Sodium droplet interface ionization mass spectrum ion source device and operation method thereof

Abstract

The invention discloses a nano-droplet interface ionization mass spectrum ion source device and an operation method thereof, and relates to the field of nano-droplet interface ionization mass spectrum ion source devices, the device comprises a nano-spray needle used for loading a sample solution and realizing a nano-spray process; the metal electrode is inserted into the nano-spray needle, is in direct contact with the sample solution and is used for providing a high-voltage electric field required by nano-droplet interface ionization for the sample solution; and the ionization chamber is pre-filled with inert auxiliary gas and is used for protecting the interface ionization process of the nano liquid drops. High-sensitivity detection of polycyclic aromatic hydrocarbon in samples such as small-size single cells is realized, and higher signal intensity is obtained; better detection response to weak polar compounds (especially compounds with aromatic skeleton structures) in a mixed sample is realized; the detection of the weak polar compound polycyclic aromatic hydrocarbon in the single cell is realized.

Description

Technical Field

[0001] The present application relates to the field of nanodroplet interface ionization mass spectrometry ion source devices, and in particular to a nanodroplet interface ionization mass spectrometry ion source device and an operating method thereof. Background Art

[0002] Mass spectrometry has attracted great interest in the field of bioanalysis due to its high sensitivity, low sample consumption, and excellent structural identification capabilities. However, the most commonly used ionization techniques for trace biological samples, namely electrospray ionization (ESI) and nanoelectrospray ionization (nanoESI), face significant challenges in measuring low-polarity compounds due to their inherent low ionization efficiency. Dielectric barrier discharge ionization (DBDI), direct analysis in real time (DART), and atmospheric pressure chemical ionization (APCI) utilize discharge and proton transfer to ionize compounds. However, the analysis of trace weakly polar compounds in single cells still faces problems such as low detection sensitivity, poor ionization efficiency, and complex matrix interference.

[0003] In recent years, droplet-based reactions performed during electrospray have attracted extensive attention due to their unique chemical and physical properties, especially their ability to accelerate chemical reactions, reduce activation energy, and improve ionization efficiency. Stimulated Raman fluorescence microscopy has confirmed the presence of a strong electric field (~10 9 V / m), is believed to play a key role in driving chemical reactions. Reactions carried out on the surface of a droplet are typically several orders of magnitude faster than those carried out in the bulk solution. Non-spontaneous reactions that would not occur in a typical bulk solution can also be achieved on the surface of the droplet. In addition, the electric field on the droplet surface can induce the formation of free radicals and ion pairs, such as H2O + and H2O - Previous studies on catalytic interfacial reactions have also shown that as the droplet size decreases, the reaction rate constant and thermodynamic equilibrium constant both increase significantly.

[0004] Compared with ESI, nanoESI produces nanodroplets with much smaller size (typically less than 200 nm) and higher desolvation and ionization efficiency. Applying a higher voltage can promote the formation of a finer Taylor cone at the tip of the nanospray needle, thereby producing smaller droplets. Due to the higher specific surface area and surface charge density, these smaller droplets have a stronger surface electric field. In theory, this is conducive to promoting interfacial reactions. However, due to the small initial size of the nanodroplets, nanoESI is currently a common method for studying non-accelerated reactions. The generation time of nanodroplets is limited for the progress of chemical reactions. To date, few studies have explored the potential of nanoESI to accelerate chemical reactions.

[0005] Therefore, based on the ability of nanodroplets to accelerate chemical reactions, solving the problems of low ionization efficiency, poor sensitivity, and multiple impurity interference in the mass spectrometry analysis of trace weak polar compounds in single cells is a current research direction.

[0006] The Chinese patent application number 202210265956.9 discloses a nano-photoionization mass spectrometry ion source device and its operation method. This patent combines the advantages of Nano-ESI and APPI to design a nano-photoionization mass spectrometry ion source device suitable for the analysis of small-volume weakly polar compounds such as polycyclic aromatic hydrocarbons. By optimizing various parameters, efficient ionization detection of single trace weakly polar compounds, highly sensitive analysis of weakly polar compounds in mixed components, and selective ionization detection of weakly polar compounds in single-cell samples are achieved. However, the sensitivity of the patent for the limit of detection (LOD) and limit of quantification (LOQ) of polycyclic aromatic hydrocarbons is still not ideal, resulting in obvious limitations in its use. Summary of the Invention

[0007] In response to the above-mentioned deficiencies in the prior art, the present invention provides a nanodroplet interface ionization mass spectrometry ion source device and an operating method thereof, which solve the problems of low ionization efficiency, poor sensitivity, and excessive impurity interference in the mass spectrometry analysis of trace weakly polar compounds in single cells.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0009] In one aspect, a nanodroplet interface ionization mass spectrometry ion source device is provided, comprising:

[0010] Nanospray needle, used to load sample solution and realize nanospray process;

[0011] The metal electrode is inserted into the nanospray needle and is in direct contact with the sample solution, and is used to provide the sample solution with the high-voltage electric field required for nanodroplet interface ionization;

[0012] The ionization chamber is pre-filled with an inert auxiliary gas to protect the ionization process of the nanodroplet interface;

[0013] The tip of the nanospray needle is located inside the ionization chamber.

[0014] Furthermore, the inner diameter of the tip of the spray needle is 1 μm.

[0015] Furthermore, a gas inlet and a gas outlet for inert auxiliary gas to enter and exhaust are provided on the ionization chamber, and the gas inlet and the gas outlet are located on opposite sides of the ionization chamber respectively; the flow rate of the inert auxiliary gas is controlled to be 2.0 L / min.

[0016] Furthermore, the metal electrode provides a high voltage electric field of +6.0 kV to the sample solution.

[0017] Furthermore, the nanospray needle is made of boron glass.

[0018] Furthermore, the inert auxiliary gas is nitrogen.

[0019] Furthermore, the metal electrode is an inert metal material.

[0020] Furthermore, the inert metal material is copper wire.

[0021] The beneficial effects of this device are as follows: by employing the above-mentioned technical solution, when high pressure is applied to the nanospray needle, the sample solution in the nanospray needle forms nanoscale droplets at the tip of the needle. The sample undergoes an interfacial reaction on the surface of the nanodroplets, achieving ionization and ultimately enabling mass spectrometric detection of the analyte. This process eliminates the need for a UV lamp, simplifies the device structure, and improves the sensitivity of the limit of detection (LOD) and limit of quantification (LOQ) of polycyclic aromatic hydrocarbons. It also reduces the interference of oxygen in the air on the nanodroplet interfacial reaction, ensuring the smooth generation of free radical cations in the sample.

[0022] On the other hand, a method for operating an ion source device based on nanodroplet interface ionization mass spectrometry is provided, comprising the following steps:

[0023] Inject the sample solution from the tail end of the nanospray needle so that the sample solution fills the tip of the nanospray needle;

[0024] Connect, insert one end of the metal electrode from the tail end of the nanospray needle until the end of the metal electrode contacts the sample solution, seal the tail end of the nanospray needle with an insulating end cap, and connect the end of the metal electrode outside the nanospray needle to an external high-voltage power supply;

[0025] Gas inlet, passing inert auxiliary gas into the ionization chamber;

[0026] Detection, turn on the high voltage power supply to achieve detection.

[0027] Furthermore, the inert auxiliary gas is high-purity nitrogen, and the flow rate is controlled to be 2.0 L / min; the voltage of the high-voltage power supply is +6.0 kV.

[0028] The beneficial effects of this method are: achieving highly sensitive detection of polycyclic aromatic hydrocarbons in small-volume single-cell samples and obtaining higher signal intensity; having a better detection response to weakly polar compounds in mixed samples (especially compounds with aromatic skeleton structures); and achieving the detection of polycyclic aromatic hydrocarbons, a weakly polar compound, in single cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure of this device;

[0030] Figure 2 The following are the detection results of six PAHs under NII mass spectrometry: (a) fluorene; (b) anthracene; (c) pyrene; (d) benzo[a]anthracene; (e) indeno[1,2,3-cd]pyrene; (f) dibenzo[a,h]anthracene;

[0031] Figure 3 The results of the detection of nine PAHs under NII mass spectrometry are as follows: (a) naphthalene; (b) acenaphthylene; (c) acenaphthene; (d) phenanthrene; (e) fluoranthene; (f) (g) Benzo[a]fluoranthene; (h) Benzo[b]fluoranthene; (i) Benzo[k]fluoranthene;

[0032] Figure 4 The detection results of four substituted polycyclic aromatic hydrocarbons under NII mass spectrometry are as follows: (a) 9-methylanthracene; (b) 9-nitroanthracene; (c) 9-chloroanthracene; (d) 7,12-dimethylbenz[a]anthracene;

[0033] Figure 5 The detection results of four heterocyclic compounds under NII mass spectrometry are as follows: (a) phenothiazine; (b) phenoxazine; (c) 2,3-pyridinedicarboxylic anhydride; (d) benzo[b]naphtho[1,2-d]thiophene;

[0034] Figure 6 The results of NII mass spectrometry analysis of HeLa cells treated with PAHs are shown in Figure 2. PAH concentrations include 20, 40, 60, 80, and 100 nM. PAHs include acenaphthene, fluorene, anthracene, pyrene, benzo[a]anthracene, benzo[a]fluoranthene, indeno[1,2,3-cd]pyrene, and dibenzo[a,h]anthracene.

[0035] Figure 7 The results of NII mass spectrometry analysis of K562 cells treated with PAHs were shown in Figure 2. PAH concentrations were 20, 40, 60, 80, and 100 nM. PAHs included acenaphthene, fluorene, anthracene, pyrene, benzo[a]anthracene, benzo[a]fluoranthene, indeno[1,2,3-cd]pyrene, and dibenzo[a,h]anthracene.

[0036] Figure 8 These are the detection results of HepG2 cells treated with PAHs under NII mass spectrometry: PAH concentrations include 20, 40, 60, 80, and 100 nM; PAHs include acenaphthene, fluorene, anthracene, pyrene, benzo[a]anthracene, benzo[a]fluoranthene, indeno[1,2,3-cd]pyrene, and dibenzo[a,h]anthracene. DETAILED DESCRIPTION

[0037] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0038] The present application discloses a nanodroplet interface ionization (NII) mass spectrometry ion source device for performing mass spectrometry detection experiments. The mass spectrometry detection experiments were performed on an Orbitrap Elite Mass Spectrometer (Thermo Scientific, San Jose, CA, USA). The device parameters can be set to: capillary temperature of 400°C; spray voltage of +6.0 kV.

[0039] Example 1:

[0040] like Figure 1 As shown, the nanodroplet interface ionization mass spectrometry ion source device includes:

[0041] The nanospray needle is used to load the sample solution and perform the nanospraying process. Made of glass, specifically borosilicate glass, the tip of the nanospray needle has an inner diameter of 1 μm. The sample solution is injected through the tip of the nanospray needle, filling the tip. Alternatively, the tip of the nanospray needle can be aspirated through capillary action, with solution added to the tip to aid conductivity.

[0042] The metal electrode, inserted into the nanospray needle and in direct contact with the sample solution, provides the high-voltage electric field required for ionization at the nanodroplet interface. The metal electrode can be made of any material, but should be chemically inert and unlikely to react with the sample solution, leading to corrosion and dissolution. It is typically made of an inert metal, specifically copper wire.

[0043] The ionization chamber is pre-filled with inert auxiliary gas to protect the ionization process of the nanodroplet interface and realize the formation of free radical cations of the sample to be tested.

[0044] In the present embodiment, high-purity nitrogen can be selected as the inert auxiliary gas, and an air path inlet and an air path outlet for the entry and discharge of high-purity nitrogen are provided on the ionization chamber. The diameter of the air path outlet is designed to be 0.5 mm, and the air path inlet and the air path outlet are respectively located on the side and below of the ionization chamber so as to maintain the circulation of inert gas in the entire ionization chamber. The inert gas flow rate is controlled to 2.0 L / min. When high-purity nitrogen is continuously introduced into the ionization chamber, the air in the ionization chamber will be effectively discharged and filled with high-purity nitrogen in the entire ionization chamber, and the concentration of nitrogen in the ionization chamber will be effectively maintained. The design of the ionization chamber can reduce the interference of oxygen in the air with the nanodroplet interface reaction, thereby ensuring the smooth generation of free radical cations of the sample to be tested.

[0045] The implementation principle of this embodiment is as follows: high-purity nitrogen passes through the gas path on the side of the ionization chamber at a flow rate of 2.0L / min, and the ionization chamber is filled with high-purity nitrogen to protect the generation of free radical cations. When a high voltage of +6.0kV is applied to the nanospray needle, nano-scale droplets are generated at the tip of the nanospray needle. There is a special electric field on the surface of the droplets, forming an electrochemical potential to drive the interfacial electrochemical reaction. The interfacial electric field can accelerate the chemical reaction rate, promote the directional arrangement of reactants and the efficient transfer of electrons. The sample molecules to be tested undergo interfacial reactions on the nanodroplets to form free radical cations, which enter the mass spectrometer for detection.

[0046] This embodiment also discloses a method for operating a nanodroplet interface ionization mass spectrometry ion source device, which includes the following steps:

[0047] S1. Inject the sample solution from the tail end of the nanospray needle so that the sample solution fills the tip of the nanospray needle;

[0048] S2. Connecting: Insert one end of the metal electrode from the tail end of the nanospray needle until the end of the metal electrode contacts the sample solution, seal the tail end of the nanospray needle with an insulating end cap, and connect the end of the metal electrode outside the nanospray needle to an external high-voltage power supply;

[0049] S3, air intake, passing inert auxiliary gas into the ionization chamber;

[0050] S4, detection, turn on the high voltage power supply to realize detection.

[0051] The detection effect of the nanodroplet interface ionization mass spectrometry ion source device will be verified through specific examples below.

[0052] (1) Comparison of detection effects between NII ion source and nanoESI ion source

[0053] To better demonstrate the advantages of the designed NII ion source for detecting weakly polar compounds, we compared the mass spectrometry signal intensity of fifteen PAHs using the NII ion source and a conventional nanoESI ion source. The PAHs used in the detection were all environmental toxicant standards, dissolved in acetonitrile to obtain 20 and 30 μM test solutions. The molecular weights were as follows: fluorene: 166; anthracene: 178; pyrene: 202; benzo[a]anthracene: 228; indeno[1,2,3-cd]pyrene: 276; dibenzo[a,h]anthracene: 278; naphthalene: 128; acenaphthylene: 152; acenaphthene: 154; phenanthrene: 178; fluoranthene: 202; Benzo[a]fluoranthene: 228; Benzo[a]fluoranthene: 252; Benzo[b]fluoranthene: 252; Benzo[k]fluoranthene: 252. In the figures, the signals of the analytes are marked with arrows.

[0054] Reference Figure 2 , showing the mass spectrometry detection results of six PAHs (20μM) in a nitrogen-protected NII ion source. The figure shows that the homemade NII ion source device has better ionization efficiency than conventional nanoESI. In NII, the detection signal-to-noise ratio (S / N) results of the six PAHs are: fluorene: 93.3; anthracene: 516.5; pyrene: 187.2; benzo[a]anthracene: 337.3; indeno[1,2,3-cd]pyrene: 275.3; dibenzo[a,h]anthracene: 321.7. In conventional nanoESI detection, no PAHs were detected. PAHs, as weakly polar compounds composed of multiple fused benzene rings, are often difficult to ionize.

[0055] Reference Figure 3 , shows the mass spectrometry detection results of nine PAHs (30μM) in a nitrogen-protected NII ion source. In NII, the detection S / N results of the nine PAHs are: naphthalene: 30.6; acenaphthylene: 78.1; acenaphthene: 87.1; phenanthrene: 68.2; fluoranthene: 262.6; The nine PAHs were nearly impossible to detect using conventional nanoESI, with no characteristic signals visible and S / N values ​​approaching 0.

[0056] Reference Figure 4 , demonstrating the detection of four substituted PAHs (20 μM) using the NII ion source: 9-methylanthracene, 9-nitroanthracene, 9-chloroanthracene, and 7,12-dimethylbenz[a]anthracene. The results demonstrate the NII's excellent detection capabilities for substituted PAHs, with all four forming radical cations.

[0057] Reference Figure 5 NII was further applied to the detection of four heterocyclic compounds (20 μM): phenothiazine, phenoxazine, 2,3-pyridinedicarboxylic anhydride, and benzo[b]naphtho[1,2-d]thiophene. The results showed that the four heterocyclic compounds successfully formed radical cations during the nanodroplet interface reaction, which were further detected by mass spectrometry. NII also has excellent ionization capabilities for heterocyclic compounds.

[0058] Example 2: This example 2 is a further supplement to Example 1:

[0059] Many pollutants in the environment can enter biological cells, bind to specific receptors in the cells, and after conversion by metabolic enzymes, ultimately affect the growth and development of the cells. Polycyclic aromatic hydrocarbons are a type of cell pollutant that is very harmful to the human body. They are converted into ionic forms of polycyclic aromatic hydrocarbons by binding to specific receptors and then metabolized to the genetic material DNA or RNA in the cells, causing cell mutations and thus cell canceration. Detecting polycyclic aromatic hydrocarbons, a weakly polar compound, in single cells is of great significance for the prevention and diagnosis of future diseases, environmental governance, and the control of harmful ingredients in food and medicine. This example continues to use NII to detect polycyclic aromatic hydrocarbons, a weakly polar compound, in HeLa, K562, and HepG2 cells after being treated and cultured with eight types of polycyclic aromatic hydrocarbons.

[0060] In this example, three different cell lines (HeLa, K562, and HepG2) were treated with a mixture of PAHs at different concentrations ranging from 20 to 100 nM. The eight PAHs, including acenaphthene, fluorene, anthracene, pyrene, benz[a]anthracene, benzo[a]fluoranthene, indeno[1,2,3-cd]pyrene, and dibenzo[a,h]anthracene, were added to the culture medium at the same concentration. The quantitative results of the eight PAHs in single cells are shown in Figure 2. Figure 6 、 Figure 7 and Figure 8 As shown in the figure, the levels of each PAH increased with increasing PAH concentration in the culture medium. The increase in PAH levels within cells showed a nonlinear trend, which may be related to changes in cellular metabolism and bioaccumulation. Furthermore, significant differences in PAH accumulation were observed among the three cell lines. K562 cells had the lowest PAH retention rate, while HeLa cells had the highest.

[0061] The limits of detection (LOD) and quantification (LOQ) of PAHs were determined by analyzing a series of serial dilutions of samples with internal standards. The sensitivity and linear dynamic range of NII were evaluated using nine different PAHs as shown in Table 1. 12) was used as an internal standard for normalizing signal intensity and calculating the linear dynamic range. The detection limits for the nine PAHs were 2-20 pM. The limits of quantification for these PAHs were 5-50 pM. Compared with previous studies (patent application number 202210265956.9), the detection and quantification limits achieved by NII were reduced by three orders of magnitude, as shown in Table 2, highlighting the significant advantage of NII in the sensitivity of PAH analysis.

[0062] Table 1: Quantification results of PAHs by NII using BaA-d at a concentration of 1 nM 12 As internal standard

[0063]

[0064]

[0065] Table 2: Comparison of PAH detection performance

[0066]

[0067] In summary, nanodroplet reaction is an interfacial chemical reaction carried out on nanoscale droplets, and these droplets often have unique physicochemical properties. The surface of the microdroplets can generate a special electric field, forming an electrochemical potential to drive the interfacial electrochemical reaction. The nanodroplet interface electric field can accelerate the chemical reaction rate, promote the directional arrangement of reactants and the efficient transfer of electrons. The nanodroplet interface ionization mass spectrometry (NII) developed by the present invention is mainly composed of an ionization chamber, a nanospray needle and a metal electrode, and is used for the accurate measurement of low-content weakly polar compounds. Under the action of high voltage, nanoscale droplets are generated at the tip of the needle, and neutral analytes undergo interfacial reactions on the nanoscale droplets to form charged ions, which enter the mass spectrometer for detection.

Claims

1. A nanodroplet interface ionization mass spectrometry ion source device, characterized in that: include: Nanospray needle, used to load sample solution and realize nanospray process; The metal electrode is inserted into the nanospray needle and is in direct contact with the sample solution, and is used to provide the sample solution with the high-voltage electric field required for nanodroplet interface ionization; The ionization chamber is pre-filled with an inert auxiliary gas to protect the ionization process of the nanodroplet interface; The tip of the nanospray needle is located inside the ionization chamber.

2. The nanodroplet interface ionization mass spectrometry ion source device according to claim 1, characterized in that: The inner diameter of the tip of the spray needle was 1 μm.

3. The nanodroplet interface ionization mass spectrometry ion source device according to claim 1, characterized in that: The ionization chamber is provided with a gas inlet and a gas outlet for inert auxiliary gas to enter and exhaust. The gas inlet and the gas outlet are located on opposite sides of the ionization chamber respectively; the flow rate of the inert auxiliary gas is controlled at 2.0L / min.

4. The nanodroplet interface ionization mass spectrometry ion source device according to claim 1, characterized in that: The metal electrode provides a high voltage electric field of +6.0 kV to the sample solution.

5. The nanodroplet interface ionization mass spectrometry ion source device according to claim 1, characterized in that: The nanospray needle is made of boron glass.

6. The nanodroplet interface ionization mass spectrometry ion source device according to claim 1, characterized in that: The inert auxiliary gas is nitrogen.

7. The nanodroplet interface ionization mass spectrometry ion source device according to claim 1, characterized in that: The metal electrode is an inert metal material.

8. The nanodroplet interface ionization mass spectrometry ion source device according to claim 7, characterized in that: The inert metal material is copper wire.

9. A method for operating the nanodroplet interface ionization mass spectrometry ion source device according to any one of claims 1 to 8, characterized in that: The following steps are involved: Inject the sample solution from the tail end of the nanospray needle so that the sample solution fills the tip of the nanospray needle; Connect, insert one end of the metal electrode from the tail end of the nanospray needle until the end of the metal electrode contacts the sample solution, seal the tail end of the nanospray needle with an insulating end cap, and connect the end of the metal electrode outside the nanospray needle to an external high-voltage power supply; Gas inlet, passing inert auxiliary gas into the ionization chamber; Detection, turn on the high voltage power supply to achieve detection.

10. The operating method according to claim 9, characterized in that: The inert auxiliary gas was high-purity nitrogen with a flow rate controlled at 2.0 L / min; the voltage of the high-voltage power supply was +6.0 kV.

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