CN113327836A

CN113327836A - Sample loading device for mass spectrometer

Info

Publication number: CN113327836A
Application number: CN202110594684.2A
Authority: CN
Inventors: 门涌帆; 王冠博; 李雯; 柴胡玲潇
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS; Shenzhen Bay Laboratory
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-08-31
Anticipated expiration: 2041-05-28
Also published as: WO2022247246A1; CN113327836B

Abstract

The invention relates to the technical field of biomedical treatment, and discloses a sample loading device for a mass spectrometer, which is arranged on the sample inlet side of the mass spectrometer and comprises a microfluidic chip, a connecting pipeline, a spray needle and a sample injection structure; the sample injection structure injects a sample to be detected and a reaction solution into the microfluidic chip; one end of the connecting pipeline is connected with the outlet end of the microfluidic chip; the spray needle is connected to the other end of the connecting pipeline, the spray needle is connected with a power supply, and the sample solution is sprayed in an electrospray mode; the spray needle generates spray to be sprayed into a sample inlet of a mass spectrometer, on one hand, the device can be adapted to a commercial mass spectrometer, on-line efficient mixing-mass spectrum sample introduction of samples is realized, the reaction time after mixing is accurately regulated and controlled, and the device is suitable for real-time mass spectrum characterization of the dynamic process of the samples at different time points in specific biochemical reaction; on the other hand, the special interface form is tight in connection, free of liquid leakage, high in ionization efficiency and sensitivity, small in dead volume and convenient to replace.

Description

Sample loading device for mass spectrometer

Technical Field

The invention relates to the technical field of biomedical treatment, in particular to a sample loading device for a mass spectrometer.

Background

Mass Spectrometry (MS) is an efficient method for the structural and kinetic properties of natural and non-natural proteins. MS has significant advantages in molecular and pharmacological analysis as well as clinical practice due to its high analytical sensitivity and specificity. However, this technique still needs to improve the performance in the following aspects: (1) dynamic (time) resolution capability. The high-order structure and interaction of the protein in a solution environment have high dynamic property, species can rapidly exchange, and a large amount of intermediates can be formed. (2) And (4) operability. Part of the method has more steps and higher requirements on experience and technology of operators, and large-scale popularization in industrial practice still needs to be simplified. (3) And (4) integration. The characterization aspects required by protein characterization are complicated, and some prior arts can only use one type of equipment, one method or one set of experience to solve the characterization problem in a single aspect, so that the economic and labor costs required for comprehensively meeting the characterization requirements are high. Although some research has been conducted on the integration of the methods, more hardware and process efforts are still required for the integration. (4) The sample preparation steps are complicated and the sample consumption is large. These problems limit further applications of mass spectrometry techniques. For example, in the conventional mass spectrometry, samples participating in a specific biochemical reaction need to be quenched at a specific time node and then subjected to offline sample injection, time resolution with a small time scale is difficult to realize, complete quenching is difficult to realize for a large number of reactions, unexpected further reaction, side reaction or reverse reaction can be introduced in subsequent processing and sample injection processes, and a measurement result is seriously interfered. Therefore, the application space of the mass spectrometry technology can be greatly expanded by the technical improvement in the aspects of sample online reaction, real-time sample introduction, dynamic regulation and control and automatic control operation.

Microfluidic devices have various advantages in the manipulation and detection of chemicals and biomolecules. The platform can realize low sample consumption, can realize automation, high flux operation and temperature control, flexible parameter customization and adjustable reaction time, and has a mass spectrum sample loading device based on a micro-flow chip, so that sample pretreatment and operation on a biochemical reaction chip can be completed on line, and downstream sample injection is carried out in real time, thereby accurately detecting the product characteristics under the condition of specific reaction time, and saving a large amount of sample waste and labor cost, therefore, the combination of the micro-flow chip and a mass spectrometer is very needed.

Among these, analyte ionization is critical for MS sample analysis, and microfluidic chip and mass spectrometer integration have many challenges, such as whether the total flow rate and solution composition of a sample after pretreatment or reaction is compatible with mass spectrometry, how to efficiently ionize a pretreated or reacted sample, and transfer analytes from a microfluidic chip to a coupling interface of a mass spectrometer. The fabrication and repeatability of glass or polymer chip-based interfaces is not straightforward, which also limits the widespread use of online electrospray mass spectrometry (ESI-MS).

Disclosure of Invention

In view of the above-mentioned shortcomings, the present invention provides a sample loading device for a mass spectrometer, which can adapt to mass spectrometry in terms of total flow rate and solution composition after pretreatment or reaction of a sample, and provide stable and high ionization efficiency, and the chip-mass spectrometry interface design is convenient, simple and expandable.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: a sample loading device for a mass spectrometer is arranged on the sample inlet side of the mass spectrometer and comprises a microfluidic chip, a connecting pipeline, a spray needle and a sample injection structure;

the sample injection structure injects a sample solution to be detected into the microfluidic chip;

one end of the connecting pipeline is connected to the outlet end of the microfluidic chip;

the spray needle is connected to the other end of the connecting pipeline, the spray needle is connected with a power supply, and the sample solution in the spray needle forms spray in an electrospray mode;

the needle produces a spray that is injected into a sample inlet of the mass spectrometer.

Furthermore, the connecting pipeline comprises a two-way connector and a sleeve, one end of the two-way connector is connected with the outlet end of the microfluidic chip through the sleeve, and the other end of the two-way connector is connected with the spray needle through the sleeve.

Further, the sleeve is made of a high polymer material, and the sleeve is in interference fit with the outlet end of the microfluidic chip.

Furthermore, an included angle between the microfluidic chip and the spray needle is an acute angle, a right angle or an obtuse angle, and the connecting pipeline and the spray needle are in the same straight line.

Further, the distance between the outlet of the spray needle and the sample inlet of the mass spectrometer is 1-100 mm.

Further, the above-described needles include, but are not limited to, microporous sleeve-like structures made of stainless steel.

Furthermore, the spray needle can be simplified into a conductive capillary which is directly connected with an outlet of the microfluidic chip without parts such as a two-way joint, a sleeve and the like.

Further, the mass spectrometer comprises a power supply, the spray needle is connected with the power supply of the mass spectrometer, and the mass spectrometer is provided with electric energy.

Furthermore, the inner diameter of the spray needle is 1-200 μm, the outer diameter is 100-3000 μm, and the length is 1-100 mm.

Further, the microfluidic chip comprises a main flow channel and an auxiliary flow channel, wherein the main flow channel and the auxiliary flow channel are used for adding different solutions;

the auxiliary flow channel is communicated with the main flow channel, and the auxiliary flow channels are provided with a plurality of auxiliary flow channels.

Furthermore, a flow guide structure is arranged in the main flow channel and used for mixing different solutions added into the main flow channel and the auxiliary flow channel.

Further, the water conservancy diversion structure includes the fishbone structure, the fishbone structure includes first fishbone structure and second fishbone structure, first fishbone structure and second fishbone structure are the axisymmetric structure, first fishbone structure and second fishbone structure are in separate the setting in the sprue.

Has the advantages that:

according to the invention, the spray needle is connected to the outlet end of the microfluidic chip by using a connecting pipeline, and the sample solution is sprayed into the sample inlet of the mass spectrometer by using the electric spraying technology, so that the electrospray efficiency and sensitivity of the mass spectrometer can be improved, and the identification of analytes with various structures can be improved; on the other hand, the structure is connected tightly, no liquid leakage can be realized, no liquid leakage can be guaranteed under the condition that a large flow velocity passes through, the dead volume is small, the replacement is convenient, the operation that the microfluidic chip or the spray needle needs to be replaced in the experiment is simplified, and the conditions that the experiment cannot be continued and the like when the pollution is reduced or the spray needle is blocked are reduced.

Drawings

The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:

FIG. 1 is a schematic diagram of a loading apparatus and mass spectrometer according to an embodiment of the invention;

FIG. 2 is a schematic view of the connection line and the needle according to an embodiment of the present invention;

FIG. 3 is a cross-sectional view of detail A of FIG. 1;

FIG. 4 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present invention;

FIG. 5 is a schematic view of the structure and solution mixing at the junction of the primary and secondary flow channels of an embodiment of the present invention;

fig. 6 is a schematic structural diagram of a microfluidic chip according to another embodiment of the present invention.

Wherein, 100, a sample loading device; 110. a microfluidic chip; 111. a main flow channel; 112. an auxiliary flow channel; 113. a fishbone structure; 113a, a first fishbone structure; 113b, a second fishbone structure; 120. connecting a pipeline; 121. a two-way joint; 122. a sleeve; 130. spraying needles; 140. a sample injection structure; 141. a sample loading pipeline; 142. a solution injector; 200. a mass spectrometer; 210. a sample inlet; 220. a power source.

Detailed Description

It is to be understood that the terminology, the specific structural and functional details disclosed herein are for the purpose of describing particular embodiments only, and are representative, but that the present invention may be embodied in many alternate forms and should not be construed as limited to only the embodiments set forth herein.

In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating relative importance or as implicitly indicating the number of technical features indicated. Thus, unless otherwise specified, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature; "plurality" means two or more. The terms "comprises" and "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that one or more other features, integers, steps, operations, elements, components, and/or combinations thereof may be present or added.

Further, terms of orientation or positional relationship indicated by "center", "lateral", "upper", "lower", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, are described based on the orientation or relative positional relationship shown in the drawings, and are only for convenience of simplifying the description of the present invention, and do not indicate that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention.

Furthermore, unless expressly stated or limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly and may include, for example, fixed connections, removable connections, and integral connections; can be mechanically or electrically connected; either directly or indirectly through intervening media, or through both elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

The invention will be further elucidated with reference to the drawings and alternative embodiments.

As shown in fig. 1, the embodiment of the present invention discloses a loading device 100 for a mass spectrometer 200, wherein the loading device 100 is disposed on a sample inlet 210 side of the mass spectrometer 200, and the loading device 100 includes a microfluidic chip 110, a connecting pipeline 120, a nozzle 130, and a sample injection structure 140; the sample injection structure 140 injects a sample solution to be tested into the microfluidic chip 110; one end of the connecting pipeline 120 is connected to the outlet end of the microfluidic chip 110; the spray needle 130 is connected to the other end of the connecting pipeline 120, the spray needle 130 is connected to a power supply 220, and the sample solution in the spray needle 130 is sprayed in an electrospray manner; the needle 130 generates a spray that is injected into the sample inlet 210 of the mass spectrometer 200.

Specifically, the principle of forming the sample solution in the needle 130 into a spray by electrospray is as follows: when fine mist droplets are ejected from the metal needle 130, a large amount of charges are obtained from the high-intensity electric field at the orifice of the metal needle 130, and the charges are uniformly distributed on the surface of the droplets due to the coulomb force acting between the charges. As the droplets are dried to remove the solvent, the volume of the droplets gradually decreases and the charge per surface area increases dramatically, making the droplets unstable and breaking apart, producing a finer spray of droplets.

In the embodiment, the nozzle 130 is connected to the outlet end of the microfluidic chip 110 by a connecting pipeline 120, and the sample solution is injected into the sample inlet 210 of the mass spectrometer 200 by the electrospray technique, so that the electrospray efficiency and sensitivity of the mass spectrometer 200 can be improved, and the identification of analytes with various structures can be improved; on the other hand, the structure is connected tightly, no liquid leakage can be realized, no liquid leakage can be guaranteed under the condition of passing at a high flow rate, the dead volume is small, the replacement is convenient, the operation of replacing the microfluidic chip 110 or the spray needle 130 in an experiment is simplified, and the conditions that the experiment cannot be continued when the pollution or the spray needle 130 is blocked are reduced.

Wherein the mass spectrometer 200 comprises a power supply 220, and the nozzle 130 is connected with the power supply 220 of the mass spectrometer 200 and is powered by the mass spectrometer 200. The sample loading device 100 is powered by the mass spectrometer 200 for electrospray, and the additional power supply 220 is not needed, so that the volume of the sample loading device 100 is reduced, and the portability and the use convenience of the sample loading device 100 are improved.

Further, the distance from the outlet of the nozzle 130 to the sample inlet 210 of the mass spectrometer 200 is 1-100 mm. The micro liquid drops formed by electrospray of the sample solution can be guaranteed to uniformly fly into the sample inlet 210 of the mass spectrometer 200, and the phenomenon that the micro liquid drops sprayed into the sample inlet 210 of the mass spectrometer 200 are not uniform due to too short distance or some formed micro liquid drops cannot fly into the sample inlet 210 of the mass spectrometer 200 due to too long distance and are wasted or polluted due to falling outside is avoided.

As shown in fig. 2 and 3, in one embodiment, the connecting line 120 includes a two-way connector 121 and a sleeve 122, one end of the two-way connector 121 is connected to the outlet end of the microfluidic chip 110 through the sleeve 122, and the other end of the two-way connector 121 is connected to the nozzle 130 through the sleeve 122. The sleeve 122 is made of a high polymer material, and the sleeve 122 is directly inserted into the outlet end of the microfluidic chip 110 through friction force, is in interference fit with the outlet end of the microfluidic chip 110, is tightly connected and does not leak liquid, and can ensure that no liquid leaks even when a large flow rate passes through the sleeve; when the sleeve 122 is blocked or needs to be cleaned, the sleeve is directly pulled out to be replaced or cleaned, so that the device is convenient to disassemble and assemble, quick and simple to replace, and the condition that the experiment and the like cannot be continued during operation is reduced.

Specifically, the material of the casing 122 may be a plurality of polymer materials suitable for mass spectrometry, such as PEEK polyether ether ketone, PEK polyether ketone, PEKK polyether ketone, PEEKK polyether ketone, PFA perfluoroalkoxy resin, FEP fluorinated ethylene propylene, ETFE ethylene-tetrafluoroethylene, PTFE polytetrafluoroethylene, or the like.

Wherein the spray needle 130 is a metal spray needle 130, the inner diameter of the spray needle 130 is 1-200 μm, the outer diameter is 100-3000 μm, and the length is 1-100 mm; the inner diameter of the sleeve 122 is 150-200 μm. Specifically, the inner diameter of the needle 130 is 30 μm, the outer diameter is 150 μm, and the length is 40 μm; the inner diameter of the sleeve 122 is 180 μm.

Further, the nozzle 130, the sleeve 122 and the two-way joint 121 are aligned after being connected, and form an acute angle, a right angle or an obtuse angle with the general flow direction of the microfluidic chip 110. The existing connection mode is that the total flowing direction of the added sample solution is a straight line, so that the sample solution needs to be designed accurately, the sealing needs to be designed in a tight connection mode, the replacement is troublesome under the condition, the accurate adjustment is needed, and the detection of the sample solution or the experiment is delayed due to long time.

As shown in fig. 4 and 5, in one embodiment, the microfluidic chip 110 includes a main channel 111 and an auxiliary channel 112, the sample injection structure 140 includes a sample injection line 141 and a solution injector 142, the sample injection line 141 is respectively inserted into inlets of the main channel 111 and the auxiliary channel 112, the other end of the sample injection line 141 is connected to the solution injector 142, and a sample solution and an additive solution are respectively injected into the main channel 111 and the auxiliary channel 112 by pressurization of the solution injector 142; the auxiliary flow channels 112 are communicated with the main flow channel 111, a plurality of auxiliary flow channels 112 are arranged, and the interval between each two auxiliary flow channels 112 is 11500 μm. The microfluidic chip 110 can realize online mixing of multiple paths of liquid, a main sample solution is added into the main flow channel 111, required adding liquid is added into the auxiliary flow channel 112, mixing is completed in a subsequent flow channel, namely, mixing and reaction of the solution can be completed in the microfluidic chip 110, continuous flow operation is performed, mass spectrum sample loading analysis is performed, the solution does not need to be mixed first and then detected, and detection operation needs to be stopped when the mixing proportion needs to be changed. And the flow rate of the solution is adjusted by adjusting the injection pressure through the solution injector 142, so that different mixing ratios can be changed in real time, and the operations of different reaction mixing ratios, reaction time and mixing effects can be realized. In the experiment of the hydrogen-deuterium exchange mass spectrum, the reaction time of the protein hydrogen-deuterium exchange can be adjusted on line in real time through flow rate control, and the operation of dynamically analyzing the protein structure on line is realized. As shown in fig. 5, the microfluidic chip 110 can achieve mixing functions at different ratios from 99:1 to 50:50 by demonstrating the mixing balance effect of the solution in the main channel 111(CH1) and the solution in the auxiliary channel 112(CH 2).

A flow guide structure is arranged in the main flow passage 111, and the flow guide structure is used for mixing different solutions added into the main flow passage 111 and the auxiliary flow passage 112. Specifically, the flow guiding structure is a fishbone structure 113(Herringbone structure), and according to the flowing direction of the solution, the fishbone structure 113 is disposed at a position after the main flow channel 111 and the first auxiliary flow channel 112 are merged. When the solution flows into the fishbone structure 113, a vortex or chaotic flow can be formed, so that the solution can be quickly and uniformly mixed, the uniform mixing efficiency is improved, and the length of a flow channel required by mixing is shortened, so that the micro-flow chip 110 can be reduced in size and is smaller. Of course, the flow directing structure is not limited to the fishbone structure 113, and other geometries for mixing are possible.

Further, the fishbone structure 113 includes a first fishbone structure 113a and a second fishbone structure 113b, the first fishbone structure 113a and the second fishbone structure 113b are axisymmetric structures, and the first fishbone structure 113a and the second fishbone structure 113b are spaced apart from each other in the main flow passage 111. The circular mixing is performed along the shape of the first and

second fishbone structures

113a and 113b, so that the mixing of the solution is more uniform and faster.

Specifically, the microfluidic chip 110 includes a main channel 111 and two auxiliary channels 112, the main channel 111 is a main channel, the two auxiliary channels 112 are respectively connected to the main channel 111, and an interval between the two auxiliary channels 112 is 4000 μm to 11500 μm depending on the number of cycles. One first fishbone structure 113a and one second fishbone structure 113b are formed into one cycle, and eight cycles are arranged in the main flow passage 111, namely eight first fishbone structures 113a and eight second fishbone structures 113b are arranged at intervals, namely the interval between two auxiliary flow passages 112 is 11500 μm. Of course, the arrangement mode of the auxiliary flow channels 112 is not limited to interval arrangement, and the auxiliary flow channels 112 may be arranged in a layered arrangement mode or in an included angle arrangement mode.

In the embodiment, the operation of mixing and loading the protein and the reaction solution on line in the research of the mass spectrum chip is realized, the mixing is fast, effective and stable, the reaction time of the protein can be controlled through flow rate adjustment, and the protein structure can be analyzed and analyzed dynamically in real time through mass spectrum analysis. The microfluidic chip 110 can realize on-line mixing of multiple paths of liquid, a main sample solution is added into the main flow channel 111, a required adding liquid is added into the auxiliary flow channel 112, the solution is rapidly and uniformly mixed and subjected to on-line reaction of the sample through the circulating first fishbone structure 113a and the second fishbone structure 113b, and the mass spectrum sample loading analysis is performed through continuous flow operation. And the flow rate of the solution is adjusted by adjusting the injection pressure through the solution injector 142, so that different mixing ratios can be changed in real time, and the operations of different reaction mixing ratios, reaction time and mixing effects can be realized. Moreover, the liquid on-line mixing proportion range and the flow rate range are large, and the mixing reaction operation of various proteins can be met.

In another embodiment, as shown in fig. 6, a main channel 111 and two auxiliary channels 112 form a set of channels, and the microfluidic chip 110 includes two independent sets of channels, but multiple sets of channels may be provided, and may be used for multiple times without cleaning, or may be used for detecting different sample solutions simultaneously. It is easier to directly design multiple sets of channels during the fabrication of the microfluidic chip 110, and the manufacturing difficulty is not greatly affected, so that raw materials and time can be saved. The microfluidic chip can be made of glass quartz, silicon chip, paper-based material, high molecular polymer (PDMS, PMMA, PC, etc.), etc. The manufacturing method of the microfluidic chip 110 specifically includes: the microfluidic chip 110 with the fishbone structure 113 is designed using drawing software, and a film is fabricated, followed by fabrication of a microfluidic chip template by photolithography. An exemplary microfluidic chip 110 was prepared by mixing Polydimethylsiloxane (PDMS) solution a and solution B at a ratio of 10:1, pouring the mixture into the previously prepared lithographic template, evacuating and defoaming, horizontally placing the resulting product in an 80-degree oven, standing for 45 minutes, and demolding to obtain a corresponding PDMS chip, and punching through holes in the inlet and outlet of the corresponding PDMS chip with a 0.75mm PDMS punch. Bonding the PDMS chip on a glass slide by a Plasma cleaning machine, putting the glass slide into an 80-degree oven, and standing for 2 hours or one night.

Specifically, the microfluidic chip 110 has three inlets, one inlet of the main channel 111 and two inlets of the auxiliary channels 112, and the 8-cycle mixing fishbone structure 113 is disposed in the middle of the main channel 111, so that the microfluidic chip 110 can realize the mixing of three paths of liquid in a continuous flow, and the liquid flowing to the outlet is fully mixed.

It should be noted that, the limitations of the steps involved in the present disclosure are not considered to limit the order of the steps without affecting the implementation of the specific embodiments, and the steps written in the foregoing may be executed first, or executed later, or even executed simultaneously, and as long as the present disclosure can be implemented, all should be considered to belong to the protection scope of the present disclosure.

The foregoing is a more detailed description of the invention in connection with specific alternative embodiments, and the practice of the invention should not be construed as limited to those descriptions. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A sample loading device for a mass spectrometer is characterized in that the sample loading device is arranged on the sample inlet side of the mass spectrometer and comprises a microfluidic chip, a connecting pipeline, a spray needle and a sample injection structure;

2. The sample loading device for the mass spectrometer as recited in claim 1, wherein the connecting line comprises a two-way connector and a sleeve, one end of the two-way connector is connected with the outlet end of the microfluidic chip through the sleeve, and the other end of the two-way connector is connected with the nozzle needle through the sleeve.

3. The sample loading device for a mass spectrometer as recited in claim 2, wherein the sleeve is made of a polymer material, and the sleeve is in interference fit with the outlet end of the microfluidic chip.

4. The sample loading device for the mass spectrometer as recited in claim 1, wherein the included angle between the microfluidic chip and the needle is an acute angle, a right angle or an obtuse angle, and the connecting pipeline and the needle are in the same straight line.

5. The sample loading device for the mass spectrometer as recited in claim 1, wherein the distance from the outlet of the nozzle needle to the sample inlet of the mass spectrometer is 1-100 mm.

6. The sample loading device for the mass spectrometer as recited in claim 1, wherein the mass spectrometer comprises a power supply, the needle is connected with the power supply of the mass spectrometer, and the spray voltage is provided by the mass spectrometer.

7. The loading device for a mass spectrometer as recited in any one of claims 1-6, wherein the nozzle needle has an inner diameter of 1-200 μm, an outer diameter of 100-3000 μm, and a length of 1-100 mm.

8. The loading device for a mass spectrometer as recited in any one of claims 1-6, wherein the microfluidic chip comprises a main channel and a secondary channel, the main channel and the secondary channel are used for adding different solutions;

9. The sample loading device for the mass spectrometer as recited in claim 8, wherein a flow guide structure is disposed in the main flow channel, and the flow guide structure is used for flow mixing of different solutions added in the main flow channel and the auxiliary flow channel.

10. The sample loading device for the mass spectrometer as defined in claim 9, wherein the flow guide structure comprises a fishbone structure, the fishbone structure comprises a first fishbone structure and a second fishbone structure, the first fishbone structure and the second fishbone structure are axisymmetric structures, and the first fishbone structure and the second fishbone structure are spaced apart in the main flow passage.