A method and device for substance analysis by directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry

Abstract

The present invention discloses a method and device for substance analysis by directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry. In the device of the present invention, the digital microfluidic system includes a digital microfluidic chip and a micro-droplet driving platform. The chip includes an upper plate, a lower plate and a conductive gasket. The upper plate includes an upper substrate and a conductive layer. A hydrophobic layer is provided on the lower surface of the conductive layer, and a number of hydrophilic sites are arranged at intervals on the surface of the hydrophobic layer. Each hydrophilic site is a pit recessed towards the conductive layer. The lower plate includes a lower substrate and a dielectric layer. An electrode array is arranged between the lower substrate and the dielectric layer, and a lower hydrophobic layer is provided on the upper surface of the dielectric layer. The matrix-assisted laser desorption / ionization mass spectrometry includes a target plate substrate, and a groove for placing the upper plate of the chip is provided on the upper surface of the target plate substrate. By directly coupling the upper plate provided with hydrophilic sites and the customized target plate and then sending them into the mass spectrometer, the present invention can reduce manual operations and achieve automated analysis, and has the potential for high-throughput, sensitive and specific analysis.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and biomedicine, and particularly relates to a method and device for analyzing substances by directly integrating a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry. Background Art

[0002] Digital microfluidic technology is a microfluidic technology based on discrete droplet manipulation. On a digital microfluidic chip, a liquid is divided into discrete micro-liter droplets, and the droplets are precisely positioned and manipulated by methods such as manipulating an electric field, mechanical force, or temperature. Among them, dielectric-wetting-based digital microfluidics is a technology that uses the dielectric effect to control the position, merging, separation, and movement of microfluidic droplets. Its principle is to apply an alternating electric field on the surface of a microchannel to change the dielectric constant and surface wettability of the fluid, thereby achieving precise manipulation of the microfluid. Due to its unique advantages (such as high precision, programmability, fast response, and real-time control), the dielectric-wetting-based electro-manipulated digital microfluidic technology has been widely used in fields such as biological and chemical analysis. It can be used for various experimental operations such as sample mixing, reaction control, cell manipulation, enzyme reactions, and gene analysis. In chemical and biological reactions (such as cell lysis and protein extraction) before mass spectrometry analysis, the high precision, high flexibility, and automation characteristics of digital microfluidic technology provide new possibilities for experimental operations, helping to improve experimental efficiency and reduce reagent waste.

[0003] Mass spectrometry is a chemical analysis technology that can be used to analyze the molecular structure and composition of compounds. Its principle is to determine the molecular weight and structure of a compound by analyzing the movement trajectory and mass-to-charge ratio of ions in an electromagnetic field. Among them, matrix-assisted laser desorption ionization mass spectrometry is one of the most commonly used laser desorption ionization mass spectrometry methods. In this mass spectrometry method, an analyte is co-crystallized with a matrix solution that absorbs and transfers laser energy to the analyte to form an ionic compound. These ionic compounds are accelerated by an electric field and captured and analyzed by a detector. Matrix-assisted laser desorption ionization mass spectrometry has the advantages of nanosecond-level rapid analysis, high sensitivity, and high throughput, and has currently been used for the analysis of proteins and bacterial identification in biological samples, as well as the detection of biomarkers and metabolites.

[0004] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) has achieved remarkable results in the field of microbial identification, especially in clinical microbiological examinations. With its advantages of simple operation, high accuracy, high throughput, rapid identification, rich database resources, and low cost, this technology has been widely used in global microbial laboratories. Compared with traditional microbial identification methods, MALDI-TOF MS can significantly shorten the identification time, providing strong support for the accurate and rapid identification of bacteria and fungi, and is of great significance for improving the efficiency of clinical diagnosis and treatment. The microbial database based on MALDI-TOF MS technology has become a common method for clinically identifying bacteria, shortening the identification time to minutes. This database contains more than 4,000 strains, with an accuracy rate of 90% at the species level.

[0005] However, currently, when most studies combine two independent and complete systems, namely the microfluidic system and matrix-assisted laser desorption / ionization mass spectrometry, it is necessary to manually transfer the sample to the target plate of matrix-assisted laser desorption / ionization mass spectrometry for off-line analysis before mass spectrometry. Even when using a microfluidic device to prepare a sample array, the mass spectrometry surface also needs to be manually transferred from the microfluidic device to the mass spectrometry system. Therefore, how to use matrix-assisted laser desorption / ionization mass spectrometry for microfluidic integration to complete on-line analysis of samples without manual intervention is the main problem currently faced. To achieve on-line analysis of the two, the existing design idea of the combined device of microfluidics and matrix-assisted laser desorption / ionization mass spectrometry is to process and design a fixed microchannel to achieve the manipulation of continuous droplets, but the technical difficulty and processing technology of this design are undoubtedly complex. Summary of the Invention

[0006] The object of the present invention is to provide a method and device for material analysis that directly integrates a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry. After directly coupling the upper plate provided with hydrophilic sites and a customized target plate and sending them into the mass spectrometer, manual operations can be reduced, and automated analysis can be achieved. This method has the potential for high-throughput, sensitive, and specific analysis.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides an analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, including:

[0009] A digital microfluidic system includes a digital microfluidic chip and a micro-droplet driving platform; the digital microfluidic chip includes an upper plate, a lower plate and a conductive gasket therebetween, the upper plate includes an upper substrate and a conductive layer provided on the lower surface of the upper substrate, an upper hydrophobic layer is provided on the lower surface of the conductive layer, and a plurality of hydrophilic sites are arranged at intervals on the surface of the upper hydrophobic layer, and each hydrophilic site is a pit recessed inward toward the conductive layer; the lower plate includes a lower substrate and a dielectric layer provided on the upper surface of the lower substrate, an electrode array is arranged between the lower substrate and the dielectric layer, and a lower hydrophobic layer is provided on the upper surface of the dielectric layer;

[0010] Matrix-assisted laser desorption / ionization mass spectrometry includes a target plate substrate, and a groove for placing the upper plate of the digital microfluidic chip is provided on the upper surface of the target plate substrate.

[0011] In the above analysis device directly integrating the digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, the depth of each pit is 49 - 51 microns;

[0012] The lateral area of each pit is 4 - 16 mm 2 。

[0013] In the above analysis device directly integrating the digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, when preparing the upper plate, before forming the upper hydrophobic layer on the lower surface of the conductive layer, a cover sheet is fixed at the pre-designed hydrophilic sites on the lower surface of the conductive layer, and after forming the upper hydrophobic layer, the cover sheet is peeled off to form the pit at the position of the cover sheet;

[0014] The material of the upper hydrophobic layer is polytetrafluoroethylene, and the upper hydrophobic layer is fabricated by spin coating on the lower surface of the conductive layer;

[0015] The material of the cover sheet is polydimethylsiloxane (PDMS).

[0016] In the above analysis device directly integrating the digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, the electrode array includes a reservoir electrode, a driving electrode, a ground electrode and a contact electrode; a plurality of reservoir electrodes are arranged in a two-dimensional array to form a two-dimensional reservoir electrode array, there are voids in the two-dimensional reservoir electrode array, and a plurality of driving electrodes are arranged in the voids of the two-dimensional reservoir electrode array, and the edge of each reservoir electrode is connected to the driving electrode located in the void of the two-dimensional reservoir electrode array through the corresponding driving electrode, each reservoir electrode, driving electrode and ground electrode are respectively connected to a contact electrode, and are connected to the micro-droplet driving platform through the contact electrode; the ground electrode is grounded; the conductive layer in the upper plate is connected to the ground electrode through the conductive gasket.

[0017] In the above analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, several of the hydrophilic sites are located at the corresponding positions of the driving electrodes on the upper plate.

[0018] In a second aspect, the present invention provides an analysis method for directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry. Using the analysis device described in any one of the above, the method includes the following steps:

[0019] Attach droplets containing the sample to be measured and the matrix solution to the upper plate at the hydrophilic sites of the microfluidic chip. Place the upper plate in the groove of the target plate with the hydrophilic sites facing outside the groove, and send it into the matrix-assisted laser desorption / ionization mass spectrometry for analysis.

[0020] In the above analysis method for directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, the sample to be measured is bacteria, verapamil, or insulin;

[0021] The matrix solution is a solution containing 2,5-dihydroxybenzoic acid or a solution containing α-cyano-4-hydroxycinnamic acid.

[0022] As an example, when performing quantitative analysis on a verapamil solution, mix the matrix solution with verapamil solutions of different concentrations at the hydrophilic sites on the upper plate of the digital microfluidic chip;

[0023] Preferably, the matrix solution is composed of 2,5-dihydroxybenzoic acid, acetonitrile, deionized water, and trifluoroacetic acid. The concentration of 2,5-dihydroxybenzoic acid is 20 mg / mL, and the volume ratio of acetonitrile, deionized water, and trifluoroacetic acid is 50%:47.5%:2.5%. The volume ratio of the matrix solution to the verapamil solution is 1:1. The sample is dropped 3 times for each concentration, 1 μL each time, and each sample is irradiated with laser 5 times. The data is collected and averaged. The machine parameters of the matrix-assisted laser desorption / ionization mass spectrometry are designed as follows: voltage 10.5x, laser frequency 1000 Hz, laser intensity 100%, method LP_700 - 2000_Da.

[0024] As an example, when performing fingerprint identification on Escherichia coli, mix Escherichia coli, lysis solution, and matrix solution at the hydrophilic sites on the upper plate of the digital microfluidic chip;

[0025] Preferably, the lysis solution is an aqueous formic acid solution with a volume fraction of 70%, the matrix solution is a solution of α-cyano-4-hydroxycinnamic acid with a concentration of 10 mg / mL, and the solvent is composed of acetonitrile, deionized water, and trifluoroacetic acid with a volume percentage of 50%:47.5%:2.5%. 1 μL of the lysis solution and 1 μL of the matrix solution are added dropwise to each single colony of Escherichia coli; 3 samples are inoculated for each sample, and each time a single colony of Escherichia coli is inoculated. Each sample is laser-irradiated 5 times, and the data is collected and averaged; the machine parameters of the matrix-assisted laser desorption ionization mass spectrometry are designed as follows: voltage 10x, laser frequency 1000 Hz, laser intensity 100%, method MBT_PC.

[0026] As an example, further, Escherichia coli, the lysis solution, and the matrix solution are mixed according to the following steps: a) The sample solution of Escherichia coli and the lysis solution are respectively dropped into the digital microfluidic chip, and the chip is controlled to split the two droplets respectively. After mixing the two split droplets, they are moved to the hydrophilic site on the upper plate of the microfluidic chip; b) The matrix solution is dropped into the digital microfluidic chip, and the chip is controlled to split the droplet, and the droplet is moved to the hydrophilic site on the upper plate of the microfluidic chip to converge with the droplet in step a).

[0027] Based on the above technical solutions, the present invention has the following beneficial effects:

[0028] The present invention directly integrates a digital microfluidic chip with matrix-assisted laser desorption ionization mass spectrometry, combines the advantages of the two, and achieves the goals of automation, high throughput, rapid operation, low sample consumption, and accurate analysis.

[0029] (1) Automation and high throughput: The direct coupling of microfluidic technology and mass spectrometry can achieve automated sample processing and analysis, as well as high-throughput operation. When using a pipette to inject samples and solvents into different electrodes, the chip controls the movement and operation process of the droplets through programming, realizing automated pretreatment of biological samples, improving the repeatability, stability, and the number of samples processed in the experiment. After directly coupling the upper plate and the customized target plate and sending them into the mass spectrometer, manual operation is reduced, and automated analysis is achieved. Compared with the prior art, the present invention can flexibly and dynamically process different trace samples through the discrete droplet chip and target plate design, and has application potential in terms of equipment use efficiency and processing complex samples.

[0030] (2) Rapid operation: Microfluidic technology has the characteristics of rapid response and real-time control. By applying an electric field or mechanical force, the position and movement of droplets can be quickly manipulated. This can greatly shorten the sample pretreatment time and improve the operation efficiency. Rapid operation can also reduce sample loss and variation, and improve the consistency and repeatability of the results.

[0031] (3) Low sample consumption: Microfluidic technology has the advantages of small sample volume and reagent conservation in sample processing. Through the droplet operation module of the microfluidic chip, trace samples and reagents can be precisely mixed, reducing the consumption of samples and reagents. This is particularly important for the research of rare samples or expensive reagents, not only saving costs but also maximizing the utilization of limited sample resources.

[0032] (4) Accurate analysis: Matrix-assisted laser desorption ionization mass spectrometry has uniform ionization, and the ionized fragments carry only a single charge. It has high sensitivity, high resolution, high analysis speed, high specificity, and high reproducibility, with a wide mass range for determination. Description of the Drawings

[0033] Figure 1 It is a side view of the digital microfluidic chip in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0034] Figure 2 It is a front view of the digital microfluidic chip in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0035] Figure 3 It is a schematic diagram of the target plate structure in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0036] Figure 4 It is a schematic diagram for showing the position of hydrophilic sites in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0037] Figure 5 It is the actual parameters of the customized target plate in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0038] Figure 6 It is a process flow chart for the preparation of hydrophilic sites in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0039] Figure 7 It is a physical photo of the original target plate provided in the embodiment of the present invention.

[0040] Figure 8 It is a physical photo of the target plate in the substance analysis device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption ionization mass spectrometry provided in the embodiment of the present invention.

[0041] Figure 9It is a physical photo of placing a digital microfluidic chip in a groove of a target plate provided in the invention embodiment. Among them, the position of the square frame in the figure indicates the position of the hydrophilic site.

[0042] Figure 10 It is the concentration gradient curve of verapamil under different target plate combinations in Embodiment 3 of the present invention.

[0043] Figure 11 It is the fingerprint spectrum identification evaluation method and the fingerprint spectra under different target plate combinations in Embodiment 4 of the present invention.

[0044] Figure 12 It is the on-chip processing flow of digital microfluidic bacteria in Embodiment 5 of the present invention.

[0045] Figure 13 It is the bacteria identification results of the combined target plate and the original target plate in Embodiment 5 of the invention.

[0046] Figures 1 - 3 The marks in it are as follows:

[0047] 100 - upper substrate; 101 - conductive layer; 102 - upper hydrophobic layer; 103 - hydrophilic site; 104 - droplet;

[0048] 110 - lower substrate; 111 - dielectric layer; 112 - electrode array; 113 - lower hydrophobic layer;

[0049] 1120 - reservoir electrode; 1121 - driving electrode; 1122 - ground electrode; 1123 - contact electrode;

[0050] 12 - conductive gasket;

[0051] 30 - target plate;

[0052] 300 - groove;

[0053] 301 - hole slot. Detailed implementation manners

[0054] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0055] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by terms such as "upper" and "lower" is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the system or component referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present invention.

[0056] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "assembly", "setting", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0057] Next, with reference to the drawings, the substance analysis device and method of a direct integration digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry provided by the embodiments of the present invention will be described in detail.

[0058] Existing digital microfluidic chips include an upper plate, a lower plate, and a conductive gasket therebetween. The upper plate includes an upper substrate and a conductive layer provided on the lower surface of the upper substrate. The lower surface of the conductive layer is provided with an upper hydrophobic layer. The lower plate includes a lower substrate and a dielectric layer on the upper surface of the lower substrate. An electrode array is arranged between the lower substrate and the dielectric layer, and the upper surface of the dielectric layer is provided with a lower hydrophobic layer. When in use, the droplet sample is between the upper hydrophobic layer and the lower hydrophobic layer, and operations such as sample movement and mixing are realized under the action of electrodes. The sample consumption is small and the processing method is simple. However, when used in combination with matrix-assisted laser desorption / ionization mass spectrometry, the operator needs to use a pipette to manually transfer the solution collected after preprocessing the digital microfluidic chip to the target plate of the matrix-assisted laser desorption / ionization mass spectrometry and dry and crystallize it. This is because, in order to facilitate the movement of droplets, the sample droplets in the existing microfluidic chips are located between the upper plate and the lower plate and both the upper and lower sides of the droplets are hydrophobic layers. When the sample needs to be transferred to the target plate, the preprocessed droplets need to be collected outside the chip and then manually spotted. As described in the background art, in order to realize the online analysis of samples by combining a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, the prior art mainly designs fixed microchannels on the digital microfluidic chip. For example, CN 115445677A discloses a microfluidic chip combined with MALDI-TOF-MS analysis, which has a concentration gradient generation network, a micro-SPE channel loaded with magnetic beads, and a chip-mass spectrometry interface, but its structure is complex and it is only applicable to the analysis of protein fingerprints.

[0059] To solve the above problems, in the first part, the present invention provides an analytical device that directly integrates a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry. The device includes:

[0060] As Figures 1 - 2 shown, the digital microfluidic system includes a digital microfluidic chip and a microdroplet driving platform. The digital microfluidic chip includes an upper plate, a lower plate, and a conductive gasket therebetween. The upper plate includes an upper substrate 100 and a conductive layer 101 provided on the lower surface of the upper substrate. The lower surface of the conductive layer 101 is provided with an upper hydrophobic layer 102, and a plurality of hydrophilic sites 103 are arranged at intervals on the surface of the upper hydrophobic layer 102. Each hydrophilic site is a concave pit that is recessed inward in the direction of the conductive layer 101; the lower plate includes a lower substrate 110 and a dielectric layer 111 provided on the upper surface of the lower substrate. An electrode array 112 is arranged between the lower substrate 110 and the dielectric layer 111, and a lower hydrophobic layer 113 is provided on the upper surface of the dielectric layer;

[0061] As Figure 3 shown, the matrix-assisted laser desorption / ionization mass spectrometry includes a target plate 30, and a groove 300 for placing the upper plate 10 of the digital microfluidic chip is provided on the upper surface of the target plate.

[0062] Based on the above technical solutions, the present invention sets the hydrophilic sites 103 on the upper plate. During use, the droplet 104 is moved to the hydrophilic sites 103 on the upper plate. Since the hydrophilic sites have wettability compared with the hydrophobic layer, the sample droplet adheres to the hydrophilic sites 103 on the upper plate through surface tension. When the upper plate is coupled with the target plate, the droplet 104 can enter the matrix-assisted laser desorption / ionization mass spectrometry with the target plate, so as to directly perform on-line analysis.

[0063] In the above embodiment, in order to better make the droplet adhere to the hydrophilic site, the depth of each said concave pit (i.e., the distance between the bottom of the concave pit and the surface of the upper hydrophobic layer 102) is 49 - 51 microns, such as 50 microns;

[0064] The lateral area of each said concave pit is 4 - 16 mm 2 (equivalent to the size of 1 - 4 electrodes), which can be adjusted according to specific experimental requirements, such as 4 mm 2 .

[0065] For convenience of operation, when preparing the upper plate, before forming the upper hydrophobic layer 102 on the lower surface of the conductive layer 101, a cover sheet is fixed at each hydrophilic site pre-designed on the lower surface of the conductive layer 101. After the upper hydrophobic layer 102 is formed, the cover sheet is peeled off to form a pit at the position of the cover sheet. Specifically, the material of the upper hydrophobic layer 102 is polytetrafluoroethylene (the material of the lower hydrophobic layer is the same as that of the upper hydrophobic layer). The upper hydrophobic layer is fabricated on the lower surface of the conductive layer 101 by spin coating. For example, after spin coating polytetrafluoroethylene, thermal cross-linking is performed to form the upper hydrophobic layer. In the practice of the present invention, it is found that when cover sheets of different materials are selected to prepare the hydrophilic sites, the hydrophilicity of the finally obtained hydrophilic sites is different. Preferably, the material of the cover sheet is PDMS. Moreover, if the thickness of the PDMS film is too thin, it will be difficult to remove the film block and affect the regular shape at the junction of the hydrophobic layer and PDMS; if it is too thick, the spin coating of Teflon will result in uneven spin coating of the chip center and the hydrophobic layer cannot be formed. For example, each cover sheet is a square PDMS film with a thickness of 0.5 mm and a size of 2 mm×2 mm.

[0066] In the above embodiment, as Figure 2 shown, the electrode array 112 includes a reservoir electrode 1120, a driving electrode 1121, a ground electrode 1122, and a contact electrode 1123; eight reservoir electrodes 1120 are arranged in a 4×2 two-dimensional array to form a two-dimensional reservoir electrode array. There are voids in the two-dimensional reservoir electrode array, and multiple driving electrodes 1121 are arranged in the voids of the two-dimensional reservoir electrode array. Moreover, the edge of each reservoir electrode 1120 is connected to the driving electrode 1121 located in the void of the two-dimensional reservoir electrode array through the corresponding driving electrode. Each reservoir electrode 1120, driving electrode 1121, and ground electrode 1122 are respectively connected to a contact electrode 1123 and are connected to the micro-droplet driving platform 2 through the contact electrode 1123; the ground electrode 1122 is grounded; the conductive layer 101 in the upper plate is connected to the ground electrode 1122 through the conductive gasket 12.

[0067] It can be understood that the sample addition position can be set on the reservoir electrode 1120. During use, the reagent and the sample solution are respectively placed on different reservoir electrodes. First, the micro-droplet driving platform is controlled to energize the reservoir electrode containing the solution. The reservoir electrode generates a dielectrophoretic wetting force. Then, the driving electrode connected to one side of the reservoir electrode containing the solution is energized simultaneously. The driving electrode generates a dielectrophoretic wetting force to pull the solution, making the solution form a long strip. Then, the reservoir electrode is powered off. At this time, the solution is not affected by the dielectrophoretic wetting force of the reservoir electrode and is only affected by the dielectrophoretic wetting force of one side driving electrode, thereby realizing sample injection.

[0068] When the sample solution needs to be split, first control the micro-droplet driving platform to energize the liquid storage pool electrode containing the reagent or sample solution. The liquid storage pool electrode generates a dielectric wetting force. Then, simultaneously energize the driving electrodes connected to both sides of the liquid storage pool electrode containing the reagent or sample solution. The driving electrodes on both sides generate a dielectric wetting force and pull the solution to both sides, causing the solution to form a strip shape. Then, cut off the power supply of the liquid storage pool electrode. At this time, the solution is not affected by the dielectric wetting force of the liquid storage pool electrode and is only affected by the dielectric wetting forces of the driving electrodes on both sides. Thus, the driving electrodes on both sides pull the solution to both sides, and finally the solution is divided into two independent droplets and are respectively located on the driving electrodes on both sides.

[0069] When the movement of the droplet needs to be controlled, according to the movement path, control the micro-droplet driving platform to energize the driving electrodes located on the movement path of the droplet in sequence, and respectively drive the droplet to move along the set movement path until the droplet is driven to the hydrophilic site, or the reagent and the sample solution are respectively driven to the hydrophilic site for mixing.

[0070] In the above embodiments, as Figure 4 shown, several hydrophilic sites can be respectively located at the corresponding positions of the driving electrode 1121 in the upper plate, that is, the corresponding positions on the surface of the upper hydrophobic layer in the upper plate corresponding to the driving electrode 1121. Preferably, the hydrophilic sites can be set at different position electrodes according to experimental needs, but preferably at the peripheral electrodes, so as not to interfere with the movement of the droplet during the experiment, resulting in the droplet being fixed by contacting the hydrophilic site. The positions of the hydrophilic sites can be adjusted accordingly according to the experimental design.

[0071] In the above embodiments, optionally, the material of the upper substrate can be a transparent material, such as glass or acrylic plate;

[0072] The material of the conductive layer can be indium tin oxide;

[0073] The material of the lower substrate can be one of glass, silicon, paper, polyester film and printed circuit board;

[0074] The liquid storage pool electrode, the driving electrode, the ground electrode and the contact electrode are made of polysilicon, metal or their oxides;

[0075] The driving electrode is a chromium electrode or an indium tin oxide electrode.

[0076] In the above embodiments, optionally, the material of the dielectric layer is Parylene C, SU 8 photoresist, SiO2, Si3N4, Al2O3, polydimethylsiloxane or parylene.

[0077] It can be understood that the device of the present invention may further include a host computer for controlling the micro-droplet driving platform.

[0078] In the above embodiments, the target plate is the target plate for dropping samples in matrix-assisted laser desorption / ionization mass spectrometry, and its material can be stainless steel, preferably 316 stainless steel. Compared with other stainless steels, its advantages in preparing mass spectrometry target plates are mainly reflected in its excellent corrosion resistance, high-temperature stability, mechanical strength, and low magnetic properties.

[0079] In the second part, the present invention provides an analysis method that directly integrates a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, including the following steps: making droplets containing the sample to be measured and the matrix solution adhere to the upper plate at the hydrophilic sites of the microfluidic chip, placing the upper plate in the groove of the target plate with the hydrophilic sites facing outside the groove, and feeding it into the matrix-assisted laser desorption / ionization mass spectrometry (MALDI-TOF MS) for analysis. In the present invention, the sample is pre-treated on the chip and transferred to the upper plate with hydrophilic sites, and the upper plate is directly coupled with a customized target plate and fed into the mass spectrometer, so as to realize the on-line analysis of the sample to be measured, reduce manual operations, and improve the repeatability, stability of the experiment and the number of samples processed.

[0080] In the above embodiments, the sample to be measured is bacteria, verapamil or insulin; the bacteria can specifically be Escherichia coli or Klebsiella pneumoniae;

[0081] The matrix solution is a solution containing 2,5-dihydroxybenzoic acid or a solution containing α-cyano-4-hydroxycinnamic acid;

[0082] The analysis can specifically be quantitative analysis or identification of bacterial species; specifically, when the sample to be measured is verapamil or insulin, the analysis is quantitative analysis; when the sample is bacteria, the analysis is fingerprint identification;

[0083] In a specific embodiment of the present invention, when performing quantitative analysis on a verapamil solution, the matrix solution is mixed with verapamil solutions of different concentrations at the hydrophilic sites of the upper plate of the digital microfluidic chip; for example, the matrix solution is mixed with verapamil solutions of different concentrations and dropped at the hydrophilic sites of the upper plate of the microfluidic chip.

[0084] Preferably, the matrix solution is composed of 2,5-dihydroxybenzoic acid, acetonitrile, deionized water and trifluoroacetic acid. The concentration of 2,5-dihydroxybenzoic acid is 20 mg / mL, and the volume ratio of acetonitrile, deionized water and trifluoroacetic acid is 50%:47.5%:2.5%. The volume ratio of the matrix solution to the verapamil solution is 1:1. The sample is dropped 3 times at each concentration, 1 μL each time, and each sample is irradiated with laser 5 times. The data is collected and averaged. The machine parameters of the matrix-assisted laser desorption / ionization mass spectrometry are designed as follows: voltage 10.5x, laser frequency 1000 Hz, laser intensity 100%, method LP_700-2000_Da. More specifically, the concentration of the verapamil solution is 0.000286 - 0.1573 mMol / L, such as 0.1573, 0.0286, 0.01573, 0.00286, 0.001573, 0.000286 mMol / L, and the solvent is acetone.

[0085] In another specific embodiment of the present invention, when performing fingerprint identification on Escherichia coli, Escherichia coli, a lysis solution and a matrix solution are mixed at the hydrophilic sites on the upper plate of the digital microfluidic chip.

[0086] Preferably, the lysis solution is an aqueous formic acid solution with a volume fraction of 70%, and the matrix solution is a solution of α-cyano-4-hydroxycinnamic acid with a concentration of 10 mg / mL. The solvent is composed of acetonitrile, deionized water and trifluoroacetic acid with a volume percentage of 50%:47.5%:2.5%. 1 μL of the lysis solution and 1 μL of the matrix solution are dropped for each single colony of Escherichia coli. Each sample is inoculated with 3 replicates, each time inoculating a single Escherichia coli colony. Each sample is irradiated with laser 5 times. The data is collected and averaged. The machine parameters of the matrix-assisted laser desorption / ionization mass spectrometry are designed as follows: voltage 10x, laser frequency 1000 Hz, laser intensity 100%, method MBT_PC.

[0087] Among them, the mixing method can be directly inoculating Escherichia coli at the hydrophilic sites on the upper plate of the digital microfluidic chip, and then successively dropping the lysis solution and the matrix solution. Preferably, Escherichia coli, the lysis solution and the matrix solution are mixed according to the following steps: a) Drop the sample solution of Escherichia coli and the lysis solution into the digital microfluidic chip respectively, control the chip to split the two droplets respectively, mix the two split droplets and move them to the hydrophilic sites on the upper plate of the microfluidic chip; b) Drop the matrix solution into the digital microfluidic chip, control the chip to split the droplet, and move the droplet to the hydrophilic sites on the upper plate of the microfluidic chip to converge with the droplet in step a).

[0088] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0089] Unless otherwise specified, the methods used in the following embodiments are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial sources.

[0090] The sources of the various materials and reagents in the following embodiments are as follows:

[0091] Trifluoroacetic acid, acetonitrile, and ultrapure water are all from Sigma-Aldrich, USA;

[0092] 2,5-Dihydroxybenzoic acid (purity > 99.0%) and α-cyano-4-hydroxycinnamic acid (purity > 98.0%) are both from Tokyo Chemical Industry Co., Ltd., Shanghai;

[0093] The main agent and curing agent of polydimethylsiloxane are from Dow Corning Sylgard 184, USA;

[0094] Polytetrafluoroethylene is from Shanghai Gisco Special Coating Co., Ltd.;

[0095] Chrome-plated glass is from Luoyang Guluo Glass Co., Ltd.;

[0096] Parylene C is manufactured by Suzhou Marqi Nano Technology Co., Ltd. on behalf of others;

[0097] Phosphate buffer solution is from Shanghai Macklin Biochemical Co., Ltd.;

[0098] Verapamil (purity > 98%) is from Shanghai Macklin Biochemical Co., Ltd.;

[0099] Acetone is from Beijing Tongguang Fine Chemical Co., Ltd.;

[0100] Formic acid is from Tianjin Fuchen Chemical Reagent Co., Ltd.;

[0101] Escherichia coli and Klebsiella pneumoniae are from Aerospace Center Hospital;

[0102] LB solid medium is from Beijing Solarbio Science & Technology Co., Ltd.;

[0103] Deionized water is from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0104] The chromium electrode is manufactured by Changsha Chromium Plate Factory on behalf of others.

[0105] Example 1. Analytical Device Integrating Digital Microfluidic Chip and Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry Directly - I. Device Structure

[0106] As Figures 1 - 3 shown, the analytical device integrating digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry provided in this example includes a digital microfluidic system and a matrix-assisted laser desorption / ionization mass spectrometry;

[0107] The digital microfluidic system includes a digital microfluidic chip 1 and a micro-droplet driving platform 2. The digital microfluidic chip 1 includes an upper plate 10, a lower plate 11 and a conductive gasket 12 therebetween. The upper plate 10 includes an upper substrate 100 made of glass and a conductive layer 101 disposed on the lower surface of the upper substrate. The conductive layer 101 is an indium tin oxide layer with a thickness of 185 nm. An upper hydrophobic layer 102 made of polytetrafluoroethylene is provided on the lower surface of the conductive layer 101. A number of hydrophilic sites 103 are arranged on the surface of the upper hydrophobic layer 102. Each hydrophilic site is a pit indented towards the conductive layer. The depth of each pit is 49 - 51 microns, and the lateral area of each pit is 4 - 16 mm 2 ; The lower plate 11 includes a lower substrate 110 made of glass and a dielectric layer 111 disposed on the upper surface of the lower substrate. The dielectric layer is made of Parylene C with a thickness of 7 microns. An electrode array 112 is arranged between the lower substrate 110 and the dielectric layer 111. A lower hydrophobic layer 113 made of polytetrafluoroethylene is provided on the upper surface of the dielectric layer; The electrode array 112 includes a reservoir electrode 1120 (50 mm 2 ), a driving electrode 1121 (4 mm 2, a chromium electrode), a ground electrode 1122, and a contact electrode 1123; eight reservoir electrodes 1120 are arranged in a 4×2 two-dimensional array to form a two-dimensional reservoir electrode array. There are gaps in the two-dimensional reservoir electrode array. A plurality of driving electrodes 1121 are arranged in the gaps of the two-dimensional reservoir electrode array. And the edge of each reservoir electrode 1120 is connected to the driving electrode 1121 located in the gap of the two-dimensional reservoir electrode array through a corresponding driving electrode. Each reservoir electrode 1120, driving electrode 1121, and ground electrode 1122 are respectively connected to a contact electrode 1123 and connected to the micro-droplet driving platform 2 through the contact electrode 1123; the ground electrode 1122 is grounded; the conductive layer 101 in the upper plate is connected to the ground electrode 1122 through a conductive gasket 12; a number of hydrophilic sites are located at the corresponding positions of the driving electrodes 1121 in the upper plate; the conductive gasket 12 is a 100-micron-thick conductive tape; the micro-droplet driving platform 2 is DropBot v3.0, developed by Sci-Bots Inc. in Canada. It is a highly flexible digital microfluidic system equipped with a 60W 12V power supply, supporting an input voltage of 100 - 240VAC, and suitable for laboratory standards worldwide. The platform provides an intuitive graphical interface through the open-source MicroDrop, enabling users to control experimental operations visually;

[0108] The matrix-assisted laser desorption / ionization mass spectrometry includes a target plate substrate 30 made of 316 stainless steel. The upper surface of the target plate substrate 30 is provided with a groove 300 for placing the upper plate of the digital microfluidic chip. The actual parameters are shown in Figure 5 , and the hole 301 is used for fixedly installing the identification device; the matrix-assisted laser desorption / ionization mass spectrometry uses an Autoflex Speed mass spectrometer produced by Bruker Corporation. This is an advanced device adopting innovative MALDI-TOF and TOF / TOF technologies. The device is configured with smartbeam-II laser technology and multiple detection modes including single linear mode, high-resolution reflectance mode, and tandem mass spectrometry mode. In addition, it also includes a 384-well stainless steel target plate for high-throughput analysis. The present invention is all based on the linear mode detection in the positive ion mode.

[0109] II. Preparation

[0110] 1. Fabrication of hydrophilic sites on the upper plate of the digital microfluidic chip

[0111] According to Figure 6 the flowchart shown, prepare the hydrophilic sites. The specific steps are as follows:

[0112] (1) As Figure 6As shown in A-C, the main agent of polydimethylsiloxane (PDMS) with a volume ratio of 10:1 was mixed with the PDMS curing agent evenly. After being evenly spread in a petri dish and left standing without bubbles, it was placed in a high-temperature oven at 80 °C for thermal cross-linking for 120 minutes, and then cooled for 12 hours for standby.

[0113] (2) As shown in Figure 6 D-E, the PDMS film was cut into film blocks of 2 mm×2 mm×0.5 mm, and was compacted and fixed on the upper plate of the digital microfluidic chip.

[0114] (3) The modified upper plate was placed in a spin coater (KW-4C bench-top spin coater of Beijing Saidekaisi Electronics Co., Ltd.) in the area outside the PDMS film block ( Figure 6 F) and polytetrafluoroethylene was spin-coated according to the set program. It was placed in a high-temperature oven at 100 °C for thermal cross-linking for 20 minutes. After cooling for 2 h, the PDMS film block was removed to form a hydrophilic site ( Figure 6 H). The unprocessed upper plate of the digital microfluidic chip is shown in Figure 6 I. Figure 6 I.

[0115] 2. Customization of matrix-assisted laser desorption / ionization mass spectrometry target plate

[0116] As shown in Figure 7 the original target plate, a groove was machined in the center of the substrate with a milling cutter (the texture in the groove is the machining trace), which is used to place the upper plate of the hydrophilic site of the digital microfluidic. The size of the groove matches the upper plate of the digital microfluidic chip. Three holes are used to fixedly install the identification device to realize the integrated integration of digital microfluidics and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry application. The physical photo of the customized target plate is shown in Figure 8 The physical photo of placing the digital microfluidic chip in the groove of the customized target plate is shown in Figure 9 .

[0117] Example 2. Hydrophilic effect of hydrophilic sites fabricated with different material cover sheets

[0118] In this example, the effects of hydrophilic sites fabricated with different material cover sheets were compared, and the steps are as follows:

[0119] (1) Fabrication of hydrophilic sites: According to the preparation steps in Example 1, 7 different material tapes and PDMS films were respectively cut into 2 mm×8 mm and covered on the upper plate of the digital microfluidic. After modification, the upper plate was spin-coated with polytetrafluoroethylene, placed in a high-temperature oven at 100 °C for thermal cross-linking for 20 minutes, and after cooling for 2 h, the tape and PDMS film block were removed respectively.

[0120] (2) Evaluation of hydrophilic effect: Visible observation of gum residue was used. And 1 μL of ultrapure water was spotted on the hydrophilic site, and the contact angle was measured by a water contact angle measuring instrument to evaluate the hydrophilic effect. The experiment was repeated three times.

[0121] (3) Results: According to the evaluation index, PDMS was finally selected as the material for manufacturing the hydrophilic sites. And the contact angle values of 1 μL of ultrapure water on the left and right sides of the hydrophilic sites and the contact angle values of the left and right sides of the polytetrafluoroethylene hydrophobic layer were measured to prove the hydrophilic effect of the hydrophilic sites. The experimental results are shown in Table 1.

[0122] Table 1. Evaluation of the hydrophilic effect of hydrophilic sites made of different materials

[0123]

[0124] Based on the Young-Laplace equation, the hydrophilicity of the hydrophilic sites made of different materials was evaluated as follows:

[0125] 1) When the contact angle θc = 0, complete wetting;

[0126] 2) When the contact angle θc < 90°, partial wetting or wetting, hydrophilic;

[0127] 3) When the contact angle θc = 90°, the dividing line between wetting and non-wetting;

[0128] 4) When the contact angle θc > 90°, non-wetting, hydrophobic;

[0129] 5) When the contact angle θc = 180°, complete non-wetting;

[0130] It can be seen from the results in Table 1 that, on the premise of no colloid residue visible to the naked eye, when PDMS was selected, the contact angle values of ultrapure water on the left and right sides of the hydrophilic sites were the smallest. Therefore, when PDMS was selected as the material for manufacturing the hydrophilic sites, the hydrophilic effect was the best.

[0131] Example 3. Concentration gradient experiment of verapamil under different target plate combinations

[0132] The mass spectrometry intensity of verapamil solutions with different concentrations was tested according to the following steps:

[0133] (1) Preparation of verapamil solutions with different concentrations: 1.3 mg of verapamil weighed by an electronic balance was put into a test tube containing 1 ml of acetone solution to prepare a 2.86 mMol / L verapamil solution. The solution was diluted proportionally (diluted with acetone) to 2.86, 1.573, 0.286, 0.1573, 0.0286, 0.01573, 0.00286, 0.001573, 0.000286 mMol / L verapamil solutions and stored at 4 °C for standby.

[0134] (2) Matrix-assisted laser desorption / ionization mass spectrometry detection: Mix the 2,5-dihydroxybenzoic acid matrix solution (DHB 20 mg / mL: acetonitrile 50%, deionized water 47.5%, trifluoroacetic acid 2.5%) and the verapamil solution in a volume ratio of 1:1. Respectively on the original target plate (as Figure 7 shown), custom target plate (as Figure 8 shown, spotted on the plane of the outer target plate of the groove), and the hydrophilic sites on the upper plate of the digital microfluidic chip on the custom target plate (as Figure 9 shown, the hydrophilic sites marked by the square). Assemble the custom target plate and the upper plate with the final mixed liquid droplets using conductive tape and dry to crystallize the substances. Drop 3 samples of each concentration, 1 μL each time. Laser each sample 5 times and collect data to calculate the average value. Matrix-assisted laser desorption / ionization mass spectrometry machine parameter design: voltage 10.5x, laser frequency 1000 Hz, laser intensity 100%, method LP_700-2000_Da. Use the concentration as the abscissa and the absolute signal intensity at that concentration as the ordinate to establish a concentration gradient curve.

[0135] (3) Results: The experimental results are as Figure 10 shown. It can be seen that the concentration gradient curves of verapamil under the original target plate and the custom target plate are highly consistent. Although the intensity of verapamil on the upper plate decreases, it can also detect the same low concentration range of 3 orders of magnitude as the two target plates.

[0136] Example 4. Fingerprint identification experiment of Escherichia coli under different target plate combinations

[0137] (1) Preparation of fresh Escherichia coli single colonies: Inoculate fresh pure Escherichia coli on LB solid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and agar 15 g / L) using the streak plate method and culture in a 37°C incubator for 15 h.

[0138] (2) Matrix-assisted laser desorption / ionization mass spectrometry detection: Respectively on the original target plate (as Figure 7 shown), custom target plate (as Figure 8 shown, spotted on the plane of the outer target plate of the groove), and inoculate Escherichia coli single colonies on the upper plate of the digital microfluidic chip on the custom target plate (as Figure 9As shown, inoculate at the hydrophilic sites marked by the square. Use 1 μL of 70% formic acid aqueous solution to lyse the bacteria, and add 1 μL of α-cyano-4-hydroxycinnamic acid (HCCA 10 mg / mL: 50% acetonitrile, 47.5% deionized water, 2.5% trifluoroacetic acid) as the matrix solution to transfer laser energy. Each target plate combination is inoculated with the sample 3 times, and each time a single Escherichia coli colony is inoculated. Laser the sample 5 times for each sample to collect data. Matrix-assisted laser desorption ionization mass spectrometry machine parameter design: voltage 10x, laser frequency 1000 Hz, laser intensity 100%, method MBT_PC.

[0139] (3) Fingerprint identification: Use the commercial mass spectrometry database MBT RTC 4.0 (MALDI Biotyper Real Time Classification 4.0) for comparison to accurately identify specific microorganisms, thereby determining the microbial species level.

[0140] (4) Verify known Klebsiella pneumoniae: Inoculate a single colony of Klebsiella pneumoniae (cultured on LB solid medium) on the digital microfluidic chip on the custom target plate (inoculate at the hydrophilic sites). Use 1 μL of 70% formic acid aqueous solution to lyse the bacteria, and add 1 μL of α-cyano-4-hydroxycinnamic acid (HCCA 10 mg / mL: 50% acetonitrile, 47.5% deionized water, 2.5% trifluoroacetic acid) as the matrix solution to transfer laser energy. Conduct the experiment 3 times, and laser the sample 5 times for each sample to collect data. Matrix-assisted laser desorption ionization mass spectrometry machine parameter design: voltage 10x, laser frequency 1000 Hz, laser intensity 100%, method MBT_PC. Use the commercial mass spectrometry database MBT RTC 4.0 (MALDI Biotyper Real Time Classification 4.0) to identify whether the strain is Klebsiella pneumoniae. The results are shown in Table 2. This method identifies the bacterial species - Klebsiella pneumoniae, which is consistent with the known Klebsiella pneumoniae strain provided clinically.

[0141] Table 2. Fingerprint experiment of Klebsiella pneumoniae on the chip

[0142]

[0143] Results: The results are shown in Table 3 and Figure 11 as shown, where Figure 11 Table 4 in Figure 11 is the evaluation method for fingerprint identification, Figure 11 A is the fingerprint of the original target plate, Figure 11C is the combined fingerprint of the custom-made target plate and the upper plate of digital microfluidics. The integration of the upper plate and the custom-made target plate can detect the species level of a certain low-concentration bacteria, and the scores are all above 2.3, reflecting the high repeatability, high sensitivity and high accuracy of this method.

[0144] Table 3. Repeatability experiment of fingerprint

[0145]

[0146] The evaluation method of grade and score can be seen in Figure 11 Table 4 in it.

[0147] Example 5. Identification experiment of Escherichia coli fingerprint under the integrated ion source of DMF and MALDI-MS

[0148] (1) Preparation of fresh Escherichia coli single colony: Inoculate fresh pure Escherichia coli on LB solid medium by the streak plate method and culture it in a constant temperature incubator at 37 °C for 15 h.

[0149] (2) Preparation of colony phosphate buffer solution: Select a loopful of colonies into a test tube, add phosphate buffer solution to make a sample solution and dilute it (the order of magnitude of the number of colonies is 10 6～7 ). Inject 2 μL into the digital microfluidic chip and split the droplet into 1 μL.

[0150] (3) Lysis: Inject 2 μL of 70% formic acid aqueous solution by volume into the digital microfluidic chip and split the droplet into 1 μL. Mix the two solutions and cycle three times to obtain 2 μL of mixed droplets. And move the droplets to the position of the lower plate electrode corresponding to the hydrophilic site on the upper plate.

[0151] (4) Transport the matrix solution to the hydrophilic site: Inject 2 μL of HCCA matrix solution (the same as in Example 4) into the digital microfluidics, split the droplet into 1 μL, and move the droplet to the position of the lower plate electrode corresponding to the hydrophilic site on the upper plate.

[0152] (5) Assembly: Assemble the custom-made target plate and the upper plate with the final mixed droplets using conductive tape and dry.

[0153] (1) Mass spectrometry analysis: Send the coupled target plate into a Bruker MALDI-TOF-MS machine. Machine parameter design: voltage 10x, laser frequency 1000 Hz, laser intensity 100%, method MBT_PC.

[0154] (2) Fingerprint identification: Compare with the commercial mass spectrometry database MBT RTC 4.0 (MALDI Biotyper Real Time Classification 4.0) to accurately identify specific microorganisms, so as to determine the species level of microorganisms.

[0155] In this embodiment, as Figure 12 shown, the lysis solution, the colony phosphate buffer solution, and the HCCA matrix solution ( Figure 12 -A) are sequentially injected. After first splitting and moving the lysis solution and the colony phosphate buffer solution, three mixings are performed ( Figure 12 -B). Then the mixed solution is transferred to the hydrophilic site within the dotted line ( Figure 12 -C). Finally, the HCCA matrix solution is split and moved to the hydrophilic site ( Figure 12 -D).

[0156] Result: As Figure 13 shown, the bacterial identification results ( Figure 13 A) under the DMF and MALDI-TOF-MS integrated ion source are consistent with the identification results of the off-chip strains ( Figure 13 B), realizing the automated and accurate identification of this method.

[0157] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use, or improvement of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. An analytical device directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, characterized in that, Comprising: A digital microfluidic system, including a digital microfluidic chip and a micro-droplet driving platform; the digital microfluidic chip includes an upper plate, a lower plate and a conductive gasket therebetween, the upper plate includes an upper substrate and a conductive layer disposed on the lower surface of the upper substrate, a hydrophobic layer is disposed on the lower surface of the conductive layer, and a plurality of hydrophilic sites are arranged at intervals on the surface of the hydrophobic layer, and each hydrophilic site is a pit recessed inward toward the conductive layer; the lower plate includes a lower substrate and a dielectric layer disposed on the upper surface of the lower substrate, an electrode array is arranged between the lower substrate and the dielectric layer, and a lower hydrophobic layer is disposed on the upper surface of the dielectric layer; Matrix-assisted laser desorption ionization mass spectrometry, including a target plate substrate, and a groove for placing the upper plate of the digital microfluidic chip is provided on the upper surface of the target plate substrate.

2. The analytical device directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 1, characterized in that: The depth of each pit is 49-51 microns; The lateral area of each of the pits is 4 to 16 mm 2 .

3. The analytical device integrating a direct integration digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to any one of claims 1-2, characterized in that: When preparing the upper plate, before forming the upper hydrophobic layer on the lower surface of the conductive layer, a cover sheet is fixed at the pre-designed hydrophilic sites on the lower surface of the conductive layer, and the cover sheet is peeled off after forming the upper hydrophobic layer, so as to form the pit at the position of the cover sheet; The material of the upper hydrophobic layer is polytetrafluoroethylene, and the upper hydrophobic layer is fabricated on the lower surface of the conductive layer by spin coating; The material of the cover sheet is polydimethylsiloxane.

4. The analytical device integrating a direct integration digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to any one of claims 1-2, characterized in that: The electrode array includes a reservoir electrode, a driving electrode, a ground electrode and a contact electrode; A plurality of reservoir electrodes are arranged in a two-dimensional array to form a two-dimensional reservoir electrode array, there are gaps in the two-dimensional reservoir electrode array, a plurality of driving electrodes are arranged in the gaps of the two-dimensional reservoir electrode array, and the edge of each reservoir electrode is connected to the driving electrode located in the gap of the two-dimensional reservoir electrode array through the corresponding driving electrode, each reservoir electrode, driving electrode and ground electrode are respectively connected to a contact electrode, and are connected to the micro-droplet driving platform through the contact electrode; the ground electrode is grounded; the conductive layer in the upper plate is connected to the ground electrode through the conductive gasket.

5. The analytical device directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 4, characterized in that: A number of the hydrophilic sites are located at the corresponding positions of the driving electrodes on the upper plate.

6. An analytical method for directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry, characterized in that, Using the analysis device according to any one of claims 1-5, comprising the following steps: Making droplets containing a sample to be measured and a matrix solution adhere to the upper plate at the hydrophilic sites of the microfluidic chip, placing the upper plate in the groove of the target plate substrate with the hydrophilic sites facing outward from the groove, and feeding it into the matrix-assisted laser desorption ionization mass spectrometry for analysis.

7. The analysis method of directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 6, characterized in that: The sample to be measured is bacteria, verapamil or insulin; The matrix solution is a solution containing 2,5-dihydroxybenzoic acid or a solution containing α-cyano-4-hydroxycinnamic acid.

8. The analytical method of directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 6 or 7, characterized in that: When performing quantitative analysis on a verapamil solution, the matrix solution and verapamil solutions with different concentrations are respectively mixed at the hydrophilic sites on the upper plate of the digital microfluidic chip.

9. The analysis method of directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 8, wherein: The matrix solution is composed of 2,5-dihydroxybenzoic acid, acetonitrile, deionized water and trifluoroacetic acid. The concentration of 2,5-dihydroxybenzoic acid is 20 mg / mL, and the volume ratio of acetonitrile, deionized water and trifluoroacetic acid is 50%:47.5%:2.5%. The volume ratio of the matrix solution to the verapamil solution is 1:

1. The sample is dropped 3 times for each concentration, 1 μL each time, and each sample is irradiated with laser 5 times, and the data is collected and averaged.

10. The analysis method of directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 6 or 7, characterized in that: When performing fingerprint identification on Escherichia coli, Escherichia coli, lysis solution and matrix solution are mixed at the hydrophilic sites on the upper plate of the digital microfluidic chip.

11. The analytical method of directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 10, characterized in that: The lysis solution is an aqueous formic acid solution with a volume fraction of 70%. The matrix solution is a solution of α-cyano-4-hydroxycinnamic acid with a concentration of 10 mg / mL. The solvent is composed of acetonitrile, deionized water and trifluoroacetic acid with a volume percentage of 50%:47.5%:2.5%. 1 μL of the lysis solution and 1 μL of the matrix solution are dropped for each single colony of Escherichia coli. Each sample is inoculated with 3 samples, and each time a single Escherichia coli colony is inoculated. Each sample is irradiated with laser 5 times, and the data is collected and averaged.

12. The analytical method of directly integrating a digital microfluidic chip and matrix-assisted laser desorption / ionization mass spectrometry according to claim 10, wherein: Mix Escherichia coli, lysis solution and matrix solution according to the following steps: a) Drop the sample solution of Escherichia coli and the lysis solution into the digital microfluidic chip respectively. Control the chip to split the two droplets respectively, mix the two split droplets and move them to the hydrophilic sites on the upper plate of the microfluidic chip; b) Drop the matrix solution into the digital microfluidic chip, control the chip to split the droplet, and move the droplet to the hydrophilic sites on the upper plate of the microfluidic chip to converge with the droplet in step a).

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