A device for detecting enzyme activity by coupling a nano kit with electrospray ionization mass spectrometry, a detection method and its application

Abstract

The present application provides a device, detection method and application of a nano kit coupled with electrospray ionization mass spectrometry for detecting enzyme activity, so as to determine the activity of enzymes in single living cells. Specifically disclosed is that a nano capillary containing a substrate corresponding to the enzyme to be detected is inserted into a living cell, the cytoplasm is electrochemically extracted, and the substrate in the capillary is hydrolyzed; after the mixture is electrosprayed at the tip of the capillary, the product and the substrate are distinguished in terms of molecular weight, thereby realizing the detection of enzyme activity in a single cell. The established detection device and method will provide a specific analysis method for further studying the situation of enzyme activity in a single cell.

Description

Technical Field

[0001] The present application relates to the field of single cell detection and analysis, and specifically to a nanometer test kit coupled with electrospray ionization mass spectrometry, a detection method and an application thereof. Background Art

[0002] Angiotensin-converting enzyme 2 (ACE2) is a type I integral membrane protein that acts as a carboxypeptidase to hydrolyze angiotensin II to angiotensin. Angiotensin-converting enzyme 2 (ACE2) is not only an enzyme but also a functional receptor on the cell membrane of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is well known that ACE2 plays an important role in hypertension, cardiac function, heart function, and diabetes.

[0003] Current studies have shown that ACE2 is a specific receptor for SARS-CoV-2, mediating the entry of the virus into host cells. Therefore, a comprehensive study of ACE2, including its catalytic activity, is very important to fully understand its physiological role. Many methods have been developed to determine ACE2 activity in cell populations, however, measurement at the single-cell level remains challenging. Given the importance of cellular heterogeneity in understanding the role of ACE2, a robust strategy needs to be established to measure ACE2 activity in single living cells.

[0004] Currently, probe-based optical methods are popular for analyzing enzyme activity in single living cells. Although this strategy is stable, the design and synthesis of specific probes that recognize enzymes is complex and time-consuming. Previously, the prior art also had a detection strategy based on a nano-kit for measuring enzyme activity in a single living cell. In the disclosed method, a nanopipette carrying the components of the kit is inserted into a living cell. These components are electrochemically pumped into the cell to react with the target enzyme to generate products and byproducts of hydrogen peroxide. The activity of the target enzyme in a cell is determined by electrochemically detecting hydrogen peroxide using a ring electrode at the orifice of the nano-microcapillary tube. This method can be adapted to the measurement of a variety of enzymes by selecting a suitable kit, but the byproduct of hydrogen peroxide is required, which limits the application of this nano-kit-based analysis. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a device for detecting enzyme activity by coupling a nano kit with an electrospray ionization mass spectrometer, the device comprising a nano kit, an electrochemical workstation, an operating system and an electrospray ionization mass spectrometer, wherein:

[0006] The nano kit comprises: a microcapillary and a reagent, wherein the reagent is a substrate of the enzyme to be tested and a metal salt solution, and the microcapillary is used to contain the reagent;

[0007] The electrochemical workstation includes an Ag / AgCl electrode, a silver wire and a data acquisition system, wherein the Ag / AgCl electrode is used as a reference electrode, the silver wire is placed in the microcapillary, and connects the substrate of the enzyme in the microcapillary with the electrochemical workstation, the silver wire, the reagent, the Ag / AgCl electrode and the data acquisition system form a complete loop, and the data acquisition system is used to record the electrochemical test results;

[0008] The operating system includes a microscope, a micromanipulator and a patch clamp amplifier, wherein:

[0009] The microscope is used to locate cells, the microcapillary containing the substrate is fixed to the mechanical arm of the micromanipulator through the electrode holder of the patch clamp, the micromanipulator is used to operate the tip of the microcapillary to move above the single cell to be tested and insert the single cell to be tested, the electrochemical workstation is used to apply negative voltage to electrochemically extract cytoplasm from the cell, so that the enzyme contacts the enzymatic substrate to react, after the reaction, the microcapillary pipette is placed at the inlet of the electrospray ionization mass spectrometer, the electrospray ionization mass spectrometer is used to record the mass spectrometry signals of the substrate and the product in the microcapillary, and the activity of the enzyme to be detected is calculated based on the collected mass spectrometry signals.

[0010] In one embodiment, the microcapillary is drawn from a borosilicate glass tube by a P-2000 laser puller, wherein the laser puller parameters are: BF100-58-10; outer diameter 1.00 mm, inner diameter 0.58 mm, and the opening of the microcapillary is 2-3 μm.

[0011] In one embodiment, the electrospray ionization mass spectrometry adopts a positive ion mode, a drying temperature of 300° C., a drying gas flow rate of 2.0 L / min, a nebulizer pressure of 0 Psig, and an applied voltage of 4000V.

[0012] The present application also provides a method for detecting enzyme activity using the electrospray ionization mass spectrometry device, comprising the following steps:

[0013] Prepare cells to be tested, prepare substrate solution of the enzyme to be tested, and use a microloader micro-injector and a pipette to transfer the enzymatic substrate solution into a microcapillary;

[0014] The microcapillary is fixed to the mechanical arm of the micromanipulator through the electrode holder of the patch clamp, the cell to be tested is positioned through a microscope using the micromanipulator, a microcapillary filled with an enzymatic substrate solution is inserted into the single cell to be tested, a silver wire is placed in the microcapillary, the substrate in the microcapillary is connected to an electrochemical workstation, and a negative voltage is applied through the electrochemical workstation to electrochemically extract the cytoplasm in the cell, so that the enzyme in the cytoplasm reacts with the enzymatic substrate in the microcapillary;

[0015] The microcapillary pipette is placed at the entrance of the electrospray ionization mass spectrometer, and the solution enters the electrospray ionization mass spectrometer for analysis. The electrospray ionization mass spectrometer is used to collect signals of the solution in the capillary, and the activity of the detected enzyme is calculated based on the mass spectrometry signal intensity of the substrate and product in the solution after the reaction.

[0016] In one embodiment, the preparation of the cells to be tested is as follows: after completing the subculture operation on the cells, taking 20 μl of the cell suspension and adding it to a culture dish, adding culture medium and incubating for 24 hours at 37°C and 5% carbon dioxide environment; before conducting the test, removing the culture medium and replacing it with PBS solution to maintain the normal physiological state of the cells, so as to facilitate positioning operation under a microscope.

[0017] In one embodiment, the enzyme reacts with the enzymatic substrate by mixing the enzyme and the substrate in a 100 μM metal salt solution, and reacting at 37° C. and saturated water vapor for 3-5 hours.

[0018] In one embodiment, a high voltage is applied to the microcapillary to cause the solution in the tube to spray out to form a fine electrospray. An electrode is inserted at the rear end of the microcapillary and a high voltage of 4000V is applied to cause the solution in the tube to spray out to form a fine electrospray, which enters the electrospray ionization mass spectrometer for analysis.

[0019] In one embodiment, the capillary pore size, negative pressure, and voltage application time can be controlled by a signal acquisition system of an electrochemical workstation.

[0020] In one embodiment, the activity of the enzyme to be detected is obtained by calculating the corresponding concentrations according to the mass spectrometry signal intensities of the substrate and product in the solution after the reaction, thereby calculating the conversion rate; and calculating the data U that can represent the enzyme activity according to the standard formula of the international unit of enzyme activity according to the conversion rate, the volume of the solution in the capillary, and the reaction time:

[0021]

[0022] The definition of the international unit of enzyme activity is the amount of enzyme that can convert 1 micromole of substrate in 1 minute under specific conditions, or the amount of enzyme that converts 1 micromole of related groups in the substrate. The unit is μmol / min.

[0023] In the present application, V is the volume of the solution in the capillary, c is the concentration of the substrate, α is the conversion rate of the substrate, M is the molar molecular weight of the substrate, and t is the reaction time.

[0024] The present application also provides an application of the coupled electrospray ionization mass spectrometry device in single cell detection.

[0025] Beneficial Effects

[0026] The present application provides a nanoscale enzyme activity detection device coupled with electrospray ionization mass spectrometry, a detection method and its application to determine the enzyme activity in a single cell. The key to the present application is to achieve sampling of single cells with the help of a micromanipulation system, and to achieve measurement of enzyme activity in a single cell by coupling high-sensitivity electrospray ionization mass spectrometry with nano kit technology. The loading of cytosol, the reaction of the target enzyme and the subsequent mass spectrometry detection are integrated in a capillary, which is suitable for single-cell analysis. Since the detection method of the present application relies on the mass difference between the substrate and the product, it can be applied to the analysis of various enzymes without restriction. More importantly, by integrating all these kits into one capillary, this method can be easily developed to measure multiple enzyme activities in a cell.

[0027] In order to ensure the accuracy of the experiment and improve the sensitivity of the detection, first of all, the detection device and method provided by the present application, in which the detection solution used is relatively simple in composition, reduces the influence of the matrix effect. Secondly, a small amount of cytoplasm is extracted into the solution in the capillary during the detection, which plays a dilution role and reduces the signal intensity of non-target substances in the cell. Finally, the reaction time is limited to be as long as possible during the detection, so that the signal in the cell can be amplified as much as possible.

[0028] The present application uses an electrochemical workstation to apply negative voltage to achieve electrochemical sequential extraction of the cytoplasm in the cell, while recording the current during the extraction process, judging the diameter of the microcapillary, the size of the negative pressure, and the time of applying the voltage to control the repeatability and accuracy of the detection.

[0029] The present application uses electrospray ionization mass spectrometry to collect signals from the solution in the capillary. Since the detection method of the present application can be applied to the detection of enzyme activity in a single cell, the volume of the solution in the capillary is relatively small. In order to avoid the influence on the signal, an electrode is directly inserted into the rear end of the capillary, and a high voltage of 4000V is applied to spray the solution in the tube to form a fine electrospray, which enters the electrospray ionization mass spectrometry for analysis, so that trace detection in a single cell can be achieved.

[0030] The present application uses a micromanipulator to operate a single cell. The micromanipulator is controlled by piezoelectric ceramics. Due to the high sensitivity of piezoelectric ceramics, high-precision operations can be achieved. There are 9 gears in total. It is very suitable for achieving fine operations on single cells. By controlling the micromanipulator, the capillary filled with solution is moved to the top of a single cell and inserted, so as to achieve accurate identification and positioning of a single cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A detection device and principle diagram of an embodiment of the present application

[0032] Figure 2A-2B In one embodiment of the present application ( Figure 2A) Standard Ang II and ( Figure 2B )Mass spectrum of Ang 1-7

[0033] Figure 3A-3B In one embodiment of the present application, different concentrations of ( Figure 3A )Ang II and ( Figure 3B ) Peak intensity of Ang 1-7

[0034] Figure 4 The conversion rate of Ang II in one embodiment of the present application

[0035] Figure 5A-5D The mass spectrometry analysis results of the mixtures with different enzymatic reaction times filled into the capillaries in one embodiment of the present application ( Figure 5A , Figure 5C ) and conversion rate ( Figure 5B , Figure 5D )

[0036] Fig. 6A Mass spectrometry of Ang II enzyme activity in A549 cells in one embodiment of the present application

[0037] Figure 6B The conversion rate of Ang II enzyme in A549 cells in one embodiment of the present application

[0038] Fig. 7A Imaging of microcapillaries inserted into a living cell in one embodiment of the present application

[0039] Figure 7B Mass spectra collected from the reaction mixture inside the capillary

[0040] Figure 7C Individual Hace2 (ACE2+) cells and cell enzyme activity during inflammation

[0041] Fig.7D Statistics of enzyme activity from single cells Error bars present standard deviation from single cell analysis

[0042] Figure 8 The results of detecting enzyme activity in a single A549 cell in an embodiment of the present application DETAILED DESCRIPTION

[0043] The present invention is further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0044] Reagents and instruments

[0045] Angiotensin-converting enzyme 2 (ACE2), angiotensin II (Ang II), and angiotensin 1-7 (Ang 1-7) were purchased from MCE (MedChemExpress) Company (Shanghai, China). hACE2-CHO cells were purchased from Antaiji Biotechnology Co (Beijing, China). Transforming growth factor β3 (TGFB3) and interleukin 1β (IL1β) were purchased from Novoprotein Co. (CA, USA). Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich. Borosilicate glass tubes (BF100-58-10) used in the experiments were purchased from Sutter Instrument. (California, USA). Ultrapure water was from a Milli-Q purification system with a resistivity of ≥18.25 MΩ·cm.

[0046] Cell culture

[0047] hACE2-CHO cells were cultured in DMEM (dulbecco's modified eagle medium) / high glucose medium supplemented with 15% FBS (fetal bovine serum) and 1% antibiotics (penicillin / streptomycin). The cells were cultured in a cell culture incubator containing 5% CO2 at 37°C in a humidified atmosphere.

[0048] instrument

[0049] The nanospray mass spectrometer was composed of a drawn microcapillary, a metal wire and an electrospray ionization mass spectrometer (Agilent G6530BQ-TOF, USA). The positive ion mode was used, the drying temperature was 300°C, the drying gas flow rate was 2.0 L / min, and the nebulizer pressure was 0 Psig. The applied voltage was 4000 V. The microscope Olympus X73 (Olympus, Japan) was used. Micromanipulation was performed using a 4r micromanipulator (Eppendorf, Germany). Single-cell electrochemical tests were recorded using a MultiClamp700B patch clamp amplifier and an Axon Digidata 1550 data acquisition system (Molecular Devices, USA), in which an Ag / AgCl electrode was used as a quasi-reference electrode. A microloader micro-injector was produced by Eppendorf.

[0050] All glass microcapillaries used in the experiments were pulled from borosilicate glass tubing (BF100-58-10; OD 1.00 mm, ID 0.58 mm) using a P-2000 laser puller (Sutter Instrument, CA, USA). The opening of the microcapillaries was 2-3 μm.

[0051] In one embodiment, the present application provides a method for detecting enzyme activity using the coupled electrospray ionization mass spectrometry device, comprising the following steps:

[0052] Prepare cells to be tested, prepare substrate solution of the enzyme to be tested, and use a microloader micro-injector and a pipette to transfer the enzymatic substrate solution into a microcapillary;

[0053] The microcapillary is fixed to the mechanical arm of the micromanipulator through the electrode holder of the patch clamp, the cell to be tested is positioned through a microscope using the micromanipulator, a microcapillary filled with an enzymatic substrate solution is inserted into the single cell to be tested, a silver wire is placed in the microcapillary, the substrate in the microcapillary is connected to an electrochemical workstation, and a negative voltage is applied through the electrochemical workstation to electrochemically extract the cytoplasm in the cell, so that the enzyme in the cytoplasm reacts with the enzymatic substrate in the microcapillary;

[0054] The microcapillary pipette is placed at the inlet of the electrospray ionization mass spectrometer, and the reagent enters the electrospray ionization mass spectrometer for analysis. The electrospray ionization mass spectrometer is used to collect signals from the solution in the capillary, and the activity of the detection enzyme is calculated based on the mass spectrometry signal intensity of the substrate and product in the solution after the reaction.

[0055] Since cells are only a dozen to several dozen microns in size, a microscope is needed to see the cells clearly. In one embodiment, 20X and 40X objective lenses are used.

[0056] In one embodiment, the microcapillary is fixed to the mechanical arm of the micromanipulator by means of the electrode holder of the patch clamp. The micromanipulator is controlled by piezoelectric ceramics. Due to the high sensitivity of piezoelectric ceramics, high-precision operations can be achieved, with a total of 9 gears. It is very suitable for achieving fine operations on single cells.

[0057] In one embodiment, the capillary filled with the solution is moved to the top of a single cell and inserted by controlling the micromanipulator. After the cells are subcultured, 20 μl of the suspension is added to a culture dish, and the culture medium is added and incubated for 24 hours at 37 degrees Celsius and 5% carbon dioxide. Before testing, the culture medium is removed and replaced with a PBS solution to maintain the normal physiological state of the cells and facilitate observation under a microscope. At this time, a negative pressure of -1 V is applied by means of the silver wire in the capillary (silver wire, solvent, reference electrode, and data acquisition system constitute a complete circuit) during electrochemical work, and the cytoplasm of a single cell is extracted through electroosmosis, thereby obtaining the target enzyme.

[0058] In one embodiment, the repeatability of the experiment is ensured by controlling the capillary pore size, the negative pressure, and the time of applying the voltage.

[0059] In one embodiment, the enzymatic substrate is angiotensin II (Ang II), and the enzyme is angiotensin converting enzyme 2 (ACE2).

[0060] After extraction, the cytoplasm of a single cell (containing the target enzyme) will react with the substrate solution infused into the capillary. The composition of the substrate solution is as simple as possible, with only 100 μg / mL Ang II and 100 μM ZnCl2. ZnCl2 is because the catalytic action of the target enzyme requires the help of the metal ion Zn 2+ The solution is simple to avoid the influence of matrix effect on the intensity of mass spectrometry signal. Under the condition of 37℃ and saturated water vapor, the solution in the capillary is prevented from volatilizing and affecting the signal, and the reaction is carried out for 3 hours. The reaction time is as long as possible to amplify the signal generated by a single cell.

[0061] The nanospray mass spectrometer was used to collect signals from the solution in the capillary. Since the volume of the solution in the capillary was small, in order to avoid the influence of HPLC on the signal, an electrode was directly inserted into the rear end of the capillary, and a high voltage of 4000V was applied to spray the solution in the tube to form a fine electrospray, which entered the mass spectrometer for analysis.

[0062] Obtaining enzyme activity: First, detect the mass spectrometry signal intensity of the standard samples of substrates and products of different concentrations to obtain a standard curve of signal intensity versus concentration. According to the signal intensity of the substrate and product in the solution after the reaction, read the corresponding concentration to calculate the conversion rate. According to the conversion rate, the volume of the solution in the capillary, and the reaction time, the data U that can represent the enzyme activity can be calculated according to the standard formula of the international enzyme activity unit.

[0063] Standard measurement

[0064] The present application specifically utilizes the mass difference between substrate and product to measure ACE2 activity in single living cells.

[0065] like Figure 1As shown, angiotensin II (AngII) is a polypeptide sequence (MW 1046.18) containing 8 amino acids (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) as a substrate for ACE2. After inserting the capillary into a live hACE2-CHO cell with overexpressed ACE2 enzyme, the cytoplasmic matrix containing ACE2 enzyme was electrochemically extracted into the nanocapillary. Due to the extremely slow evaporation of the liquid from the micropores, the mixture of Ang II and ACE2 enzyme can be retained at the tip of the capillary, allowing the reaction to continue for several hours. The catalytic process removes Phe from Ang II to generate Ang 1-7 with a molecular weight of 899.00. After applying high voltage in the capillary to start electrospray, the mass spectrum was recorded. Ang 1-7 and Ang II were distinguished in the spectrum based on the difference in molecular weight. Finally, their peak intensities were used to calculate the activity of ACE2.

[0066] The mass spectra of standard Ang II and Ang 1-7 were measured by filling the chemicals into a microcapillary (inner diameter 2-3 μm) after ESI-MS. The peaks with mass-to-charge ratios of 1046.54 and 523.77 were associated with Ang II ( Figure 2A ), while 899.47, 450.24 and 300.50 were associated with Ang 1-7 ( Figure 2B ). Different concentrations of Ang II ( Figure 3A ) and Ang 1-7( Figure 3B ) are listed in Figure 3A-3B In the results, it was shown that the ionization efficiency of Ang 1-7 was slightly higher than that of Ang II.

[0067] Enzyme activity analysis and quantitative enzyme activity conversion rate

[0068] To validate the enzyme activity analysis, Ang II was reacted with pure ACE2 enzyme in a buffer at 37°C. Then, the mixtures with different reaction times were filled into capillaries for mass spectrometry analysis.

[0069] Removal of ACE2 enzyme from the solution resulted in only the peak of Ang II being observed after 2 h of incubation ( Figure 4 ). These results show the sequential conversion of Ang II by the ACE2 enzyme.

[0070] At 0.5 min, only the peak corresponding to Ang II was observed, indicating only the substrate in the mixture. After 10 min of reaction, peaks related to Ang II (1046.54 and 523.77) and Ang 1-7 (450.24 and 300.50) appeared, indicating that Ang II was partially converted to Ang 1-7 ( Figure 5A). As the reaction time prolonged, the peak associated with Ang II gradually decreased, while the peak associated with Ang1-7 increased.

[0071] The peak intensities of Ang II and Ang 1-7 at different reaction times were measured. The conversion rate was quantified using the peak intensities of Ang II and Ang 1-7 ( Figure 5B ). A nearly linear increase in conversion was observed, which showed a continuous reaction between ACE2 and Ang II. After 3 hours of reaction, about 40% of Ang II was converted into Ang 1-7. Since the initial concentration of Ang II was 100 μg / ml and the volume of the mixture in the capillary was 10 μL, the catalytic activity of ACE2 was calculated to be 1.765 μU, and the enzymatic reaction was quantified by quantifying the conversion rate.

[0072] It is also further explained that the device and detection method of the present application can detect the activity of the target enzyme by scanning the substrate and the reactant.

[0073] For the hACE-CHO cells used in this experiment, the enzymatic activity of ACE2 in the cell lysate was first detected. The cell lysate was obtained by digesting a flask full of hACE2-CHO cells with 2mL of pancreatin at 37℃ for 2min, adding 8mL of DMEM / high glucose medium to terminate the digestion, and transferring it to a 15mL centrifuge tube. Centrifuge at 2000r / min for 2min to obtain a cell mass of about 106 cells. Pour out the culture medium, add 200μL of ultrapure water to blow away the cell mass, centrifuge at 2000r / min for 2min, discard the solution part, and obtain the cell mass. Wash three times until colorless. Add 200μL of ultrapure water, put it in a 4℃ refrigerator to swell for 4h, and use an ultrasonic disruptor with a power of 70W, ultrasonic time of 5s, pause time of 9s, and total working time of 5min to break the cell suspension in an ice water bath. Centrifuge at 13500r / min and 4℃ for 10 min, discard the cell debris precipitate, take the supernatant, and obtain hACE2-CHO cell lysate containing ACE2. Add 50μl hACE2-CHO cell lysate to 100μM substrate, with a total volume of 500μl, and react at 37℃. The mass spectrometry results after 15min reaction are as follows: Figure 5C As shown. The relationship between conversion rate and reaction time (Figure 5D) shows that more than 80% of Ang II was converted by the enzyme within 1 hour. The enzyme activity of the cell lysate was calculated to be 1.06mU, which is much higher than the enzyme activity of the commercial ACE2 enzyme. For comparison, A549 cells that do not express ACE2 enzyme were used in the control experiment ( Figure 6A-6B). After 3 hours, only 3.14% of Ang II was converted to Ang 1-7, showing significantly low enzyme activity in A549 cells. Both experimental results support the detection of enzyme activity from ACE2-expressing cells. The main reason for the low enzyme activity of A549 cells is that the cell lysis solution used in this example contains more complex intracellular components, which may lead to the instability of the substrate structure, resulting in decomposition and the generation of products.

[0074] Detection of enzyme activity in single cells

[0075] In single-cell analysis, a mixture of 100 μg / mL Ang II and 100 μM ZnCl2 was loaded into the capillary. Then, with the help of the micromanipulation system, the capillary tip was positioned to the live hACE2-CHO cells. A voltage of -1 V was applied to the Ag / AgCl line inside the capillary to electrochemically sort the cytosol for 3 minutes. After that, the capillary was placed at 37 °C for 3 h to allow Ang II to react with the ACE2 enzyme. Finally, the capillary was placed in front of the electrospray ionization mass spectrometer inlet to start the nanospray.

[0076] With the help of a micromanipulation system, the tip of the capillary is inserted into a cell ( Fig. 7A ). The cytosol in the cells was sorted into the capillary using an electrochemical pump. Considering that the sorting amount of single-cell ACE2 was less than that of the cell lysate, the reaction time was extended to 3 h to maximize the conversion rate of Ang II. During this period, no obvious liquid loss was observed in the capillary with the same reaction volume. The buffer evaporated slowly from the capillary orifice, ensuring the reaction between the sorting enzyme from the single cell and the substrate in the capillary. Then, the mass spectra were collected ( Figure 7B ), showing peaks for Ang 1-7 (450.24 and 899.47). Since Ang 1-7 is not a cell content, the observation of Ang1-7 from the mixture within the capillary provides direct evidence for the reaction between ACE2 and Ang II. The results show that the enzyme loaded by a single cell can react with the pre-filled substrate to complete the reaction. The same detection method was applied to a single live A549 cell, and no peaks associated with Ang1-7 were observed ( Figure 8 ), which further supports the results of single hACE2-CHO cells.

[0077] Unlike the partial conversion of Ang II by the enzyme in the cell lysate, the enzyme loaded on the single cells was insufficient and could convert a small amount of substrate. As a result, the peak intensity of Ang II was almost the same, which was not shown in the spectrum. To calculate the enzyme activity in the single cells, the production of Ang 1-7 was determined based on the calibration curve of the standard peak intensity (Figure 2). Figure 7C The ACE2 activity of 8 cells is listed. The average enzyme activity of a single cell was determined to be 4.2 ± 1.4 nU. The calculated relative standard deviation was 33.3%, which is significantly larger than the reaction with the enzyme in the cell lysate ( Figure 5D The observed large deviation from single-cell analysis provides clear evidence for cellular heterogeneity in ACE2 activity. Compared with existing electrochemical detection, this method does not require any electrochemical byproducts and can be applied to almost all enzymatic reactions to determine their activity.

[0078] Correlation between enzyme activity and inflammatory activity

[0079] Coronavirus can specifically recognize and bind to ACE2 on the cell membrane, then enter the cell and trigger an inflammatory response. To study the changes in ACE2 activity under inflammation, hACE2-CHO cells were treated with inflammatory factors (tgfb3 and IL1B) for 24 hours. Afterwards, the ACE2 catalytic activity of a single cell was measured using the method of this application. The activity from 10 cells is also listed in Figure 7C The average results and standard deviations of ACE2 enzyme activity of hACE2-CHO cells with and without inflammation treatment are shown in Fig. Fig.7D , in order to better see the deviation between parallel experiments, and to compare whether the presence or absence of inflammatory treatment has an effect on the activity of the target enzyme in single cells. Statistical analysis of activities with and without inflammation did not show significant differences. This result provides clues that the inflammatory state may not affect the catalytic activity of ACE2 in cells. Previous proteomic studies have shown that the expression of ACE2 is not changed in COVID-19 patients. Therefore, it may only serve as a carrier of SARS-CoV-2.

[0080] Furthermore, information on the independence of its activity from cellular inflammation will provide more information on the role of this enzyme in disease.

[0081] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A nano kit coupled to electrospray ionization mass spectrometry for detecting enzyme activity, characterized in that: The device comprises a nano reagent kit, an electrochemical workstation, an operating system and an electrospray ionization mass spectrometer, wherein: The nano kit comprises: a microcapillary and a reagent, wherein the reagent is a substrate of the enzyme to be tested and a metal salt solution, and the microcapillary is used to contain the reagent and extract the cytoplasm; The electrochemical workstation includes an Ag / AgCl electrode, a silver wire and a data acquisition system, wherein the Ag / AgCl electrode is used as a reference electrode, the silver wire is placed in the microcapillary, and the reagent in the microcapillary is connected to the electrochemical workstation, the silver wire, the reagent, the Ag / AgCl electrode and the data acquisition system form a complete loop, and the data acquisition system is used to record the electrochemical test results; The operating system includes a microscope, a micromanipulator and a patch clamp amplifier, wherein: The microscope is used to locate cells. The microcapillary containing the substrate is fixed to the mechanical arm of the micromanipulator through the electrode holder of the patch clamp. The micromanipulator is used to operate the tip of the microcapillary to move above the single cell to be tested and insert the single cell to be tested. The electrochemical workstation is used to apply negative electric pressure to electrochemically extract cytoplasm from the cell, so that the enzyme to be tested contacts with the substrate of the enzyme to be tested to react. After the reaction, the microcapillary is placed at the inlet of an electrospray ionization mass spectrometer. The electrospray ionization mass spectrometer is used to record the mass spectrometer signals of the substrate and the product in the microcapillary, and the activity of the enzyme to be tested is calculated based on the collected mass spectrometer signals.

2. The device according to claim 1, characterized in that The microcapillary is drawn from a borosilicate glass tube by a P-2000 laser puller, wherein the parameters of the laser puller are: BF100-58-10; outer diameter 1.00 mm, inner diameter 0.58 mm, and the opening of the microcapillary is 2-3 μm.

3. The device according to claim 1, characterized in that The electrospray ionization mass spectrometer adopted a positive ion mode, a drying temperature of 300° C., a drying gas flow rate of 2.0 L / min, a nebulizer pressure of 0 Psig, and an applied voltage of 4000 V.

4. A method for detecting enzyme activity using the coupled electrospray ionization mass spectrometry device according to claim 1, characterized in that: The following steps are involved: Prepare cells to be tested, prepare substrate solution of the enzyme to be tested, and use a microloader micro-injector and a pipette to transfer the substrate solution of the enzyme to be tested into a microcapillary; The microcapillary is fixed to the mechanical arm of the micromanipulator through the electrode holder of the patch clamp, the micromanipulator is used to locate the cell to be tested through a microscope, the microcapillary filled with an enzymatic substrate solution is inserted into the single cell to be tested, a silver wire is placed in the microcapillary, the reagent in the microcapillary is connected to an electrochemical workstation, and a negative voltage is applied through the electrochemical workstation to electrochemically extract the cytoplasm in the cell, so that the enzyme in the cytoplasm reacts with the enzymatic substrate in the microcapillary; The microcapillary after the reaction is placed at the inlet of the electrospray ionization mass spectrometer, the mass spectrometer is set to a high voltage, and the electrospray ionization mass spectrometer is used for analysis. The electrospray ionization mass spectrometer is used to collect signals from the solution in the capillary, and the activity of the detected enzyme is calculated based on the mass spectrometer signal intensity of the substrate and product in the solution after the reaction.

5. The method according to claim 4, characterized in that The preparation of the cells to be tested is as follows: after completing the subculture operation of the cells, 20 μl of the cell suspension is added to a culture dish, and culture medium is added to incubate at 37° C. and 5% carbon dioxide for 24 hours; Before testing, the culture medium was removed and replaced with PBS solution to maintain the normal physiological state of the cells, making it easier to locate and operate under a microscope.

6. The method according to claim 4, characterized in that The enzyme reacts with the enzymatic substrate by mixing the enzyme and the substrate in a 100 μM metal salt solution, and reacting at 37° C. and under saturated water vapor for 3-5 hours.

7. The method according to claim 4, characterized in that A high voltage is applied to the microcapillary to eject the solution in the tube to form a fine electrospray. An electrode is inserted at the rear end of the microcapillary and a high voltage of 4000V is applied to eject the solution in the tube to form a fine electrospray, which enters the electrospray ionization mass spectrometer for analysis.

8. The method according to claim 4, characterized in that The capillary pore size, negative pressure, and voltage application time can be controlled through the signal acquisition system of the electrochemical workstation.

9. The method according to claim 4, characterized in that The activity of the enzyme to be tested is calculated by reading the corresponding concentrations according to the mass spectrometry signal intensities of the substrate and product in the solution after the reaction, thereby calculating the conversion rate; based on the conversion rate, the volume of the solution in the capillary, and the reaction time, the data U that can represent the enzyme activity is calculated according to the standard formula of the international enzyme activity unit.

10. Use of the electrospray ionization mass spectrometer according to claim 1 in single cell detection.

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