Automatic electrospray spraying device and method for mass spectrometry imaging sample preparation

Abstract

The present invention discloses an automatic electrospray spraying device and method for preparing samples for mass spectrometry imaging, wherein the automatic electrospray spraying device for preparing samples for mass spectrometry imaging includes: a spray capillary; an injection mechanism whose outlet end is connected to the inlet end of the spray capillary; a high-voltage power supply electrically connected to the spray capillary; and a robotic arm for driving the movement of the spray capillary. This solution generates micron-sized droplets through the electrospray principle to achieve derivatization reagent and matrix spraying. Compared with the original technology, it has an order of magnitude improvement in droplet fineness, ensuring that the derivatization reaction does not destroy the in-situ information, and ensuring that the grain size of the matrix crystal is small and evenly distributed, ensuring the quality of mass spectrometry imaging. In addition, this solution also uses a robotic arm to operate the spraying process, making the spraying process more flexible and efficient, and can automatically absorb and replace reagents, realizing fully automatic multi-reagent spraying sample preparation, such as superimposed spraying of derivatization reagent and matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry imaging, and in particular to an automatic electrospray spraying device and method for preparing mass spectrometry imaging samples. Background Art

[0002] Applications of mass spectrometry in medicine: Histopathology is a crucial tool for medical diagnosis, particularly for critical diseases like cancer, where pathology is crucial for determining the type. However, traditional histopathology relies on optical observation after staining sections, providing relatively limited information. Mass spectrometry, on the other hand, provides chemical information, such as metabolite changes, that can further aid in tissue differentiation. For example, in 2001, Stoeckli et al. first demonstrated the enormous potential of mass spectrometry in tumor research. This paper discussed how mass spectrometry could be used to reveal the chemical structure of glioblastoma tissue sections. Subsequently, mass spectrometry has been shown to be capable of directly analyzing tissues to locate gliomas and grade their malignancy.

[0003] MALDI mass spectrometry imaging technology: MALDI mass spectrometry imaging is one of the most advanced techniques in mass spectrometry imaging. MALDI mass spectrometry molecular imaging is performed using a mass spectrometer, controlled by specialized mass spectrometry imaging software, that analyzes the standard molecular weights of chemical and biological molecules by measuring mass-to-charge ratios. The tissue being studied is frozen and sectioned to obtain extremely thin slices. First, the need for derivatization is determined based on the analyte. If derivatization is required, a derivatization reagent is applied to the surface of the tissue slice in the form of tiny droplets, and the temperature is controlled to allow the derivatization reaction to occur. Otherwise, this step is omitted. The tissue slice is then evenly coated with a matrix and placed on the target of the mass spectrometer. The sample is observed on a computer screen, and the MALDI system's mass spectrometry imaging software is used to select the portion to be imaged. The image size is first defined, and the image is then divided into a two-dimensional array of points based on the size of the points to determine the spacing between the laser beams. The laser beam passes through this raster pattern and strikes the tissue section on the target disk. Software controls the acquisition of mass spectrometric data. In the mass spectrometer, the laser beam continuously scans the tissue section. Molecules released by the tissue sample under the stimulation of the laser beam are identified by the mass spectrometer, obtaining mass-to-charge ratio (m / z) information for each point on the sample. The molecular weight information at each point is then converted into pixels on the image. At each point, all mass spectrometric data are averaged to produce a complete mass spectrum representing the distribution of compounds in that area. The instrument gradually acquires mass spectrometric data for each tissue section, ultimately obtaining a complete set of mass spectrometric data with spatial information. This completes "molecular imaging" of the tissue sample. By setting the m / z range, the types of biomolecules present in that tissue region can be determined, and peak height or peak area can be selected to represent the relative abundance of biomolecules. The colored spots in the image represent the location of the compounds, and the depth of each spot's color is related to the signal detected by the laser at each point or pixel.

[0004] Traditional MALDI imaging sample preparation technology: MALDI imaging requires frozen sections and cannot be embedded with embedding reagents such as OCT, as the polymers in OCT will seriously interfere with mass spectrometry. After the tissue sections are transferred to a conductive glass slide, they are vacuum dried and derivatized as needed (spraying the derivatization reagent and controlling the reaction temperature). Then, the matrix is ​​sprayed and sent to the instrument for analysis.

[0005] Problems with traditional sample preparation technology: Pre-derivatization treatment of tissue sections requires maintaining their in-situ information, so derivatization reagents need to be applied to the tissue surface in the form of tiny droplets. Currently, there are no commercial instruments specifically for in-situ derivatization of tissue sections on the market. Preparation operations are all based on commercial matrix spraying instruments, which have large spray droplets that destroy the in-situ information. At the same time, the instrument cannot meet the derivatization reaction conditions such as temperature, affecting or destroying the derivatization effect. During the drying and crystallization process of the matrix on the surface of the tissue section, larger crystal particles will be formed, which will destroy the uniformity of the matrix coating, affect the mass spectrometry imaging results, and reduce the signal quality and spatial resolution. Commercial instruments use ultrasonic atomization to deposit the matrix on the tissue surface, which improves the crystallization quality, but still does not meet the analytical accuracy of 50 microns, and is only suitable for spraying a single matrix on the entire slice, which is time-consuming. At the same time, the instrument is unstable and often has accidents such as blockage.

[0006] That is to say, in traditional sample preparation technology, the method of spraying derivatization reagents and matrices has problems such as derivatization reagent droplets greatly destroying in situ information, inability to control the derivatization reaction temperature, uneven distribution of the matrix on the tissue surface, and crystallization of large particles, which seriously affect the quality of mass spectrometry imaging, and the preparation process is too time-consuming. Summary of the Invention

[0007] In view of this, the present invention provides an automatic electrospray spraying device for mass spectrometry imaging sample preparation. The device generates micron-sized droplets through the electrospray principle to achieve derivatization reagent and matrix spraying. Compared with the original technology, the device has an order of magnitude improvement in droplet fineness, ensuring that the derivatization reaction does not destroy the in-situ information and that the matrix crystal grain size is small and evenly distributed. At the same time, it is equipped with a heating plate to control the temperature, meet the derivatization reaction conditions and matrix drying requirements, and ensure the quality of mass spectrometry imaging.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] An automatic electrospray coating device for mass spectrometry imaging sample preparation, comprising:

[0010] Spray capillary;

[0011] an injection mechanism whose outlet end is connected to the inlet end of the spray capillary;

[0012] A high voltage power supply is electrically connected to the spray capillary.

[0013] Preferably, it further comprises a first connecting member;

[0014] The first connecting member is a conductor, and one end of the first connecting member is connected to the outlet end of the injection mechanism, and the other end is connected to the inlet end of the spray capillary, and is electrically connected to the high-voltage power supply.

[0015] Preferably, the first connecting member comprises a two-way connection.

[0016] Preferably, the outlet end of the spray capillary is a closed structure.

[0017] Preferably, the injection mechanism comprises a syringe pump;

[0018] The outlet end of the syringe capillary of the injection pump is communicated with the inlet end of the spray capillary.

[0019] Preferably, it also includes:

[0020] A heating plate for heating the slide.

[0021] Preferably, it also includes a robotic arm;

[0022] The spray capillary is arranged at the end of the mechanical arm.

[0023] Preferably, it further comprises a connecting piece;

[0024] The connecting piece is an insulator, and one end of the connecting piece is provided with a docking structure for cooperating with the end of the mechanical arm, and the other end is provided with a mounting structure for cooperating with the spray capillary.

[0025] An automatic electrospray coating method for mass spectrometry imaging sample preparation, using the automatic electrospray coating device for mass spectrometry imaging sample preparation as described above for automatic spray coating, the method comprising the following steps:

[0026] S1. According to the sample preparation process, the robotic arm drives the spray capillary downward into the derivatization reagent or matrix reagent bottle, and the injection mechanism is driven to allow the spray capillary to absorb the derivatization reagent or matrix reagent;

[0027] S2. The spray capillary is moved to an initial position outside the range of the glass slide by the robotic arm, and the high-voltage power supply is turned on to drive the injection mechanism so that the spray capillary slowly pushes out the derivatization reagent or matrix reagent. Wait until the spray capillary spray is stable; at the same time, the temperature of the heating plate is adjusted according to the reaction temperature requirement of the derivatization reagent or the drying temperature requirement of the matrix reagent;

[0028] S3. The robotic arm drives the spray capillary to a set height above the glass slide and moves horizontally according to the set program to complete the derivatization reagent or matrix spraying.

[0029] Preferably, after step S3, the method further includes:

[0030] S4. The spray capillary is moved out of the slide area by the robotic arm, and the high voltage power supply and the injection mechanism are turned off;

[0031] S5. The robot arm drives the spray capillary to the waste liquid area to discharge the remaining matrix, and then to the pure water area to repeatedly suck and empty the spray capillary through the injection mechanism to clean it;

[0032] S6. After cleaning the spray capillary, return to S1 according to the sample preparation requirements, absorb new reagents for the next round of spraying, and repeat S1 to S6. When all sample preparation and spraying tasks are completed, the process ends.

[0033] As can be seen from the above technical solution, the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the present invention generates micron-sized droplets through the electrospray principle to achieve derivatization reagent and matrix spraying. Compared with the existing technology, it has an order of magnitude improvement in droplet fineness, ensuring that the derivatization reaction does not destroy the in-situ information and that the matrix crystal grain size is small and evenly distributed. At the same time, temperature control can ensure that the derivatization reaction and matrix drying proceed smoothly, ensuring the quality of mass spectrometry imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 A simplified structural diagram of an automatic electrospray coating device for mass spectrometry imaging sample preparation provided by an embodiment of the present invention;

[0036] Figure 2 A simplified structural diagram of an automatic electrospray coating device for mass spectrometry imaging sample preparation provided by another embodiment of the present invention;

[0037] Figure 3 A structural diagram of an automatic electrospray coating device for mass spectrometry imaging sample preparation provided by an embodiment of the present invention;

[0038] Figure 4 A flow chart of an automatic electrospray coating method for preparing mass spectrometry imaging samples provided by an embodiment of the present invention.

[0039] Among them, 1 is a high-voltage power supply, 2 is a first connecting piece, 3 is a spray capillary, 4 is a syringe pump, 5 is a robotic arm, 6 is a glass slide, 7 is a heating plate, 8 is an electrospray, 9 is a tissue section, and 10 is a second connecting piece. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] The automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention is as follows: Figure 1 Shown, including:

[0042] Spray capillary 3;

[0043] An injection mechanism whose outlet end is connected to the inlet end of the spray capillary 3;

[0044] A high-voltage power supply 1 is electrically connected to the spray capillary 3 .

[0045] It should be noted that the injection mechanism can not only realize the spraying of the spray capillary 3, but also realize the suction of the spray capillary 3, that is, the injection mechanism can also drive the spray capillary 3 to absorb the derivatization reagent or matrix reagent, as described below for details. In addition, the high-voltage power supply 1 is used to provide a high-voltage electric field for the spray capillary 3, wherein the high-voltage power supply 1 is electrically connected to the portion of the spray capillary 3 near its outlet end. In addition, the present solution is based on the principle of electrospraying. First, the derivatization reagent or matrix reagent is driven by the injection mechanism to fill the spray capillary 3, and then, driven by the high-voltage power supply 1 and the injection mechanism, the derivatization reagent or matrix reagent in the spray capillary 3 is ejected to form an electrospray 8 (such as Figure 2 In other words, this solution allows the derivatization reagent and the matrix reagent to be dispersed into micron-sized droplets by electrospraying. This means that this solution can achieve electrospraying of the derivatization reagent and the matrix, ensuring that the derivatization reaction does not destroy the in-situ information and that the matrix crystals are small and uniformly distributed.

[0046] As can be seen from the above technical solutions, the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention can realize the spraying of derivatization reagents and matrices by generating micron-sized droplets through the electrospray principle. Compared with the existing technology, it has an order of magnitude improvement in the fineness of droplets, ensuring that the derivatization reaction does not destroy the in-situ information, and helps to ensure that the grain size of the matrix crystals is small and evenly distributed, thereby ensuring the quality of mass spectrometry imaging.

[0047] In this program, if Figure 1 As shown, the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention further includes a first connecting member 2;

[0048] The first connector 2 is a conductor, one end of which is connected to the outlet of the injection mechanism (i.e., connected and conductive), and the other end of which is connected to the inlet of the spray capillary 3 and electrically connected to the high-voltage power supply 1. This design not only improves the connection between the outlet of the injection mechanism and the inlet of the spray capillary 3, but also facilitates electrical conductivity between the high-voltage power supply 1 and the spray capillary 3.

[0049] Specifically, the first connecting member 2 includes a two-way connection. The first connecting member 2 of this solution is designed in this way, which has the characteristics of simple structure and convenient connection. In addition, both ends of the two-way connection are connected to the sleeve and corresponding components through PEEK connectors.

[0050] Furthermore, in order to improve the electrospray effect of the spray capillary 3; accordingly, as Figure 2 As shown, the outlet end of the spray capillary 3 is a closed structure.

[0051] Furthermore, if Figure 1 As shown, the injection mechanism includes an injection pump 4;

[0052] The outlet end of the syringe capillary of the syringe pump 4 is connected to the inlet end of the spray capillary 3. Specifically, the outlet end of the output capillary of the syringe pump 4 is connected to the inlet end of the spray capillary 3. This solution uses the syringe pump 4 as the injection mechanism, which features uniform and smooth injection and precise flow control.

[0053] In order to further optimize the above technical solution, Figure 1 As shown, the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention also includes:

[0054] A heating plate 7 is provided for heating the glass slide 6, wherein the heating plate 7 is disposed below the glass slide 6. By adding the heating plate 7, this solution facilitates raising the temperature of the tissue slice 9, meeting the derivatization reaction conditions, ensuring smooth derivatization, accelerating the evaporation of the matrix, and preventing the mass spectrometry imaging sample preparation process from being too time-consuming.

[0055] In this program, if Figure 1 As shown, the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention further includes a robotic arm 5;

[0056] The spray capillary 3 is mounted at the end of a robotic arm 5. This design allows the robotic arm 5 to drive the spray capillary 3 to spray a designated area, completing the derivatization reagent and matrix coating of tissue sections. This facilitates rapid and flexible spraying of the derivatization reagent and matrix. Furthermore, the robotic arm 5 drives the spray capillary 3, allowing for flexible control of the spraying area and number of layers, thereby enabling parameterized control and programming of the derivatization reagent and matrix spraying.

[0057] Specifically, if Figure 1 As shown, the automatic electrospray spraying device for mass spectrometry imaging sample preparation provided by the embodiment of the present invention further includes a second connecting member 10;

[0058] The second connector 10 is an insulator, and one end of it is provided with a docking structure for mating with the end of the robotic arm 5, and the other end is provided with a mounting structure for mating with the spray capillary 3. In other words, the spray capillary 3 is fixed to the end of the robotic arm 5 via the second connector 10. Taking the end of the robotic arm 5 as an example, the docking structure of the second connector 10 can be designed as: a slot that matches the size of the two-finger clamp to facilitate the grip of the two-finger clamp; the mounting structure of the second connector 10 can be designed as a vertical hole to facilitate the insertion and fixation of the spray capillary 3. Of course, the docking structure and mounting structure of the second connector 10 are not limited to this, and can also be other forms of structures, which will not be described here. In addition, the second connector 10 can be a connecting block.

[0059] The embodiment of the present invention also provides an automatic electrospray coating method for mass spectrometry imaging sample preparation, which uses the automatic electrospray coating device for mass spectrometry imaging sample preparation as described above to perform automatic spraying, such as Figure 4 As shown, the method includes the following steps:

[0060] S1. According to the sample preparation process, the robotic arm drives the spray capillary downward into the derivatization reagent or matrix reagent bottle, and the injection mechanism is actuated to cause the spray capillary to absorb the derivatization reagent or matrix reagent, i.e., the derivatization reagent or matrix reagent is filled in the spray capillary; of course, the injection mechanism is closed after the derivatization reagent or matrix reagent is absorbed;

[0061] S2. The spray capillary is moved to an initial position outside the range of the glass slide by the robotic arm, and the high-voltage power supply is turned on to drive the injection mechanism so that the spray capillary slowly pushes out the derivatization reagent or matrix reagent. Wait until the spray capillary spray is stable; at the same time, the temperature of the heating plate is adjusted according to the reaction temperature requirement of the derivatization reagent or the drying temperature requirement of the matrix reagent;

[0062] S3. The robotic arm moves the spray capillary to a set height above the glass slide and moves horizontally according to a set program to complete the spraying of the derivatization reagent or matrix. Because this solution utilizes the aforementioned automated electrospray spraying device for mass spectrometry imaging sample preparation, it also has corresponding beneficial effects. The details can be found in the previous description and will not be repeated here.

[0063] In this solution, after step S3, the following steps are further included:

[0064] S4, the spray capillary is moved out of the slide area by the robotic arm, the high voltage power supply is turned off, and the injection mechanism is stopped;

[0065] S5. The robot arm drives the spray capillary to the waste liquid area to discharge the remaining matrix, and then to the pure water area to repeatedly suck and empty the spray capillary through the injection mechanism to clean it. This solution is designed in this way to facilitate the cleaning of the spray capillary.

[0066] After cleaning the spray capillary in S6, the process can return to S1 and draw new reagents for the next spraying round, repeating S1 through S6. The process ends when all sample preparation spraying tasks are complete. Of course, depending on sample preparation requirements, the derivatization reagent and matrix can be sprayed sequentially, achieving a stacked spraying of the derivatization reagent and matrix. In other words, the derivatization reagent is drawn and sprayed in the first spraying round, and the matrix reagent is then drawn and sprayed in the next spraying round.

[0067] The present invention will be further described below with reference to specific embodiments:

[0068] Mass spectrometry imaging is a crucial detection and visualization technique in biochemical analysis and research, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging represents one of the most advanced technologies. Sample preparation is a crucial and time-consuming step in MALDI mass spectrometry imaging, crucial for analytical quality. Ideal sample preparation involves selecting whether to perform derivatization. If necessary, the derivatization reagent is sprayed onto the tissue section surface using tiny droplets, the reaction temperature is controlled, and the section is allowed to dry after the reaction. Otherwise, this step can be omitted. Currently, commercial equipment is lacking for in situ derivatization of tissue sections. An alternative approach is to use commercial matrix spraying equipment, but this approach suffers from issues such as droplet destruction, which can significantly disrupt in situ information, and the inability to control the reaction temperature, hindering the smooth progress of the reaction. Subsequently, the matrix must be evenly applied to the tissue section surface. However, current matrix spraying methods often suffer from uneven matrix distribution on the tissue surface, resulting in large particle crystallization, which severely impacts mass spectrometry imaging quality and is time-consuming.

[0069] The present invention is to achieve automated, high-quality spraying of derivatization reagents and substrates, thereby providing a sample preparation technology, method, and device for MALDI mass spectrometry imaging that sprays uniformly, quickly, flexibly, and fully automatically. The derivatization reagent and substrate are dispersed into micron-sized droplets by electrospraying, ensuring that the derivatization reaction does not destroy the in-situ information and that the grain size of the substrate crystals is small and uniform. A heating plate controls the temperature to ensure that the derivatization reaction proceeds smoothly and that the substrate evaporates. By driving the electrospray nozzle through a robotic arm, the spraying area and the number of spray layers can be flexibly controlled, thereby achieving parameterized control and programming of the derivatization reagent and substrate spraying. By integrating and controlling the robotic arm, high-voltage power supply, heating plate, and injection pump, switching between multiple derivatization reagents and multiple substrates and automatic spraying can be achieved, further expanding the application of this technology.

[0070] In addition, mass spectrometry: a method of separating moving ions according to their mass-to-charge ratio using electric and magnetic fields and then detecting them, can identify substances by their mass-to-charge ratio.

[0071] Mass spectrometry imaging technology: Mass spectrometry imaging is an imaging method based on mass spectrometry. This method uses mass spectrometry to directly scan biological samples and image them. It can simultaneously analyze the spatial distribution characteristics of hundreds of molecules on the same tissue section or tissue chip. Simply put, mass spectrometry imaging technology uses mass spectrometry, coupled with specialized mass spectrometry imaging software, to generate images using a mass spectrometer that analyzes the standard molecular weight of chemical and biological molecules by measuring mass-to-charge ratios.

[0072] Tissue sectioning: Biological sample tissue is frozen and cut into thin slices and adhered to glass slides for observation of tissue morphology, cell morphology, imaging analysis, etc.

[0073] MALDI: Matrix-assisted laser desorption / ionization is a new mass spectrometry ionization technology.

[0074] Derivatization: Derivatization is a chemical transformation that converts a compound into a substance with a similar chemical structure. It is widely used in instrumental analysis. Its primary function is to convert difficult-to-analyze substances into similar, but easier-to-analyze substances, facilitating quantification and separation. The reagents used in derivatization are liquid reagents and are pre-mixed at the time of use.

[0075] Matrix: In MALDI technology, the matrix is ​​a substance that co-crystallizes with the analyte, absorbs the incident laser energy and transfers it to the analyte molecules, while also preventing direct irradiation and potential damage to the sample. It plays a crucial role in the desorption and ionization process. The matrix is ​​a liquid reagent that is pre-mixed for use.

[0076] MALDI imaging technology: By scanning the prepared sample slices in a laser array, mass spectrometry analysis results with spatial information are obtained, which can be visualized as imaging results.

[0077] Electrospray: A high-voltage electric field is added at the outlet of the capillary. Under the action of the electric field force and the Coulomb force, the liquid reagent will produce Taylor cone spray at the outlet of the capillary and atomize into tiny charged droplets with a diameter of microns.

[0078] The present invention is a technology, system and device for pre-treatment of tissue slice derivatization and matrix coating using MALDI mass spectrometry imaging technology. Its function is to drive the derivatization reagent or matrix to fill the spray capillary through an injection pump, and then, driven by high voltage electricity, the derivatization reagent or matrix in the spray capillary is sprayed out through the electrospray principle. The spray capillary is driven by a mechanical arm and sprayed on a designated area. The temperature is controlled by a heating plate to complete the derivatization and matrix coating preparation of the tissue slice. The heating plate can increase the temperature of the tissue slice to meet the derivatization reaction conditions, accelerate the volatilization of the matrix, and control the crystallization quality. The overall structure of the present invention is as follows Figure 1 shown.

[0079] Through the combined control of a robotic arm, syringe pump, heater, and high-voltage power supply, parametric control can be achieved for spraying a specific amount of derivatization reagent and matrix in a designated area. Different derivatization reagents and matrices can be freely switched, automating complex pretreatment processes. The system can be further expanded to include the spraying of other reagents, such as enzymatic hydrolysis reagents, to achieve comprehensive sample pretreatment capabilities.

[0080] The spray capillary described in this solution is a pipe structure with a small inner diameter, such as Figure 2 As shown, the inner diameter is usually 0.3 to 1 mm, the length is 10 cm, and the front opening is narrowed to 0.01 mm, which is the spray outlet. The rear end is connected to the connector of the syringe pump. The connector of the syringe pump is a metal conductor, which is connected to the high-voltage power supply so that the liquid in the spray capillary is conductive to the high-voltage power supply. The spray capillary is fixed to the end of the robotic arm and connected to the connector of the robotic arm. The connector of the robotic arm is an insulator to prevent short circuit. In addition, large-volume equipment such as the syringe pump, robotic arm and high-voltage power supply are all placed on the table. The spray capillary and its connector are installed at the end of the robotic arm. The spray capillary is connected to the high-voltage power supply through a flexible wire and to the syringe pump through a flexible capillary.

[0081] Working principle: When a high voltage electric field is applied at the outlet of the capillary, the reagent will be sprayed at the outlet of the capillary under the action of Coulomb force and atomized into tiny droplets with electric charge, which is called electrospray. Figure 2 As shown in the figure, the spray formation principle is that the electrostatic Coulomb force overcomes the surface tension of the droplets, causing them to break up into fine droplets. This phenomenon is called the Taylor cone. The droplet size and Taylor cone width can be adjusted by adjusting parameters such as the electric field strength, the inner diameter of the capillary opening, and the viscosity of the reagent. When the robotic arm drives the spray capillary horizontally, a Taylor cone-covered spray area is formed below. By adjusting the robotic arm's movement direction, number of movements, the time between the two movements, and the distance between the spray capillary opening and the tissue section, the size, thickness, and speed of the spray area can be adjusted.

[0082] like Figure 3 and Figure 4As shown, the workflow is as follows: the heating plate is turned on and preheated. The tissue sections are placed on the glass slide, dried, and placed in a designated position, and the temperature is controlled by the heating plate below. The derivatization reagent and the matrix are pre-mixed liquid reagents, stored in reagent bottles, and placed in designated positions. Driven by the robotic arm, the spray capillary extends into the reagent bottle, and driven by the injection pump, the derivatization reagent or matrix reagent is sucked into the capillary. Afterwards, the spray capillary moves to the initial position outside the range of the glass slide, turns on the high-voltage power supply, and waits for a few seconds until the spray stabilizes. Afterwards, the spray capillary moves to the set height above the glass slide, and moves horizontally according to the set program to complete the spraying of the derivatization reagent or matrix. After the spraying is completed, the spray capillary moves out of the glass slide area and turns off the high-voltage power supply. The robotic arm drives the spray capillary to the waste liquid area to discharge the remaining reagent, and to the pure water area to repeatedly pump and empty the spray capillary through the injection pump. If there is a need to spray more than one reagent, repeat the above process. The process is as follows Figure 4 shown.

[0083] Processing: The spray nozzle of the spray capillary is realized by drawing with a capillary needle puller. The high-voltage power supply connector (i.e., the second connector) is a commodity, the heating plate is a commodity, and the robotic arm connector is processed by 3D printing.

[0084] Advantages of the present invention:

[0085] The present invention uses the electrospray principle to generate micron-sized droplets to achieve derivatization reagent and matrix spraying, which has an order of magnitude improvement in droplet fineness compared to existing technologies; spraying is achieved by a robotic arm driving the spray, which is more flexible, more efficient and more intelligent than existing technologies; through the combined control of the robotic arm, electrospray and drive pump, the derivatization reagent and matrix spraying task can be parameterized and customized, and easily expanded to the mixed spraying of multiple reagents or multiple matrices, fully automatically realizing complex sample pretreatment tasks; and through the heating plate, temperature control is achieved to meet the derivatization reaction conditions and accelerate the evaporation of the matrix.

[0086] The key points and points to be protected of the present invention are:

[0087] 1. Realize comprehensive pre-treatment tasks such as derivatization, matrix spraying and enzymatic hydrolysis by electrospraying;

[0088] 2. Use the robotic arm to drive the electrospray to achieve parameterized setting of the spraying task;

[0089] 3. A fully automatic MALDI imaging tissue section sample preparation device is realized through the integrated control of the robotic arm, high-voltage power supply, syringe pump, spray capillary and heating plate.

[0090] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0091] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An automatic electrospray spraying device for mass spectrometry imaging sample preparation, characterized in that: include: spray capillary (3); An injection mechanism whose outlet end is connected to the inlet end of the spray capillary (3); wherein the injection mechanism is also used to drive the spray capillary (3) to absorb the derivatization reagent or the matrix reagent; a high-voltage power supply (1) electrically connected to the spray capillary (3); The automatic electrospray coating device further comprises a heating plate (7), which is arranged below the glass slide (6) and is used to heat the glass slide (6); The automatic electrospray spraying device further comprises a mechanical arm (5), the spray capillary (3) is arranged at the end of the mechanical arm (5), and the mechanical arm (5) is at least used to drive the spray capillary (3) to spray in a designated area; The automatic electrospray spraying device further includes a second connecting member (10); The second connecting member (10) is an insulator, and one end of the second connecting member is provided with a docking structure for cooperating with the end of the mechanical arm (5), and the other end of the second connecting member is provided with a mounting structure for cooperating with the spray capillary (3).

2. The automatic electrospray coating device for mass spectrometry imaging sample preparation according to claim 1, characterized in that: Also includes a first connecting member (2); The first connecting member (2) is a conductor, one end of which is connected to the outlet end of the injection mechanism, the other end of which is connected to the inlet end of the spray capillary (3), and is electrically connected to the high-voltage power supply (1).

3. The automatic electrospray coating device for mass spectrometry imaging sample preparation according to claim 2, characterized in that: The first connecting member (2) comprises a two-way connection.

4. The automatic electrospray coating device for mass spectrometry imaging sample preparation according to claim 1, characterized in that: The outlet end of the spray capillary (3) is a closed structure.

5. The automatic electrospray coating device for mass spectrometry imaging sample preparation according to claim 1, characterized in that: The injection mechanism includes an injection pump (4); The outlet end of the syringe capillary of the injection pump (4) is communicated with the inlet end of the spray capillary (3).

6. An automatic electrospray coating method for mass spectrometry imaging sample preparation, characterized in that: Automatic spraying is performed using the automatic electrospray spraying device for mass spectrometry imaging sample preparation according to claim 1, the method comprising the following steps: S1. According to the sample preparation process, the robotic arm drives the spray capillary downward into the derivatization reagent or matrix reagent bottle, and the injection mechanism is driven to allow the spray capillary to absorb the derivatization reagent or matrix reagent; S2. The spray capillary is moved to an initial position outside the range of the glass slide by the robotic arm, and the high-voltage power supply is turned on to drive the injection mechanism so that the spray capillary slowly pushes out the derivatization reagent or matrix reagent. Wait until the spray capillary spray is stable; at the same time, the temperature of the heating plate is adjusted according to the reaction temperature requirement of the derivatization reagent or the drying temperature requirement of the matrix reagent; S3. The robotic arm drives the spray capillary to a set height above the glass slide and moves horizontally according to the set program to complete the derivatization reagent or matrix spraying.

7. The automatic electrospray coating method for mass spectrometry imaging sample preparation according to claim 6, characterized in that: After step S3, the method further includes: S4. The spray capillary is moved out of the slide area by the robotic arm, and the high voltage power supply and the injection mechanism are turned off; S5. The robot arm drives the spray capillary to the waste liquid area to discharge the remaining matrix, and then to the pure water area to repeatedly suck and empty the spray capillary through the injection mechanism to clean it; S6. After cleaning the spray capillary, return to S1 according to the sample preparation requirements, absorb new reagents for the next round of spraying, and repeat S1 to S6. When all sample preparation and spraying tasks are completed, the process ends.

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