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CN120356819B - Digital micro-fluidic-mass spectrometry online coupling ion source and online analysis method - Google Patents

Digital micro-fluidic-mass spectrometry online coupling ion source and online analysis method

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Publication number
CN120356819B
CN120356819B CN202510851426.6A CN202510851426A CN120356819B CN 120356819 B CN120356819 B CN 120356819B CN 202510851426 A CN202510851426 A CN 202510851426A CN 120356819 B CN120356819 B CN 120356819B
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spray
online
capillary
mass spectrometry
ion source
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CN120356819A (en
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张帅龙
赵梦磊
李航
郭宗良
刘皓冰
李宗灏
符荣鑫
李博宇
郭力瑗
侯佳禄
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Beijing Institute of Technology BIT
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    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
    • H01J49/0404—Capillaries used for transferring samples or ions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/165—Electrospray ionisation
    • H01J49/167—Capillaries and nozzles specially adapted therefor

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plasma & Fusion (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a digital microfluidic-mass spectrometry online coupling ion source and an online analysis method, which belong to the technical field of ion sources and online analysis methods, and are characterized in that an upper cover plate of the digital microfluidic chip is provided with 1mm of micropores, a transfer capillary tube with the outer diameter of 1mm is inserted into the micropores of the upper cover plate of the digital microfluidic chip, a spray capillary tube with the outer diameter of 360 micrometers and the inner diameter of 100 micrometers or 50 micrometers is nested in the transfer capillary tube, a spray electrode is a metal wire and is arranged in the transfer capillary tube, an adaptation structure is printed by a 3D printer, and the digital microfluidic-mass spectrometry online coupling ion source is used for fixing the transfer capillary tube, the spray capillary tube and the spray electrode.

Description

Digital micro-fluidic-mass spectrometry online coupling ion source and online analysis method
Technical Field
The invention relates to an ion source and an online analysis method, in particular to a digital micro-fluidic-mass spectrometry online coupling ion source and an online analysis method, and belongs to the technical field of ion sources and online analysis methods.
Background
The mass spectrometry technology is used as a high-sensitivity, high-precision and high-resolution molecular detection method, has the advantages of small sample consumption, high analysis speed, high specificity, capability of simultaneously separating and identifying and the like, is widely applied to a plurality of industries and scientific research fields such as chemical engineering, biological medicine, life science, clinical medicine, food sanitation, environmental science, material science and the like, becomes a gold standard and mainstream analysis tool for biochemical analysis in the related fields, and plays a vital role in accurate measurement of mass and material structure. However, traditional mass spectrometry typically relies on manual sample preparation, which is inefficient and complex in flow, and difficult to meet on-the-fly analysis requirements. In addition, the sample to be measured is often required to be transferred between different containers for manually preparing the sample, so that the sample is relatively lost and is easy to pollute, and the accuracy of a mass spectrum result is affected.
Based on this, we have devised a new ion source to solve the above-mentioned problems.
Disclosure of Invention
The invention mainly aims to provide a digital micro-fluidic-mass spectrometry online coupling ion source and an online analysis method.
The aim of the invention can be achieved by adopting the following technical scheme:
the digital microfluidic-mass spectrometry online coupling ion source and the online analysis method comprise a digital microfluidic chip, wherein a 1mm micropore is arranged on an upper cover plate of the digital microfluidic chip;
the outer diameter of the transfer capillary tube is 1mm, and the transfer capillary tube is inserted into a micropore of the upper cover plate of the digital microfluidic chip;
A spray capillary having an outer diameter of 360 microns and an inner diameter of 100 microns or 50 microns, nested within the transfer capillary;
the spray electrode is a metal wire and is arranged in the transfer capillary;
And the adapting structure is printed by a 3D printer and is used for fixing the transfer capillary, the spray capillary and the spray electrode.
Preferably, the digital microfluidic chip is used for mixing, reacting, separating and detecting micro droplets.
Preferably, the transverse distance between the spray capillary and the mass spectrum sample inlet is 3mm, and the longitudinal distance is 2mm.
Preferably, the voltage applied by the spray electrode is 3kV.
The digital microfluidic-mass spectrometry online coupled ion source online analysis method comprises the following steps of injecting a sample into a digital microfluidic chip;
driving the liquid drops on the chip to move onto the spray electrode, enabling the liquid drops to enter the transfer capillary by utilizing capillary action, and enabling the liquid drops to be in contact with the spray capillary and the metal electrode;
and applying 3kV voltage to the metal wire of the spray electrode to generate electrospray, and transmitting the ionized sample to a mass spectrometer for analysis.
Preferably, the method further comprises the step of selecting a surfactant at a suitable concentration, wherein the surfactant is poloxamer, and the concentration is 0.05%.
Preferably, the sample is a small molecule standard, a mixed solution or a miltefosine and N-benzoyl-L-arginine ethyl ester solution with different concentrations.
Preferably, in the analysis process, the method further comprises the step of verifying the system performance, wherein the verification comprises the step of testing the influence of different distances between the spray capillary and the mass spectrum sample inlet on signals and optimizing distance parameters;
Testing the influence of spray capillaries with different inner diameters and different voltages on signals, and optimizing the inner diameters and voltage parameters of the spray capillaries;
testing the repeatability and sensitivity of the system, and evaluating the overall performance of the system.
Preferably, the repeatability test is that 10 experiments are carried out on each group of samples of miltefosine of 40, 80 and 120 mug/mL, and the variation coefficient is less than 8 percent.
Preferably, the sensitivity test is to analyze 1-1000ng/mL miltefosine and N-benzoyl-L-arginine ethyl ester solution by adopting the system, the linearity of the miltefosine and the N-benzoyl-L-arginine ethyl ester solution is good, and the detection limit is 1ng/mL;
the shape and size of the adapting structure are designed according to the positions and shapes of the transfer capillary, the spray capillary and the spray electrode;
The materials of the transfer capillary, the spray capillary and the spray electrode are materials which can withstand the experimental environment and do not affect the sample property and the analysis result, such as quartz and stainless steel.
The beneficial technical effects of the invention are as follows:
According to the digital microfluidic-mass spectrometry online coupling ion source and the online analysis method, the digital microfluidic chip can complete a series of operations such as mixing, reacting, separating and detecting of tiny liquid drops on a single platform, and high integration and automation of a sample processing flow are realized. Compared with the traditional complicated process that samples need to be transferred between different containers in manual sample preparation, the technology greatly reduces manual operation steps, remarkably improves analysis efficiency, and simultaneously effectively reduces sample loss and pollution risk caused by manual operation.
By optimizing the surfactant (poloxamer, concentration 0.05%), the interference on the mass spectrum signal is avoided under the premise of ensuring the normal driving of the liquid drops. Experiments show that the driving rate of the liquid drops under the concentration is equivalent to that of the liquid drops under the concentration of 0.1%, and the accuracy of subsequent mass spectrometry can be ensured.
Drawings
FIG. 1 is a schematic diagram of a digital microfluidic-mass spectrometry online coupled ion source and system for online coupling of digital microfluidic and mass spectrometry according to a preferred embodiment of the present invention;
FIG. 2 is a graph of the lateral and longitudinal distances of an optimized spray capillary from a mass spectrometry sample inlet for a preferred embodiment of a digital microfluidic-mass spectrometry online coupled ion source and online analysis method according to the present invention;
FIG. 3 is a graph of optimized spray capillary inner diameter and spray voltage for a preferred embodiment of a digital microfluidic-mass spectrometry online coupled ion source and online analysis method according to the present invention;
FIG. 4 is a graph showing the effect of PF68 of different concentrations on the movement speed of droplets in digital microfluidic according to a preferred embodiment of the digital microfluidic-mass spectrometry online coupled ion source and online analysis method of the present invention;
FIG. 5 is a graph of the effect of PF68 of 0.05% on mass spectrometry signal for a preferred embodiment of a digital microfluidic-mass spectrometry online coupled ion source and an online analysis method according to the present invention;
FIG. 6 is a diagram comparing a preferred embodiment of the method of the present invention with the Nano-ESI method for an online coupled ion source and online analysis method of digital microfluidic-mass spectrometry;
FIG. 7 is a diagram of a reproducibility analysis of a preferred embodiment of a digital microfluidic-mass spectrometry online coupled ion source and online analysis method according to the present invention;
Fig. 8 is a graph of detection performance analysis of a preferred embodiment of the digital microfluidic-mass spectrometry online coupled ion source and online analysis method according to the present invention.
Detailed Description
In order to make the technical solution of the present invention more clear and obvious to those skilled in the art, the present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Firstly, a 1mm micropore is punched on an upper cover plate of a digital microfluidic chip, then a capillary tube with the outer diameter of 1mm is inserted to be used as a transfer capillary tube, a capillary tube with the outer diameter of 360 micrometers and the inner diameter of 100 (50) micrometers is nested in the transfer capillary tube to be used as a spray capillary tube, a metal wire is used as a spray electrode, and a 3D printer is used for printing an adapting structure to fix the ion source. The structure is shown in fig. 1. Then verifying the influence of different distances between the spray capillary and a mass spectrum sample inlet, different inner diameters of the spray capillary and different voltages on signals, and then verifying the overall performance of the system by using a plurality of small molecular standards (miltefosine, N-benzoyl-L-ethyl arginine and the like);
Experiment one spray capillary and mass spectrum sample inlet different distance to influence the signal;
we used 100ug/mL miltefosine as standard, injected 4 uL standard into the chip, then driven the liquid drop to move to the spray electrode, because of capillary action, the liquid drop will enter the transfer capillary and contact with the spray capillary and the metal electrode, at this time, by applying 3kV voltage to the metal wire to generate electrospray, the distance between the spray capillary and the mass spectrum sample inlet is optimized according to the signal size. As shown in FIG. 2, the signal intensity is maximum and the ionization effect is best when the transverse and longitudinal distances between the spray capillary and the mass spectrum sample inlet are 3 mm and 2mm respectively. The subsequent experiments all employ this set of distance parameters;
experimental two spray capillary inner diameter and voltage size influence on signal;
similar to experiment I, we still used miltefosine as a standard, and evaluated the effect of spray capillaries with outer diameters of 360um, inner diameters of 100, 75 and 50um, and voltage levels (2-4 kV) on the signal, and the results were shown in FIG. 3, with the highest intensity at an inner diameter of 100 um and a spray voltage level of 3 kV.
The interference of the surfactant on the mass spectrum signal is explored in the third experiment;
Since digital microfluidic driving droplet movement requires a certain concentration of surfactant, and the sensitivity of mass spectrum is extremely high, it is necessary to explore different concentrations of surfactant, and because poloxamer is a non-ionic surfactant, it has small interference to the mass spectrum signal, we first tested the effect of different concentrations (0.0125, 0.025, 0.05, 0.1%) of surfactant on droplet driving, as shown in fig. 4, when the surfactant concentration is 0.05%, the droplet driving rate is far higher than the first two groups, and is equivalent to the result of 0.1%, and fig. 5 shows that 0.05% of surfactant does not interfere much with the mass spectrum signal, so that 0.05% of poloxamer can support normal driving of droplet, but does not interfere with the mass spectrum signal, and the subsequent experiments also use the parameter.
Performing online coupling system evaluation of experimental four-digit microfluidic and mass spectrum;
After optimization of experimental conditions, we tested the performance of the system. Firstly, the reserpine and other mixed solutions are injected into a chip, then the chip is driven to a spraying position for online mass spectrometry, and then the same sample is analyzed by using Nano-ESI, as shown in figure 6, the mass spectrograms of the reserpine and the sample are similar, so that the effect of the reserpine and the sample is equivalent to that of the Nano-ESI. Then we use miltefosine of 40, 80 and 120ug/mL to conduct repeatability test, and each group of samples is subjected to 10 experiments, and the results are shown in figure 7, and the variation coefficients are all less than 8%, which shows that the system has good repeatability. Finally, the sensitivity of the system is evaluated by respectively using miltefosine and N-benzoyl-L-arginine ethyl ester, and in an experiment, the system is adopted to analyze N-benzoyl-L-arginine ethyl ester solutions, imatinib and miltefosine with different concentrations (1-1000 ng/mL). At least 3 replicates were run per measurement. The average ionic strength during MS analysis was recorded. As shown in fig. 8, the linearity of the two is good (r2= 0.975,0.976,0.988), the detection limit is 1ng/mL, and the insertion is the corresponding tandem mass spectrum.
The above description is merely a further embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and any person skilled in the art will be able to apply equivalents and modifications according to the technical solution and the concept of the present invention within the scope of the present invention disclosed in the present invention.

Claims (8)

1.数字微流控-质谱在线耦合离子源，其特征在于，包括数字微流控芯片，所述数字微流控芯片的上盖板设有1mm的微孔；1. A digital microfluidics-mass spectrometry online coupled ion source, comprising a digital microfluidics chip, wherein the upper cover of the digital microfluidics chip is provided with a 1 mm microhole; 转移毛细管，其外径为1mm，插入所述数字微流控芯片上盖板的微孔；A transfer capillary tube with an outer diameter of 1 mm is inserted into the microhole of the cover plate of the digital microfluidic chip; 喷雾毛细管，其外径360微米，内径为100微米或50微米，嵌套于所述转移毛细管内；A spray capillary with an outer diameter of 360 μm and an inner diameter of 100 μm or 50 μm is nested in the transfer capillary; 喷雾电极，为一根金属丝，设置于所述转移毛细管内；The spray electrode is a metal wire and is disposed in the transfer capillary; 适配结构，由3D打印机打印制成，用于固定所述转移毛细管、喷雾毛细管和喷雾电极；An adapting structure, printed by a 3D printer, for fixing the transfer capillary, the spray capillary and the spray electrode; 数字微流控芯片用于对微小液滴进行混合、反应、分离和检测操作；Digital microfluidic chips are used to mix, react, separate, and detect tiny droplets; 所述喷雾毛细管与质谱进样口的横向距离为3mm，纵向距离为2mm。The lateral distance between the spray capillary and the mass spectrometer injection port is 3 mm, and the longitudinal distance between the spray capillary and the mass spectrometer injection port is 2 mm. 2.根据权利要求1所述的数字微流控-质谱在线耦合离子源，其特征在于，所述喷雾电极施加的电压为3kV。2. The digital microfluidics-mass spectrometry online coupled ion source according to claim 1, characterized in that the voltage applied by the spray electrode is 3 kV. 3.数字微流控-质谱在线耦合离子源在线分析方法，基于权利要求1-2任意一项所述的数字微流控-质谱在线耦合离子源，其特征在于，包括以下步骤：将样本注入数字微流控芯片；3. A digital microfluidics-mass spectrometry online coupled ion source online analysis method, based on the digital microfluidics-mass spectrometry online coupled ion source according to any one of claims 1-2, characterized in that it comprises the following steps: injecting a sample into a digital microfluidics chip; 驱动芯片上的液滴移动至喷雾电极上，利用毛细作用使液滴进入转移毛细管，并与喷雾毛细管和金属电极接触；The droplets on the driving chip move to the spray electrode, and the droplets use capillary action to enter the transfer capillary and contact the spray capillary and the metal electrode; 给喷雾电极的金属丝施加3kV电压产生电喷雾，将样本离子化后传输至质谱仪进行分析。A voltage of 3 kV was applied to the metal wire of the spray electrode to generate electrospray, which ionized the sample and then transmitted it to the mass spectrometer for analysis. 4.根据权利要求3所述数字微流控-质谱在线耦合离子源在线分析方法，其特征在于，在注入样本前，还包括选择合适浓度表面活性剂的步骤，所述表面活性剂为泊洛沙姆，浓度为0.05%。4. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 3, characterized in that before injecting the sample, it also includes a step of selecting a surfactant with a suitable concentration, and the surfactant is poloxamer with a concentration of 0.05%. 5.根据权利要求4所述的数字微流控-质谱在线耦合离子源在线分析方法，其特征在于，所述样本为小分子标准品、混合溶液或不同浓度的米替福新和N-苯甲酰-L-精氨酸乙酯溶液。5. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 4, characterized in that the sample is a small molecule standard, a mixed solution, or a solution of miltefosine and N-benzoyl-L-arginine ethyl ester at different concentrations. 6.根据权利要求5所述的数字微流控-质谱在线耦合离子源在线分析方法，其特征在于，在分析过程中，还包括对系统性能进行验证的步骤，所述验证包括测试喷雾毛细管与质谱进样口不同距离对信号的影响，优化距离参数；6. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 5, characterized in that during the analysis process, a step of verifying system performance is further included, wherein the verification includes testing the effect of different distances between the spray capillary and the mass spectrometer inlet on the signal and optimizing the distance parameter; 测试不同内径喷雾毛细管和不同电压大小对信号的影响，优化喷雾毛细管内径和电压参数；Test the effects of different inner diameter spray capillaries and different voltages on the signal, and optimize the spray capillary inner diameter and voltage parameters; 测试表面活性剂对质谱信号的干扰，确定合适的表面活性剂浓度；测试系统的重复性和灵敏度，评估系统整体性能。Test the interference of surfactants on mass spectrometry signals and determine the appropriate surfactant concentration; test the repeatability and sensitivity of the system and evaluate the overall performance of the system. 7.根据权利要求6所述的数字微流控-质谱在线耦合离子源在线分析方法，其特征在于，所述重复性测试是对40、80、120μg/mL的米替福新每组样本进行10次实验，变异系数均小于8%。7. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 6, characterized in that the repeatability test is performed 10 times for each group of samples at 40, 80, and 120 μg/mL of miltefosine, and the coefficient of variation is less than 8%. 8.根据权利要求7所述的数字微流控-质谱在线耦合离子源在线分析方法，其特征在于，灵敏度测试是采用该系统对1-1000ng/mL的米替福新和N-苯甲酰-L-精氨酸乙酯溶液进行分析，二者线性度良好，检测限为1ng/mL；8. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 7, characterized in that the sensitivity test is performed using the system to analyze 1-1000 ng/mL miltefosine and N-benzoyl-L-arginine ethyl ester solutions, and the linearity of the two is good, with a detection limit of 1 ng/mL; 所述适配结构的形状和尺寸根据转移毛细管、喷雾毛细管和喷雾电极的位置和形状进行设计；The shape and size of the adapting structure are designed according to the position and shape of the transfer capillary, the spray capillary and the spray electrode; 所述转移毛细管、喷雾毛细管和喷雾电极的材料均为能够耐受实验环境且不影响样本性质和分析结果的材料。The materials of the transfer capillary, spray capillary and spray electrode are all materials that can withstand the experimental environment and do not affect the sample properties and analysis results.
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