Mass spectrometry static nanoelectrospray device capable of real-time temperature control

Abstract

This invention relates to the field of mass spectrometry analysis technology, specifically to a real-time temperature-controlled static nanojet mass spectrometry sample introduction device. It includes a base with a rotating platform rotatable relative to the base. A heat transfer component is fixedly mounted on the rotating platform, containing a hollow ceramic sleeve. The device also includes a temperature control component, comprising a TEC semiconductor chip and a heat sink. The cold end of the TEC semiconductor chip is thermally connected to the heat transfer component, and the hot end is thermally connected to the heat sink. The TEC semiconductor chip achieves bidirectional switching between cooling and heating by changing the direction and magnitude of the input current. Driven by the rotating platform, the entire device can move left and right, facilitating capillary sample introduction operations. It integrates real-time continuous temperature monitoring and rapid temperature response functions. Through the synergistic effect of the temperature control component and the heat transfer component, it enables efficient temperature control and real-time detection of samples.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry analysis technology, and specifically to a static nanojet mass spectrometry sample introduction device with real-time temperature control. Background Technology

[0002] A mass spectrometer is centered around an ion source, a mass analyzer, and an ion detector. The ion source is a device that ionizes sample molecules under high vacuum conditions. The ionized molecules then enter the mass analyzer under the influence of an accelerating electric field. The mass analyzer separates ions of different masses that enter simultaneously according to their mass-to-charge ratio (m / z). The separated ions then sequentially enter the ion detector, where signal processing generates a mass spectrum. There are various types of ion sources, mass analyzers, and ion detectors.

[0003] Traditional mass spectrometry relies on denaturing agents or high temperatures, which can cause conformational folding and breakage of non-covalent interactions in biomolecules such as proteins, failing to reflect their true biological functional state. Non-denaturing mass spectrometry is a key technology developed to overcome the limitations of traditional mass spectrometry in destroying the native state of biomolecules. Static nanospray ion sources are commonly used in non-denaturing mass spectrometry detection. Their principle is as follows: a sample solution is loaded into a glass capillary, and a high voltage of 1–3 kV is applied to the tip. A stable Taylor cone is formed under low flow rate and without auxiliary gas, directly generating gaseous ions through electrospray ionization. Ionization is completed under atmospheric pressure throughout the process, making it highly suitable for biomolecules such as proteins, antibodies, nucleic acids, and complexes. Static nanospray ion sources offer advantages such as tolerance to high salt, buffer solutions, non-denaturing additives, preservation of non-covalent interactions, low consumption, high stability, and high sensitivity.

[0004] However, existing sample introduction devices for static nanojet ion sources adapted to non-denaturing mass spectrometry analysis lack continuously adjustable and real-time temperature control functions. Temperature fluctuations can easily cause denaturation of biomacromolecules such as proteins, destroy their natural conformations, and the sample introduction operation is cumbersome, affecting the accuracy of detection.

[0005] In other words, existing sample introduction devices generally lack the ability to control sample temperature in real time, continuously, and precisely. This prevents programmed heating and cooling of samples during experiments, limiting research on temperature-dependent biological processes (such as protein denaturation). Due to the lack of effective temperature control, samples are easily affected by ambient temperature fluctuations during sample introduction. This unstable temperature environment can easily cause denaturation of biomolecules such as proteins, disrupting their natural spatial conformation and non-covalent interactions, thus making it impossible to detect their true biological functional state by mass spectrometry. Traditional static nanojet sample introduction devices involve limited operating space and cumbersome processes when changing or installing glass capillaries. This not only increases the difficulty of operation but may also introduce errors due to repeated sample loading, affecting the accuracy and repeatability of detection. It is also not conducive to continuous analysis of the same sample under multiple conditions, reducing experimental efficiency.

[0006] Therefore, there is an urgent need for a compact, easy-to-assemble and disassemble mass spectrometer static nanojet injection device with real-time and precise temperature control, so as to achieve stable control and real-time monitoring of sample temperature, effectively protect the natural conformation of biological samples, and avoid sample denaturation. Summary of the Invention

[0007] The purpose of this invention is to provide a static nanojet mass spectrometer injection device with real-time temperature control in order to solve the problems existing in the prior art.

[0008] To address the problems existing in the prior art, the present invention adopts the following technical solution:

[0009] A real-time temperature-controlled static nanojet mass spectrometer includes:

[0010] The base serves as the fundamental load-bearing foundation.

[0011] A rotating platform is mounted on the base via a limiting structure and can rotate relative to the base. A groove is provided in the middle of the rotating platform.

[0012] A heat transfer assembly is embedded and fixed in the groove. The heat transfer assembly includes an aluminum heat exchanger and a hollow ceramic sleeve for placing a glass capillary tube containing a sample. The aluminum heat exchanger has a through-hole in the center, and the hollow ceramic sleeve is fitted into the through-hole. The aluminum heat exchanger has a sensor placement hole for placing a temperature sensor.

[0013] A temperature control component includes a TEC level 3 semiconductor chip and a heat sink. With the outline center of the heat transfer component as a reference, the TEC level 3 semiconductor chip and the heat sink are stacked sequentially from top to bottom. The cold end of the TEC level 3 semiconductor chip faces downward and is bonded and fixed to the top surface of the aluminum heat exchanger with thermally conductive adhesive. The heat sink is attached and fixed to the hot end of the TEC level 3 semiconductor chip with thermally conductive adhesive, forming a temperature control conduction structure.

[0014] The support structure includes a support column and a temperature control support member for supporting the temperature control component. The temperature control support member is mounted above the radiator via the support column. The support member passes through the temperature control support member and is locked by a locking member, so that the temperature control support member is pressed and limited above the radiator.

[0015] As an improvement to the technical solution of the mass spectrometry static nanojet injection device with real-time temperature control of the present invention, the rotating platform is an integrally formed layered structure, and the surface of the lower layer is provided with a circular track groove.

[0016] The base has multiple stepped limiting assembly holes evenly distributed around its perimeter. Each limiting assembly hole contains a spring and a steel ball. The steel ball is positioned at the outer end of the spring, and the top of the steel ball protrudes from the upper surface of the base. Along the outer periphery of the circular track groove, the circular track groove has multiple preset locking positions to fix the rotating platform relative to the base at a fixed point.

[0017] The circular track groove is adapted to the steel ball and spring in the limiting assembly hole. The steel ball can roll along the circular track groove and can be locked into the preset locking position to realize the angle limit of the rotating platform.

[0018] The upper layer of the rotating platform is an irregularly shaped boss, and the boss has a groove for installing the heat transfer component.

[0019] As an improvement to the technical solution of the mass spectrometry static nanojet injection device with real-time temperature control of the present invention, four locking positions are evenly distributed along the circumference of the circular track groove, so that the preset angle of rotation of the rotating platform is 90° each time.

[0020] As an improvement to the technical solution of the mass spectrometer static nanojet injection device with real-time temperature control of the present invention, the upper section of each stepped limiting assembly hole is a square hole and the lower section is a circular hole. The spring is placed in the circular hole and the steel ball is adapted to be installed in the square hole.

[0021] As an improvement to the technical solution of the mass spectrometer static nanojet injection device with real-time temperature control of the present invention, the protrusion is provided with an assembly hole for inserting the support column.

[0022] As an improvement to the technical solution of the mass spectrometer static nanojet injection device with real-time temperature control of the present invention, the support column has a coarse and fine segmented structure. The coarse end of the support column is inserted and fixed in the boss, and the fine end of the support column is provided with an external thread for threaded connection with the locking member, so that the temperature control support is limited above the heat sink, thereby realizing the overall fixation of the temperature control component and the heat transfer component.

[0023] As an improvement to the technical solution of the mass spectrometer static nanojet injection device with real-time temperature control of the present invention, the base is provided with an installation through hole for mounting on the mass spectrometer, and the center of the base is provided with a central through hole for assembling the rotating platform; the rotating platform and the base are coaxially assembled, and the rotating platform rotates with the center of the base as the rotation center; a plurality of limiting assembly holes are evenly distributed around the central through hole.

[0024] As an improvement to the technical solution of the mass spectrometer static nanojet injection device with real-time temperature control of the present invention, the sensor probe of the temperature sensor is attached to the outer wall of the hollow ceramic sleeve to collect temperature data around the sample and feed it back to the temperature control component.

[0025] The temperature control component adjusts the direction and magnitude of the power supply current to the TEC level 3 semiconductor chip according to the preset temperature;

[0026] When heating is required, a positive current is input to the TEC level 3 semiconductor chip, and the heat is transferred to the sample through the aluminum heat exchanger.

[0027] When cooling is required, the direction of the current input to the TEC level 3 semiconductor chip is switched so that the cold end absorbs heat to cool the sample.

[0028] When the temperature reaches the preset temperature value, the temperature control component maintains a stable temperature and starts the mass spectrometer to analyze the sample.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention features a small and compact size, and is easy to assemble and disassemble. It can be mounted on a 40*40mm platform, allowing for rapid and precise adjustment of the relative distance between the sample capillary glass tube and the mass spectrometer inlet. The rotating platform enables the entire device to move left and right, facilitating sample introduction via the capillary glass tube. Furthermore, the device integrates real-time continuous temperature monitoring and rapid temperature response. Through the synergistic effect of the temperature control and heat transfer components, it achieves efficient temperature control and real-time detection of the sample.

[0031] 2. In this invention, the sample loading operation is simplified by using a rotating structure, and the sample is cooled and heated by a temperature control component, so as to achieve stable control and real-time monitoring of the sample temperature, effectively protect the natural conformation of biological samples and avoid sample denaturation. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the overall structure of the present invention;

[0033] Figure 2 This is a schematic diagram of the front structure of the present invention;

[0034] Figure 3 This is a schematic diagram of the left-side structure of the present invention;

[0035] Figure 4 This is a top view of the structure of the present invention;

[0036] Figure 5 This is a cross-sectional view of the center position of the heat transfer component of the present invention;

[0037] Figure 6 This is a 1 / 3 position cross-sectional view of the heat transfer component of the present invention;

[0038] Figure 7 This is a physical illustration of the present invention.

[0039] Figure 8 Temperature accuracy curves for measuring the same myoglobin standard at different temperatures using the present invention;

[0040] Figure 9 Total ion chromatograms of the same myoglobin standard obtained by continuous mass spectrometry at different temperatures using the present invention;

[0041] Figure 10 Mass spectra of the same myoglobin standard collected continuously at different temperatures using the present invention;

[0042] Figure 11 This is a molecular weight distribution spectrum obtained by continuously collecting data on the same myoglobin standard at different temperatures using the present invention, after deconvolution processing.

[0043] Figure 12 This is a mass spectrum of deheme-free myoglobin obtained by continuous acquisition of the same myoglobin standard at different temperatures using the present invention.

[0044] Figure 13 This is a mass spectrum of heme obtained by continuously collecting the same myoglobin standard at different temperatures using the present invention.

[0045] Explanation of reference numerals in the attached diagram: 1 - Base; 2 - Rotating platform; 3 - Heat transfer assembly; 31 - Aluminum heat exchanger; 32 - Ceramic sleeve; 33 - Sensor placement hole; 34 - Glass capillary tube; 4 - Temperature control assembly; 41 - TEC level 3 semiconductor chip; 42 - Heat sink; 5 - Support column; 6 - Temperature control support component; 7 - Screw. Detailed Implementation

[0046] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments.

[0047] like Figures 1 to 10 As shown, the present invention provides a static nanojet mass spectrometer injection device with real-time temperature control, comprising:

[0048] Base 1, which serves as the basic load-bearing base;

[0049] The rotating platform 2 is mounted on the base 1 by a limiting structure and can rotate relative to the base 1. A groove is provided in the middle of the rotating platform 2.

[0050] The heat transfer component 3 is embedded and fixed in the groove. The heat transfer component 3 includes an aluminum heat exchanger 31 and a hollow ceramic sleeve 32 for placing a glass capillary tube 34 containing a sample. The aluminum heat exchanger 31 has a through-hole in the center, and the hollow ceramic sleeve 32 is fitted into the through-hole. The aluminum heat exchanger 31 has a sensor placement hole 33 for placing a temperature sensor.

[0051] The temperature control component 4 includes a TEC level semiconductor chip 41 and a heat sink 42. With the outline center of the heat transfer component 3 as a reference, the TEC level semiconductor chip 41 and the heat sink 42 are stacked from top to bottom. The cold end of the TEC level semiconductor chip 41 faces down and is bonded and fixed to the top surface of the aluminum heat exchanger 31 with thermally conductive adhesive. The heat sink 42 is attached and fixed to the hot end of the TEC level semiconductor chip 41 with thermally conductive adhesive to form a temperature control conduction structure.

[0052] The support structure includes a support column 5 and a temperature control support 6 for supporting the temperature control assembly 4. The temperature control support 6 is mounted above the radiator 42 via the support column 5. The support 6 passes through the temperature control support 6 and is locked by a locking member, so that the temperature control support 6 is pressed and limited above the radiator 42. Preferably, the locking member is a screw 7.

[0053] This invention addresses the problems of existing static nanojet injection devices, such as the lack of bidirectional real-time temperature control, the tendency of temperature fluctuations to cause denaturation of biomolecules, and the limited space for capillary sample loading and cumbersome disassembly and maintenance. It utilizes the TEC semiconductor chip 41 to achieve bidirectional continuous temperature control for both cooling and heating, and forms a closed-loop temperature control with the help of temperature sensing elements to stabilize and protect the natural conformation of the sample. It meets the multiple requirements of convenient sample loading, accurate gradient temperature control, and easy disassembly and maintenance, and enables the same sample to be detected by non-denaturing mass spectrometry at multiple temperature gradients without replacement.

[0054] In some embodiments of the present invention, the rotating platform 2 is a layered structure integrally formed, and a circular track groove is formed on the surface of the lower layer.

[0055] Multiple stepped limiting assembly holes are evenly opened around the base 1. A spring and a steel ball are installed in each limiting assembly hole. The steel ball is set at the outer end of the spring, and the top of the steel ball protrudes from the upper surface of the base 1. Along the outer periphery of the circular track groove, multiple preset locking positions are opened in the circular track groove to make the rotating platform 2 relative to the fixed point of the base 1.

[0056] The circular track groove is adapted to the steel ball and spring in the limiting assembly hole. The steel ball can roll along the circular track groove and can be locked into the preset position to realize the angle limit of the rotating platform 2.

[0057] The upper part of the rotating platform 2 is an irregularly shaped boss, and a groove for installing the heat transfer component 3 is provided inside the boss.

[0058] In some embodiments of the present invention, the upper section of each stepped limiting assembly hole is a square hole and the lower section is a circular hole. A spring is placed in the circular hole and a steel ball is fitted in the square hole.

[0059] In some embodiments of the present invention, four locking positions are evenly distributed along the circumference of the circular track groove, so that the preset angle of rotation of the rotating platform 2 is 90° each time.

[0060] In detail, in this invention, the base is a small plate-like structure with mounting holes at the four corners for mounting the entire device on a 40*40mm platform. An assembly hole is located in the center of the base, and stepped limiting holes with springs and steel balls are evenly arranged around the perimeter. A circular track groove is fitted to the bottom of the rotating platform. These two components work together to allow the rotating platform 2 to rotate freely 360°, with automatic locking every 90°. During sample loading, rotating the platform 90° laterally creates operating space, facilitating the insertion of glass capillaries. After loading, rotating it back to its original position allows for precise alignment of the mass spectrometer inlet, significantly simplifying operation and solving the problems of limited operating space and difficulty in capillary disassembly and alignment in traditional devices.

[0061] In some embodiments of the present invention, the boss is provided with an assembly hole for inserting a support column.

[0062] In some embodiments of the present invention, the support column has a segmented structure with thick and thin sections. The thick end of the support column is inserted and fixed in the boss, and the thin end of the support column is provided with an external thread for threaded connection with the locking member, so that the temperature control support is limited above the heat sink, thereby realizing the overall fixation of the temperature control component and the heat transfer component.

[0063] In some embodiments of the present invention, the base is provided with a mounting through hole for mounting on a mass spectrometer, and the center of the base is provided with a central through hole for mounting a rotating platform; the rotating platform and the base are coaxially mounted, and the rotating platform rotates around the center of the base; a plurality of limiting mounting holes are evenly distributed around the central through hole.

[0064] In some embodiments of the present invention, the sensor probe of the temperature sensor is attached to the outer wall of the hollow ceramic sleeve to collect temperature data around the sample and feed it back to the temperature control component.

[0065] The temperature control component adjusts the direction and magnitude of the power supply current to the TEC level semiconductor chip according to the preset temperature;

[0066] When heating is required, a positive current is input to the TEC level 3 semiconductor chip, and the heat is transferred to the sample through the aluminum heat exchanger.

[0067] When cooling is required, the direction of the current input to the TEC level 3 semiconductor chip is switched so that the cold end absorbs heat to cool the sample.

[0068] When the temperature reaches the preset value, the temperature control component maintains a stable temperature and starts the mass spectrometer to analyze the sample.

[0069] In this invention, an integrated temperature control system of aluminum heat transfer and TEC semiconductor chip 41 is constructed. It uses a cuboid aluminum heat exchanger 31 as a temperature equalization carrier, with a hollow ceramic sleeve 32 nested inside to support a glass capillary 34. A sensor hole is reserved in the aluminum body to collect the sample temperature in real time. At the same time, the TEC semiconductor chip 41 and heat sink 42 are stacked on top of the aluminum heat exchanger 31. The cold end of the chip is attached to the aluminum body, which can cool and heat in both directions, realize continuous and precise temperature control in a wide range of -10℃ to 90℃, stably maintain the natural conformation of biological samples, and support uninterrupted mass spectrometry acquisition of the same sample at multiple temperature gradients.

[0070] Furthermore, a groove is made on the upper boss of the rotating platform 2, and a rectangular aluminum heat exchanger 31 is embedded in the groove; a hollow ceramic sleeve 32 is fitted into the central through hole of the aluminum heat exchanger 31, and a glass capillary tube 34 is placed in the inner cavity of the ceramic, so that the aluminum material conducts heat evenly and stably; the aluminum heat exchanger 31 has a reserved sensor placement hole 33, and the built-in temperature sensor collects the temperature of the sample area in real time, realizing real-time temperature monitoring and providing a data basis for precise temperature control.

[0071] In some embodiments of the present invention, the hollow ceramic sleeve 32 is arranged to extend through the heat transfer component 3 along the axial direction, and one end of the glass capillary 34 is inserted into the hollow ceramic sleeve 32 and extends to the outside of the heat transfer component 3 to facilitate circuit grounding, while the other end extends out of the heat transfer component 3 by 1-2 mm to form a nano-spray tip.

[0072] In some embodiments of the present invention, the mass spectrometer static nanojet injection device with real-time temperature control further includes a temperature control support 6, which is disposed on the side of the heat sink 42 away from the TEC semiconductor chip 41.

[0073] The TEC semiconductor chip 41 and heat sink 42 are stacked from top to bottom, with the aluminum heat exchanger 31 as the center. The cold end of the TEC chip is tightly bonded to the aluminum heat exchanger 31 with thermally conductive adhesive, and the hot end is bonded to the heat sink 42. By changing the direction of the chip's power supply current, it is possible to both cool the sample to maintain the low temperature and protect the protein's native conformation, and heat it to achieve continuous gradient temperature control from -10℃ to 90℃, eliminating sample denaturation problems caused by temperature fluctuations. The same capillary tube can complete continuous mass spectrometry acquisition at multiple temperature gradients without replacement, improving experimental efficiency.

[0074] In some embodiments of the present invention, the mass spectrometer static nanojet injection device with real-time temperature control further includes a support column 5, which passes through the rotating platform 2 and one end of the support column 5 is fixedly connected to the temperature control support 6. The support column 5 is used to press and fix the temperature control support 6, the heat sink 42, the TEC semiconductor chip 41 and the heat transfer component 3.

[0075] In this invention, since the two segmented support columns 5 are inserted and fixed on the rotating platform 2, together with the temperature control support 6 with clearance holes and the locking screws 7, the heat sink 42, semiconductor chip and aluminum heat exchanger 31 are pressed from top to bottom, ensuring that each temperature control component fits tightly and heat transfer is stable; at the same time, the whole device is miniaturized and can be directly mounted on a 40*40mm standard experimental platform, the parts are standardized, and disassembly, cleaning, replacement and maintenance are convenient.

[0076] In this invention, a rotating platform 2 with spring-loaded steel ball holders is provided. After rotation, the rotating platform 2 can free up sufficient space for easy insertion and removal of glass capillary tubes 34. It is combined with aluminum heat exchange components and hollow ceramic sleeves 32 to form a heat transfer carrier. Relying on the TEC semiconductor chip 41, bidirectional continuous temperature control for cooling and heating is achieved. With the help of temperature sensing elements, a closed-loop temperature control is formed to stabilize and protect the natural conformation of the sample. All temperature control and heat transfer components are then pressed and fixed by support columns 5 and temperature control support components 6. The whole machine is miniaturized and adapted to mass spectrometry platforms, taking into account multiple requirements such as convenient sample loading, accurate gradient temperature control and easy disassembly and maintenance. It can realize multi-temperature gradient non-denaturing mass spectrometry detection of the same sample without replacement.

[0077] In detail, this invention uses a rotating platform 2 to rotate the entire device 360° to facilitate the insertion of the glass capillary 34. Before sample injection, the platform is rotated 90° to any side, and the glass capillary 34 containing the sample is inserted into the heat transfer component 3. Then, it is rotated back to its original position, and the sample is cooled and heated by the temperature control component 4. Finally, the sample is analyzed by mass spectrometry. This invention can maintain low temperatures to ensure the non-denaturation of biological samples and can also continuously control the temperature of the same sample at different temperatures to explore the effects of sample injection at different temperatures on sample properties.

[0078] The present invention will be better illustrated by the following examples.

[0079] Example 1

[0080] like Figures 1 to 7 As shown, this embodiment provides a static nanojet mass spectrometer injection device with real-time temperature control, including: a base 1, a rotating platform 2, a heat transfer component 3, a temperature control component 4, a support column 5, a temperature control support component 6, and multiple fasteners such as screws 7. Preferably, the support column 5 is provided as two columns.

[0081] The rotating platform 2 and the base 1 are assembled by a spring-ball limiting rotation structure. The base 1 is a plate-shaped structure with M3 mounting through holes at the four corners, a central through hole in the center, and four stepped limiting assembly holes evenly distributed around it. Springs and steel balls are installed in the holes respectively, and the top of the steel ball protrudes from the upper surface of the base 1. The lower part of the rotating platform 2 is a regular hexagonal structure with a circular track groove on the lower surface, which is adapted to the spring steel ball of the base 1. The steel ball can roll along the circular track groove to achieve 360° rotation of the rotating platform 2, and can be locked into a preset position to achieve 90° fixed-point limiting rotation. In use, the rotating platform 2 can drive the upper whole device to rotate on the base 1, which facilitates the insertion and position adjustment of the glass capillary tube 34.

[0082] The upper part of the rotating platform 2 has an irregular boss structure with a groove on the boss. The heat transfer component 3 is directly embedded and fixed in the groove. The heat transfer component 3 includes a cuboid aluminum heat exchanger 31, a hollow ceramic sleeve 32, a sensor placement hole 33, and a glass capillary tube 34. The aluminum heat exchanger 31 has a through-hole in the center, and the hollow ceramic sleeve 32 is fitted into the through-hole. The glass capillary tube 34 containing the sample is placed in the cavity inside the hollow ceramic sleeve 32. The aluminum heat exchanger 31 has a sensor placement hole 33 for placing a temperature feedback sensor to realize real-time acquisition of the temperature around the sample.

[0083] Specifically, the capillary glass tube tip cannot extend too far out of the ceramic sleeve in the heat transfer component during measurement, ensuring that the upper temperature control component does not collide with the mass spectrometer inlet; and because there is very little liquid in the capillary, if it extends too far out and does not cover most of the liquid in the capillary, the temperature fluctuation will be too large; the upper part of the rotating platform 2 is an irregular boss structure, and its upper part can be a semicircle that is then cut off to make a small rectangular plane. This is to facilitate the adjustment of the distance to the mass spectrometer, and also because the capillary is inserted from the small rectangular plane, while the arc shape is difficult to operate and takes up space.

[0084] The temperature control component 4 includes a TEC level 3 semiconductor chip 41 and a heat sink 42. Based on the outline center of the heat transfer component 3, the TEC level 3 semiconductor chip 41 and the heat sink 42 are stacked from top to bottom, with the cold end of the TEC level 3 semiconductor chip 41 facing down. It is bonded and fixed to the top surface of the aluminum heat exchanger 31 with thermally conductive adhesive. The heat sink 42 is attached and fixed to the hot end of the TEC level 3 semiconductor chip 41 with thermally conductive adhesive, forming a temperature control conduction structure.

[0085] The support column 5 has a segmented structure with thick and thin sections, and there are two columns in total. The thick end of the support column 5 is inserted and fixed in the vertical mounting hole of the irregular boss of the rotating platform 2, and the thin end has an external thread on its outer circumference. The temperature control support 6 has a clearance through hole corresponding to the position of the two support columns 5. The thin end of the support column 5 passes through the clearance through hole of the temperature control support 6 and is locked and fixed by screws 7, so that the temperature control support 6 is pressed and limited above the radiator 42, realizing the overall pressing and fixing of the temperature control component 4 and the heat transfer component 3.

[0086] During assembly, the base 1 is first installed on the adapter platform with screws, and then the rotating platform 2 is aligned and assembled with the base 1. The spring steel ball and the circular track groove cooperate to achieve a limited rotational connection. The aluminum heat exchanger 31 of the heat transfer component 3 is embedded in the groove of the upper boss of the rotating platform 2. The hollow ceramic sleeve 32 is placed into the central through hole of the aluminum heat exchanger 31, and the temperature sensor is installed into the sensor placement hole 33. The cold end of the TEC level semiconductor chip 41 is fixed to the center of the upper end face of the aluminum heat exchanger 31 with thermally conductive adhesive, and then the heat sink 42 is fixed to the hot end of the TEC level semiconductor chip 41 with thermally conductive adhesive. The thick ends of the two support columns 5 are inserted into the vertical assembly holes of the rotating platform 2, the temperature control support 6 is fitted into the thin end of the support column 5, and locked with screws 7 to complete the assembly of the whole machine.

[0087] When using the device, the operator first rotates the rotating platform 2 90° to one side and inserts the glass capillary 34 containing the sample into the hollow ceramic sleeve 32, ensuring that the tip of the glass capillary 34 extends 1-2 mm out of the sleeve. Then, the rotating platform 2 is rotated back to its original position, and the distance between the tip of the glass capillary 34 and the mass spectrometer inlet is finely adjusted to the optimal detection position of 5-10 mm.

[0088] Temperature monitoring is achieved through a temperature sensor embedded in the sensor placement hole 33 of the aluminum heat exchanger 31. The sensor probe is attached to the outer wall of the hollow ceramic sleeve 32, collecting real-time temperature data around the sample and feeding it back to the temperature control component. The temperature control component adjusts the direction and magnitude of the power supply current to the TEC three-stage semiconductor chip 41 according to the set temperature: when heating is required, a positive current is input, and heat is conducted to the sample through the aluminum heat exchanger 31; when cooling is required, the current direction is switched, and the cold end absorbs heat to cool the sample; after the temperature reaches the set value, the system automatically maintains the temperature stability; once the temperature stabilizes, the mass spectrometer can be started for sample analysis.

[0089] During disassembly and maintenance, the screws 7 on the support column 5 can be loosened and the temperature control support 6 can be removed, allowing the heat sink 42 and the TEC level semiconductor chip 41 to be disassembled in sequence, facilitating the cleaning, replacement, or repair of each component. The device has a compact overall structure, is easy to assemble and disassemble, and has standardized design for each component, making it highly versatile.

[0090] Furthermore, as a specific embodiment of the present invention, the total height of the rotating platform is 12mm. Its lower part is a regular hexagonal structure with an inscribed circle diameter of 28mm and a height of 2mm. The bottom surface has a circular track groove with an outer diameter of 25.5mm, a groove width of 1.5mm, and a groove depth of 0.2mm. The upper part of the platform has an irregular boss with a height of 10mm. The boss has a groove with a length of 9mm, a width of 16mm, and a depth of 7mm for installing heat transfer components, and also has a vertical mounting hole with a diameter of 6mm and a depth of 5mm.

[0091] The rectangular aluminum heat exchanger in the heat transfer assembly measures 9mm*16mm*6mm, with a central through-hole diameter of 3mm. The hollow ceramic sleeve has an outer diameter of 3mm, an inner diameter of 1mm, and a length of 16mm. The aluminum heat exchanger also has a sensor placement hole that is 1mm long, 3mm wide, and 10mm deep.

[0092] In the support structure, the support column has a 6mm diameter and 15mm length at the thicker end, a 5mm diameter at the thinner end, and a 4mm long M3 threaded section. The overall dimensions of the temperature control support are 60mm*46mm*4mm, and it has a 5mm diameter clearance through hole.

[0093] In this way, by setting the base size to 40mm*40mm and precisely designing the dimensions and assembly relationships of each component, the entire device is small in size and compact in structure, which can be easily mounted on the three-dimensional moving platform of the mass spectrometer without occupying too much operating space. Since the present invention is suspended on the three-dimensional moving platform of the mass spectrometer during installation, in order to maintain the durability of the three-dimensional platform and the space for sample injection operation, the smaller the size, the better. However, the size of the three-dimensional moving platform is determined by the moving platform of the static nano-jet source. Therefore, the side length of the base in the present invention is generally 10-100mm, which needs to be adjusted according to the actual sample injection method and the size of the moving platform.

[0094] Example 2

[0095] like Figures 8 to 13 As shown, this embodiment uses native mass spectrometry as an example. Using myoglobin as the test sample, the static nanojet mass spectrometer of this invention, with real-time temperature control, was used to acquire mass spectrometry data at a continuous temperature gradient of 20℃ to 90℃. The effect of different injection temperatures on the mass spectrum of the biological sample was verified. The specific experiment is as follows:

[0096] 1. Experimental Materials and Instruments

[0097] Test sample: Myoglobin standard, CAS-No: 100684-32-0;

[0098] Experimental setup: The present invention includes a real-time temperature-controlled static nanojet mass spectrometer and a self-made sample introduction platform;

[0099] Detection instrument: Bruker's timsTOF high-resolution mass spectrometer.

[0100] 2. Experimental Methods

[0101] Sample loading: Dissolve the myoglobin standard in pure water, select ammonium acetate solution as buffer solution, continue ultrafiltration and desalting, and then load it into the glass capillary tube 34. The sample loading operation is completed by rotating the rotating platform 2 of this device by 90°. Then the rotating platform 2 is reset to the detection position.

[0102] Temperature control settings: Before needle insertion, the temperature is set to 20℃ using the device's temperature control component. After the temperature program is ready, needle insertion is performed, and after 30 seconds, temperature recording begins. The continuous test temperature gradient is set to 20℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ using the device's temperature control component. Relying on the synergistic effect of the device's TEC level 3 semiconductor chip 41 and aluminum heat exchanger 31, data acquisition begins after each temperature point stabilizes to ±0.5℃. Each temperature point is maintained for 3 minutes during the experiment.

[0103] Temperature accuracy test: Using the standard solution to be tested, the solution temperature at the capillary tip was measured using a combination of a USB-TC01 thermocouple from NI (National Instruments) and a T thermocouple from Kempson. The temperature inside the aluminum heat exchanger was fed back by the thermocouple from the temperature control device, and a plot was generated. Figure 8 Temperature accuracy curve.

[0104] Mass spectrometry acquisition: Non-denaturing mass spectrometry mode was used. Without changing the sample, positive ion mode was employed, and the instrument was continuously powered on throughout the test. This resulted in the acquisition of total ion current signals and raw mass spectrometry signals at various continuous temperature points. These signals were then deconvolved to obtain protein molecular weight distribution data, which were finally processed to generate... Figure 9 Total ion chromatogram Figure 10 Original mass spectrum Figure 11 Deconvolution molecular weight distribution map Figure 12 Myoglobin protein spectrum after heme removal Figure 13 Hemoglobin spectrum.

[0105] 3. Experimental Results

[0106] To verify the temperature control accuracy of the real-time temperature-controlled static nanojet mass spectrometer described in this invention under actual sample introduction conditions, a TEC semiconductor chip controller was used as the set / feedback temperature reference. Simultaneously, a temperature recording probe was placed at the solution position at the capillary nozzle tip to collect the solution temperature at the nozzle tip in real time. During the experiment, the controller temperature was gradually increased from approximately 10 °C to approximately 90 °C according to a gradient program. The changes in the controller feedback temperature and the capillary nozzle tip solution temperature over time were recorded synchronously. The temperature curves are shown below. Figure 8 As shown.

[0107] like Figure 8 As shown, the temperature curve fed back by the TEC controller and the temperature curve of the solution at the tip of the capillary nozzle exhibit a highly consistent trend throughout the heating process, both displaying stable stepwise heating characteristics. Within each temperature plateau segment, the temperature of the solution at the tip of the capillary nozzle quickly follows the temperature fed back by the controller and tends to stabilize; during the temperature transition phase, no significant lag or overshoot occurs between the temperature of the solution at the nozzle tip and the temperature fed back by the controller. Analysis of the data within the common acquisition time range shows that the average absolute difference between the two is 0.60 ℃, the root mean square error (RMSE) is 0.63 ℃, the average deviation is +0.60 ℃, and the maximum absolute difference is 1.05 ℃. These results demonstrate that the device of this invention can effectively transfer the temperature control function of the TEC semiconductor chip to the solution region at the tip of the capillary nozzle, achieving accurate control of the sample end temperature. Even during a continuous gradient temperature increase from approximately 10°C to 90°C, the actual temperature of the solution at the nozzle tip remains consistent with the temperature fed back by the controller, indicating that the device of this invention has high temperature control accuracy and good temperature response stability, and can meet the requirements for real-time, continuous, and precise temperature control of the sample end during Native Mass static nanojet injection.

[0108] Figure 9This invention was used to obtain total ion current maps of the same protein sample at different temperatures during continuous acquisition. Throughout the acquisition process, the signal showed no sudden interruption, drop, or baseline drift, demonstrating that the mechanical structure and temperature control components of the device operate stably within a wide temperature range of 20-90℃, without vibration, temperature runaway, or other issues interfering with signal and data acquisition, thus meeting the requirements for long-term continuous acquisition using native MS. Increased temperature significantly improved the nanospray signal intensity, increasing it by nearly 10 times. This is because increased temperature reduces the viscosity and surface tension of the sample solution, resulting in better solution flow within the glass capillary, more stable spraying, higher atomization efficiency, and significantly improved ionization efficiency. Simultaneously, the device exhibits good temperature control uniformity, ensuring continuous temperature changes in the sample solution without localized overheating or overcooling affecting the spray state. The changes in signal complexity are consistent with the thermal conformational behavior of myoglobin: the signal is stable and the peak shape is simple in the low temperature range; in the intermediate temperature range, the signal begins to strengthen and the fluctuations become larger, indicating that as the temperature increases, the protein begins to undergo slight conformational loosening, and at the same time, the ionization efficiency increases, and the signal complexity increases; in the high temperature range, the signal intensity reaches its peak, the fluctuations are the most obvious, and the peak shape is the most complex. At this time, the protein begins to partially unfold, generating more ions with different charge states and different conformations. At the same time, the spray efficiency is further improved at high temperature, so the signal intensity and complexity of the BPC plot increase simultaneously.

[0109] Figure 10 This is a mass spectrum obtained by continuously acquiring the same protein standard at different temperatures using the present invention. Figure 11 This is a deconvolution-processed molecular weight distribution spectrum of the same protein standard collected continuously at different temperatures using this invention. The theoretical molecular weight of this myoglobin is 17.6 kDa, and its native state is predominantly low-charged: m / z ≈ 2196, corresponding to the +8 valence state; m / z ≈ 2510, corresponding to the +7 valence state. At low temperatures, only two main peaks, +7 and +8, are present, with no high-charged state peaks, indicating that the protein completely maintains its native folded conformation without denaturation / unfolding. As the temperature increases, a +9 peak appears and the signal gradually strengthens. Examination of the spectrum revealed a trace amount of high-charged state signal, with a peak of +13. At high temperatures, the peak baseline slightly broadens, consistent with the slight conformational loosening behavior of myoglobin at thermal denaturation temperatures. The deconvolution-processed molecular weight distribution spectrum shows a signal of 17566 Da in all temperature ranges, indicating that the protein bulk is not degraded, the overall structure remains stable, and heating did not cause irreversible aggregation. As the temperature increases, more fragments are detected, and the tailing of the main peak at 17566 becomes severe. This is because the temperature increases induce local conformational changes in the protein, forming dimers, oxidizing, or exposing regions that bind to salt ions, resulting in a series of characteristic peaks of Na⁺ / K⁺ adducts. Figure 10 and Figure 11The overall protein mass distribution demonstrates that the temperature-controlled static nanojet injection device can induce and detect the temperature response behavior of protein non-covalent complexes.

[0110] Figure 12 This image shows mass spectra of deheme-bound myoglobin obtained by continuous sampling of the same myoglobin standard at different temperatures using this invention. Deheme-bound myoglobin is the protein component formed after myoglobin loses its heme cofactor, and its signal intensity reflects the degree of dissociation of heme from myoglobin. At 20 °C and 40 °C, the deheme-bound myoglobin signal is weak, indicating that myoglobin mainly remains in a heme-bound state at low temperatures. As the temperature increases, especially after 50 °C, the deheme-bound myoglobin signal gradually strengthens, suggesting that myoglobin begins to undergo thermally induced dissociation. When the temperature is further increased to 70, 80, and 90 °C, the deheme-bound myoglobin peak becomes more pronounced, indicating that higher temperatures significantly promote the detachment of the heme cofactor from myoglobin. This figure directly demonstrates that by controlling the temperature of the injection solution, the process of myoglobin transitioning from an intact bound state to a deheme-bound state can be observed under native mass spectrometry conditions.

[0111] Figure 13 This invention provides mass spectra of heme obtained by continuously collecting samples of the same myoglobin standard at different temperatures. Heme is a non-covalently bound cofactor in myoglobin, and the appearance and enhancement of its free signal can serve as direct evidence of the dissociation of the myoglobin-heme complex. At low temperatures, the free heme signal is weak, indicating that heme is mainly bound to myoglobin. As the temperature increases, the heme-related peaks gradually enhance, suggesting that heating causes heme to be released from the myoglobin binding site. At high temperatures, the heme signal further enhances and interacts with the myoglobin. Figure 12 The increasing trend in the deheme myoglobin signal corresponds to this. Both indicate that heating can induce the dissociation of the myoglobin-heme non-covalent complex, generating deheme myoglobin and free heme. This figure further validates the ability of the temperature-controlled native MS method to detect changes in protein-prosthetic interactions at the level of small molecule prosthetic groups.

[0112] 4. Experimental Conclusions

[0113] This embodiment demonstrates, through continuous temperature-controlled mass spectrometry testing of myoglobin standards using non-denaturing mass spectrometry, that: the device can achieve continuous, accurate, and stable temperature control within the range of 20℃ to 90℃; the device can effectively protect the natural conformation of biological samples, adapt to the needs of non-denaturing mass spectrometry detection, and is especially suitable for thermally unstable protein samples; the device can achieve continuous acquisition of the same sample under different temperature gradients without the need to change samples or repeat sample loading, making operation convenient and significantly improving the efficiency of non-denaturing mass spectrometry analysis.

[0114] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. A static nanojet mass spectrometer injection device with real-time temperature control, characterized in that, include: The base serves as the fundamental load-bearing foundation. A rotating platform is mounted on the base via a limiting structure and can rotate relative to the base. A groove is provided in the middle of the rotating platform. A heat transfer assembly is embedded and fixed in the groove. The heat transfer assembly includes an aluminum heat exchanger and a hollow ceramic sleeve for placing a glass capillary tube containing a sample. The aluminum heat exchanger has a through-hole in the center, and the hollow ceramic sleeve is fitted into the through-hole. The aluminum heat exchanger has a sensor placement hole for placing a temperature sensor. A temperature control component includes a TEC level 3 semiconductor chip and a heat sink. With the outline center of the heat transfer component as a reference, the TEC level 3 semiconductor chip and the heat sink are stacked sequentially from top to bottom. The cold end of the TEC level 3 semiconductor chip faces downward and is bonded and fixed to the top surface of the aluminum heat exchanger with thermally conductive adhesive. The heat sink is attached and fixed to the hot end of the TEC level 3 semiconductor chip with thermally conductive adhesive, forming a temperature control conduction structure. The support structure includes a support column and a temperature control support member for supporting the temperature control component. The temperature control support member is mounted above the radiator via the support column. The support member passes through the temperature control support member and is locked by a locking member, so that the temperature control support member is pressed and limited above the radiator.

2. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 1, characterized in that, The rotating platform is a one-piece, layered structure, with a circular track groove on the surface of the lower layer. The base has multiple stepped limiting assembly holes evenly distributed around its perimeter. Each limiting assembly hole contains a spring and a steel ball. The steel ball is positioned at the outer end of the spring, and the top of the steel ball protrudes from the upper surface of the base. Along the outer periphery of the circular track groove, the circular track groove has multiple preset locking positions to fix the rotating platform relative to the base at a fixed point. The circular track groove is adapted to the steel ball and spring in the limiting assembly hole. The steel ball can roll along the circular track groove and can be locked into the preset locking position to realize the angle limit of the rotating platform. The upper layer of the rotating platform is an irregularly shaped boss, and the boss has a groove for installing the heat transfer component.

3. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 2, characterized in that, Four locking positions are evenly distributed along the circumference of the circular track groove, so that the preset angle of rotation of the rotating platform is 90° each time.

4. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 2, characterized in that, Each of the stepped limiting assembly holes has a square hole at the top and a circular hole at the bottom. The spring is placed in the circular hole, and the steel ball is fitted into the square hole.

5. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 2, characterized in that, The boss has an assembly hole for the support column to pass through.

6. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 1, characterized in that, The support column has a segmented structure with thick and thin sections. The thick end of the support column is inserted and fixed in the boss, and the thin end of the support column is provided with an external thread for threaded connection with the locking member, so that the temperature control support is limited above the heat sink, thereby realizing the overall fixation of the temperature control component and the heat transfer component.

7. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 1, characterized in that, The base has a mounting through hole for mounting on a mass spectrometer, and the center of the base has a central through hole for assembling the rotating platform; the rotating platform and the base are coaxially assembled, and the rotating platform rotates around the center of the base; a plurality of limiting mounting holes are evenly distributed around the central through hole.

8. The real-time temperature-controlled static nanojet mass spectrometer injection device according to claim 1, characterized in that, The sensor probe of the temperature sensor is attached to the outer wall of the hollow ceramic sleeve to collect temperature data around the sample and feed it back to the temperature control component. The temperature control component adjusts the direction and magnitude of the power supply current to the TEC level 3 semiconductor chip according to the preset temperature; When heating is required, a positive current is input to the TEC level 3 semiconductor chip, and the heat is transferred to the sample through the aluminum heat exchanger. When cooling is required, the direction of the current input to the TEC level 3 semiconductor chip is switched so that the cold end absorbs heat to cool the sample. When the temperature reaches the preset temperature value, the temperature control component maintains a stable temperature and starts the mass spectrometer to analyze the sample.

Images