WO2022247246A1 - Sample loading device for mass spectrometer - Google Patents

Abstract

The present invention relates to the technical field of biomedical treatment. Disclosed is a sample loading device for a mass spectrometer. The sample loading device is provided at a sample inlet side of the mass spectrometer. The sample loading device comprises a microfluidic chip, a connecting pipeline, a spray needle, and a sample injection structure; the sample injection structure injects a sample to be tested and a reaction solution into the microfluidic chip; one end of the connecting pipeline is connected to an outlet end of the microfluidic chip; the spray needle is connected to the other end of the connecting pipeline and is connected to a power supply, and a sample solution is formed into spray in an electrospray manner; the spray needle generates the spray for being sprayed into a sample inlet of the mass spectrometer. On the one hand, the device can be adapted to a commercialized mass spectrometer to achieve online sample high-efficiency mixing-mass spectrum sample injection and accurate regulation and control of reaction time after mixing, and is suitable for real-time mass spectrum characterization of a sample dynamic process at different time points in a specific biochemical reaction; on the other hand, a special interface form is used, so that connection is tight without liquid leakage, ionization efficiency and sensitivity are high, dead volume is small, and replacement is convenient.

Description

Sample Loading Devices for Mass Spectrometers technical field

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

Background technique

Mass spectrometry (MS) is a powerful method for targeting the structural and dynamic properties of native and unnatural proteins. Due to its high analytical sensitivity and specificity, MS has significant advantages in molecular and pharmacological analysis as well as in clinical practice. However, this technology still needs to improve performance in the following areas: (1) Dynamic (time) resolution capability. The high-order structure and interaction of proteins in solution environment are highly dynamic, and species can be rapidly interconverted, and a large number of intermediates can also be formed. However, existing methods are difficult to provide characterization data in the time dimension. (2) Operability. Some methods have many steps and require high experience and technical requirements for operators, so they still need to be simplified for large-scale promotion in industrial practice. (3) Integration. The characterization required for protein characterization is complicated, and some existing technologies can only fight on their own, using a type of equipment, a method or a set of experience to solve a single aspect of the characterization problem, fully meeting the economic and human requirements required for characterization The cost is huge. Although some research work has explored method integration, more hardware and method efforts are still needed to achieve the goal of integration. (4) The sample preparation steps are complicated and the sample consumption is large. These problems limit the further application of mass spectrometry. For example, in the traditional mass spectrometry method, the samples participating in specific biochemical reactions need to be quenched at a specific time point and then injected offline. It is difficult to achieve time resolution on a small time scale, and it is difficult to achieve complete quenching for a large number of reactions. The process can introduce unintended further reactions, side reactions or reverse reactions, which seriously interfere with the assay results. Therefore, technological improvements in online sample reaction, real-time sample injection, dynamic regulation, and automatic control operations can greatly expand the application space of mass spectrometry.

Microfluidic devices offer various advantages in manipulating and detecting chemicals and biomolecules. The platform can achieve low sample consumption, automation, high-throughput operation and temperature control, flexible parameter customization and adjustable reaction time. With a mass spectrometer sample loading device based on a microfluidic chip, samples can be completed online. The pretreatment and biochemical reaction chip are operated, and samples are injected downstream in real time, so that the product characteristics under specific reaction time conditions can be accurately detected, and a lot of sample waste and labor costs are saved. Therefore, the combination of microfluidic chips and mass spectrometers is very needs.

technical problem

Among them, analyte ionization is very important for MS sample analysis. There are many challenges in the integration of microfluidic chip and mass spectrometer, such as whether the total flow rate and solution composition of the sample after pretreatment or reaction are compatible with the mass Efficient ionization of processed or reacted samples, and coupling interface for transfer of analytes from the microfluidic chip to the mass spectrometer. The fabrication and reproducibility of glass or polymer chip-based interfaces is not easy, which also limits the widespread use of online electrospray mass spectrometry (ESI-MS).

technical solution

In view of the above-mentioned deficiencies, the object of the present invention is to provide a sample loading device for mass spectrometer, the device scheme can be adapted to the mass spectrometer on the total flow rate and solution composition after the sample is pretreated or reacted, and provides Stable and high ionization efficiency, and the chip-mass spectrometer interface design is convenient, simple and scalable.

In order to achieve the above object, the technical solution provided by the present invention is: a sample loading device for a mass spectrometer, the sample loading device is arranged on the side of the sample inlet of the mass spectrometer, and the sample loading device includes a microfluidic chip , connecting pipelines, injection needles and sample injection structures;

The sample injection structure injects the sample solution to be tested into the microfluidic chip;

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

The spray needle is connected to the other end of the connecting pipeline, the spray needle is powered on, and the sample solution in the spray needle is sprayed by means of electrospray;

The needle generates a spray spray into the injection port of the mass spectrometer.

Further, the connecting pipeline includes a two-way joint and a sleeve, one end of the two-way joint is connected to the outlet end of the microfluidic chip through the sleeve, and the other end of the two-way joint is connected through the sleeve A tube is connected to the needle.

Further, the material of the sleeve is a polymer material, and the sleeve is interference-fitted with the outlet end of the microfluidic chip.

Further, the 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 on the same straight line.

Further, the distance from the outlet of the needle to the inlet of the mass spectrometer is 1-100 mm.

Further, the spray needle mentioned above includes but is not limited to a microporous sleeve-like structure made of stainless steel.

Further, the spray needle can be simplified as a conductive capillary directly connected to the outlet of the microfluidic chip, without the need for two-way joints, sleeves and other parts.

Further, the mass spectrometer includes a power supply, the needle is connected to the power supply of the mass spectrometer, and the mass spectrometer provides electric energy.

Further, 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 includes a main channel and an auxiliary channel, and the main channel and the auxiliary channel are used to add different solutions;

The auxiliary flow channel is connected to the main flow channel, and there are multiple auxiliary flow channels.

Further, a diversion structure is provided in the main channel, and the diversion structure is used for mixing different solutions added in the main channel and the auxiliary channel.

Further, the flow guide structure includes a fishbone structure, and the fishbone structure includes a first fishbone structure and a second fishbone structure, and the first fishbone structure and the second fishbone structure are axisymmetric structures, so The first fishbone structure and the second fishbone structure are arranged at intervals in the main channel.

Beneficial effect

In the present invention, a connecting pipeline is used to connect the spray needle to the outlet end of the microfluidic chip, and then the sample solution is sprayed into the sample inlet of the mass spectrometer by electrospray technology. Such arrangement can improve the electrospray efficiency and the efficiency of the mass spectrometer on the one hand. Sensitivity, and improve the identification of analytes with diverse structures; on the other hand, the structure is tightly connected, no leakage, and it can also ensure no leakage under the condition of high flow rate, the dead volume is small, and it is easy to replace. It simplifies the operation that needs to replace the microfluidic chip or spray needle in the experiment, and reduces the situation that the experiment cannot be continued when the contamination or the spray needle is blocked.

Description of drawings

The included drawings are used to provide further understanding of the embodiments of the present invention, and constitute a part of the specification, are used to illustrate the implementation mode of the present invention, and together with the text description, explain the principle of the present invention. Apparently, the drawings in the following description are only some embodiments of the present invention, and those skilled in the art can obtain other drawings according to these drawings without any creative effort. In the attached picture:

Fig. 1 is a schematic structural view of a sample loading device and a mass spectrometer according to an embodiment of the present invention;

Fig. 2 is a structural schematic diagram of a connecting pipeline and an injection needle according to an embodiment of the present invention;

Fig. 3 is a sectional view of part A in Fig. 1;

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

Fig. 5 is a schematic diagram of the structure and solution mixing of the confluence of the main channel and the auxiliary channel according to 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.

Among them, 100, sample loading device; 110, microfluidic chip; 111, main flow channel; 112, auxiliary flow channel; 113, fishbone structure; 113a, first fishbone structure; 113b, second fishbone structure; 120, connection 121, two-way joint; 122, casing; 130, injection needle; 140, sample injection structure; 141, sample loading pipeline; 142, solution injector; 200, mass spectrometer; 210, sample inlet; 220, power supply.

Embodiments of the present invention

It should be understood that the terminology used and specific structural and functional details disclosed herein are merely representative for describing specific embodiments, but the invention may be embodied in many alternative forms and should not be construed as merely Be limited by the examples set forth herein.

In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating relative importance, or implicitly indicating the quantity of indicated technical features. Therefore, unless otherwise specified, the features defined as "first" and "second" may explicitly or implicitly include one or more of these features; "plurality" means two or more. The term "comprising" and any variations thereof mean non-exclusive inclusion, possible presence or addition of one or more other features, integers, steps, operations, units, components and/or combinations thereof.

Also, Center, Horizontal, Top, Bottom, Left, Right, Vertical, Horizontal, Top, Bottom, Inner, Outer The terms indicating the orientation or positional relationship are described based on the orientation or relative positional relationship shown in the drawings, and are only for the convenience of describing the simplified description of the present invention, rather than indicating that the referred device or element must have a specific orientation. , constructed and operated in a particular orientation and therefore should not be construed as limiting the invention.

In addition, unless otherwise clearly specified and limited, the terms "mounted", "connected" and "connected" should be interpreted in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection , can also be an electrical connection; it can be a direct connection, an indirect connection through an intermediary, or an internal communication between two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

The present invention will be further described below with reference to the accompanying drawings and optional embodiments.

As shown in Figure 1, the embodiment of the present invention discloses a sample loading device 100 for a mass spectrometer 200, the sample loading device 100 is arranged on the side of the sample inlet 210 of the mass spectrometer 200, the sample loading device 100 includes a microfluidic chip 110, a connecting pipeline 120, a needle 130, and a sample injection structure 140; the sample injection structure 140 injects the sample solution to be tested into the microfluidic chip 110; one end of the connecting pipeline 120 is connected to At 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 the power supply 220, and the spray needle 130 is made to The sample solution in the sample solution forms a spray; the spray needle 130 generates a spray and sprays it into the sample inlet 210 of the mass spectrometer 200 .

Specifically, the principle of spraying the sample solution in the spray needle 130 by means of electrospray is: when the fine mist droplets are sprayed out from the metal spray needle 130, they are obtained from the high-intensity electric field at the mouth of the metal spray needle 130. A large number of charges, due to the Coulomb force between the charges, these charges are evenly distributed on the surface of the droplet. When the droplet is dried to remove the solvent, the volume of the droplet decreases gradually, so the charge per unit surface area increases sharply, making the droplet unstable and splitting, resulting in a finer droplet spray.

In this embodiment, the nozzle 130 is connected to the outlet port of the microfluidic chip 110 by using a connecting pipeline 120, and then the sample solution is sprayed into the sample inlet 210 of the mass spectrometer 200 by electrospray technology. The electrospray efficiency and sensitivity of the mass spectrometer 200 improve the identification of analytes with diverse structures; The volume is small, and it is easy to replace, which simplifies the operation of replacing the microfluidic chip 110 or the injection needle 130 in the experiment, and reduces the situation that the experiment cannot be continued when the pollution or the injection needle 130 is blocked.

Wherein, the mass spectrometer 200 includes a power supply 220 , the needle 130 is connected to the power supply 220 of the mass spectrometer 200 , and the mass spectrometer 200 provides electricity. The sample loading device 100 is powered by the mass spectrometer 200 to perform electrospray without additional power supply 220 , which reduces the volume of the sample loading device 100 and increases the portability and ease of use of the sample loading device 100 .

Further, the distance from the outlet of the injection needle 130 to the sample inlet 210 of the mass spectrometer 200 is 1-100 mm. Ensure that the tiny droplets formed by the electrospray of the sample solution can evenly fly to the sample inlet 210 of the mass spectrometer 200, and prevent the tiny droplets sprayed into the sample inlet 210 of the mass spectrometer 200 from being uneven, Or the distance is too long so that some formed tiny droplets cannot fly into the sample inlet 210 of the mass spectrometer 200 and fall outside to cause waste or pollution.

As shown in Figures 2 and 3, in one embodiment, the connecting pipeline 120 includes a two-way joint 121 and a sleeve 122, and one end of the two-way joint 121 is connected to the microfluidic chip 110 through the sleeve 122. The outlet end of the two-way joint 121 is connected to the spray needle 130 through the sleeve 122 . The sleeve 122 is made of a polymer material, and the sleeve 122 is directly inserted into the outlet end of the microfluidic chip 110 through frictional force, and has an interference fit with the outlet end of the microfluidic chip 110, so that the connection is tight and leak-free. , even at a high flow rate, it can ensure no leakage; when the sleeve 122 is blocked or needs to be cleaned, it can be directly pulled out for replacement or cleaning. It is easy to disassemble and replace, and the replacement is fast and simple, reducing the failure to continue during operation The case of experiments etc.

Specifically, the material of the sleeve 122 can be PEEK polyether ether ketone, PEK polyether ketone, PEKK polyether ketone ketone, PEEKK polyether ether ketone ketone, PEKEKK polyether ketone ether ketone ketone, PFA perfluoroalkoxy Resin, FEP fluorinated ethylene propylene, ETFE ethylene-tetrafluoroethylene or PTFE polytetrafluoroethylene and other polymer materials suitable for mass spectrometry experiments.

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 spray 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 needle 130 , the sleeve 122 and the two-way connector 121 are on the same straight line after being connected, and the included angle with the overall flow direction of the microfluidic chip 110 is an acute angle, a right angle or an obtuse angle. The current connection method is to make the overall direction of the flow of the added sample solution be a straight line, which requires a more precise design, and the seal also requires a tight connection design. In this case, replacement is very troublesome, and precise Adjustment takes a long time, which will delay the detection of the sample solution or the progress of the experiment.

As shown in Figures 4 and 5, in one embodiment, the microfluidic chip 110 includes a primary channel 111 and an auxiliary channel 112, and the sample injection structure 140 includes a sample loading pipeline 141 and a solution injector 142, and the upper The sample pipeline 141 is respectively inserted into the inlets of the main flow channel 111 and the auxiliary flow channel 112, and the other end of the sample loading pipeline 141 is connected to the solution injector 142, and the sample solution and the added solution are respectively injected into the Among the main channel 111 and the auxiliary channel 112 ; the auxiliary channel 112 is connected to the main channel 111 , there are multiple auxiliary channels 112 , and the interval between each auxiliary channel 112 is 11500 μm. The microfluidic chip 110 can realize the on-line mixing of multiple liquids. The main sample solution is added to the main channel 111, and the required additional liquid is added to the auxiliary channel 112 to complete the mixing in the subsequent channel, that is, in the microfluidic The mixing and reaction of the solution can be completed in the chip 110, and the continuous flow operation can be used for mass spectrometry sample loading analysis. It is not necessary to mix the solution first and then use it for detection. To change the mixing ratio, the detection operation needs to be stopped. And by adjusting the injection pressure through the solution injector 142 to adjust the flow rate of the solution, different mixing ratios can be changed in real time to achieve different reaction mixing ratios, reaction times and mixing effects. In the hydrogen-deuterium exchange mass spectrometry experiment, through flow rate control, the protein hydrogen-deuterium exchange reaction time can be adjusted online in real time, realizing the operation of online dynamic analysis of protein structure. As shown in Figure 5, it shows the mixing balance effect of the solution in the main channel 111 (CH1) and the solution in the auxiliary channel 112 (CH2). The microfluidic chip 110 can achieve different ratios from 99:1 to 50:50. the mixing function.

Wherein, the main channel 111 is provided with a guide structure, and the guide structure is used for mixing different solutions added in the main channel 111 and the auxiliary channel 112 . Specifically, the diversion structure is a herringbone structure 113 (Herringbone structure). According to the direction of solution flow, the herringbone structure 113 is arranged after the main channel 111 and the first auxiliary channel 112 merge. Location. When the solution flows into the fishbone structure 113, a vortex or chaotic flow will be formed, which can quickly mix uniformly, improve the efficiency of uniform mixing, and shorten the length of the flow channel required for mixing, so that the size of the microfluidic chip 110 can be reduced even more. for small. Of course, the flow guide structure is not limited to the fishbone structure 113, and other geometric structures for mixing are also 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 The structure 113a and the second fishbone structure 113b are arranged at intervals in the main channel 111 . Circular mixing is performed along the shapes of the first fishbone structure 113a and the second fishbone structure 113b, so that the mixing of the solution is more uniform and faster.

Specifically, the microfluidic chip 110 includes a main flow channel 111 and two auxiliary flow channels 112, the main flow channel 111 is a main line flow channel, and the two auxiliary flow channels 112 are respectively connected to the main flow channel 111 Among them, the interval between the two auxiliary channels 112 is 4000 μm-11500 μm, depending on the number of cycles. A first fishbone structure 113a plus a second fishbone structure 113b is a cycle, and eight cycles are set in the main channel 111, that is, eight first fishbone structures 113a and eight second fishbone structures 113b are spaced apart The setting, that is, the interval between the two auxiliary flow channels 112 is set to 11500 μm. Of course, the arrangement of the auxiliary flow channels 112 is not limited to the arrangement at intervals, and the arrangement of the auxiliary flow channels 112 may also be arranged in layers, or arranged at an included angle.

In this example, in the research of mass spectrometer chip, the operation of mixing protein and reaction solution while loading sample is realized online, and the mixing is fast, effective and stable. By adjusting the flow rate, the reaction time of protein can also be controlled, and the real-time dynamic process can be carried out. Mass spectrometry elucidates protein structure. The microfluidic chip 110 can realize the online mixing of multi-channel liquids. The main sample solution is added to the main channel 111, and the required additional liquid is added to the auxiliary channel 112, and a circulating first herringbone structure 113a is added. The second fishbone structure 113b quickly and uniformly mixes the solution and reacts the sample online, and performs continuous flow operation for mass spectrometry sample loading analysis. And by adjusting the injection pressure through the solution injector 142 to adjust the flow rate of the solution, different mixing ratios can be changed in real time to achieve different reaction mixing ratios, reaction times and mixing effects. Moreover, the liquid online mixing ratio range and flow rate range are relatively large, which can meet the mixing reaction operation of various proteins.

As shown in Figure 6, in another embodiment, one main flow channel 111 and two auxiliary flow channels 112 form a set of flow channels, and the microfluidic chip 110 includes two independent sets of flow channels. Of course, multiple flow channels can also be set. The group of flow channels can be used multiple times without cleaning, and different sample solutions can be tested at the same time. It is relatively easy to directly design multiple sets of flow channels during the manufacture of the microfluidic chip 110 , and has little influence on the manufacturing difficulty, which can save raw materials and time. The material of the microfluidic chip can be glass quartz, silicon wafer, paper-based material and high molecular polymer (PDMS, PMMA and PC, etc.). The manufacturing method of the above-mentioned microfluidic chip 110 specifically includes: designing the microfluidic chip 110 with the fishbone structure 113 using graphics software, and making a film, and then making a microfluidic chip template by photolithography. The exemplary microfluidic chip 110 is mixed with Polydimethylsiloxane (Polydimethylsiloxane, PDMS) A liquid and B liquid at a ratio of 10:1, and then poured into the photolithographic template prepared above, after vacuum degassing , place it horizontally in an oven at 80 degrees, let it stand for 45 minutes, and remove the mold to obtain the corresponding PDMS chip. Use a 0.75 mm PDMS puncher to punch through holes at the inlet and outlet. Bond the PDMS chip on a glass slide with a Plasma plasma cleaning machine, put it in an 80-degree oven, and place it for 2 hours or overnight.

Specifically, the microfluidic chip 110 has three inlets, one inlet of the main channel 111 and two inlets of the auxiliary channel 112, and eight circular mixed herringbone structures 113 are arranged in the middle of the main channel 111, the microfluidic The chip 110 can realize the mixing of three liquids in continuous flow, and the liquid flowing to the outlet has been fully mixed.

It should be noted that the limitations of the steps involved in this plan are not considered as limiting the order of the steps without affecting the implementation of the specific plan. The steps written in the front can be executed first. It can also be performed later, or even simultaneously, as long as the solution can be implemented, it should be regarded as belonging to the protection scope of the present invention.

The above content is a further detailed description of the present invention in conjunction with specific optional embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. For those of ordinary skill in the technical field of the present invention, without departing from the concept of the present invention, some simple deduction or replacement can be made, which should be regarded as belonging to the protection scope of the present invention.

Claims (10)

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

   The sample injection structure injects the sample solution to be tested into the microfluidic chip;

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

   The spray needle is connected to the other end of the connecting pipeline, the spray needle is powered on, and the sample solution in the spray needle is sprayed by means of electrospray;

   The needle generates a spray spray into the injection port of the mass spectrometer.
2. The sample loading device for a mass spectrometer according to claim 1, wherein the connecting pipeline includes a two-way joint and a sleeve, and one end of the two-way joint passes through the sleeve and the microfluidic chip The outlet end of the two-way joint is connected, and the other end of the two-way joint is connected with the spray needle through the sleeve.
3. The sample loading device for a mass spectrometer according to claim 2, wherein the material of the sleeve is a polymer material, and the sleeve is interference-fitted with the outlet end of the microfluidic chip.
4. The sample loading device for a mass spectrometer according to claim 1, wherein the 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 The needles are on the same straight line.
5. The sample loading device for a mass spectrometer according to claim 1, wherein the distance from the outlet of the injection needle to the sample inlet of the mass spectrometer is 1-100mm.
6. The sample loading device for a mass spectrometer according to claim 1, wherein the mass spectrometer includes a power supply, the spray needle is connected to the power supply of the mass spectrometer, and the spray voltage is provided by the mass spectrometer.
7. The sample loading device for a mass spectrometer according to any one of claims 1-6, wherein the inner diameter of the injection needle is 1-200 μm, the outer diameter is 100-3000 μm, and the length is 1-100 mm.
8. The sample loading device for a mass spectrometer according to any one of claims 1-6, wherein the microfluidic chip includes a main flow channel and an auxiliary flow channel, and the main flow channel and the auxiliary flow channel are used to add different The solution;

   The auxiliary flow channel is connected to the main flow channel, and there are multiple auxiliary flow channels.
9. The sample loading device for a mass spectrometer according to claim 8, wherein a diversion structure is arranged in the main flow channel, and the flow guide structure is used to combine the main flow channel and the auxiliary flow channel Different solutions flow and mix.
10. The sample loading device for a mass spectrometer according to claim 9, wherein the flow guide structure includes a fishbone structure, and the fishbone structure includes a first fishbone structure and a second fishbone structure, and the The first fishbone structure and the second fishbone structure are axisymmetric structures, and the first fishbone structure and the second fishbone structure are arranged at intervals in the main channel.