CN116698957A - Enzyme activity detection device and method based on mass spectrum - Google Patents
Enzyme activity detection device and method based on mass spectrum Download PDFInfo
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- G01N27/628—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 using heat to ionise a gas and a beam of energy, e.g. laser enhanced ionisation
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Abstract
The application relates to an enzyme activity detection device and method based on mass spectrum. The device combines the operation of applying the enzyme substrate with the mass spectrum detection process through the integrated enzyme substrate applying unit and the mass spectrum detection unit, does not need to carry out complex pretreatment steps on the tissue slice, simplifies the operation process of the detection method, and effectively reduces detection errors caused by the pretreatment process in the prior art. The enzyme activity detection method can realize instant reaction-detection-imaging under normal pressure environment, furthest maintains the original state of the biological tissue slice, realizes in-situ detection imaging of the tissue slice, and improves the efficiency and accuracy of enzyme activity detection.
Description
Technical Field
The application relates to the technical field of enzyme activity detection, in particular to an enzyme activity detection device and method based on mass spectrum.
Background
Enzymes are a very important class of biocatalysts, involved in most physiological processes in living beings, the abundance and activity of which are closely related to the pathology of many diseases. Thus, detection of enzyme activity and high resolution imaging analysis would provide a powerful tool for elucidating a range of disease mechanisms.
The localization detection of enzymes on biological tissues is an important method for revealing the physiological functions of enzymes, and the detection of enzymes is mainly focused on two dimensions: first, the relative content and distribution of the enzyme are determined. The method is based on immunohistochemistry, and uses the antigen-antibody specific binding principle to make the color-developing agent of the labeled antibody develop through chemical reaction, so as to realize the localization, qualitative and relative quantification of enzymes in tissues or cells (chem. Rev.2011,111, 6130-6185), but the method only can obtain static distribution information of the enzymes, and is difficult to evaluate the activity of the enzymes;
and secondly, detecting the activity of the enzyme in the organism. The method is mainly realized by relying on an enzyme histochemical method, namely, by utilizing the characteristic that the enzyme has the function of catalyzing a certain reaction, a specific product is generated by utilizing the catalysis of the enzyme by applying a specific substrate to the enzyme on biological tissues or cells, and then the activity of the enzyme can be positioned or quantitatively analyzed by detecting the product by various detection means.
Most of the detection means commonly used at present are based on color reaction. The biological tissue slice or cell is generally placed in a solution containing an enzyme substrate for incubation, the enzyme substrate forms a primary reaction product under the catalysis of the enzyme, the obtained primary reaction product is combined with a certain capturing agent to form a visible or chromogenic precipitate, and finally the detection of the enzyme activity on the tissue or cell is realized through optical image acquisition. The current main detection means include fluorescence detection (biotechnol.j.2019, 14,1800144), metal ion precipitation, azo dye coupling method, and the like. The method generally needs to perform secondary reaction, is complex in operation, and is easy to cause diffusion of a catalytic product and further cause result deviation due to multiple reactions, and an imaging result is generally required to be observed by a microscope after color development, so that the detection difficulty is increased. Other enzyme histochemical means, such as the use of isotopes, pigments, metal-labeled substrates, allow the catalytic products of the enzyme to be detected without the need for secondary color reactions. However, the substrate labels are expensive and therefore not versatile.
The mass spectrum detection is a detection means with high efficiency, high flux and high precision, and can realize effective reading of the molecular information of the object to be detected. Molecular imaging on the tissue level can be realized based on the imaging technology developed by the mass spectrum detection method. The combination of mass spectrum imaging technology and enzyme histochemistry to realize enzyme activity imaging of biological tissue level is a current research hotspot. Research has been reported to detect enzyme activity on biological tissues using mass spectrometry imaging methods, typically using matrix-assisted laser desorption ionization mass spectrometry imaging techniques. The method comprises the steps of firstly spraying enzyme substrates on tissue slices to be detected, then placing the tissue slices in an environment with constant temperature and pressure and proper humidity for incubation, and finally detecting the tissue slices by using mass spectrum. However, there are many limitations to laser desorption ionization mass spectrometry, and it is necessary to apply a matrix to a sample to be tested to assist in ionization of molecules, so that it is also necessary to spray-coat the matrix onto a tissue slice, and then dry the tissue slice, and then detect the tissue slice using mass spectrometry. The method needs to carry out a complex pretreatment process, has a complex operation process and has high environmental requirements. In addition, this multiple processing of tissue slices affects the in situ detection of enzymes, and the spraying of the substrate may result in spatial diffusion shifts of the detection products. Meanwhile, as the requirements of different types of to-be-detected objects on the used matrixes are different, the screening of the matrixes also greatly increases the actual workload. More importantly, the prior art can only realize the detection of single enzyme activity, and the simultaneous monitoring and imaging of multiple enzyme activities cannot be realized at present.
There is therefore a need to develop a more convenient and efficient method of mass spectrometry imaging.
Disclosure of Invention
It is a primary object of the present application to overcome at least one of the above-mentioned drawbacks of the prior art and to provide a mass spectrometry-based enzyme activity detection device that is more simple and efficient to operate.
Another main object of the present application is to overcome at least one of the above drawbacks of the prior art, and to provide a method for performing an enzymatic activity detection based on mass spectrometry that is simple and efficient to operate.
In order to achieve the above purpose, the application adopts the following technical scheme:
the application provides an enzyme activity detection device based on mass spectrum, which comprises:
the tissue bearing unit is used for bearing the tissue slice to be detected;
an enzyme substrate application unit comprising a liquid-feeding end for applying an enzyme substrate, said liquid-feeding end being located at said tissue-bearing unit;
the mass spectrum detection unit comprises a mass spectrometer and a detection end connected with the mass spectrometer, wherein the detection end is positioned at the tissue bearing unit and is used for extracting and desorbing molecules of an object to be detected on the tissue slice to form ionized products, and the ionized products are sent to the mass spectrometer to finish mass spectrum detection.
According to one embodiment of the present application, the enzyme substrate applying unit includes a first solution supplying mechanism having a liquid outlet connected to the liquid feeding end, and the first solution supplying mechanism stores an enzyme substrate solution therein.
According to one embodiment of the application, the liquid feeding end is a liquid feeding pipe or a spray pipe, or other effective liquid feeding modes.
According to one embodiment of the application, the detection end comprises a primary capillary and a secondary capillary. The liquid inlet of the primary capillary tube is connected with the detection solvent to which high voltage is applied, and the liquid outlet of the primary capillary tube is arranged at the position of the tissue slice on the tissue bearing unit. The liquid inlet of the secondary capillary tube is arranged at the position of the tissue slice on the tissue bearing unit, and the secondary capillary tube and the primary capillary tube form an extraction system together, so that the formed stable liquid bridge can realize extraction of molecules to be detected on the tissue slice. The liquid outlet of the secondary capillary tube is arranged at the inlet of an ion transmission tube of the mass spectrometer, and ionized molecules to be detected are sent into the mass spectrometer for detection through the ion transmission tube under the action of an electric field.
According to one embodiment of the present application, the detection end further includes a second solution supply mechanism, the second solution supply mechanism stores the detection solvent, and the second solution supply mechanism is provided with a high-voltage electric access device, and is used for applying high-voltage electricity to the detection solvent in the second solution supply mechanism, so as to form a high-voltage electric field for transmitting ions.
According to one embodiment of the application, the drive unit is further comprised. The tissue bearing unit is arranged on the driving unit, and the driving unit drives the tissue bearing unit to move according to a set path.
According to one embodiment of the present application, the driving unit is a three-axis driving mechanism, and the three-axis driving mechanism is used for driving the tissue bearing unit to move along the x-axis, the y-axis or the z-axis.
According to one embodiment of the application, the tissue-bearing unit comprises a substrate having at least one tissue slice to be measured.
In particular, the application also provides an enzyme activity detection method based on the enzyme activity detection device, which comprises the following processing steps:
the liquid feeding end of the enzyme substrate applying unit is aligned with the tissue slice on the tissue carrying unit, and an enzyme substrate solution is applied to the tissue slice through the liquid feeding end and reacts under the catalysis of enzymes in the tissue slice to generate a specific product;
the primary capillary in the mass spectrum detection unit is aligned with the tissue slice on the tissue bearing unit, and a detection solvent is injected into the tissue slice after the catalytic reaction is completed;
the detection solvent contacts the tissue slice to extract the molecules of the object to be detected on the surface of the tissue slice into the detection solvent in a molecular desorption diffusion mode, the molecules of the object to be detected are conveyed to an ion transmission pipe inlet of the mass spectrometer through a secondary capillary, ionization occurs to the molecules of the object to be detected under normal pressure, and the ionized molecules are enabled to enter the mass spectrometer in the mass spectrum detection unit for detection under the action of a high-voltage electric field. According to one embodiment of the application, the driving unit is used for adjusting the position of the tissue bearing unit, and enzyme activity detection is respectively carried out on a plurality of tissue slices to be detected on the tissue bearing unit.
According to one embodiment of the present application, in the process that the detection solvent contacts the tissue slice to extract the molecules of the analyte on the surface of the tissue slice in a molecular desorption diffusion form, the analyte is a residual enzyme substrate and the specific product generated by the substrate under the catalysis of the enzyme.
According to one embodiment of the application, the driving unit is used for adjusting the position of the tissue bearing unit, the tissue slices to be detected on the tissue bearing unit are subjected to line-by-line and row-by-row full-sequence detection, and the mass spectrograms measured in each line and each row are assembled and reconstructed to obtain a visual image.
Compared with the prior art, the enzyme activity detection device and the enzyme activity detection method based on mass spectrum have the advantages that:
according to the mass spectrum-based enzyme activity detection device, the operation of applying the enzyme substrate is combined with the mass spectrum detection process through the integrated enzyme substrate applying unit and the mass spectrum detection unit, so that complex pretreatment steps on tissue slices are not needed, and the operation flow of the detection method is simplified. The enzyme activity detection method can realize instant reaction-detection-imaging under normal pressure environment, furthest maintains the original state of the tissue slice, realizes in-situ detection imaging of the tissue slice, and improves the efficiency and accuracy of enzyme activity detection.
Further, the enzyme activity detection device of the present application is not limited to an applied enzyme substrate, and a substrate solution containing one or more enzymes may be introduced from the enzyme substrate applying unit, so that simultaneous detection and imaging of activities of a plurality of enzymes may be achieved.
Drawings
Some specific embodiments of the application will be described in detail hereinafter by way of example and not by way of limitation with reference to the accompanying drawings. The same reference numbers will be used throughout the drawings to refer to the same or like parts or portions. It will be appreciated by those skilled in the art that the drawings are not necessarily drawn to scale. In the accompanying drawings:
FIG. 1 is a schematic diagram of a mass spectrometry-based enzyme activity detection device according to one embodiment of the present application;
FIG. 2 is a schematic diagram showing the structure of the enzyme activity detecting apparatus shown in FIG. 1 at A;
FIG. 3 is a schematic diagram showing the structure of the enzyme activity detecting apparatus shown in FIG. 1 at B;
FIG. 4 is a schematic workflow diagram of a mass spectrometry-based enzyme activity detection method according to one embodiment of the application;
fig. 5 is a mass spectrometry imaging of acetylcholine and choline in accordance with one embodiment of the application;
FIG. 6 is an imaging of acetylcholinesterase activity according to one embodiment of the present application;
fig. 7 is a mass spectrum signal diagram of acetylcholine and choline in accordance with an embodiment of the application.
The reference numerals are explained as follows:
1. the tissue carrying unit 11, the substrate 12 and the tissue slice to be detected;
2. an enzyme substrate applying unit 21, a liquid feeding end 22, and a first solution supplying mechanism;
3. a mass spectrum detection unit 31, a primary capillary 32, a secondary capillary 33, a second solution supply mechanism 34, and a mass spectrometer;
4. the lifting platform comprises a driving unit, 411, a first guide rail, 412, a first servo motor, 421, a second guide rail, 422, a second servo motor, 431, a third guide rail, 432, a third servo motor, 44, a first movable plate, 45, a second movable plate, 46, a positioning jig, 461, a fourth guide rail, 462, a fixed adjusting block, 463, an adjusting column, 464, a positioning frame, 47 and a lifting platform.
Detailed Description
The following description of the embodiments of the present application will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the application are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the description of the present application, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present application. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In addition, the technical features of the different embodiments of the present application described below may be combined with each other as long as they do not collide with each other.
In addition, in the drawings, the length, width, and size of the region thereof are exaggerated for clarity, and the arrangement position of the device is adjusted.
Example 1:
FIG. 1 shows the structure of an enzyme activity detection device in one embodiment. The enzyme activity detection device in the embodiment is designed based on mass spectrometry detection, wherein an ionization source adopts a nano-spray desorption electrospray ionization source, and belongs to a normal pressure mass spectrometry detection method.
Atmospheric pressure mass spectrometry (Ambient Mass Spectrometry, AMS) detection is a technique for mass spectrometry at atmospheric pressure, which has the following advantages over conventional vacuum mass spectrometry:
high sensitivity: the normal pressure mass spectrum technology generally adopts an electrospray ion source (Electrospray Ionization, ESI), has high ion generation efficiency and wide molecular weight range, can realize ionization process under normal pressure environment, and has higher sensitivity when detecting low-concentration substances or high-molecular weight compounds; diversified applications: the normal pressure mass spectrum has good adaptability, can be applied to analysis of various samples, such as the fields of food, environment, medicine and the like, and can meet the requirements of different fields on sample analysis;
in short, the normal pressure mass spectrometry technology has become an important branch in the mass spectrometry field, and the characteristics of simplicity, high efficiency and sensitivity have brought much convenience to scientific research and industrial application in various fields. The enzyme activity detection device combines the operation of applying the enzyme substrate with the mass spectrum detection process based on the normal pressure mass spectrum detection, does not need to carry out complex pretreatment steps on the tissue slices, simplifies the operation process of the detection method, and can reduce detection errors caused by the pretreatment process in the prior art.
Specifically, the enzyme activity detection device of the present embodiment includes:
a tissue carrying unit 1 for carrying a tissue slice to be detected;
an enzyme substrate applying unit 2 comprising a liquid feeding end 21 for applying an enzyme substrate, said liquid feeding end 21 being located at said tissue carrying unit 1;
the mass spectrum detection unit 3 comprises a mass spectrometer 34 and a detection end connected with the mass spectrometer 34, wherein the detection end is positioned at the tissue bearing unit 1 and is used for desorbing and extracting molecules of an object to be detected on the tissue slice to form ionized products, and the ionized products are sent to the mass spectrometer 34 to finish mass spectrum detection.
Nano-spray desorption electrospray ionization is the process by which the molecules to be measured on the tissue slice form charged ions. And transferring the molecules to be detected from the surface of the tissue slice to a detection solvent through solvent extraction, and generating charged liquid drops through nano-upgrading spraying under the action of a high-voltage electric field, wherein the charged liquid drops contain the molecules to be detected. Then, under the action of an electric field, the ionized molecules to be detected undergo desolvation to finally form gas-phase ions, namely the ionized products, and the gas-phase ions are sent to a mass spectrometer for detection.
The tissue carrying unit 1 adopts a substrate 11 made of a base material such as glass, and a tissue slice 12 to be measured is arranged on the substrate 11. The detection end of the mass spectrum detection unit 3 adopts a nano-spray desorption electrospray ionization source which is developed on the basis of the electrospray ionization source.
In one embodiment, as shown in fig. 2, the detection end includes a primary capillary 31 and a secondary capillary 32, the liquid inlet of the primary capillary 31 is connected to the detection solvent to which high voltage is applied, the liquid outlet of the primary capillary 31 is disposed at the position of the tissue slice on the tissue carrying unit 1, and the liquid inlet of the secondary capillary 32 is disposed at the position of the tissue slice on the tissue carrying unit 1 and contacts with the liquid outlet of the primary capillary 31 to form a passage. The outlet of the secondary capillary 32 is located at the ion transfer tube inlet of the mass spectrometer through which the ionised products are fed into the mass spectrometer 34. The primary capillary 31 and the secondary capillary 32 are nano capillaries, and a certain included angle is formed between the primary capillary 31 and the secondary capillary 32 to form a stable liquid bridge, and the capillaries are close to a tissue slice to be detected and leave a certain gap, so that the liquid bridge is ensured to be in contact with the tissue slice, and the transfer of molecules to be detected on the tissue slice into a detection solvent is realized.
The angle between primary capillary 31 and secondary capillary 32 affects the size, shape and stability of the liquid bridge formed between the end of the capillary and tissue slice 12 to be measured. At the same time, the size of the liquid bridge formed also affects the resolution of mass spectrometry imaging. In this embodiment, the included angle between the primary capillary 31 and the secondary capillary 32 is set to 60 ° to 120 °, so that a better detection effect is achieved in the range of the included angle, and the stability of the liquid bridge is affected when the included angle exceeds the range, so that the accuracy of the detection result is affected.
The primary capillary 31 and the secondary capillary 32 are not in direct contact with the tissue slice 12 to be measured, and the tail ends of the capillaries are close to the tissue slice 12 to be measured but leave a certain gap. The detection solvent forms a stable liquid bridge among the primary capillary 31, the secondary capillary 32 and the tissue slice 12 to be detected, molecules (enzyme substrate residues and specific products) in the tissue slice 12 to be detected after enzyme catalysis are transferred into the detection solvent from the surface of the tissue slice, and then the detection solvent is conveyed to an inlet of a mass spectrometer by the secondary capillary 32 to generate charged liquid drops through electrospray ionization. Under the action of an electric field, ionized products in the charged liquid drops are desolvated to form gas-phase ions, and finally the gas-phase ions are conveyed into a mass spectrometer for detection.
Specifically, during detection, the detection solvent is sent out from the liquid outlet of the primary capillary 31 and is sucked by the secondary capillary 32, the detection solvent forms a stable liquid bridge on the surface of the tissue, the object to be detected (enzyme substrate and specific product generated after the enzyme substrate is catalyzed) on the surface of the tissue slice is extracted into the detection solvent in a molecular desorption diffusion mode, the detection solvent is sent to the ion transmission pipe inlet of the mass spectrometer 34 through the secondary capillary 32 to generate charged liquid drops through electrospray ionization, the ionized product contained in the charged liquid drops is desolvated under the action of an electric field to form gas-phase ions, and finally the gas-phase ions are sent to the mass spectrometer 34 to be detected by mass spectrum, the enzyme substrate and the catalytic product of the enzyme applied to the tissue are detected by mass spectrum, and the detection result is processed to realize imaging.
In addition, it should be noted that the imaging of the mass spectrometer 34 can be performed at the display end of the instrument, or can be performed by an external computer, and this part of the imaging technology is already mature, and is not described in detail herein, and is known.
When the positions of the liquid feeding end 21 of the enzyme substrate applying unit 2 and the detection end of the mass spectrum detecting unit 3 are adjusted, fine microscopic operations can be performed by matching with a high-power camera. The high power camera is used for observing images on a microscopic level. The high-power camera can be connected to a computer, so that real-time monitoring is performed, the corresponding nano capillary can be aligned to the tissue slice to be detected on the tissue bearing unit 1, and the detection precision and accuracy are improved. The high-power camera can be taken out from the outside and can also be fixed above the tissue bearing unit 1 of the enzyme activity detection device through a bracket.
In order to enable the application of high voltage electricity to the detection solvent, in one embodiment, the detection end further comprises a second solution supply mechanism 33, wherein the detection solvent is stored in the second solution supply mechanism 33, and the second solution supply mechanism 33 is provided with high voltage electricity access means for applying high voltage electricity to the detection solvent in the second solution supply mechanism 33. The voltage used for the high voltage in this embodiment is 3.8kV, and the specific value can be generally set according to the actually detected tissue slice 12 to be measured. Typically, the high voltage power is an external power source of the mass spectrometer, and is connected to a metal needle of the injector serving as the second solution supply mechanism 33 through a line, so as to form a high voltage electric field at the inlet of the mass spectrometer 34.
In one embodiment, the enzyme substrate applying unit 2 comprises a first solution supply mechanism 22, wherein a liquid outlet of the first solution supply mechanism 22 is connected with the liquid supply end 21, and the first solution supply mechanism 22 stores an enzyme substrate solution. In the present embodiment, the first solution supply means 22 employs a syringe, and the solution supply end 21 employs a nano-capillary, that is, an enzyme substrate solution is uniformly and stably applied to a tissue slice at a constant flow rate by connecting the nano-capillary to the syringe and storing the enzyme substrate solution in the syringe. The applied enzyme substrate is also capable of reacting under the catalysis of the enzyme within the tissue slice to produce a specific product. The catalytic efficiency of the enzyme (conversion rate between enzyme substrate and specific product) is reflected by detecting the amount of enzyme substrate (residue) and specific product (how much of this amount is represented by the signal intensity detected by mass spectrum), which is affected by the enzyme activity in the tissue section, and the catalytic efficiency of the enzyme (conversion rate between enzyme substrate and specific product) is different from place to place, and the substrate application amount is constant. Therefore, in the detection process, the liquid feeding end 21 is contacted with the group fabric before the detection end, and a certain time is reserved to ensure that the enzyme is catalyzed normally.
The liquid feeding end 21 can realize stable direct-current liquid feeding by using a nano capillary tube combined with a syringe. In addition, the application of the enzyme substrate to the tissue slice may also be accomplished by spraying the substrate with a spraying device, such as a modified nano-capillary, applying a high pressure air stream to effect the spraying of the substrate onto the tissue slice.
The substrate application by the enzyme substrate application unit 2 is not limited to a single substrate, and substrate mixing and feeding of a plurality of enzymes can be simultaneously realized by a syringe, and simultaneous detection imaging of a plurality of enzyme activities can be realized. This means that the enzyme activity detection device is not limited to the enzyme substrate to be applied, and that the substrate solution containing one or more enzymes can be fed from the enzyme substrate applying unit 2, that is, simultaneous detection of the activities of a plurality of enzymes and imaging can be achieved.
In one embodiment, the enzyme activity detection device further comprises a drive unit 4. The tissue bearing unit 1 is arranged on the driving unit 4, and the driving unit 4 drives the tissue bearing unit 1 to move according to a set path. In order to facilitate the positional adjustment of the tissue support unit 1, the drive unit 4 preferably employs a three-axis drive for driving the tissue support unit 1 in the x-axis, y-axis or z-axis.
As shown in fig. 1, the driving unit 4 includes a z-axis driving mechanism disposed along a z-direction (vertical), and the z-axis driving mechanism includes a first rail 411 and a first servo motor 412. The first guide rail 411 is vertically arranged, the lifting platform 47 is in sliding fit with the first guide rail 411, and the lifting platform 47 is in transmission connection with a power shaft of the first servo motor 412. The lifting platform 47 is driven by the first servo motor 412 to translate along the first guide rail 411, and an x-axis driving mechanism and a y-axis driving mechanism are arranged on the lifting platform 47. The y-axis driving mechanism includes a second guide rail 421 and a second servo motor 422, the second guide rail 421 is fixed on the surface of the lifting platform 47 along the y-direction, and the second guide rail 421 is provided with a first movable plate 44 in sliding fit with the second guide rail 421. The second servo motor 422 is fixed on the lifting platform 47, and a power shaft of the second servo motor 422 is in transmission connection with the first movable plate 44, and the first movable plate 44 moves along the second guide rail 421 under the driving of the second servo motor 422. The x-axis driving mechanism includes a third rail 431 and a third servo motor 432, the third rail 431 is fixed on the surface of the first movable plate 44 along the x-direction, and the third rail 431 is provided with a second movable plate 45 slidably engaged therewith. The third servo motor 432 is fixed on the first movable plate 44, and a power shaft of the third servo motor 432 is in transmission connection with the second movable plate 45, and the second movable plate 45 is driven by the third servo motor 432 to move along the third guide rail 431.
It will be appreciated that the selective design of the drive unit 4 may be made as desired. The complete area of the tissue slice 12 to be measured can be scanned by performing drive control according to the movement path by the movement in the plane formed by the X axis and the Y axis. The structural design of the Z axis is to be able to adjust the up and down position of the tissue carrying unit 1, so as to limit the position adjustment of the tissue 12 to be tested thereon, which is all the protection of the present application.
To the extent that the above-described object can be achieved, the drive unit 4 can be designed, even in some cases, without setting the Z-axis drive mechanism. The position of the tissue 12 to be measured can be adjusted manually by external adjustment, or the positions of the primary capillary 31, the secondary capillary 32 and the liquid feeding end 21 can be adjusted so that the distance between the primary capillary and the tissue slice 12 to be measured can be adjusted relatively, and the purpose of adjusting the position in the Z-axis direction can also be achieved.
The tissue carrying unit 1 is mounted on the second movable plate 45 through a positioning jig 46, as shown in fig. 2, a positioning frame 464 for positioning the tissue carrying unit 1 is provided on the surface of the positioning jig 46, and based on a three-axis driving mechanism, the position adjustment of the tissue carrying unit 1 in three directions along the x-axis, the y-axis and the z-axis is realized. For example, the tissue carrying unit 1 is driven by the z-axis driving mechanism to lift, so that contact and separation between the tissue and the detection end and the liquid feeding end 21 are realized. The x-axis driving mechanism and the y-axis driving mechanism adjust the position of the tissue bearing unit 1 under the control of an external motion controller (such as a numerical control platform) so as to move relative to the detection end and the liquid feeding end 21, so that mass spectrum detection of the tissue groups in a plurality of tissues 12 to be detected on a substrate 11 can be realized, and the whole operation of the device is more efficient. Meanwhile, the detection process is automatically carried out according to a set program, no additional operation is needed, and complete automatic detection is realized. In the detection process, the processes of enzyme substrate application, enzyme catalysis, mass spectrum detection, detection record and the like are sequentially carried out, and the corresponding imaging result can be obtained after the detection result is subjected to post-treatment.
In a further embodiment, as shown in fig. 3, a fourth guide rail 461, a fixed adjusting block 462, and an adjusting column 463 are provided on the second movable plate 45, and the bottom of the positioning jig 46 is slidably engaged with the third guide rail 461. The third guide rail 461 is disposed along the y direction, the fixed adjusting block 462 is fixed on the second movable plate 45, a threaded through hole is formed in the fixed adjusting block 462, the adjusting column 463 passes through the threaded through hole to be in threaded engagement with the second movable plate 45, and the adjusting column 463 is parallel to the third guide rail 461. The positioning jig 46 is provided with a screw pair, the adjusting column 463 penetrates through the screw through hole and then is in screw fit with the screw pair, fine adjustment of the position of the positioning jig 46 in the y direction is achieved through rotation of the adjusting column 463, adjustment and correction of the initial position of the tissue bearing unit 1 on the positioning jig 46 are achieved, it is guaranteed that the tissue slice 12 to be detected is right opposite to the detection end and the liquid feeding end 21, and follow-up motion control is facilitated.
The liquid supply end 21, the primary capillary tube 31 and the secondary capillary tube 32 are arranged on the tissue slice 12 to be measured, can be suspended through a peripheral bracket, and can be clamped through an adjustable combined clamp and then arranged above the tissue 12 to be measured. Among them, a combined clamp composed of a triaxial (XYZ triaxial) translation stage and an R-axis adjustment stage is preferable, and both the triaxial translation stage and the R-axis adjustment stage are commercially available. The R-axis adjusting table is fixed on the side surface of the Z-direction table in the triaxial translation table, the structure is similar to the existing XYZR-axis fine-tuning table, and the structure can realize the fine tuning of the positions of the various nano-capillaries and is regarded as a preferable scheme. Of course, if this scheme is not adopted, the suspension and position adjustment of each nano-capillary above the tissue to be measured can also be performed through other brackets, and the description is omitted here.
Example 2:
the present embodiment provides an enzyme activity detection method, based on the enzyme activity detection apparatus described in embodiment 1, as shown in fig. 4, comprising the following processing steps:
the liquid feeding end 21 of the enzyme substrate applying unit 2 is aligned with the group fabric on the tissue carrying unit 1, and enzyme substrate solution is applied to the group fabric through the liquid feeding end 21, and reacts under the catalysis of enzymes in the tissue slices to generate specific products;
the primary capillary 31 in the mass spectrum detecting unit 3 is aligned with the tissue on the tissue bearing unit 1, the detecting solvent with high-voltage electricity applied is injected into the tissue slice after the catalytic reaction is completed, and the detecting solvent is sent into the secondary capillary 32;
the detection solvent contacts the tissue slice to extract an object to be detected on the surface of the tissue slice into the detection solvent in a molecular desorption diffusion mode;
the detection solvent is delivered through secondary capillary 32 to an ion transport tube at the inlet of mass spectrometer 34 and electrospray ionization occurs at the outlet of secondary capillary 32 to form charged droplets. Under the action of the electric field, the ionized molecules of the object to be detected enter a mass spectrum detector and are sent to a mass spectrometer 34 in the mass spectrum detection unit 3 for mass spectrum detection.
In the process that the detection solvent contacts the tissue material to extract the object to be detected on the surface of the tissue slice in a molecular desorption diffusion mode, the object to be detected is a residual enzyme substrate and the newly generated specific product.
In one embodiment, the driving unit 4 adjusts the position of the tissue carrying unit 1, so that enzyme activity detection can be performed on a plurality of tissue slices 12 to be detected on the tissue carrying unit 1 respectively. In the present embodiment, the tissue slice 12 to be measured is exemplified by a tissue slice of a certain organism, and the tissue carrying unit 1 may be used for detecting a single tissue slice, or a plurality of tissue slices may be provided on the tissue carrying unit 1. Each tissue slice is detected sequentially, and each tissue slice is only required to be placed in parallel and then detected one by one.
In one embodiment, the driving unit 4 adjusts the position of the tissue carrying unit 1, performs line-by-line and column-by-column full-sequence detection on the tissue slice 12 to be detected on the tissue carrying unit 1, and gathers and displays each mass spectrum detection result to form a visual image.
In a specific embodiment, the detection of acetylcholinesterase (AchE) in a brain tissue section of a mouse (corresponding to the aforementioned tissue section to be measured) is exemplified. Acetylcholinesterase can specifically catalyze acetylcholine (Ach, 146.1178 m/z) as an enzyme substrate to produce choline (Ch, 104.1072 m/z), and the catalytic reaction formula of acetylcholinesterase is as follows:
therefore, the mouse brain tissue is sliced and then used as a tissue slice 12 to be detected of the tissue bearing unit 1, the enzyme substrate applying unit 2 applies the acetylcholine serving as the enzyme substrate to the mouse brain tissue slice, and then the mass spectrometer 34 detects the acetylcholine and the choline signal of the enzymolysis product, so that the activity imaging of the acetylcholinesterase is finally realized.
In this experimental example, the scanning mode of the mass spectrometer 34 adopts the positive ion mode when the enzyme activity detection device is in operation; the detection solvent adopts acetonitrile/water (1:1) mixed solvent, and the application flow rate is 3 mu L/min; the enzyme substrate solution adopts acetylcholine solution (NH 4HCO 3), and the application flow rate is 0.05 mu L/min; the imaging resolution was 100 μm.
Since the amount of enzyme substrate applied to the mouse brain tissue slices is uniform, the amounts of acetylcholine and choline in the different areas of the mouse brain tissue slices are determined by the catalytic ability of the acetylcholinesterase, i.e., the conversion efficiency of acetylcholine and choline reflects the activity of the acetylcholinesterase. Therefore, the activity of acetylcholinesterase in different areas on the brain tissue section of the mouse can be calculated through the signal intensity change of the acetylcholinesterase and the choline. Mass spectrum signals of acetylcholine and choline are shown in fig. 7, mass spectrum imaging diagram of acetylcholine and choline (LPC is endogenous to brain tissue section of mice) is shown in fig. 5, and an acetylcholinesterase activity imaging diagram obtained by data processing is shown in fig. 6.
In summary, according to the enzyme activity detection device based on mass spectrometry, the operation of applying the enzyme substrate is combined with the mass spectrometry detection process through the integrated enzyme substrate applying unit 2 and the mass spectrometry detection unit 3, so that complex pretreatment steps on tissue are not needed, the operation flow of a detection method is simplified, and detection errors caused by the pretreatment process in the prior art can be effectively reduced. The enzyme activity detection method can realize instant reaction-detection-imaging under normal pressure environment, furthest maintains the original state of the biological tissue slice, realizes in-situ detection imaging of the tissue slice, and improves the efficiency and accuracy of enzyme activity detection.
The above embodiments are only for illustrating the technical concept and features of the present application, and are intended to enable those skilled in the art to understand the present application and to implement the same, but are not intended to limit the scope of the present application, and all equivalent changes or modifications made according to the spirit of the present application should be included in the scope of the present application.
Claims (12)
1. An enzyme activity detection device based on mass spectrometry, comprising:
a tissue carrying unit (1) for carrying a tissue slice to be detected;
an enzyme substrate application unit (2) comprising a liquid feeding end (21) for applying an enzyme substrate, said liquid feeding end (21) being located at said tissue bearing unit (1);
the mass spectrum detection unit (3) comprises a mass spectrometer (34) and a detection end connected with the mass spectrometer (34), wherein the detection end is positioned at the tissue bearing unit (1) and is used for carrying out nano-spray desorption ionization on the tissue to form ionized products, and the ionized products are sent to the mass spectrometer (34) to finish mass spectrum detection.
2. The mass spectrometry-based enzyme activity detection apparatus according to claim 1, wherein the enzyme substrate applying unit (2) comprises a first solution supply mechanism (22), a liquid outlet of the first solution supply mechanism (22) is connected to the liquid supply end (21), and an enzyme substrate solution is stored in the first solution supply mechanism (22).
3. The mass spectrometry-based enzyme activity detection device according to claim 1, wherein the liquid supply end (21) is a direct-flow liquid supply pipe or a spray pipe.
4. The mass spectrometry-based enzyme activity detection device according to claim 1, wherein the detection end comprises a primary capillary (31) and a secondary capillary (32), a liquid inlet of the primary capillary (31) is connected with a detection solvent to which high-voltage electricity is applied, a liquid outlet of the primary capillary (31) is arranged at a position of a tissue slice on the tissue carrying unit (1), an inlet of the secondary capillary (32) is arranged at a position of a tissue slice on the tissue carrying unit (1), and an outlet of the secondary capillary (32) is aligned with the mass spectrometer (34).
5. The mass spectrometry-based enzyme activity detection device according to claim 4, wherein the detection end further comprises a second solution supply mechanism (33), the second solution supply mechanism (33) storing the detection solvent therein, the second solution supply mechanism (33) being provided with high-voltage electrical access means for applying high-voltage electricity to the detection solvent in the second solution supply mechanism (33).
6. The mass spectrometry-based enzyme activity detection device according to claim 1, further comprising a driving unit (4), wherein the tissue bearing unit (1) is disposed on the driving unit (4), and the driving unit (4) drives the tissue bearing unit (1) to move along a set path.
7. The mass spectrometry-based enzyme activity detection device according to claim 6, wherein the driving unit (4) is a three-axis driving mechanism for driving the tissue bearing unit (1) to move along the x-axis, the y-axis or the z-axis.
8. The mass spectrometry-based enzyme activity detection device according to any one of claims 1 to 7, wherein the tissue bearing unit (1) comprises at least one substrate (11), each substrate (11) having at least one tissue slice (12) to be measured.
9. An enzyme activity detection method, characterized in that it is based on the enzyme activity detection device according to any one of claims 1 to 8, and comprises the following processing steps:
the liquid feeding end (21) of the enzyme substrate applying unit (2) is aligned with the tissue slice on the tissue carrying unit (1), and an enzyme substrate solution is applied to the tissue slice through the liquid feeding end (21), and the enzyme substrate solution and the enzyme in the tissue slice are subjected to catalytic reaction to generate a specific product;
a primary capillary (31) in the mass spectrum detection unit (3) is aligned with a tissue slice on the tissue bearing unit (1), and a detection solvent with high voltage (3 kV) applied is injected into the tissue slice after the catalytic reaction is completed; the detection solvent is contacted with the tissue slice, then the object to be detected on the surface of the tissue slice is extracted into the detection solvent in a desorption diffusion mode, and the detection solvent is sent into a mass spectrometer (34) in a mass spectrum detection unit (3) through a secondary capillary (32), and under the action of a high-voltage electric field, the molecules to be detected are ionized and sent into the mass spectrometer for detection.
10. The enzyme activity detection method according to claim 9, characterized in that the driving unit (4) adjusts the position of the tissue carrying unit (1), and the enzyme activity detection is performed on a plurality of tissue slices (12) to be detected on the tissue carrying unit (1) respectively.
11. The method according to claim 9, wherein the analyte molecules are extracted from the surface of the tissue slice into the detection solvent in the form of desorption diffusion when the detection solvent contacts the tissue slice, and the analyte molecules are the residual enzyme substrate and the newly generated specific product.
12. The enzyme activity detection method according to claim 9, wherein the driving unit (4) adjusts the position of the tissue carrying unit (1), performs line-by-line and row-by-row full-sequence detection on the tissue slice (12) to be detected on the tissue carrying unit (1), and assembles and reconstructs the mass spectrograms measured in each line and each row to obtain a visual image.
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