Digital microfluidics-mass spectrometry online coupled ion source and online analysis method

Abstract

The present invention discloses a digital microfluidic-mass spectrometry online coupled ion source and an online analysis method, belonging to the technical field of ion sources and online analysis methods. The present invention comprises a digital microfluidic chip, an upper cover plate of the digital microfluidic chip is provided with a 1 mm microhole, a transfer capillary with an outer diameter of 1 mm, which is inserted into the microhole of the upper cover plate of the digital microfluidic chip, a spray capillary with an outer diameter of 360 μm and an inner diameter of 100 μm or 50 μm, which is nested in the transfer capillary, a spray electrode which is a metal wire arranged in the transfer capillary, and an adapter structure printed by a 3D printer for fixing the transfer capillary, the spray capillary and the spray electrode.

Description

Technical Field

[0001] The present invention relates to an ion source and an online analysis method, in particular to a digital microfluidics-mass spectrometry online coupled ion source and an online analysis method, belonging to the technical field of ion sources and online analysis methods. Background Art

[0002] As a molecular detection method with high sensitivity, high precision and high resolution, mass spectrometry technology has many advantages such as small sample usage, fast analysis speed, strong specificity, and the ability to perform simultaneous separation and identification. It is widely used in many industries and scientific research fields such as chemistry and chemical engineering, biomedicine, life sciences, clinical medicine, food hygiene, environmental science and materials science. It has become the gold standard and mainstream analytical tool for biochemical analysis in related fields, and plays a vital role in the accurate measurement of the amount and structure of substances. However, traditional mass spectrometry analysis usually relies on manual sample preparation, which is inefficient and has a complex process, making it difficult to meet the needs of immediate analysis. In addition, manual sample preparation often requires transferring the sample to be tested between different containers, resulting in large sample loss and susceptibility to contamination, thereby affecting the accuracy of the mass spectrometry results.

[0003] Based on this, we designed a new ion source to solve the above problems. Summary of the Invention

[0004] The main purpose of the present invention is to provide a digital microfluidics-mass spectrometry online coupled ion source and an online analysis method.

[0005] The purpose of the present invention can be achieved by adopting the following technical solutions:

[0006] A digital microfluidics-mass spectrometry online coupled ion source and online analysis method, comprising: a digital microfluidics chip, wherein the upper cover of the digital microfluidics chip is provided with a 1 mm microhole;

[0007] 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;

[0008] 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;

[0009] The spray electrode is a metal wire and is disposed in the transfer capillary;

[0010] The adapting structure is printed by a 3D printer and is used to fix the transfer capillary, the spray capillary and the spray electrode.

[0011] Preferably, the digital microfluidic chip is used to perform mixing, reaction, separation and detection operations on tiny droplets.

[0012] Preferably, the lateral distance between the spray capillary and the mass spectrometer inlet is 3 mm, and the longitudinal distance is 2 mm.

[0013] Preferably, the voltage applied by the spray electrode is 3 kV.

[0014] The digital microfluidics-mass spectrometry online coupled ion source online analysis method comprises the following steps: injecting a sample into a digital microfluidics chip;

[0015] 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;

[0016] 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.

[0017] Preferably, before injecting the sample, the method further includes the step of selecting a surfactant of appropriate concentration, wherein the surfactant is poloxamer with a concentration of 0.05%.

[0018] Preferably, 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.

[0019] Preferably, the analysis process further includes a step of verifying the system performance, 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;

[0020] 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;

[0021] 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.

[0022] Preferably, the repeatability test is performed 10 times on each group of samples at 40, 80, and 120 μg / mL of miltefosine, and the coefficient of variation is less than 8%.

[0023] Preferably, the sensitivity test is to use the system to analyze 1-1000 ng / mL of 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;

[0024] 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;

[0025] 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, such as quartz and stainless steel.

[0026] Beneficial technical effects of the present invention:

[0027] The digital microfluidics-mass spectrometry online coupled ion source and online analysis method provided by this invention enable a digital microfluidics chip to complete a series of operations, including mixing, reaction, separation, and detection of tiny droplets, on a single platform, achieving a highly integrated and automated sample processing process. Compared to traditional manual sample preparation, which requires cumbersome sample transfer between different containers, this technology significantly reduces the number of manual steps, significantly improving analysis efficiency, and effectively reducing the risk of sample loss and contamination caused by manual operation.

[0028] By optimizing the surfactant (poloxamer, 0.05%), interference with the mass spectrometry signal was avoided while ensuring normal droplet actuation. Experiments showed that at this concentration, the droplet actuation rate was comparable to that of a 0.1% concentration, while also ensuring the accuracy of subsequent mass spectrometry analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of a digital microfluidics-mass spectrometry online coupling system according to a preferred embodiment of the digital microfluidics-mass spectrometry online coupling ion source and online analysis method of the present invention;

[0030] Figure 2 A diagram showing the lateral and longitudinal distances between the optimized spray capillary and the mass spectrometer inlet according to a preferred embodiment of the digital microfluidics-mass spectrometry online coupled ion source and online analysis method of the present invention;

[0031] Figure 3 A diagram of optimized spray capillary inner diameter and spray voltage according to a preferred embodiment of the digital microfluidics-mass spectrometry online coupled ion source and online analysis method of the present invention;

[0032] Figure 4 This is a diagram showing the effect of different concentrations of PF68 on the droplet movement speed in digital microfluidics according to a preferred embodiment of the digital microfluidics-mass spectrometry online coupled ion source and online analysis method of the present invention;

[0033] Figure 5 This is a diagram showing the effect of 0.05% PF68 on mass spectrometry signals according to a preferred embodiment of the digital microfluidics-mass spectrometry online coupled ion source and online analysis method of the present invention;

[0034] Figure 6 A comparison diagram of a digital microfluidics-mass spectrometry online coupled ion source and online analysis method according to a preferred embodiment of the present invention and a Nano-ESI method;

[0035] Figure 7A repeatability analysis diagram of a preferred embodiment of a digital microfluidics-mass spectrometry online coupled ion source and online analysis method according to the present invention;

[0036] Figure 8 This is a detection performance analysis diagram of a preferred embodiment of the digital microfluidics-mass spectrometry online coupled ion source and online analysis method of the present invention. DETAILED DESCRIPTION

[0037] In order to make the technical solution of the present invention more clear and specific to those skilled in the art, the present invention is further described in detail below with reference to embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0038] First, a 1mm microhole is made on the cover of the digital microfluidic chip, and then a capillary with an outer diameter of 1mm is inserted as a "transfer capillary", which is nested with a capillary with an outer diameter of 360 microns and an inner diameter of 100 (50) microns as a "spray capillary", and a metal wire as a spray electrode. The adapter structure is printed with a 3D printer to fix the above ion source. The structure is shown in the figure. Figure 1 The effects of different distances between the spray capillary and the mass spectrometer inlet, different inner diameters of the spray capillary, and different voltages on the signal were then verified. The overall performance of the system was then verified using small molecule standards (miltefos, N-benzoyl-L-arginine ethyl ester, etc.).

[0039] Experiment 1: The effect of different distances between the spray capillary and the mass spectrometer inlet on the signal;

[0040] We used 100ug / mL of miltefosine as a standard, injected 4 uL of the standard into the chip, and then drove the droplets to move to the spray electrode. Due to capillary action, the droplets entered the transfer capillary and contacted the spray capillary and the metal electrode. At this time, an electrospray was generated by applying a 3kV voltage to the metal wire. The distance between the spray capillary and the mass spectrometer inlet was optimized according to the signal size. The results are shown in Figure 2. Figure 2 As shown in the figure, when the horizontal and vertical distances between the spray capillary and the mass spectrometer inlet are 3 and 2 mm respectively, the signal intensity is the highest and the ionization effect is the best. Therefore, this set of distance parameters was used in subsequent experiments.

[0041] Experiment 2: The influence of the inner diameter of the spray capillary and the voltage on the signal;

[0042] The experimental steps are similar to those in Experiment 1. We still use miltefosine as the standard and evaluate the effect of spray capillaries with an outer diameter of 360 μm, inner diameters of 100, 75, and 50 μm, and voltage (2-4 kV) on the signal. The results are as follows: Figure 3 As shown in the figure, the intensity is highest when the inner diameter is 100 um and the spray voltage is 3 kV.

[0043] Experiment 3 explored the interference of surfactants on mass spectrometry signals;

[0044] Since digital microfluidics requires a certain concentration of surfactant to drive droplet movement, and mass spectrometry is extremely sensitive, it is very necessary to explore different concentrations of surfactants. We chose poloxamer here because it is a non-ionic surfactant that has little interference with mass spectrometry signals. We first tested the effects of different concentrations (0.0125, 0.025, 0.05, 0.1%) of surfactants on droplet driving, such as Figure 4 As shown in Figure 2, when the surfactant concentration is 0.05%, the droplet driving rate is much higher than the first two groups and is comparable to the result of 0.1%. Figure 5 It was shown that 0.05% surfactant would not cause excessive interference to the mass spectrometry signal, so 0.05% poloxamer could support the normal driving of droplets without interfering with the mass spectrometry signal. This parameter was also used in subsequent experiments.

[0045] Experiment 4: Evaluation of the online coupling system of digital microfluidics and mass spectrometry;

[0046] After optimizing the experimental conditions, we tested the performance of the system. First, we injected a mixed solution of reserpine and other substances into the chip, then drove it to the spray area for online mass spectrometry analysis, and then used Nano-ESI to analyze the same sample. Figure 6 As shown in the figure, the mass spectra of the two are very similar, indicating that the system is comparable to Nano-ESI. We then used 40, 80, and 120 μg / mL of miltefosine for repeatability testing, with 10 experiments per group of samples. The results are shown in the figure. Figure 7 As shown, the coefficient of variation is less than 8%, indicating that the system has good repeatability. Finally, we used miltefosine and N-benzoyl-L-arginine ethyl ester to evaluate the sensitivity of the system. In the experiment, the system was used to analyze N-benzoyl-L-arginine ethyl ester solution, imatinib and miltefosine with different concentrations (1~1000ng / mL). At least 3 parallel experiments were performed for each measurement. The average ion intensity during MS analysis was recorded. The results are shown in Figure 8 As shown in the figure, the linearity of the two methods was good (R²=0.975, 0.976, 0.988), and the detection limit was 1 ng / mL. The corresponding tandem mass spectra were inserted.

[0047] The above is only a further embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which fall within the scope of protection of the present invention.

Claims

1. Digital microfluidics-mass spectrometry online coupled ion source, characterized in that, It comprises a digital microfluidic chip, wherein the upper cover plate of the digital microfluidic chip is provided with a 1 mm microhole; 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; 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; The spray electrode is a metal wire and is disposed in the transfer capillary; An adapting structure, printed by a 3D printer, for fixing the transfer capillary, the spray capillary and the spray electrode; Digital microfluidic chips are used to mix, react, separate, and detect tiny droplets; 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. The digital microfluidics-mass spectrometry online coupled ion source according to claim 1, characterized in that: The voltage applied to the spray electrode was 3 kV.

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: The method comprises the following steps: injecting a sample into a digital microfluidic chip; 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; 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. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 3, characterized in that: Before injecting the sample, the method further includes the step of selecting a surfactant with a suitable concentration, wherein the surfactant is poloxamer with a concentration of 0.05%.

5. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 4, characterized in that: The samples are small molecule standards, mixed solutions or solutions of miltefosine and N-benzoyl-L-arginine ethyl ester at different concentrations.

6. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 5, characterized in that: The analysis process also includes a step of verifying the system performance, which includes testing the effect of different distances between the spray capillary and the mass spectrometer inlet on the signal and optimizing the distance parameters; 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; 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. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 6, characterized in that: The repeatability test was performed 10 times on each group of samples at 40, 80, and 120 μg / mL of miltefosine, and the coefficient of variation was less than 8%.

8. The digital microfluidics-mass spectrometry online coupled ion source online analysis method according to claim 7, characterized in that: Sensitivity testing was performed using the system to analyze 1-1000 ng / mL solutions of miltefosine and N-benzoyl-L-arginine ethyl ester, with good linearity and a detection limit of 1 ng / mL. 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; 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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