Digital micro-fluidic chip integrated with electrochemical sensor

Abstract

The utility model discloses a digital micro-fluidic chip integrated with an electrochemical sensor, which comprises a plurality of control units, each control unit at least comprises an electrode control structure, and the electrode control structure is communicated with at least one electrochemical sensing electrode. The utility model 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, and automatic processing of samples is realized through a digital micro-fluidic technology; in-situ rapid high-sensitivity electrochemical detection of sample liquid drops is realized through the electrochemical electrode array. Each individual electrochemical electrode in the electrochemical electrode array can be specifically modified, so that detection of a specific sample is realized, and the applicability is very wide.

Description

TECHNICAL FIELD

[0001] The utility model relates to the field of chip, especially integrate electrochemical sensor's digital micro -fluidic chip. BACKGROUND

[0002] Micro total analysis system (mTAS) in the application of chemistry and biology arouses people's great interest, and these integrated, miniaturized, automated analysis systems are also called lab-on-a-chip (LOC). Microfluidic technology as one of the important branches obtains extensive attention and a large number of researches, and the main advantage of the technology is to improve the automation of traditional biochemical experiment process, reduce the consumption of sample and reagent, improve the operability of sample etc. Digital microfluidic technology based on electrowetting-on-dielectric (EWOD) as droplet control principle realizes the precise control of discrete droplets with microliter to nanoliter volume through a series of electrode arrays protected by insulation layer and hydrophobic layer, has the advantages of less reagent consumption, small overall size easy to integrate of traditional microfluidic chip, and without micro pump, micro valve and other structures or power source, and the generalization degree and automation degree of chip are higher. However, in practical application, after a series of reactions and processing, the most important step for the product is rapid and accurate detection, and the conventional digital microfluidic chip is difficult to realize this function. The most commonly used detection method of digital microfluidic is optical method, such as visual observation, fluorescence, chemiluminescence etc., and currently there are related researches on the combination of electrochemical sensing and digital microfluidic, but these researches are focused on the combination of single electrochemical sensor and digital microfluidic technology, however, complex applications involve a large number of droplets and different applications have different electrochemical detection requirements, and different droplets in the same application may also need to face their own specific electrochemical detection requirements, these unique requirements require electrochemical sensing electrodes to have their own specific size, shape, structure and modification, and single electrochemical electrode cannot meet these requirements, but optical detection method often relies on the light emission or light absorption behavior of reagent itself, and the system for accurate detection and collection of light signal is usually relatively large, which is not conducive to integration and miniaturization.

[0003] In summary, in the prior art, there is lack of an electrochemical electrode control structure based on transistor switch matrix, which meets various electrochemical detection requirements by specific design of each electrochemical electrode in the electrochemical electrode array. UTILITY MODEL CONTENT

[0004] In view of the above defects of the prior art, the technical problem to be solved by the utility model is to provide an electrochemical electrode control structure based on a transistor switch matrix, by specifically designing the control of each electrochemical electrode in an electrochemical electrode array, while meeting various electrochemical detection requirements.

[0005] Based on the above technical problem, the utility model provides a digital micro -fluidic chip of integrated electrochemical sensor, including a plurality of control units, at least including an electrode control structure in the control unit, at least one electrochemical sensing electrode is connected on the electrode control structure.

[0006] Preferably, the control unit includes oppositely arranged upper and lower plates, the electrode control structure is arranged on the upper plate, the electrode driving structure is arranged on the lower plate, and a hydrophobic layer is arranged on the upper surface of the lower plate.

[0007] Preferably, the upper plate is provided with an upper plate substrate, the upper plate substrate is sequentially provided with an electrode control structure layer, an electrochemical sensing layer and a ground electrode layer in the direction towards the lower 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 is separated from the electrochemical sensing layer by a dielectric layer, one end of the working electrode of the electrochemical sensing electrode is connected with the electrode control structure, and the other end of the working electrode is connected with the ground electrode layer and the lower surface of the upper plate.

[0008] Preferably, the lower surface of the upper plate except the working electrode is provided with a hydrophobic layer.

[0009] Preferably, the lower surface of the upper plate has a working electrode surface modifier.

[0010] Preferably, the electrode control structure is set as a transistor switch, and the reference electrodes of the electrochemical sensing electrodes of each control unit are connected in series with each other.

[0011] Preferably, the electrode control structure is set as a TFT thin film transistor.

[0012] Preferably, the working electrode of the electrochemical sensing electrode is connected with the source electrode current of the TFT.

[0013] Preferably, the gates of the TFTs in the plurality of control units are connected in series with each other.

[0014] Preferably, the drains of the TFTs in the plurality of control units are connected in series with each other.

[0015] The utility model has the advantages of:

[0016] (1) The application provides a special digital microfluidic chip, integrates an electrochemical sensor into the digital microfluidic chip, and can form a matrix of the electrochemical sensor, realizes automatic processing of a sample through a digital microfluidic technology, and realizes in-situ rapid high-sensitivity electrochemical detection of the sample droplet through an electrochemical electrode array. Each single electrochemical electrode in the electrochemical electrode array can be modified to realize detection of a specific sample, and the applicability is very wide.

[0017] (2) The application provides a large-scale electrochemical sensor matrix constructed based on a transistor switch matrix. The transistor switch matrix can be a thin film transistor (TFT), a field effect transistor (FET), a transistor or other switch transistor, can realize large-scale complex droplet manipulation and concentration detection, and can gradually decompose large complex liquid experiment operation to form a simple and easy-to-operate automatic operation system.

[0018] (3) The electrochemical sensor structure of the application is independently controlled, very efficient and easy to maintain. The electrochemical sensing electrode array based on the transistor switch matrix designed by the application connects the reference electrodes (RE) of each column of electrochemical electrodes in series, can realize control of the signals of n reference electrodes through n paths, and the counter electrodes (CE) in the electrochemical electrodes adopt the same control scheme as the reference electrodes. The switch of each electrochemical working electrode in the array is a transistor switch. Taking a TFT thin film transistor as an example, when a positive voltage is applied to the gate, an electric field in a direction upwards is generated, and the electrons in the active layer move to the insulating layer under the action of the electric field, forming an electron accumulation layer at the interface (front channel) between the active layer and the insulating layer. At this time, a positive voltage is applied between the source and the drain, and the electrons in the electron accumulation layer move to the drain, forming a conduction current. Therefore, only when the gate and the drain apply effective voltages at the same time, the source and the drain form a conduction current. Connecting the source of the TFT with the working electrode in the electrochemical electrode can control the on-off current of the electrochemical working electrode through the TFT. The gate of each row of TFTs in the TFT array is connected in series, and the drain of each column of TFTs is connected in series, so that m+n paths of electrical signals can be used to control m*n TFT switches, and then m*n electrochemical sensors can be controlled. Each single electrochemical electrode in the electrochemical sensor array can be controlled and used independently.

[0019] (4) Based on the present application, the electrochemical sensing sensitivity can be further increased by specific modification. In order to enhance the electrochemical signal, the working electrode surface of the present application can be modified by electrochemical deposition, slurry printing, thermal evaporation and other processes to realize the enhancement of the electrochemical signal. And the working electrode of each electrochemical electrode can be specially modified to realize the signal collection of specific samples, for example, the working electrode is modified by glucose oxidase to realize the detection of glucose concentration in the droplet. The working electrode is modified by specific antibody markers to realize the detection of specific antigens in the droplet. Some electrochemical electrodes are designed as interdigital electrodes, which can be used for impedance detection and other applications.

[0020] (5) The present application also provides a specific digital droplet microfluidic chip preparation method, which can quickly form the digital droplet microfluidic chip of the present application. The present application realizes the rapid preparation of the structure of the present application.

[0021] (6) The present application also constructs an automatic sugar sequencing system through the digital microfluidic chip of the present application. The digital microfluidic chip realizes the complete automation of sugar sequencing, and the efficiency of sugar sequencing is completely improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a cross-sectional structure schematic diagram of the digital microfluidic chip control unit of the embodiment one of the present application.

[0023] Figure 2 is a connection schematic diagram of the electrochemical detection electrode array based on the transistor switch array in the embodiment one of the present application.

[0024] Figure 3 is Figure 2 is an enlarged schematic diagram of the local part A.

[0025] Figure 4 is one of the digital microfluidic chip upper plate preparation flowcharts of the embodiment two of the present application.

[0026] Figure 5 is the second digital microfluidic chip upper plate preparation flowchart of the embodiment two of the present application.

[0027] Figure 6 is the third digital microfluidic chip upper plate preparation flowchart of the embodiment two of the present application.

[0028] Figure 7 is the fourth digital microfluidic chip upper plate preparation flowchart of the embodiment two of the present application.

[0029] Figure 8 is the fifth digital microfluidic chip upper plate preparation flowchart of the embodiment two of the present application.

[0030] Figure 9is a flowchart of embodiment three of the present application.

[0031] Figure 10 is a flowchart of embodiment three of the present application. DETAILED DESCRIPTION

[0032] The present application is further described below in conjunction with the accompanying drawings and embodiments: Embodiment 1

[0033] An embodiment one is provided in the present application, specifically, a digital microfluidic chip is provided in the present embodiment, which comprises a plurality of control units, and at least one electrode control structure is included in the control unit, and the electrode control structure is particularly a transistor in the present embodiment, which can be particularly set as a TFT structure, and one electrochemical sensing electrode is connected to each TFT transistor, so that one electrochemical sensing electrode can be controlled by one electrode control structure, i.e., the TFT transistor switch, the array arrangement of the electrochemical sensing electrode can be realized, the electrochemical sensing of the droplet can be realized, and each electrochemical sensing electrode can be controlled respectively. The core device of the electrochemical detection is the electrochemical sensing electrode, and the integration of the electrochemical sensing electrode on the digital microfluidic chip is the premise and important basis for the chip to have the electrochemical detection. The three-electrode system is commonly used in the electrochemical sensing electrode detection, which comprises 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 controlled 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, so as to obtain the function relationship curve between the current and the potential.

[0034] Specifically, the specific structure of the control unit in the present embodiment can include oppositely arranged upper and lower plates, as shown in Figure 1 The electrode control structure is a TFT structure, and the main function of the electrode control structure here is to control the electrochemical sensor, and the TFT structure can be used in the present embodiment, and other types of transistors can also be used to realize the same technical effect in other embodiments, which are arranged on the upper plate, and the droplet electrode driving structure 13 is arranged on the lower plate, and the upper surface of the lower plate is provided with a hydrophobic layer 11.

[0035] The upper plate is provided with an upper plate substrate 1, and the lower plate is provided with a lower plate substrate 14. The upper plate substrate 1 is sequentially provided with an electrode control structure layer in the direction towards the lower plate. The electrode control structure layer includes a TFT gate 2, a TFT drain 4, and 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. An electrochemical sensing electrode is arranged on the electrochemical sensing layer. The electrode control structure is arranged on the electrode control structure layer and is spaced from the electrochemical sensing layer by a dielectric layer 10. The electrode control structure is in communication with one end of the working electrode 8 of the electrochemical sensing electrode, and the other end of the working electrode 8 is in communication with the ground electrode layer 6 and the lower surface of the upper plate. The lower surface of the upper plate, except for the working electrode, is provided with a hydrophobic layer 11. The ground electrode layer 6 and the hydrophobic layer 11 are also spaced by a dielectric layer.

[0036] The working electrode has a working electrode surface modifier 12 on the lower surface. The working electrode surface modifier 12 can expand the detection range of the electrochemical sensing. The electrode control structure is a transistor switch. The digital microfluidic chip integrating the electrochemical electrode array is composed of upper and lower plates. The upper plate is provided with an electrochemical electrode array based on a transistor switch array. For example, the upper plate substrate 1 is made of glass or other materials. The 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 (commonly used silicon oxide, silicon nitride, etc.) between the gate 2 and the active layer. Each electrochemical electrode is composed of a working electrode (WE) 8, a counter electrode (CE) 9, and a reference electrode (RE) 5. The ground electrode 6 of the upper plate forms an electric field with the droplet electrode driving structure array of the lower plate, which is used for droplet control. The dielectric layer 10 and the hydrophobic layer 11 are covered on the ground electrode 6. The hydrophobic layer 11 is the same as the lower plate substrate 14, but the electrochemical three-electrode surface needs to be exposed. The dielectric layer 6 and the hydrophobic layer 10 on the electrochemical electrode surface are removed by dry etching or wet etching, so that the droplet can fully contact the electrochemical electrode, and the electrochemical sensing signal can be collected. The lower plate is provided with an electrode driving structure, i.e., a droplet driving electrode 13.

[0037] As 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 with each other. The working electrodes 8 of the electrochemical sensing electrodes are in current communication with the sources 7 of the TFTs. The gates 2 of the TFTs in the plurality of control units are connected in series with each other. The drains 4 of the TFTs in the plurality of control units are connected in series with each other.

[0038] The transistor switch matrix-based electrochemical sensing electrode array of the present application connects the reference electrodes (RE) 5 of each column of electrochemical electrodes in series, which can realize the control of the signals of n rows to n×n reference electrodes, and the counter electrodes (CE) 9 in the electrochemical electrodes adopt the same control scheme as the reference electrodes 5. The switch of each electrochemical working electrode in the array is a transistor switch. Taking the TFT thin film transistor as an example, the structure of the TFT is as shown in Figure 3 When a positive voltage is applied to the gate 2, an electric field in the upward direction is generated, and the electrons in the active layer move to the insulating layer under the action of the electric field, forming an electron accumulation layer 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 to the drain, forming a conduction current. Therefore, only when the gate 2 and the drain 4 are simultaneously applied with an effective voltage, a conduction current is formed between the source 7 and the drain. Therefore, in this embodiment, the source of the TFT is connected to the working electrode in the electrochemical electrode, and the current of the electrochemical working electrode can be controlled by the TFT. The gate of each row of TFTs in the TFT array is connected in series, and the drain of each column of TFTs is connected in series, so that m+n electrical signals can be used to control m×n TFT switches, and further to control 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 for the control of the electrochemical array. The large-scale array is controlled in a scanning manner, and only one electrode is tested at a specific moment, so the pressure on the electrochemical workstation is not large. Embodiment 2

[0039] 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 integrated with an electrochemical electrode array. The preparation process of the TFT thin film transistor array on the upper plate is the same as the conventional TFT preparation process, and the present application focuses on the preparation of the TFT-based electrochemical sensing electrode array. The specific process flow is as shown in Figure 4

[0040] As Figures 4-8 , the specific preparation method of the above digital microfluidic chip is as follows:

[0041] (1) After the preparation of the electrode control structure on the upper plate is completed, a first dielectric layer is prepared on the electrode control structure, and the connection points of the electrode control structure and the electrochemical sensor are exposed on the first dielectric layer;

[0042] Specifically, the following steps are included:

[0043] (i1) After the preparation of the TFT array on the upper plate is completed, 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).​

[0044] (i2) Cover other locations using photoresist 16 by photolithography process, expose the TFT source above.

[0045] (i3) Remove the dielectric layer silicon nitride above the source by etching process such as ICP dry etching process.

[0046] (i4) Use the stripping process, put the sample into acetone, isopropyl alcohol and deionized water respectively for 5 minutes, and peel off the photoresist.

[0047] (2) Generate the electrochemical sensor on the first dielectric layer; this step includes:

[0048] (i5) Prepare the electrochemical three-electrode and ground electrode for droplet driving by lift-off process, the specific process is to prepare a photoresist pattern on the sample surface by photolithography process, commonly use double-layer resist or reverse resist such as AZ5214, expose the position where the electrode needs to be prepared, and cover the photoresist on other positions.

[0049] (i6) Deposit metal on the top by electron beam evaporation.

[0050] (i7) Again use the stripping process, put the sample into acetone, isopropyl alcohol and deionized water respectively for 5 minutes, and peel off the photoresist, get the patterned electrochemical three-electrode and ground electrode.

[0051] (3) Form a second dielectric layer on the electrochemical sensor, and expose the three-electrode of the electrochemical sensor; this step includes:

[0052] (i8) Prepare a dielectric layer such as silicon nitride material on it by thin film deposition process such as plasma enhanced chemical vapor deposition (PECVD).

[0053] (i9) Cover other locations using photoresist by photolithography process, expose the electrochemical three-electrode above.

[0054] (i10) Remove the dielectric layer silicon nitride above the electrochemical three-electrode by etching process such as ICP dry etching process.

[0055] (i11) Again use the stripping process, put the sample into acetone, isopropyl alcohol and deionized water respectively for 5 minutes, and peel off the photoresist.

[0056] (4) Prepare a hydrophobic layer, and expose the three-electrode of the chemical sensor;

[0057] Specifically includes:

[0058] (i12) Hydrophobic layer material such as Teflon film, cytop or other hydrophobic material is prepared on the sample surface by spin coating and drying, thermal evaporation and other methods.

[0059] (i13) The hydrophobic layer above the electrochemical three-electrode is removed by local plasma, exposing the three-electrode to enable contact with the test droplet.

[0060] (5) Modification is made on the surface of the working electrode.

[0061] In this embodiment, specific materials are modified on the surface of the working electrode, and the type and material of the modification can be controlled according to different chemical sensing needs. In this embodiment, the following steps can be used: (i14) Gold nanoparticles are deposited on the surface of the working electrode by electrodeposition to enhance the electrochemical signal. Silver is deposited on the surface of the reference electrode by electrodeposition or thermal evaporation, and then immersed in ferric chloride solution to make part of the silver into silver chloride, obtaining an Ag / AgCl reference electrode. In other embodiments, Ag / AgCl glue is applied to the surface of the reference electrode to obtain a more stable Ag / AgCl reference electrode to achieve the corresponding technical effect.

[0062] In this embodiment, the material of the lower plate substrate 14 of the platform is glass, resin or the like, and an electrode driving structure array 13 for driving liquid droplets is prepared thereon. The electrode driving structure is mainly used for driving the movement of liquid droplets. In this embodiment, the driving layer is used to realize the driving control of the liquid droplets. In other embodiments, other driving structures can be used to achieve the same technical effect. The electrode array surface is modified with a dielectric layer 10 (such as silicon oxide, silicon nitride, aluminum oxide, parylene, SU8 photoresist, and other insulating materials, which are not limited by the present application) and a hydrophobic layer 11 (such as Teflon, cytop, fdts, and other hydrophobic materials, which are not limited by the present application). The main function of the lower plate is to drive and control the liquid droplets. The construction method of the liquid droplet electrode driving structure array has many kinds: such as based on PCB printed circuit board, ITO patterned electrode glass, electrode array based on active matrix TFT, and the like, which are not limited by the present application. Through the above described method, the corresponding electrochemical sensing array can be quickly prepared. In other embodiments, other materials and methods can be used to achieve the same technical effect under different needs. Embodiment 3:

[0063] In this embodiment, the application also provides a method for sugar sequencing using the digital microfluidic chip. Sugar is the most abundant biomolecule in nature and is essential for almost all known organisms. It plays an important role in a wide range of biological processes. The complexity of sugar chain structure makes the analysis of sugar chain structure much more difficult than that of nucleic acids and proteins, which greatly limits the functional research. In order to accurately obtain the information of glycan connection mode and configuration, highly specific non-reducing end exoglycosidases are often used for sugar chain sequencing, but the operation and detection process of this method is very tedious. Here, we propose a new strategy for sugar sequencing, which integrates glycosidase library and electrochemical detection function in digital microfluidic chip, realizes the full-process automation of the information of each monosaccharide in the sugar chain in order, such as type, number, connection mode, etc. This system is expected to provide a new path for accurate and efficient sugar sequencing in the field of sugar science. Exoglycosidase digestion assay is one of the commonly used techniques for determining the structure of complex carbohydrates. Because exoglycosidases have specificity for monosaccharide units and linkage position and orientation (alpha and beta), they can recognize positional and linkage isomers, so they can not only reveal the sequence, but also reveal the related connection mode and telomere configuration information. Specifically, non-reducing end specific exoglycosidases can hydrolyze glycosidic bonds of specific configuration and connection mode between specific monosaccharides, and can realize the sequential hydrolysis of sugar chains. According to the type of glycosidase and the composition of monosaccharide after hydrolysis, the complete and accurate sugar chain sequence including glycosidic bond information can be obtained. However, the operation process of using glycosidase for sugar chain sequencing is very tedious, and it is necessary to separate, purify and detect the product after each specific glycosidase hydrolysis. In addition, the large amount of enzyme consumption leads to high cost, so it is usually combined with some other high-throughput analysis and detection methods. Bwanali et al. reported a method for sequencing N-glycans using exoglycosidase combined with capillary electrophoresis technology. Rudd et al. reported a method for sequencing N-glycans in serum using exoglycosidase 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 a capillary, making N-glycan structures containing alpha2-6-linked sialic acids easy to separate, detect and quantify. Only nanoliter volume of enzyme or lectin is consumed for each analysis. This work completed the measurement of specific glycan biomarkers in the serum of patients with rheumatoid arthritis, and discovered 117 N-glycan structures and 36 enzyme digestion products. Although integrating various separation and detection tools can help to realize the basic process of sugar chain sequencing based on exoglycosidase, these schemes have not fundamentally solved the problems of tedious operation process, poor universality and large amount of enzyme consumption.Digital microfluidics technology based on electrowetting-on-dielectric (EWOD) principle can precisely manipulate discrete droplets with volume from microliter to nanoliter through a series of electrode arrays protected by insulating and hydrophobic layers. It has the advantages of low reagent consumption and small overall size. On the digital microfluidic chip, the electrode array manipulates the movement, fusion and splitting of droplets through electric field force, without the need for micro-pumps, micro-valves and other structures or power sources. Therefore, the degree of generalization and integration of the chip is higher. Since the droplets are controlled by the electrode array, in other words, we can control the droplets by controlling the electrode through electrical signals, therefore digital microfluidic technology has strong automation potential. However, it needs to be combined with mass spectrometry to verify the specific information of the degradation products, and it cannot realize the full-process automation of glycan sequencing in a true sense.

[0064] Here we propose a new glycan sequencing strategy and establish a digital microfluidic system integrated with electrochemical detection. The system can realize the new strategy of full-process automated glycan sequencing based on exoglycosidase library. As shown in Figure 9 , when facing a glycan to be tested, it is mixed and reacted with each enzyme in the glycosidase library and the product is separated. Only the exoglycosidase that matches the monosaccharide at the outer end of the glycan chain will react with the glycan and cut off the monosaccharide at the outer end of the glycan chain. Therefore, only the product corresponding to the correct enzyme will contain the monosaccharide cut off by the enzyme. This reaction process of glycan and glycosidase can be fully automated on a digital microfluidic chip. In order to find the product containing monosaccharide, the separated product will be transported to the electrochemical detection electrode inside the chip. The working electrode surface is deposited with gold nanostructure, which can quickly and accurately detect whether there is monosaccharide in the product, and through the analysis of electrochemical signal, the concentration information of monosaccharide in the product can be obtained. By the presence or absence of monosaccharide in the product, the corresponding active glycosidase can be found, and according to the glycosidase, the information of the monosaccharide at the end of the glycan chain, such as the type, connection mode and telomere configuration, can be inferred. By repeating the above operation, the glycan chain with the first monosaccharide cut off at the end is reacted with each enzyme in the glycosidase library and the product is detected, and the monosaccharide information at the end of the glycan chain at this time can be obtained. Repeat the cycle until the glycan chain is completely hydrolyzed. Through the glycosidase in each cycle, the information of the type and sequence of monosaccharides in the glycan chain can be obtained, and through the concentration ratio of monosaccharide product in each cycle, the number of each monosaccharide in the glycan chain can be inferred. Thus, the complete sequencing of the glycan chain is completed. The system is expected to provide a scheme for accurately and efficiently sequencing glycan in the field of glycoscience.

[0065] Specifically, the sugar sequencing method of the patent comprises the following steps: 1) driving the oligosaccharide droplet to mix with the first glycosidase droplet by using the electrode driving structure to perform enzyme cutting of the oligosaccharide; wherein the enzyme cutting can be performed in the manner of mixing the oligosaccharide droplet with the magnetic beads and then mixing with the first glycosidase; 2) separating the enzyme-cut oligosaccharide chain from the monosaccharide product by using a magnet; 3) moving the droplet of the enzyme-cut product to the electrochemical sensor by using the electrode driving structure to perform concentration detection; 4) repeating the above steps 1) to 3) until the oligosaccharide chain is completely hydrolyzed. Specifically, in the embodiment of the present application, chitosan is taken as an example, which is composed of two monosaccharides of glucosamine and N-acetylglucosamine, and the glycosidic bond in it is of the beta 1-4 configuration. As shown in the following formula: Figures 9-10 Specifically, the following steps can be adopted:

[0066] ① The oligosaccharide droplet is mixed with the first glycosidase droplet to perform enzyme cutting of the oligosaccharide.

[0067] ② The enzyme-cut oligosaccharide chain is separated from the monosaccharide product by using a magnet.

[0068] ③ The droplet of the product of the first-step enzyme cutting is moved to the electrochemical electrode to perform concentration detection.

[0069] ④ The oligosaccharide chain is mixed with the second glycosidase droplet to perform enzyme cutting.

[0070] ⑤ The enzyme-cut oligosaccharide chain is separated from the monosaccharide product by using a magnet.

[0071] ⑥ The droplet of the product of the second-step enzyme cutting is moved to the electrochemical electrode to perform concentration detection.

[0072] The above steps can be continuously repeated until the oligosaccharide chain is completely hydrolyzed. Each of the above steps can be adopted.

[0073] However, it is worth noting that although the present application provides a specific use method and scenario for sugar sequencing, the digital microfluidic chip of the present application is not only used for sugar sequencing, but also can realize many other required use scenarios. The use scenario can be adjusted according to actual needs to meet the corresponding requirements.

[0074] The above describes the preferred embodiments of the present application in detail. It should be understood that those skilled in the art can make many modifications and changes without creative labor according to the concept of the present application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment on the basis of the prior art according to the concept of the present application shall be within the protection scope determined by the claims.

Claims

1. An integrated electrochemical sensor digital microfluidic chip, characterized by: The application relates to a control unit comprising a plurality of control units, at least one electrode control structure is arranged in the control units, and at least one electrochemical sensing electrode is connected to the electrode control structure; the control unit comprises oppositely arranged upper and lower electrode plates, the electrode control structure is arranged on the upper electrode plate, an electrode driving structure is arranged on the lower electrode plate, and a hydrophobic layer is arranged on the upper surface of the lower electrode plate.

2. The integrated electrochemical sensor digital microfluidic chip of claim 1, wherein: An upper electrode plate substrate is arranged on the upper electrode plate, and an electrode control structure layer, an electrochemical sensing layer and a ground electrode layer are sequentially arranged on the upper electrode plate substrate in the direction towards the lower electrode 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 is separated from the electrochemical sensing layer by a dielectric layer, one end of a working electrode of the electrochemical sensing electrode is connected to the electrode control structure, and the other end of the working electrode is connected to the ground electrode layer and the lower surface of the upper electrode plate.

3. The integrated electrochemical sensor digital microfluidic chip of claim 2, wherein: A hydrophobic layer is arranged on the lower surface of the upper electrode plate except the working electrode.

4. The digital microfluidic chip integrated with electrochemical sensors of claim 3, wherein: The lower surface of the upper electrode plate is provided with a working electrode surface modifier.

5. The integrated electrochemical sensor digital microfluidic chip of claim 1, wherein: the plurality of electrodes are configured to perform a plurality of electrochemical reactions; and the plurality of electrodes are configured to perform a plurality of electrochemical reactions. The electrode control structure is arranged as a transistor switch, and the reference electrodes of the electrochemical sensing electrodes of each control unit are connected in series.

6. The integrated electrochemical sensor digital microfluidic chip of claim 5, wherein: The electrode control structure is arranged as a TFT thin film transistor.

7. The integrated electrochemical sensor digital microfluidic chip of claim 6, wherein: The working electrode of the electrochemical sensing electrode is connected to the source current of the TFT.

8. The integrated electrochemical sensor digital microfluidic chip of claim 7, wherein: The gates of the TFTs in the plurality of control units are connected in series.

9. The integrated electrochemical sensor digital microfluidic chip of claim 8, wherein: The drains of the TFTs in the plurality of control units are connected in series.

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