Figure 4-Figure 8 The specific preparation method of the digital microfluidic chip is as follows:

[0061] (1) After the electrode control structure on the upper electrode plate is prepared, a first dielectric layer is prepared on the electrode control structure, and a connection point between the electrode control structure and the electrochemical sensor is exposed on the first dielectric layer;

[0062] Specifically, the following steps are included:

[0063] (i1) After the TFT array of the upper plate is prepared, a dielectric layer such as silicon nitride material is prepared thereon by a thin film deposition process such as plasma enhanced chemical vapor deposition (PECVD).

[0064] (i2) Through the photolithography process, use photoresist 16 to cover other positions and expose the top of the TFT source.

[0065] (i3) The dielectric silicon nitride layer above the source is removed by an etching process such as an ICP dry etching process.

[0066] (i4) Using the degumming process, the sample was placed in acetone, isopropanol and deionized water for 5 minutes respectively to remove the photoresist.

[0067] (2) Generating the electrochemical sensor on the first dielectric layer; this step includes:

[0068] (i5) The three electrochemical electrodes and the ground electrode for droplet driving are prepared by the lift-off process. The specific process is to prepare a photoresist pattern on the sample surface by a photolithography process. Double-layer glue or reverse glue such as AZ5214 is commonly used to expose the position where the electrode needs to be prepared, and cover the other positions with photoresist.

[0069] (i6) Deposit metal on top by electron beam evaporation.

[0070] (i7) Then, using the degumming process, the sample was placed in acetone, isopropanol, and deionized water for 5 minutes, respectively, and the photoresist was stripped off to obtain a patterned electrochemical three-electrode and a ground electrode.

[0071] (3) forming a second dielectric layer on the electrochemical sensor and exposing the three electrodes of the electrochemical sensor; this step includes:

[0072] (i8) Preparing a dielectric layer such as silicon nitride material thereon by a thin film deposition process such as plasma enhanced chemical vapor deposition (PECVD).

[0073] (i9) Through the photolithography process, photoresist is used to cover other positions and expose the top of the electrochemical three electrodes.

[0074] (i10) The dielectric silicon nitride layer above the electrochemical three electrodes is removed by an etching process such as an ICP dry etching process.

[0075] (i11) Then, using the degumming process, the sample was placed in acetone, isopropanol, and deionized water for 5 minutes respectively to remove the photoresist.

[0076] (4) preparing a hydrophobic layer and exposing the three electrodes of the chemical sensor;

[0077] Specifically include:

[0078] (i12) Preparing a hydrophobic layer material on the sample surface by spin coating, drying, thermal evaporation, etc., such as Teflon film, cytop or other hydrophobic materials.

[0079] (i13) The hydrophobic layer above the three electrochemical electrodes is removed by local plasma, exposing the three electrodes so that they can contact the test droplets.

[0080] (5) Modification of the surface of the working electrode.

[0081] In this embodiment, a specific material is modified on the surface of the working electrode, and the modification type and material can be controlled by different chemical sensing needs. For example, in this embodiment, the following steps can be adopted, (i14) such as depositing gold nanoparticles on the surface of the working electrode by electrodeposition to enhance the electrochemical signal, depositing silver on the surface of the reference electrode by electrodeposition or thermal evaporation, and then soaking in ferric chloride solution to convert part of the silver into silver chloride to obtain an Ag / AgCl reference electrode. In other embodiments, or applying Ag / AgCl glue on the surface of the reference electrode to obtain a more stable Ag / AgCl reference electrode to obtain the corresponding technical effect. .

[0082] In this embodiment, the lower plate substrate 14 of the platform is made of glass, resin, etc., and an electrode drive structure array 13 for droplet drive is prepared thereon. The electrode drive structure here is mainly used for driving the droplet. In this patent embodiment, the drive layer is used to realize the drive control of the droplet. In other embodiments, other drive structures can also be used to achieve the same technical effect. The surface of the electrode array is modified with a dielectric layer 10 (such as insulating materials such as silicon oxide, silicon nitride, aluminum oxide, parylene, SU8 photoresist, etc., which are not limited by the present invention) and a hydrophobic layer 11 (such as hydrophobic materials such as Teflon, cytop, fdts, etc., which are not limited by the present invention). The main function of the lower plate is the drive and control of the droplet. There are many methods for constructing the droplet electrode drive structure array: such as based on PCB printed circuit board, ITO patterned electrode glass, electrode array based on active matrix TFT, etc., which are not limited by the present invention. Through the method described above, the corresponding electrochemical sensor array can be quickly prepared. In other embodiments, other materials and methods can be used as needed to achieve the same technical effects under different requirements.

[0083] Embodiment three:

[0084] In this embodiment, the present invention also provides a method for realizing sugar sequencing using the digital microfluidic chip of the present invention. Sugar is the most abundant biological molecule in nature and is indispensable to almost all known organisms. It plays an important role in a wide range of biological processes. The complexity of the sugar chain structure makes the analysis of the sugar chain structure much more difficult than nucleic acids and proteins, which greatly limits the functional research on it. In order to accurately obtain information such as the connection mode and configuration of polysaccharides, highly specific non-reducing end exoglycosidases are often used for sugar chain sequencing, but the operation and detection process of this method can be very cumbersome. Here, we propose a new strategy for sugar sequencing, integrating the glycosidase library with the electrochemical detection function in the digital microfluidic chip, and realizing the full process automation to sequentially obtain the type, number, connection mode and other information of each monosaccharide in the sugar chain one by one. This system is expected to provide a new path for accurate and efficient sugar sequencing in the field of sugar science. Determining oligosaccharide sequences by exoglycosidase cleavage is one of the commonly used techniques for determining complex carbohydrate structures. Since exoglycosidases are specific to monosaccharide units, linkage positions, and orientations (α and β), they can recognize positional and linkage isomers, so they can reveal not only sequences, but also related connection patterns and telomere configuration information. Specifically, non-reducing end-specific exoglycosidases only hydrolyze glycosidic bonds of specific configurations and connection patterns between specific monosaccharides, and can hydrolyze sugar chains sequentially. According to the type of glycosidase and the composition of monosaccharides after hydrolysis, a complete and accurate sugar chain sequence including glycosidic bond information can be obtained. However, the operation process of using glycosidases for sugar chain sequencing is very cumbersome, requiring separation, purification, and product detection operations after each step of specific glycosidase hydrolysis. In addition, the high enzyme consumption leads to high costs, so it is usually combined with some other high-throughput analytical tests. Using the detection method, Bwanali et al. reported a method for sequencing N-glycans using the exoglycosidase method combined with capillary electrophoresis technology. Rudd et al. reported a method for sequencing N-glycans in serum using the exoglycosidase method combined with liquid chromatography. "A capillary electrophoresis method integrates a unique combination of enzymes and lectins to modify sialylated N-glycans in real time in the capillary, making N-glycan structures containing α2-6-linked sialic acid easy to separate, detect and quantify. Each analysis consumes only nanoliters of enzyme or lectin." This work completed the measurement of specific sugar chain biomarkers in the serum of patients with rheumatoid arthritis, and newly discovered 117 N-glycan structures and 36 enzyme cleavage products. Although the integration of various separation and detection tools can help realize the basic process of sugar chain sequencing based on exoglycosidases, these schemes have failed to fundamentally solve the problems of cumbersome operation procedures, low versatility and high enzyme consumption.Digital microfluidics technology, which uses electrowetting-on-dielectric (EWOD) as the principle of droplet manipulation, achieves precise manipulation of discrete droplets ranging from microliters to nanoliters through a series of electrode arrays protected by insulating layers and hydrophobic layers. It has the advantages of low reagent consumption and small overall size. On the digital microfluidic chip, the electrode array controls the movement, fusion, and splitting of droplets through electric field forces, without the need for micropumps, microvalves, and other structures or power sources. Therefore, the chip has a higher degree of universality and integration. Since the droplets are controlled by the electrode array, in other words, we can control the electrodes through electrical signals to manipulate the droplets. Therefore, digital microfluidics technology has great automation potential. However, it is necessary to combine mass spectrometry and other means to verify the specific information of degradation products, and it has not been possible to truly realize the full process automation of sugar chain sequencing.

[0085] Here we propose a new sugar chain sequencing strategy and establish a digital microfluidic system with integrated electrochemical detection. The system can realize a new strategy of full-process automated sugar chain sequencing based on exoglycosidase library. The specific process is as follows Fig. 9 As shown, when faced with a glycan to be tested, it is mixed with the enzymes in the glycosidase library one by one to react and separate the products. Only the exoglycosidase that matches the monosaccharide at the outermost end of the glycan chain will react with the glycan and cut off the monosaccharide at the outermost end of the glycan chain. Therefore, only the product corresponding to the correct enzyme will contain the monosaccharide cut by the enzyme. This reaction process of glycan and glycosidase can be fully automated in the digital microfluidic chip. In order to find the product containing monosaccharides, the separated product will be transported to the electrochemical detection electrode inside the chip. The gold nanostructure is deposited on the surface of the working electrode, which can quickly and accurately detect whether there are monosaccharides in the product, and the monosaccharide concentration information in the product can be obtained by analyzing the electrochemical signal. The presence or absence of monosaccharides in the product can be used to find the corresponding active glycosidase. Based on the glycosidase, the type of monosaccharide at the end of the sugar chain, the connection mode, and the telomere configuration can be inferred. Repeat the above operation, and the sugar chain that has cut the first monosaccharide at the end is reacted with the enzymes in the glycosidase library one by one and the product is detected. The monosaccharide information at the end of the sugar chain can be obtained. Repeat the cycle until the sugar chain is completely hydrolyzed. The type of monosaccharide and the composition order of the sugar chain are obtained through the active glycosidase in each cycle. The number of each monosaccharide in the sugar chain can be inferred through the concentration ratio of the monosaccharide products in each cycle, thus completing the complete sequencing of the sugar chain. This system is expected to provide a set of solutions for accurate and efficient sugar sequencing in the field of sugar science.

[0086] Specifically, the sugar sequencing method of this patent includes the following steps: 1) using the electrode driving structure to drive the oligosaccharide droplets to mix with the first glycosidase droplets to perform enzymatic cleavage of the oligosaccharides; wherein, the enzymatic cleavage can be performed by mixing the oligosaccharide droplets with magnetic beads, and then mixing with the first glycosidase for enzymatic cleavage; 2) using a magnet to separate the oligosaccharide chains and monosaccharide products after enzymatic cleavage by droplet separation; 3) using the electrode driving structure to move the droplets of the enzymatic cleavage products to the electrochemical sensor for concentration detection; 4) repeating the above steps 1) to 3) until the oligosaccharide chains are completely hydrolyzed. Specifically, in the embodiment of the present invention, chitosan oligosaccharides are taken as an example. Chitosan oligosaccharides are composed of two monosaccharides, glucosamine and N-acetylglucosamine, and the glycosidic bonds therein are all β1-4 configurations. As follows Figures 9 and 10 As shown: The following steps can be taken specifically;

[0087] ① The oligosaccharide droplets are mixed with the first glycosidase droplets to perform enzymatic cleavage of the oligosaccharides.

[0088] ② Use a magnet to separate the oligosaccharide chains and monosaccharide products after enzyme cleavage into droplets.

[0089] ③The product droplets from the first step of enzyme cleavage move to the electrochemical electrode for concentration detection.

[0090] ④ The oligosaccharide chains are mixed with the second glycosidase droplets for enzymatic cleavage.

[0091] ⑤ Use a magnet to separate the oligosaccharide chains and monosaccharide products after enzyme cleavage into droplets.

[0092] ⑥ The product droplets from the second step of enzyme cleavage move to the electrochemical electrode for concentration detection.

[0093] The above steps can be repeated continuously until the oligosaccharide chain is completely hydrolyzed. Each of the above steps can be used to achieve sugar sequencing of oligosaccharide chains.

[0094] However, it is worth noting that although the present invention provides a specific method and scenario for sugar sequencing, the digital microfluidic chip of the present invention is not only used for sugar sequencing, but can also realize a variety of other required usage scenarios. The usage scenarios can be adjusted according to actual needs to meet the corresponding needs.

[0095] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A digital microfluidic chip, characterized in that: It includes several control units, The control unit comprises an upper electrode plate and a lower electrode plate which are arranged opposite to each other and have a hydrophobic layer on their surfaces, the upper electrode plate is provided with an electrode control structure, and the lower electrode plate is provided with an electrode driving structure, a dielectric layer and a hydrophobic layer; The upper electrode plate is provided with an electrode control structure layer and an electrochemical sensing layer in sequence in the direction toward the lower electrode plate. The electrochemical sensing electrode is provided in the electrochemical sensing layer. The electrode control structure is provided in the electrode control structure layer. At least one electrochemical sensor is connected to the electrode control structure.

2. A method for preparing a digital microfluidic chip, characterized in that: The preparation of the digital microfluidic chip as claimed in claim 1 comprises the following steps: (1) After the electrode control structure on the upper electrode plate is prepared, a first dielectric layer is prepared on the electrode control structure, and a connection point between the electrode control structure and the electrochemical sensor is exposed on the first dielectric layer; (2) forming the electrochemical sensor on the first dielectric layer; (3) forming a second dielectric layer on the electrochemical sensor and exposing three electrodes of the electrochemical sensor; (4) preparing a hydrophobic layer and exposing the three electrodes of the chemical sensor; (5) Prepare the electrode driving structure, dielectric layer and hydrophobic layer on the lower electrode plate.

3. The method for preparing a digital microfluidic chip with an integrated electrochemical sensor as claimed in claim 2, characterized in that: In the step (1), The first dielectric layer is prepared by a thin film deposition process. The connection points between the electrode control structure and the electrochemical sensor are exposed by photoresist covering and stripping.

4. The method for preparing a digital microfluidic chip with integrated electrochemical sensor according to claim 2, characterized in that: The step (2) comprises the following steps: Prepare the electrode photoresist pattern by photolithography, expose the position where the electrode needs to be prepared according to the pattern, and cover the other positions with photoresist; The photoresist is removed from the glass using a stripping process to form the three electrodes of the electrochemical sensor.

5. The method for preparing a digital microfluidic chip with integrated electrochemical sensor according to claim 2, characterized in that: The step (2) comprises the following steps: A ground electrode of the electrode driving structure is formed while the electrochemical sensor is formed on the first dielectric layer.

6. The method for preparing a digital microfluidic chip with integrated electrochemical sensor according to claim 5, characterized in that: In step (2), the following steps are included: After creating the electrochemical sensor and the ground electrode, metal is deposited over the electrodes.

7. The method for preparing a digital microfluidic chip with integrated electrochemical sensor according to claim 1, characterized in that: The step (4) further comprises the following steps: modifying the surface of the working electrode.

8. A digital microfluidic chip sugar sequencing method, characterized by: Sugar sequencing is performed using a digital microfluidic chip prepared by the method for preparing a digital microfluidic chip according to any one of claims 1 to 7.

9. The microfluidic chip sugar sequencing method according to claim 8, characterized in that: The following steps are involved: 1) using the electrode driving structure to drive the oligosaccharide droplet to mix with the first glycosidase droplet to perform enzymatic cleavage of the oligosaccharide; wherein the enzymatic cleavage can be performed by mixing the oligosaccharide droplet with magnetic beads and then mixing with the first glycosidase for enzymatic cleavage; 2) Using a magnet to separate the oligosaccharide chains and monosaccharide products after enzyme cleavage into droplets; 3) using the electrode driving structure to move the droplets of the enzyme cleavage product to the electrochemical sensor for concentration detection; 4) Repeating the above steps 1) to 3), sequentially using the electrode driving structure to drive the oligosaccharide droplet to mix with the second glycosidase droplet, the third glycosidase droplet, etc. until the oligosaccharide chain is completely hydrolyzed.

10. The microfluidic chip sugar sequencing method according to claim 8, characterized in that: The step 1) comprises the following steps: mixing the oligosaccharide solution with magnetic beads to form oligosaccharide droplets; The oligosaccharide droplet is mixed with the first glycosidase droplet to complete the enzymatic cleavage of the oligosaccharide.

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