CN110161170B - Sample processing device and biological activity screening system containing same - Google Patents

Sample processing device and biological activity screening system containing same Download PDF

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CN110161170B
CN110161170B CN201810151540.8A CN201810151540A CN110161170B CN 110161170 B CN110161170 B CN 110161170B CN 201810151540 A CN201810151540 A CN 201810151540A CN 110161170 B CN110161170 B CN 110161170B
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CN110161170A (en
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邓亦峰
林振鹏
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Guangdong Medical University
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Abstract

The invention belongs to the field of active ingredient screening, and relates to a sample processing device, a device for preparing a planar chromatography microarray and a biological activity screening system. The system comprises a planar chromatograph, a grid plate, a microporous plate, a biological activity detection unit and a mass spectrometry analysis unit; the system is used for preparing a 'planar chromatography component microarray' sample from a planar chromatogram, and carrying out a biological activity test by using the sample and focusing the sample on an obvious biological activity array unit to carry out mass spectrometry; and analyzing the bioactive components from complex components with complex components and greatly different contents by taking the dependence relationship between the bioactive strength and the biomolecular ion quasi-molecular peak strength of the corresponding array unit and the chromatographic behavior coupling degree of the array unit as indications. The system does not need to separate and purify chemical components one by one in advance, does not need to carry out comprehensive analysis and detection, does not depend on a molecular diversity compound library, can amplify samples in parallel, has good compatibility and high flux, is digital, greatly improves the screening efficiency and reduces the workload and the cost.

Description

Sample processing device and biological activity screening system containing same
Technical Field
The invention belongs to the technical field of pharmaceutical analysis and biological detection, and particularly relates to a sample processing device and a system containing the device for high-throughput screening of bioactive components in complex components by using a planar chromatographic component microarray.
Background
Natural products, traditional Chinese medicines and compound and artificially synthesized and bioengineering products thereof, such as various substance components in terrestrial animals and plants, marine organisms and microorganisms and separated biological secondary metabolites, are potential medicinal resource compound libraries. Most of the drugs have the characteristics of complex components and greatly different contents, and the problem that new drugs with obvious biological activity are screened out is always troubling people.
The high-flux drug screening technology is developed rapidly as a means for rapidly finding medicinal lead compounds, and the microarray and compound chip technology is the leading edge of the development.
The microarray and compound chip technology is an experimental method for orderly arranging compounds in a molecular diversity compound library on a planar carrier one by one according to a two-dimensional array to be subjected to biological activity reaction detection, screening biological activity compounds in the compound library and revealing the interaction between the compounds and biological activity macromolecules (RNA, DNA, protein or ligand). A plurality of experimental platforms such as small molecule compound chips (SMMs) and microfluidic chip (microfluidic-cell microarray) are developed by taking the small molecule compound chips as a framework, and an excellent technical means is provided for the medicinal development of a large number of compound libraries obtained by combinatorial chemistry [ Ma H, Horiuchi K Y.chemical microarray: a new tool for Drug screening and discovery [ J ]. Drug discovery protocol, 2006,11(13): 661 and 668 ]. However, this method is a nearly ideal Screening method, which relies heavily on the library of molecular diversity compounds [ Janzen W P. Screening technologies for small molecule discovery: the state of the art [ J ]. Chemistry & biology, 2014,21(9):1162-1170 ], which is difficult to do for the active ingredient Screening of often supposedly large and complex natural products.
Plane Chromatography (Plane Chromatography) is different from other Chromatography methods such as HPLC and the like due to the combination of chromatographic separation, differential distribution and in situ characterization features. Planar chromatography (TLC) bioautography methods have also found application in bioactive molecular screening [ e.g.
Figure RE-GDA0001635586440000011
A,Gether J,Landmark L,
Figure RE-GDA0001635586440000012
M,Detection of mutagens in complex samples by the salmonella assay applied directly on thin-layer chromatography plates,Science.215 (1982)87-89.]However, it is limited by interference or hindrance of the separation medium of planar Chromatography to the detection of bioactive reactions and chemical analysis [ Choma I M, Grzelak E M. bioautograph detection in the thin-layer Chromatography, Journal of Chromatography A.1218(2011) 2684-.]。
The West-Blot (immunoblot detection) method derived from planar chromatography transfers the biological macromolecules from the gel electrophoresis layer to the electrophilic membrane to recognize the bioactive macromolecules with specific immunoreactions. The method is widely applied to immunoassay. However, the method depends on immune specific reaction and has limited application range.
The thin layer chromatography-mass spectrometry Interface (TESI-MS Interface) from CAMAG corporation (Muttenz, Swit-Zerland) provides a method for semi-automated EXTRACTION OF chromatographic components from thin layer plates in a 4mm diameter annular region for various experiments, and US20060160127, "EXTRACTION OF MOLECULES USING FRAME", discloses a method for extracting chromatographic spot components in a liquid pool consisting OF a FRAME pressing an electrophoretic strip target region. Both are used for the extraction of local chromatographic components, with chromatographic spot spreading and cross-interference due to the capillary action of the solvent on the stationary phase.
CN 106818868A 'method for screening active ingredients in complex natural products and application' discloses a method for coherent combination of multichannel high performance liquid chromatography separation and mass spectrometry detection, and automatic collection of fractions onto a 96-well microporous plate for activity evaluation, which 'coherently combines' the conventional screening methods, but reduces the efficiency and quality: although the total flow of five chromatographic columns which are parallel to the same constant flow pump is constant, the column pressure curves of the chromatographic columns are different, the flow rates are naturally different, the column pressure is dynamically changed, and the arrangement of chromatographic fractions on a microporous plate is a running water type and does not have fixed peak collection, so that the corresponding relation among analysis and detection, activity evaluation and the fractions cannot be guaranteed; secondly, in order to adapt to the capacity of the microporous plate, the separation of the traditional Chinese medicine extract on the high performance liquid chromatography with the particle size of 5 mu m and the diameter of 4.6mm is limited to be completed within 0.40ml/min flow and 35min, so that the separation performance of HPLC and the selectivity and sensitivity of ESI-MS detection cannot be exerted; and thirdly, only the traditional Chinese medicine components with complex components and very different contents are divided into 96 fractions according to the outflow volume, the bioactivity is detected on a 96-hole microporous plate, the chemical components in the microporous pool cannot be guaranteed to be monomers, and the attribution of the bioactivity to the chemical components is difficult to determine. These all have adverse effects on the selectivity and sensitivity of analytical detection and activity evaluation, making it difficult to repeat and reproduce the experimental results, making screening and screening of active compounds difficult, and prone to screen leakage and misscreening.
CN 104792914A 'a planar chromatography and micropore plate array mapping correlation experiment method and application thereof' discloses a planar chromatography and micropore plate array mapping correlation experiment method and application thereof, the method maps planar chromatography spot components to micropore plate array units to carry out biological activity experiments and analysis detection, and active components in traditional Chinese medicines and natural products are screened under the guidance of activity. In order to make each chromatographic spot fall into the same micropore pool as much as possible, the method requires that a planar chromatographic spot strictly corresponds to a micropore plate array, and the implementation difficulty is high for a thin layer chromatogram with irregular spot shapes and positions; in the case that one chromatographic spot falls into a plurality of micropore pools and a plurality of coexisting components exist in the micropore pools, the attribution chemical components of the biological activity are difficult to determine. Furthermore, when a cavity formed by combining the thin-layer plate and the microporous plate by using the silica gel sealing ring is turned over for elution, the chromatographic spot diffusion and cross interference of different microporous pools caused by the capillary action of a solvent on a stationary phase cannot be eliminated.
Disclosure of Invention
The invention aims to provide sample processing for dividing a chromatographic thin layer of a stripped planar chromatographic fingerprint diagram.
The invention also aims to provide a device for preparing the planar chromatographic microarray, which is used for dividing and stripping a chromatographic thin layer of a planar chromatographic fingerprint pattern into a microplate according to a microplate array format and screening bioactive components.
It is still another object of the present invention to provide a bioactive screening system for high-throughput screening of bioactive components.
The invention also aims to provide a using method of the device and the system.
It is also an object of the present invention to provide a device and system that does not rely entirely on the bioactive components of a library of molecularly diverse compounds.
The invention also aims to provide a micro/semi-micro level and reproducible bioactive component screening device and system.
The invention also aims to provide a device and a system for analyzing the bioactive components from the complex components by combining chromatographic separation, bioactive detection and mass spectrometry.
The above object of the present invention is achieved by the following technical means:
in one aspect, the present invention provides a sample processing device, which is a grid plate, having a plurality of channel-shaped spaces defined by grid frames, the channel-shaped spaces having open first ends and second ends, wherein on an open plane of the first ends, the area of the grid frames defining each channel-shaped space is preferably 10% or less, more preferably 5% or less, and still more preferably 3% or less of the area of the first ends of the channel-shaped spaces; the lower limit may be 2%, for example, 2% or more, or 1%, for example, 1% or more. When the area of the grid frame for limiting each channel-type space at the first end plane is small enough and is less than or equal to 10% of the area of the first end plane of the channel-type space, the thickness of the frame of each grid frame at the first end is narrow enough to form a sharp edge, so that the sharp edge can be used for cutting the chromatographic thin layers of the planar chromatography, and all the chromatographic thin layers can be cut and separated. Theoretically, the ratio can approach 0% infinitely, but in practice, no thickness per mesh frame can be achieved.
Each channel-type space is a regular n-polygon on the first end plane, and the angle of the n-polygon interior is 180(n-2) ÷ n. At this point, the first end of the channel-shaped space can be spread over the entire surface, ensuring that the chromatographic lamellae of the planar chromatograph are completely cut.
In a preferred embodiment, the sample processing device further comprises a microplate, and the grid plate has an array format corresponding to the array format of the microwell. The open second end of the channel-shaped space is used for being butted with a micropore pool of a micropore plate.
In a preferred embodiment, the thickness of the grid frame defining each channel-shaped space is greater than the thickness of the grid frame in the open plane of the first end, and is the same as the thickness of the cell edge of the microplate.
As a preferred embodiment of the present invention, the channel-shaped space is a regular quadrilateral on the first end plane, and the channel-shaped space of the interface board is a regular quadrangular frustum, wherein the lower bottom surface of the regular quadrangular frustum is the open first end of the channel-shaped space, and the upper bottom surface is the open second end of the channel-shaped space. Furthermore, the longitudinal section of a grid frame for limiting each channel-shaped space is an isosceles trapezoid, and the width of the lower bottom edge of the isosceles trapezoid is the same as the thickness of the edge of the micropore pool of the micropore plate; the width of the upper bottom edge of the isosceles trapezoid is less than or equal to 0.15mm, so that the isosceles trapezoid is as close to an isosceles triangle as possible, and a grid frame of the upper bottom edge of the isosceles trapezoid forms a square sharp edge for cutting a chromatographic thin layer of a planar chromatograph; the width of the lower bottom edge of the isosceles trapezoid is the same as the thickness of the edge of the micropore pool of the micropore plate.
In a more preferred embodiment, the grid plate array format corresponds to a 384-microplate array format, which is a 4.5mm × 4.5mm square grid 24 × 16-horizontal array format. More specifically, the longitudinal section of the grid frame is an isosceles trapezoid with the height of 2.5 mm. The width of the lower bottom edge of the isosceles trapezoid is the same as the thickness of the edge of the 384-square-mouth micropore plate pool, and the width is 0.68-0.72 mm; the width of the upper bottom edge of the isosceles trapezoid is less than or equal to 0.15 mm.
It should be noted that the longitudinal section of the grid frame is a section cut along the longitudinal section of the channel-shaped space when the interface board is placed horizontally, and the structure of the frame is described as a longitudinal section of one side of the grid.
In the present invention, as an alternative embodiment, the mesh plate is prepared by 3D printing.
The grid plate is used for dividing and stripping a chromatographic thin layer of a planar chromatographic fingerprint image. Because the space in the grid is a quadrangular frustum pyramid, the thicknesses of grid frames on two sides of the grid plate are different, and the thickness of one side of the grid plate is small, so that a square sharp edge is formed, and the square sharp edge is used for being in butt joint with a chromatographic thin layer of a planar chromatographic fingerprint to divide and strip the chromatographic thin layer of the planar chromatographic fingerprint. The other side is relatively blunt, and the thickness is generally the same as the thickness of the edge of the micropore pool, and the other side is used for being butted with a micropore plate, and the divided and stripped chromatographic thin layer is received by the micropore pool of the micropore plate.
In another aspect, the invention provides a device for preparing a planar chromatography microarray, wherein the device for preparing the planar chromatography microarray comprises planar chromatographs in array formats corresponding to one another, the grid plate and the microporous plate.
Wherein, the planar chromatogram is used for constructing a planar chromatogram fingerprint map of a sample to be screened;
the grid plate is used for dividing and stripping a chromatographic thin layer of the planar chromatographic fingerprint image according to a micropore plate array format;
the micro-porous plate is used for positioning and receiving components of the planar chromatography and preparing a planar chromatography component microarray sample (also called a planar chromatography microarray).
The planar chromatographic microarray is an array sample obtained by separating a pretreated sample, namely a sample of an active component to be screened, by a chromatographic thin layer, dividing the sample according to a microplate array format, integrally mapping and transplanting the chromatographic component of the pretreated sample to a corresponding micropore pool of a microplate, and removing a chromatographic thin layer matrix. Each array unit of the array sample reserves the chemical composition characteristics, the composition and the chromatographic information of the corresponding area of the thin-layer chromatography, and the microarray sample integrally reserves the chemical composition, the composition and the chromatographic difference and the characteristics of the planar chromatography fingerprint chromatogram.
Wherein the planar chromatography can be selected from the group consisting of a planar chromatography without a support matrix and/or a planar chromatography with a support matrix; as an exemplary embodiment, the supported matrix-free planar chromatography is performed by gel electrophoresis, polymer membrane thin layer chromatography or electrophoresis with a membrane-transferred polymer membrane. As an exemplary embodiment, the plane chromatography with the supporting matrix is selected from a preparative thin-layer silica gel chromatography plate of a glass plate matrix or an aluminum-based thin-layer chromatography silica gel plate. As a preferred embodiment, the plane chromatography is selected from an aluminum-based thin-layer chromatography silica gel plate, in particular a high-performance aluminum-based thin-layer chromatography silica gel plate.
When the plane chromatogram is selected from an aluminum-based thin-layer chromatography silica gel plate, the plane chromatogram also contains a PVDF membrane; still more preferably, the planar chromatograph further comprises a sheet of silicone rubber and a sheet of paper; when the system operates, the silicon rubber sheet, the grid plate, the PVDF membrane, the aluminum-based thin-layer chromatography silica gel plate and the paper sheet are stacked in parallel from bottom to top in sequence.
In the device for preparing the planar chromatography microarray, the chromatogram span of the planar chromatography fingerprint corresponds to the micropore plate array format of the channel width. In a preferred embodiment, the span length and the channel width of the planar chromatogram are multiples of 4.5mm, and the planar chromatogram comprises a one-dimensional planar chromatogram with the span length of 9.0cm multiplied by 7.2 cm.
In a preferred embodiment, the microplate is a square-mouth microplate. In a more preferred embodiment, the square-mouth microplate is a 384-well square-mouth microplate having an array format of 4.5mm square, 24 × 16 horizontal arrays.
The grid plate in this system is described above in the context of the sample processing device.
As a preferred embodiment, the apparatus for preparing a planar chromatography microarray further comprises a microporous filter plate. The microporous filter plate is used for positioning and eluting a chromatographic thin layer divided and stripped by a grid interface to a microporous plate. As a preferred embodiment, the microporous filter plate is a 384 square-mouth microporous filter plate; more preferably, the 384 square-mouth microporous filter plate has a model number of Pall No. 5072.
In another aspect, the invention also provides a use method of the device for preparing the planar chromatography microarray.
1) Constructing a planar chromatography fingerprint of a sample to be screened by using a planar chromatography;
2) and dividing, stripping and positioning the chromatographic thin layer of the planar chromatographic fingerprint image into micropore pools of a micropore plate according to a micropore plate array format by adopting a grid plate, and eluting to prepare a planar chromatographic component microarray sample.
As a preferred embodiment, in step 2), the chromatographic layer peeled off and positioned in the grid plate is firstly pushed into the micropore pool of the micropore filter plate, then the micropore filter plate is aligned with the micropore plate receiving the eluent, and then the solvent is adopted to position and elute the chromatographic component on the chromatographic stationary phase into the corresponding micropore pool in the micropore plate.
In another aspect, the invention also provides a device or a method for using the device for preparing the planar chromatography microarray.
The planar chromatographic microarray is prepared through separating planar chromatographic thin layer on a microporous plate, partitioning with interface board corresponding to the microporous plate array format, integrally mapping the chromatographic components and transplanting to the microporous pool of the microporous plate to eliminate bioactive sample to be screened. Each array unit of the array sample reserves the chemical composition characteristics, the composition and the chromatographic information of the corresponding area of the thin-layer chromatography, and the microarray sample integrally reserves the chemical composition, the composition and the chromatographic difference and the characteristics of the planar chromatography fingerprint chromatogram.
In another aspect, the present invention also provides a biological activity screening system comprising the apparatus for preparing a planar chromatography microarray as described above.
In this system, in addition to the above-mentioned apparatus for preparing a planar chromatography microarray, it contains a mass spectrometry unit and a biological activity detection unit.
The mass spectrometry analysis unit is used for measuring mass spectrometry information of active ingredients in the planar chromatographic ingredient microarray sample;
the biological activity detection unit is used for detecting biological activity in the microarray sample of the planar chromatographic component.
In the system of the present invention, the mass spectrometry unit is selected from mass spectrometry units with soft ionization ion sources. More specifically, the soft ionization ion source can be selected from one or more of electrospray ionization (ESI) in liquid chromatography-mass spectrometry, Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photoionization (APPI), Matrix Assisted Laser Desorption Ionization (MALDI), or chemical ionization in gas chromatography-mass spectrometry. In a preferred embodiment, the soft ionization ion source is electrospray ionization.
In the system, the biological activity detection unit comprises one or more of an enzyme-labeling instrument, a multifunctional microporous plate detection system, a high-flux microporous plate detector and the like.
In a preferred embodiment, the system of the present invention further comprises a data processing system for performing one or more operations of digital representation of planar chromatographic components in an array manner, construction of a bioactivity distribution map, correlation of bioactivity intensity and mass spectrum information, coupling, normalization analysis, active substance selection, construction of a planar chromatographic fingerprint related to spectrum-effect, and the like.
In another aspect, the present invention also provides a method for using the above-mentioned biological activity screening system, which comprises the following steps: a biological activity screening system.
1) Constructing a planar chromatography fingerprint of a sample to be screened by using a planar chromatography;
2) dividing, stripping, positioning and eluting a chromatographic thin layer of a planar chromatographic fingerprint image into a micropore pool of a micropore plate according to a micropore plate array format by adopting a grid plate to prepare a planar chromatographic component microarray sample;
3) performing biological activity detection on the planar chromatographic component microarray sample by adopting a biological activity detection unit;
4) performing mass spectrometry on the active hotspot array unit and the adjacent area array unit thereof by adopting a mass spectrometry unit;
as a preferred embodiment, in step 2), preparing a microarray sample of the planar chromatographic components, and digitally expressing the planar chromatographic components in an array mode by using a data processing system;
in a preferred embodiment, in step 3), after the biological activity detection is completed, a data processing system is adopted to construct a biological activity distribution map;
in a preferable embodiment, in the step 4), after mass spectrometry, a data processing system is adopted to perform association, coupling and normalization analysis on the biological activity intensity and mass spectrometry information, active substances are selected, and a planar chromatographic fingerprint related to spectrum-effect is constructed.
To more fully illustrate the method of operation or use of the biological viable screening system of the present invention, further description is provided below.
The invention divides the planar chromatogram fingerprint spectrum reflecting the chemical composition characteristics of complex components according to the micropore plate array format, integrally maps the chromatogram components and transplants the chromatogram components to the corresponding micropore pool of the micropore plate, and prepares a sample of 'planar chromatogram components-microarray'; carrying out biological activity tests, active component tracking analysis and chromatographic behavior investigation on the microarray sample, digitizing the planar chromatographic component characteristics with irregular chromatographic spot shapes and position distributions in an array mode, and carrying out correlation analysis by taking the array distribution of the special attributes of the biological activity intensity and mass spectrum excimer peak intensity of the active components of the microarray unit as the chromatographic behavior; the method is characterized in that a compound molecule with interdependent biological activity intensity and mass spectrum excimer ion peak intensity and coupled chromatographic behavior in a planar chromatographic microarray sample is used as a screening indication of drug effect substance basis, and biological activity normalization belonging to the same molecule in an array unit is used as an activity contribution degree of the molecule to be taken into consideration, so that a high-throughput screening platform combining chromatographic separation, biological activity detection and chemical analysis is constructed. On the basis, the corresponding relation between the planar chromatogram, the bioactive thermodynamic diagram and the active compound is quantized and comparably expressed, and a planar chromatogram fingerprint spectrum related to spectrum-effect is constructed.
The planar chromatography referred to hereinafter may be selected from unsupported and/or supported matrix planar chromatography. Examples of the plane chromatography as the unsupported substrate are gel electrophoresis, polymer membrane thin layer chromatography, or electrophoresis with a polymer membrane; as examples of the flat chromatography having a supporting substrate, there are prepared silica gel thin layer chromatography plates of glass plate substrate or aluminum-based thin layer chromatography silica gel plates. In a preferred embodiment of the present invention, the planar chromatography is selected from an aluminum-based thin layer chromatography silica gel plate.
The sample to be screened is a complex composition extract, including but not limited to natural products, such as natural product extracts in terrestrial animals and plants, marine organisms, and microorganisms.
Or, as a preferred embodiment, the sample to be screened is a compound extract of traditional Chinese medicine, an artificially synthesized product and a bioengineering product.
The chromatographic band formed by chromatographic retention time difference distribution after the sample component to be screened is separated by planar chromatography is called planar chromatographic spot.
It should be noted that the plane referred to herein is not an absolute plane. Which may have different thicknesses.
Specifically, the method of the present invention comprises the steps of:
s1, constructing a planar chromatographic fingerprint which fully reflects and records the chemical composition difference characteristics and chromatographic behavior of a sample to be screened and has the chromatogram span and channel width consistent with the square-mouth microplate array format;
s2, dividing, stripping, positioning and eluting a chromatographic thin layer of the planar chromatographic fingerprint image into a micropore pool of a micropore plate according to a micropore plate array format to prepare a planar chromatographic component microarray sample;
s3, carrying out digital expression on the chemical and biological characteristics of the planar chromatographic components of the chromatographic spots in an array mode;
s4, performing biological activity detection on the planar chromatographic component microarray sample, constructing a biological activity hot spot diagram (Heat Map), and selecting an active hot spot array unit;
s5, carrying out mass spectrometry on the active hotspot array unit and the adjacent area array unit thereof;
s6, carrying out correlation, coupling and normalization analysis on the biological activity intensity of the active hot spot array unit and the adjacent area array unit thereof and the mass spectrum excimer ion peak intensity, and selecting a biological active component;
s7, the corresponding relation among the planar chromatogram, the bioactive thermodynamic diagram and the active compound is quantized and comparably expressed, and a planar chromatogram fingerprint related to spectrum-effect is constructed.
Wherein in S3, when the planar chromatographic spot is digitally expressed by corresponding to the microplate array, there may exist some spots exactly corresponding to a certain or certain microwell in the microplate, which we call "regular spots"; however, there are many cases where a planar chromatographic spot, whose shape and location distribution does not correspond exactly to a cell or cells of a microwell, is called an "irregular spot". The above-mentioned flat chromatographic spots include regular spots and irregular spots.
As a preferred embodiment of the present invention, the preparation method of the extract comprises: ultrasonic extraction is carried out by respectively adopting at least one solvent in 8 types of solvents proposed by Snyder (Snyder L R.Classification of the solvent properties of common liquids [ J ]. Journal of Chromatography A, 1974,92(2):223-230.), and after the extracting solutions are combined, the solvent is removed to obtain the traditional Chinese medicine or natural product extract.
In an exemplary embodiment of the invention, the solvent comprises typically petroleum ether, ethanol, tetrahydrofuran, acetone, dichloromethane, ethyl acetate, benzene, and water; and combining the extracting solutions of the 8 solvents to prepare the extract which fully reflects the chemical composition characteristics of the sample.
In a preferred embodiment, the step S1 is to construct a planar chromatogram fingerprint of the sample to be screened, and to examine the separation degree and concentration distribution of the planar chromatogram. As an alternative embodiment, the separation and concentration distribution of the planar chromatography was examined using uv light observation and iodine fumigation and sulfuric acid-vanillin reagent visualization.
In steps S1 and S2, the chromatogram span and channel width of the planar chromatogram fingerprint are consistent with the format of the microplate array. It should be emphasized that the chromatogram span and channel width of the planar chromatogram fingerprint are consistent with the micropore plate array format, so as to establish the correspondence between the divided array units of the planar chromatogram and the micropore plate micropore pool array units, but for the chromatogram layer or the elution component thereof, only the chromatogram layer or the elution component thereof is required to fall into the micropore pool corresponding to the micropore plate with the same array format, and for the shape of the micropore plate, no requirement is required, such as a round-mouth micropore plate or a square-mouth micropore plate. In order to meet the requirement, the array format of one surface of the grid plate facing the planar chromatography corresponds to the array format of the microporous plate, and a square sharp edge is adopted, so that the area contacting with the planar chromatography layer during butt joint is reduced as much as possible on the premise of ensuring the array format, and the chromatography layer is guided into the microporous plate to the greatest extent; the side of the plate that is in contact with the microplate may be square or round or of other shapes as long as it facilitates the introduction of the chromatographic layer or its eluting components into the corresponding array unit.
As an alternative, the microplate is a conventional square-well microplate. In a preferred embodiment, the square-well microplate is a commercially available 384-well square-mouth microplate array format in a 4.5mm square 24 × 16 array. Correspondingly, the span length and the channel width of the plane chromatogram in the step S1 are multiples of 4.5mm, and include a one-dimensional plane chromatogram with a span length of 9.0cm × a channel of 7.2cm and a two-dimensional plane chromatogram with a span length of 7.2cm × a channel of 7.2 cm.
In step S2, an interface board corresponding to the microplate array format is used to divide and strip the chromatographic thin layers in step S1 as an advantageous embodiment of the present invention.
In a preferred embodiment, the grid of the interface board is hollow as a rectangular frustum. In a more preferred embodiment, the grid plate is in a horizontal 24 × vertical 16 array format with 4.5mm × 4.5mm squares, and the vertical section of the grid frame is an isosceles trapezoid with a height of 2.5 mm. The width of the lower bottom edge of the isosceles trapezoid is the same as the thickness of the edge of the 384 square-mouth micropore pool, specifically 0.68-0.72mm, the wall thickness of the micropore pool of different manufacturers of commercially available square-mouth micropore plates is slightly different, for example, a Corning plate is 0.70mm, a Pall No.5072 filter plate is 0.68, and some domestic plates are about 0.72. One side of the lower bottom edge of the isosceles trapezoid is used for being butted with the micropore plate; the width of the upper base edge of the isosceles trapezoid is less than or equal to 0.15mm, so that the isosceles trapezoid is as close to an isosceles triangle as possible, and a grid frame of the upper base edge of the isosceles trapezoid forms a square sharp edge for cutting a chromatographic thin layer of a planar chromatograph.
As an alternative embodiment, the interface board is prepared by 3D printing.
In an alternative embodiment, the interface plate material is a hard, tough metal material, such as cobalt-chromium alloy or stainless steel.
For different types of planar chromatography, the division and stripping of the chromatographic thin layers are mainly performed in the following 4 ways, but other feasible schemes are not excluded:
1) when the planar chromatographic layer of colloid unsupported matrix, such as gel electrophoresis, is wet, it is parallel sandwiched between interface board and glass plate, and then placed on parallel pressing plate to make flat pressing, so that the whole planar chromatographic layer can be divided into square array and embedded in the interface.
2) For the plane chromatographic layer of polymer film without supporting base body, such as polymer film of polymer film electrophoresis, film chromatography or electrophoresis strip transfer film, it is parallelly clamped between soft metal sheet of aluminium, lead, etc. and interface board, the sharp edge of the interface board is faced to the polymer film, and placed on the parallel press plate to make flat pressing, and after the polymer is cut into square array and embedded in the interface, the soft metal sheet can be removed.
3) The preparation type thin-layer silica gel chromatographic plate for the glass plate substrate comprises a preparation type thin-layer silica gel chromatographic plate with the thickness of 2.0mm, the dried silica gel surface faces the sharp edge surface of the grid plate and is aligned, the silica gel sheet is stacked on the other surface of the interface plate, the silica gel sheet is stacked together in parallel and placed on a parallel pressing plate for flat pressing, and the whole plane chromatographic layer is pressed until the whole plane chromatographic layer is divided into a square array and embedded in the square grid array of the grid plate; and then releasing the pressure until the glass plate can just slide in parallel between the parallel pressing plate and the grid plate, and laterally knocking the glass plate to make the glass plate move in parallel at least one array unit left and right, front and back respectively, so that the glass plate and the silica gel layer are completely peeled.
4) For the aluminum-based thin-layer chromatography silica gel plate, a silicon rubber sheet, a grid plate, a PVDF (polyvinylidene fluoride) membrane, a dried aluminum-based thin-layer chromatography silica gel plate and a thin paper sheet are sequentially stacked in parallel from bottom to top, sharp edge surfaces of the grid plate are aligned with the thin-layer chromatography silica gel layer through the PVDF membrane, and the sharp edge surfaces are placed on a parallel pressing plate together to be pressed flatly under the pressure of 1-80 MPa; the thin aluminum plate with ductility and the crisp thin silica gel are different in changes under the compression of sharp edges of the grid plate, the thin silica gel is integrally and orderly split into square sheet arrays, and the square sheet arrays are peeled from the slightly deformed thin aluminum plate and attached to the PVDF film slightly embedded in the interface. The PVDF film is soft and flexible, has electrostatic adsorption, and is lined with the PVDF film to help prevent the fragmentation and scattering when the chromatographic thin layer is stripped, so that a complete and orderly arranged silica gel thin layer square lattice array is obtained.
After the thin layer division of the chromatogram and the integral orderly stripping are finished, the positioning elution is carried out. Further, the step of localized elution is: pushing the silica gel thin layer stripped and positioned in the grid plate into a micropore pool of a micropore filter plate, aligning the micropore filter plate with a micropore plate for receiving eluent, and adopting a solvent to position and elute chromatographic components on a chromatographic stationary phase into a corresponding micropore pool in the micropore plate to obtain a planar chromatographic component microarray sample. Wherein, the array format of the micro-pore filter plate corresponds to the array format of the microarray. In a preferred embodiment, the microporous filter plate corresponding to the 384-well square-well microplate is a 384-square-mouth microporous filter plate. In an exemplary embodiment of the invention, a 384 square-port micro-well filter plate is used, model number Pall No. 5072.
In order to obtain the planar chromatographic component microarray sample capable of fully reflecting the molecular diversity of the sample to be screened, the steps S1-S4 are repeated for many times, the test concentration interval of a planar chromatographic fingerprint image is optimized, a prepared sample and a planar chromatographic two-dimensional solvent system are optimized, and after optimization, the planar chromatographic component microarray sample capable of better reflecting the chemical component characteristics and the biological activity of the planar chromatographic component microarray sample array unit and considering the planar chromatographic separation degree and the planar chromatographic capacity is prepared.
Specifically, the method comprises the following steps:
A. optimizing the experimental concentration interval of the planar chromatographic fingerprint, referring to the methods of S1 and S2, carrying out step concentration spotting in a certain concentration range, transversely spotting by using a layout of 4 concentrations, preferably covering a 20-fold concentration range and paralleling 5 array units to each concentration, longitudinally unfolding 16 array units, and preparing a one-dimensional planar chromatographic component microarray sample in a transverse 20X longitudinal 16 array format. Performing a bioactivity test on the microarray sample, processing the data with an EXCEL spreadsheet, and constructing a bioactivity thermodynamic diagram; and (3) observing the distribution of the biological activity heat on the array and the corresponding concentration of the test solution, and preferably selecting a test solution concentration interval which can obviously reflect the biological activity of each array unit of the sample.
B. The prepared sample was applied in parallel at high concentration on a preparative flat panel, comprising 20 array units applied in parallel at an application amount 10 times that of a high performance flat panel on a preparative flat panel having a thin layer thickness of 2.0mm, and 16 array units were developed to prepare a microarray sample of flat chromatography components in a horizontal 20X vertical 16 array format, with reference to the methods of S1 and S2. According to the preferable test solution concentration interval in A, sampling is carried out on the microarray sample, and the proportion of the test solution (corresponding to chromatographic components) of 16 array units is combined to obtain a modulation sample which can better reflect the biological activity of the array units of the microarray sample of the planar chromatographic components and the difference characteristics of the biological activity.
C. And (4) optimizing a two-dimensional plane chromatography solvent system by referring to S1 and S2 by using the prepared sample obtained in the step (B), so that the separation degree and the capacity of the plane chromatography are comprehensively improved.
D. Integrating the results of the steps A-C, referring to S1 and S2, preparing the two-dimensional plane chromatography component microarray sample which can fully reflect the molecular diversity and the difference characteristics of each array unit from the aspects of chemical component characteristics and biological activity by using the prepared sample to be spotted and developing the sample by using an optimized two-dimensional plane chromatography solvent system.
In step S3, the chemical and biological properties of the planar chromatographic components of the chromatographic spot are expressed digitally in an array format. Specifically, biological activity detection and chemical analysis are carried out on the planar chromatographic component microarray samples, the experimental data of each array unit reflects the characteristics of the chromatographic components in the corresponding area of the planar chromatogram, and the digitization of the graphic file is realized. As a preferred embodiment, a computer is used to process these data; further, as a more preferable scheme, the specific steps are as follows: the detection data of various biological activity detection and chemical analysis carried out on the planar chromatographic component microarray sample are incorporated into computer software according to array coordinates, and the data are digitally expressed, analyzed and processed by EXCEL spreadsheet and the like.
In step S3, the spectrum spot, especially the planar chromatographic component with irregular distribution of the shape and position of the spectrum spot, is digitally expressed by an array, which is helpful for the subsequent data analysis processing step, because the chromatographic spot has irregular shape and position and the bioactive component of the spot is likely to be distributed in a plurality of array units. By taking the subsequent normalization of the biological activity attributed to the same molecule in the array unit as the selection consideration of the activity contribution degree of the molecule, the phenomena of screen leakage and screen error in the condition can be effectively avoided.
In step S4, performing bioactivity detection on the planar chromatography component microarray sample, constructing a bioactivity distribution map, and selecting an active hotspot array unit ", as a preferred embodiment, the specific steps are as follows:
and (2) dividing the test solution from the two-dimensional planar chromatographic component microarray sample obtained after optimization into reaction tanks corresponding to the microporous plates with the same array format according to array coordinates to perform various biological activity detections, including one or more of cell survival rate detection, immunoreaction, ligand reaction, probe detection, bacterial culture, zebra fish drug effect models and the like, processing data by software such as an EXCEL spreadsheet and the like to construct a biological activity Heat Map (Heat Map), and selecting an array unit with significant biological activity components.
It should be noted that the biological activity Heat Map (Heat Map) is a method that the biological activity value obtained by performing a biological activity test on a planar chromatography microarray sample is visually marked by numbers, colors and the like according to a corresponding array format so as to determine an array unit with a significant biological activity component.
In step S5, focusing on a bioactive hot spot region from a large and complex sample component, and performing liquid mass spectrometry on the active hot spot and an adjacent region, as a preferred embodiment, the specific steps are as follows:
on the optimized two-dimensional planar chromatographic component microarray sample, carrying out chromatographic analysis detection on the array unit with obvious biological activity of the planar chromatographic component microarray sample and the adjacent array units thereof to obtain chemical component information such as the excimer ion peak intensity of each array unit, the array distribution difference and the like.
In step S5, the chromatography method is selected from mass spectrometry with soft ionizing ion source, which can obtain excimer ion peaks corresponding to molecular weights of chemical components of the sample. These soft ionizing ion sources may be, for example, electrospray ionization (ESI) in liquid chromatography-mass spectrometry, Atmospheric Pressure Chemical Ionization (APCI), Atmospheric Pressure Photoionization (APPI), Matrix Assisted Laser Desorption Ionization (MALDI), or chemical ionization in gas chromatography-mass spectrometry. As a preferred embodiment, these soft ionizing ion sources are selected from the group consisting of electrospray ionization; different from the traditional mass spectrum which adopts 70 electron volt voltage to beat the compound into 'fragment' ions, the mass spectrum analysis with the soft ionization ion source adopts the technologies of induced ionization and the like, the structure of the compound is rarely damaged, the obtained quasi-molecular ions are obtained, and the spectrum peak in the mass spectrum has a corresponding relation with the mass number of each chemical component of the sample.
In step S6, correlation, coupling and normalization analysis are performed on the bioactivity intensity of the active ingredient in the microarray unit and the mass spectrum excimer ion peak intensity, specifically, a compound molecule with significant bioactivity in the area of the microarray unit and interdependence between the bioactivity intensity of the adjacent unit and the mass spectrum excimer ion peak intensity (peak height or peak area) in the planar chromatography microarray sample and coupled chromatographic behavior is used as a screening indication of drug-effect substance basis, and the bioactivity normalization belonging to the same molecule in the array unit is taken as the activity contribution of the molecule to take into consideration, so as to screen bioactive ingredients from large and complex traditional Chinese medicines and natural medicines at high flux.
It is emphasized that for chromatographic components, especially planar chromatographic components with irregular distribution of spot shapes and locations, it is possible to distribute them over a plurality of array elements. The method of normalizing the biological activity attributed to the same molecule in the array unit as the selection consideration of the activity contribution degree of the molecule can effectively avoid the phenomena of screen leakage and screen error under the condition;
as a preferable scheme, the specific steps of step S6 are as follows:
according to the corresponding relation of microarray samples, performing correlation, coupling and normalization analysis on the biological activity intensity obtained at S4, the mass spectrum excimer ion peak intensity obtained at S5 and the array distribution of the mass spectrum excimer ion peak intensity, and selecting compound molecules with the biological activity intensity-mass spectrum excimer ion peak intensity interdependence and chromatographic behavior coupling as selected compounds based on drug-effect substances; the method comprises the following steps of preliminarily screening bioactive compound molecules by a conventional method according to the mass-to-charge ratio of an excimer ion peak of a selected compound, the drug analysis and detection of a test solution of an array unit where the compound is located, and tracing to the chromatographic characteristic, the spectral characteristic, the in-situ chemical identification reaction, the sample source and the like displayed on a planar chromatogram, and combining with retrieval of a related compound spectrum library, a related compound library, literature data and the like; and (4) using the reference substance or separating and preparing the reference substance for further biological activity evaluation and confirmation.
As a more preferred embodiment, the operation of step S6 is as follows:
selecting a biological activity intensity data array with a significant biological activity array unit area and an adjacent array unit on a planar chromatographic component microarray sample as a variable 1; taking the array of intensities (peak heights or peak areas of the quasi-ion peaks) of the main quasi-ion peaks coexisting in the corresponding area array units as a variable 2; carrying out array correlation analysis on the variable 1 and the variable 2, and calculating correlation coefficients of each excimer ion peak and biological activity; screening out active ingredients according to the magnitude of the correlation coefficient; specifically, the compounds corresponding to the quasi-ion peaks are selected as bioactive substances by the correlation coefficient sequencing choose, the correlation coefficients are larger, the mutual difference is more obvious, and the reliability is higher; when the correlation coefficient is negative, the compound corresponding to the quasi-ion peak is not considered as a bioactive substance; normalizing the biological activity ascribed to the same molecule in the array unit as an activity contribution for that molecule is taken into consideration;
the correlation analysis of the two arrays can adopt one of various computer software and functions, such as a corel function and an RSQ function of Excel software, a coreref function of Matlab software and the like, which can realize the purpose of the correlation analysis of the arrays of the invention.
As an alternative implementation, in an example of the invention, correlation analysis of the Array uses the Correl (Array1, Array2) function of Excel.
Note: array1 and Array2 are variables 1 and 2.
The interdependence of the bioactivity intensity of the area with the obvious bioactivity array unit and the adjacent units in the planar chromatography microarray sample and the mass spectrum excimer ion peak intensity (peak height or peak area) is the internal correlation of the drug action; the chromatographic behavior coupling between them is the macroscopic manifestation of the chemical behavior and the biological activity of the same drug component. Both can be expressed and quantified from array correlation analysis.
In step S7, a spectrum-effect related planar chromatography fingerprint of the Chinese medicine and natural products is constructed, and as a preferred scheme, the specific steps are as follows:
the results of S1-S6 are integrated to express the corresponding relationship between the planar chromatogram fingerprint map, the activity hotspot map and the pharmacodynamic compound molecules in a quantitative, comparable and visual way, and the method comprises the following steps: the array format of the planar chromatographic component microarray sample is X, Y two-dimensional coordinates, the planar chromatographic component microarray sample is mapped to a corresponding planar chromatographic fingerprint array unit area, and a Y axis is established on the array unit to mark the activity value of the array unit and the corresponding compound component (structural formula, molecular formula or name), so that the planar chromatographic fingerprint of the traditional Chinese medicine and the natural product related to spectrum-effect is established.
Compared with the prior art, the invention has the following beneficial effects:
1) the device and the system combine the characteristics of chromatographic separation, differential distribution and in-situ recording of the planar chromatography with high throughput, digitization and compatibility of the microarray to prepare a 'planar chromatography component-microarray' sample. The sample not only retains the separation characteristic of the planar chromatogram, but also is thoroughly separated from the chromatographic matrix, the microarray units are mutually separated, the interference and the obstruction of the chromatographic matrix such as capillary diffusion, adsorption interference, background noise and the like are eliminated, and the sample can be naturally butted with the existing biological activity detection and drug analysis method and standard.
2) The device and the system aim at the characteristics of complex components and great content disparity of traditional Chinese medicines, natural products and the like, the sample extract is prepared by 8 solvents provided by Snyder, the concentration proportion of sample components is optimized and modulated on the basis of comprehensively inspecting the detection sensitivity of biological activity and the separation degree and the capacity of a plane chromatogram, and the two-dimensional plane chromatogram component microarray sample which can integrally reflect the molecular diversity and the difference characteristics of the traditional Chinese medicines and the natural products from the two aspects of chemical component characteristics and the biological activity is prepared, so that the representativeness and the screening sensitivity of the microarray sample are facilitated, the sample has integrity, repeatability and reproducibility, and the amplifiability of homologous parallel samples is ensured.
3) Biological activity tests, active ingredient tracking analysis and chromatographic behavior investigation can be carried out on the planar chromatographic component-microarray samples of the device and the system, the characteristics of the planar chromatographic components with irregular chromatographic spot shapes and positions are digitalized in an array mode, and a high-throughput screening platform combining chromatographic separation, biological activity detection and chemical analysis is constructed.
4) The device and the system carry out biological activity detection on a planar chromatographic component-microarray sample with molecular diversity difference distribution, quickly focus a screening target on a scarce biological activity component from a large and complex natural product under the condition of biological activity tracing, and purposefully carry out fine mass spectrometry on an active hot spot and an adjacent array unit region;
5) the device and the system perform correlation analysis by taking array distribution of special attributes of biological activity intensity and mass spectrum excimer ion peak intensity of a microarray unit as chromatographic behavior of active ingredients, take the compound molecules of which the biological activity intensity and the mass spectrum excimer ion peak intensity in a planar chromatographic microarray sample are interdependent and the chromatographic behavior is coupled as screening indications of drug effect substance bases, take the biological activity normalization belonging to the same molecules in the array unit as the activity contribution degree of the molecules into selection consideration, and realize the combination of chromatographic separation, biological activity detection and ESI-MS analysis in the selection of the biological activity ingredient choose.
6) The system is based on a spectrum-effect related traditional Chinese medicine and natural product plane chromatogram fingerprint diagram constructed by a plane chromatogram component microarray, expresses the traditional Chinese medicine effect substance basic characteristics of the traditional Chinese medicine and the natural product in a visual, quantitative and comparable manner by a plane chromatogram diagram, an activity heat point diagram and a pharmacodynamic compound molecule identifier and the corresponding relation among the two, and is not a plane chromatogram fingerprint diagram which is disjointed with biological activity and singly reflects the chemical component characteristics.
In conclusion, the system prepares the plane chromatogram which integrally reflects the molecular diversity and the difference characteristics of the complex components into a 'plane chromatogram component microarray' sample, and carries out a biological activity test by using the sample and focuses on a significant biological activity array unit to carry out mass spectrometry; the dependency relationship between the bioactivity intensity of the bioactive array unit and the mass spectrum excimer ion peak intensity of the corresponding array unit and the chromatographic behavior coupling degree of the dependency relationship are taken as indications, bioactive components are analyzed from natural products with complex components and different contents, the advantages of screening and screening the bioactive components by combining chromatographic separation, bioactive detection and mass spectrum analysis are played, and the screening sensitivity and selectivity are improved. Therefore, the invention does not require the prior separation and purification of chemical components one by one, does not require comprehensive chemical analysis and detection, and does not depend on molecular diversity compound libraries, specific biological activity reactions or special equipment. The system has the characteristics of parallel amplification of samples, good compatibility, high flux and digitalization, realizes the high flux screening of bioactive components of traditional Chinese medicines and natural products under the guidance of bioactivity at a micro/semi-micro level, greatly improves the screening efficiency and reduces the workload and the cost.
Drawings
FIG. 1 is a schematic diagram of a planar chromatography component microarray screening process.
FIG. 2 is a two-dimensional thin-layer chromatography fingerprint.
Fig. 3 is a schematic view of a metal mesh interface.
FIG. 4 is a diagram showing the array division and peeling effects of an aluminum-based thin-layer chromatography silica gel plate; fig. 4a and 4b show the front and back sides of a thin aluminum sheet, fig. 4c shows an array of thin square sheets of silicone gel peeled and attached to a PVDF film, and fig. 4d shows a thin square sheet of silicone gel peeled.
FIG. 5 is a graph showing the effect of array division of gel electrophoresis sheets.
FIG. 6 is a graph showing the effect of array division of PVDF membranes; FIG. 6a shows a PVDF membrane embedded in a grid of a cobalt chromium alloy array after flat pressing and after lifting off a thin lead plate, and FIG. 6b shows a PVDF membrane pushed out of the grid.
FIG. 7 is a diagram showing the effect of the silica gel layer being divided and peeled off integrally into the corresponding microwell of 384-well filter plate in a 384-well microplate array format.
FIG. 8 is a graph of a cell viability assay image taken and a bioactivities thermogram plotted from the cell viability assay results performed on a planar chromatographic component microarray sample; FIG. 8a, A549 Lung cancer cell survival Rate detection microarray sample imaging, FIG. 8b HepG2
Live cancer cell survival rate detection microarray sample image capture, fig. 8c A549 lung cancer cell survival rate detection microarray sample biological activity heat map, fig. 8d. hepg2 liver cancer cell survival rate detection microarray sample biological activity heat map.
FIG. 9a is the ESI-MS mass spectrum of the most bioactive array element in the region indicated by the box in FIG. 8c, and FIG. 9b is the ESI-MS mass spectrum of the most bioactive array element in the region indicated by the box in FIG. 8d.
FIG. 10a is a graph of the image of a G-quadruplex ligand screening assay performed on a thin layer chromatography microarray sample of example 9.
FIG. 10b is a graph of the digitized representation of biological activity of the corresponding region of the planar chromatogram in array format (circled in boxes) in example 9.
FIG. 11 is an ESI-MS mass spectrum of the array cell with the strongest excimer ion peak screened by the ligand activity assay.
Fig. 12 is a thin layer chromatography fingerprint of galangal extract constructed based on cell survival assay and mass spectrometry results performed on planar chromatography component microarray samples, which correlates with anti-cancer activity.
FIG. 13 shows a biological activity screening system in example 3 of the present invention.
FIG. 14 is a pictorial view of a metal mesh interface board of the present invention, which is a quadrilateral mesh in this exemplary figure, but this is not a limiting example; wherein a shows a second end plane (wide side surface) of the metal grid interface board, b shows a first end plane (sharp side surface) of the metal grid interface board, the thicknesses of grid frames on the two surfaces are different, the frame thickness of the second end plane is thick, and corresponds to the thickness of the micropore pool edge of the micropore plate (not shown in the figure) and is matched with the position; the thickness of the edge of the first end plane is as thin as possible; the channel-shaped space which communicates the two open ends is a regular quadrangular frustum.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
Example 1
Sample processing device
In this embodiment, a grid plate corresponding to the 384-well square-mouth microplate array format is taken as an example to illustrate the structure of the grid plate, and in the actual application process, the grid plate can be adjusted according to the array format of the microplate.
The grid plate shown in fig. 3 has a plurality of channel-shaped spaces (i.e., the internal space of each cell) defined by cell borders, the channel-shaped spaces having open first and second ends, wherein the area of the cell borders defining each channel-shaped space is 10% of the area of the first end of the channel-shaped space on the open plane of the first end. At the moment, the frame thickness of each grid frame at the first end is narrow enough to form a sharp edge, and the sharp edge can be used for cutting the chromatographic thin layers of the planar chromatography, so that all the chromatographic thin layers can be cut and separated. The first end plane of the channel-shaped space is a regular 4-sided polygon, and at the moment, the first end of the channel-shaped space can be paved on the whole plane, so that the chromatographic thin layer of the planar chromatograph is completely cut.
The sample processing device also comprises a micro-porous plate, and the array format of the grid plate is consistent with that of a 384 square-mouth micro-porous plate, in particular to a horizontal 24X longitudinal 16 array format of a 4.5mm X4.5 mm square grid. The open second end of the channel-shaped space is used for butting with the micropore pool of the micropore plate.
Each grid of the grid plate is internally provided with a regular quadrangular frustum pyramid, the longitudinal section of the frame of the grid is an isosceles trapezoid (namely the height of the grid plate when the grid plate is horizontally placed) with the height of 2.5mm, the width of the lower bottom edge of the isosceles trapezoid is the same as the thickness of the micropore pool edge of the 384 square-mouth micropore plate, specifically 0.70mm, the plane of the lower bottom edge is the plane of the open second end of the open channel, and the plane is used for being butted with the micropore plate; the width of the upper bottom edge of the isosceles trapezoid is less than or equal to 0.15mm, the plane where the upper bottom edge is located is the plane where the open first end of the open-type channel is located, the thickness of the upper bottom surface screen frame is smaller than that of the lower bottom surface screen frame, and the upper bottom surface grid frame forms a square-mouth sharp edge for cutting a chromatographic thin layer of the planar chromatogram. The grid plate is prepared by 3D printing of cobalt-chromium alloy, and can also be prepared by other hard and tough materials.
Example 2 an apparatus for preparing a planar chromatography microarray
The device comprises a planar chromatograph, a grid plate, a micropore filter plate and a micropore plate which are in one-to-one correspondence in an array format. And a biological activity detection unit and a mass spectrometry unit.
The planar chromatogram is used for constructing a planar chromatogram fingerprint map of a sample to be screened; the grid plate is used for dividing and stripping the chromatographic thin layer of the planar chromatographic fingerprint image according to a micropore plate array format; the microporous filter plate is used for positioning and eluting the chromatographic thin layer divided and stripped by the grid interface to a microporous plate; the micro-porous plate is used for positioning and receiving components of the planar chromatography and preparing a planar chromatography component microarray sample.
In this embodiment, the chromatogram span of the planar chromatogram fingerprint corresponds to the microplate array format of the channel width. Specifically, the span length and channel width of the planar chromatogram are multiples of 4.5mm, including a one-dimensional planar chromatogram having a span length of 9.0cm × channel 7.2cm and a two-dimensional planar chromatogram having a span length of 7.2cm × channel 7.2 cm. Correspondingly, the micropore plate is in a 4.5mm multiplied by 4.5mm square grid horizontal 24 multiplied by longitudinal 16 array format, the specific structure is shown in example 1, the micropore filter plate is a 384 micropore filter plate, and the micropore plate is a 384 square-mouth micropore plate. If the array format of one of the structures is changed, the array formats of the other structures are changed correspondingly.
In this embodiment, when the planar chromatography is selected from an aluminum-based thin-layer silica gel plate, the planar chromatography further contains a PVDF film; still more preferably, the planar chromatograph further comprises a sheet of silicone rubber and a sheet of paper; when the system operates, the silicon rubber sheet, the grid plate, the PVDF film, the aluminum-based thin-layer silicon rubber plate and the paper sheet are sequentially stacked in parallel from bottom to top.
Example 3A biological Activity screening System
The biological activity screening system shown in fig. 13 comprises a planar chromatograph, a grid plate, a microporous filter plate, a microporous plate, a biological activity detection unit and a mass spectrometry unit which are in one-to-one correspondence with each other in an array format.
The planar chromatogram is used for constructing a planar chromatogram fingerprint map of a sample to be screened; the grid plate is used for dividing and stripping the chromatographic thin layer of the planar chromatographic fingerprint image according to a micropore plate array format; the microporous filter plate is used for positioning and eluting the chromatographic thin layer divided and stripped by the grid interface to a microporous plate; the microporous plate is used for positioning and receiving components of the planar chromatography and preparing a planar chromatography component microarray sample; the mass spectrometry unit is used for determining mass spectrometry information of active ingredients in the planar chromatographic ingredient microarray sample; the biological activity detection unit is used for detecting biological activity in the microarray sample of the planar chromatographic component.
In this embodiment, the chromatogram span of the planar chromatogram fingerprint corresponds to the microplate array format of the channel width. Specifically, the span length and channel width of the planar chromatogram are multiples of 4.5mm, including a one-dimensional planar chromatogram having a span length of 9.0cm × channel 7.2cm and a two-dimensional planar chromatogram having a span length of 7.2cm × channel 7.2 cm. Correspondingly, the micropore plate is in a 4.5mm multiplied by 4.5mm square grid horizontal 24 multiplied by longitudinal 16 array format, the specific structure is shown in example 1, the micropore filter plate is a 384 micropore filter plate, and the micropore plate is a 384 square-mouth micropore plate. If the array format of one of the structures is changed, the array formats of the other structures are changed correspondingly.
In the embodiment, the system also comprises a data processing system which is used for carrying out operations of carrying out digital representation on the planar chromatographic components in an array mode, constructing a biological activity distribution diagram, associating, coupling and normalizing analysis of biological activity intensity and mass spectrum information, selecting active substances, constructing a planar chromatographic fingerprint related to spectrum-effect and the like.
Example 4 construction of thin-layer chromatography fingerprints sufficiently representing chemical compositions of samples of Chinese herbs and natural products and reflecting differences and characteristics of chemical components of samples
1) Taking 60-mesh rhizoma Alpiniae Officinarum dry powder as a sample, and selecting one solvent from 8 solvents extracted from Snyder, wherein the solvents are petroleum ether, ethanol, tetrahydrofuran, acetone, dichloromethane, ethyl acetate, benzene and water; respectively taking the above raw materials as solvents to carry out conventional ultrasonic extraction; mixing the extractive solutions of 8 solvents, and removing solvent by conventional method to obtain rhizoma Alpiniae Officinarum extract. Following the principle of similar solubility, the sample is extracted with a combination of solvents to make the extract more representative.
2) The span length and the channel width of the thin-layer chromatography strictly correspond to a 384-hole square-mouth micropore plate array (a transverse 24 multiplied by longitudinal 16 array with an array format of 4.5mm square), the span length and the channel width of the one-dimensional thin-layer chromatography are set to be 9.0cm multiplied by 7.2cm multiplied by a channel, the span length and the channel width of the two-dimensional thin-layer chromatography are set to be 7.2cm multiplied by 7.2cm, and the span length and the channel width are multiples of 4.5 mm. Selecting chloroform and methanol as a solvent system A, and petroleum ether and ethyl acetate as a solvent system B, wherein the ratio of chloroform to methanol is 98: 2, and the ratio of ethyl acetate to acetic acid is 70: 30: 2, and spotting 5 mu L of galangal extract methanol solution of 0.275g/mL on a Merk aluminum-based high-efficiency thin-layer plate to construct a two-dimensional thin-layer chromatography fingerprint; the invention strictly corresponds the whole plane chromatogram and the micro-array of the micro-porous plate, and keeps the integrity of the chromatographic behavior of the chemical components on the plane chromatogram, thereby laying the foundation for the subsequent quantity-effect relationship and the chromatographic behavior analysis thereof. The inventor's prior patent CN 104792914a emphasizes the mapping correspondence between the spectrum spots and the array elements, does not specify the overall correspondence between the spectrum development area and the array format, and also considers that the spot is allowed to be locally picked to align with the grid, which destroys the integrity of the planar chromatogram, and also raises the problem that the existence of multiple components in a microwell pool does not provide the basis for confirming the biological activity attribution compound. Furthermore, the method of localized screening of bioactive components by chemical methods is prone to screen leakage.
3) Observing silica gel thin layer chromatography under 254nm and 365nm ultraviolet light, developing with 10% sulfuric acid-vanillin ethanol solution reagent, and examining the separation degree and concentration distribution of the thin layer chromatography. By optimizing a chromatographic solvent system, the separation degree and the capacity of the thin-layer chromatography are optimized, and the chemical component difference and the characteristics of the galangal are fully reflected on a thin-layer chromatography fingerprint, and the result is shown in figure 2.
Embodiment 5 the whole chromatographic thin layer recorded with the planar chromatographic fingerprint is divided, stripped and eluted into a micropore pool corresponding to a micropore plate according to a square-mouth micropore plate array format to prepare a planar chromatographic component microarray sample.
1) The preparation of the grid plate adopts the 3D printing preparation of cobalt chromium alloy, obtains with 384 square-mouth micropore plate array formats unanimous, the interior space is the grid plate of regular quadrangular frustum of a prism, specifically is: a transverse 24X longitudinal 16 array format of 4.5mm X4.5 mm square grids, a grid internal space is a regular quadrangular frustum, the longitudinal section of a grid frame is an isosceles trapezoid with the height of 2.5mm, the width of the lower base edge of the isosceles trapezoid is the same as the thickness of the edge of a 384 square-mouth micropore plate micropore pool, and the width is specifically 0.70mm, and the surface is used for butt joint with a micropore plate; the width of the upper bottom edge of the isosceles trapezoid is less than or equal to 0.15mm, so that the isosceles trapezoid is as close to an isosceles triangle as possible, and a grid frame on the bottom surface forms a square-mouth sharp edge for cutting a plane chromatogram, wherein the schematic diagram is shown in FIG. 3;
2) array division and stripping of thin-layer silica gel chromatography A2 mm thick silicone rubber sheet, an interface board, a PVDF membrane (Millipore, thickness 0.2mm), an aluminum-based thin-layer silica gel plate (dry) and a thin paper sheet (PVDF membrane protective paper) are sequentially stacked in parallel from bottom to top, sharp edges of the grid plate are aligned with the thin-layer silica gel chromatography area through the PVDF membrane, and the grid plate and the thin-layer silica gel chromatography area are placed together on a parallel pressing plate to form a 8 x 10 structure3KPa pressure flat pressing; the thin aluminum plate with ductility and the fragile thin silica gel are differently changed under the pressure of the sharp edge of the interface plate, the thin silica gel is integrally and orderly split into square sheet arrays, and the square sheet arrays are peeled from the slightly deformed thin aluminum plate and attached to the PVDF film slightly embedded in the interface, and the effect is shown in figure 4. Fig. 4a and 4b show the front and back sides of a thin aluminum sheet, fig. 4c shows an array of thin square sheets of silicone gel peeled and attached to a PVDF film, and fig. 4d shows a thin square sheet of silicone gel peeled.
3) Array partitioning of preparative thin-layer Silica gel chromatography and peeling on a preparative thin-layer Silica gel chromatography plate (Merck, PLC Silica gel 60F) with a Silica gel thickness of 2.0mm254+3662mm glass plate) prepared by one-dimensional thin-layer chromatography of Alpinia officinarum with span and channel width set at 9.0cm × 7.2cm, at 0.275g/mL of Alpinia officinarum extractStrip sample application of methanol solution is carried out for 200 mu L; taking a dry thin-layer chromatography plate with a sharp edge surface of an interface plate, aligning the sharp edge surface of the interface plate with a thin-layer chromatography expansion area, stacking a silicon rubber sheet with the thickness of 2mm on the other surface of the interface, parallelly laminating the silicon rubber sheet together, placing the silicon rubber sheet on a parallel pressing plate, flatly pressing the silicon rubber sheet until the whole thin-layer chromatography layer is divided into square arrays and embedded in the square lattice array of the interface plate; and then releasing the pressure until the glass plate can just slide in parallel between the parallel pressing plate and the interface plate, laterally knocking the glass plate to make the glass plate move in parallel at least one array unit left and right, front and back respectively, so that the glass plate and the silica gel layer are completely peeled, and the effect is shown in figure 4.
4) The array division of the gel electrophoresis sheet is that the activated gel electrophoresis sheet is clamped between the grid plate and the silicon rubber plate in parallel, the sharp edge of the interface plate faces the gel electrophoresis sheet and is aligned with the chromatographic development area, the gel electrophoresis sheet is placed on a parallel pressing plate to be pressed flatly, the whole gel electrophoresis sheet is divided into square arrays and embedded in the metal grid interface, the square arrays of the gel electrophoresis sheet can be separated from the metal grid interface after micro-drying, and the effect is shown in figure 5.
5) Array division of PVDF film is parallelly sandwiched between 0.50mm thin lead plate and cobalt-chromium alloy grid plate, sharp edge of interface plate faces to polymer film, and is placed on parallel pressing plate at 15 × 103And (3) pressing under KPa pressure, dividing the polymer film into square arrays and embedding the square arrays in the interface, and then lifting off the thin lead plate, wherein the effect is as shown in figure 6. The above 2) -5) specifically exemplify several types of chromatographic thin layer dividing and stripping methods for the planar chromatography, and in the practical application process, a corresponding method may be selected according to the type of the planar chromatography, or other methods capable of realizing chromatographic thin layer dividing and stripping may be also used.
6) Thin layer chromatography component microarray sample preparation two-dimensional thin layer chromatography of galangal extract was prepared on high performance thin layer Silica gel chromatography plates (Merck, HPTLC Silica gel 60F254, Aluminum sheets): setting the span and the channel width to be 7.2cm multiplied by 7.2cm, spotting 5 mul of galangal extract methanol solution of 0.275g/ml, and performing two-dimensional development by using a solvent system A of chloroform to methanol of 97 to 3 and a solvent system B of petroleum ether to ethyl acetate to acetic acid of 70 to 30 to 2 to prepare the galangal two-dimensional thin-layer chromatography; referring to the array dividing and stripping method of the thin-layer silica gel chromatography in the example 5, thin-layer silica gel square sheets which are orderly stripped integrally and attached to the PVDF film embedded in the grid interface are obtained; aligning and butting the ports of a 384 square-port microporous filter plate (Pall, specification 5072) with grid interfaces, and lightly touching the PVDF membrane from the other side of the grid interfaces by using 16 round ports of a pipette gun (without installing a gun head), so that a silica gel layer attached to the PVDF membrane falls into a corresponding microporous pool of the 384-port filter plate, and the effect is shown in figure 7; the thin silica gel layer (FIG. 7) in the microporous filter plate was wetted with methanol (15. mu.L/well), centrifuged (269 Xg, 1min), repeated 5 times, and the chromatographic fractions were localized in corresponding microwell wells of a 384-well microplate; the 384-well microplate containing the eluted fractions was concentrated to dryness (269 Xg, 50 ℃, 80.0KPa under reduced pressure, 1.5h) on a vacuum centrifugal concentrator to prepare a thin layer chromatography fraction microarray sample.
The microarray sample not only retains the separation characteristic of planar chromatography, but also is completely separated from the chromatography matrix, the microarray units are mutually separated, the interference and the obstruction of the chromatography matrix such as capillary diffusion, adsorption interference, background noise and the like are eliminated, and the microarray sample can be naturally butted with the existing biological activity detection and drug analysis method and standard.
Example 6 cell viability assay on thin layer chromatography component microarray samples
On the galangal two-dimensional thin-layer chromatography component microarray sample prepared as in example 5, the test solution was dispensed according to the array coordinates into corresponding reaction cells of a 384-well square-mouth microplate (Corning 3701) to perform the Alamar Blue method for detecting the survival rates of human lung cancer cells a549 cells and HepG2 liver cancer cells, respectively, and the inhibition effect of galangal thin-layer chromatography components on a549 lung cancer cells and HepG2 liver cancer cells was detected with 5-Fu (pentafluorouracil) as a positive control. Referring to the Invitrogen Alma blue kit instructions and conventional experimental methods, 30. mu.L of cell suspension (1000 cells/well in logarithmic growth phase) was added to each well and placed in a 37 ℃ carbon dioxide cell incubator; culturing for 48h after adding medicine, and adding Alamar Blue 12h before detection; the measurement wavelength is 570nm, the reference wavelength is 600nm, and the growth inhibition rates of galangal two-dimensional thin-layer chromatography component microarray sample unit human lung adenocarcinoma A549 cell strain and HepG2 liver cancer cell strain are respectively calculated by taking 5-Fu as a positive control. The image of microarray sample for survival rate detection of a549 lung cancer cell and HepG2 liver cancer cell is shown in fig. 8a and fig. 8c, respectively.
Example 7 construction of bioactive Hot Point map by digital representation of Alpinia galanga thin layer chromatography component characteristics with irregular chromatographic spot shape and position distribution in array and computer data processing
The cell viability test results of each array unit of the thin layer chromatography component microarray sample in example 5 are digitally expressed in an array format for the cell viability of the chromatographic components in the corresponding area of the planar chromatography, and a bioactivity heat dot map is constructed by EXCEL. Viability assay microarray samples for a549 lung cancer cells and HepG2 liver cancer cells in the bioactivities of the dot blot diagrams of fig. 8b and 8d, respectively.
Example 8
1) Combined screening and screening of bioactive components by chromatographic separation, bioactivity detection and ESI-MS analysis
a) Based on examples 4-7, array units with significant cell activity on two-dimensional thin-layer chromatography microarray samples and their adjacent array units were selected for ESI-MS analysis based on the bio-activity heat map. The A549 cell activity hot spot array unit is shown as a circled area in fig. 8c, and the ESI-MS mass spectrum of the highest biological activity array unit is shown as fig. 9 a; the hot spot array unit of HepG2 cell activity is outlined in box in fig. 8d as region 1 and region 2, and the ESI-MS mass spectrum of the highest bioactive array unit in region 2 is shown in fig. 9 b.
b) The peak intensity of each main excimer ion peak coexisting in the ESI-MS mass spectrum of the hot spot Array unit is taken as Array unit data Array1, the cell proliferation inhibition rate of the corresponding Array unit is taken as Array unit data Array2, and the correlation analysis is carried out by using a statistical function CORREL (Array1, Array2) of Excel.
c) The correlation analysis result shows that:
■ the main excimer ion peak related to the A549 cell activity hot spot array unit shown in FIG. 8c is 327[ M-H]-、255[M-H]-And 283[ M-H]-An Array of peak heights of their excimer peaks (Array1) and an Array of cell proliferation inhibition rate data of corresponding Array units (Array2) were found to have correlation coefficients of 0.827, -0.256, and-0.224, respectively. The results of the calculations are shown in Table 1.
TABLE 1
Figure RE-GDA0001635586440000191
■ FIG. 8d shows major excimer ion peak 327[ M-H ] associated with HepG2 cell active hot spot region 1]-、255 [M-H]-And 283[ M-H]-The correlation coefficients of the peak height data Array (Array1) of their excimer ion peaks and the cell proliferation inhibition rate data Array (Array2) of the corresponding Array units were 0.712, -0.288, and-0.268, respectively. The results of the calculations are shown in Table 2.
TABLE 2
Figure RE-GDA0001635586440000192
■ FIG. 8d shows that the main excimer ion peak associated with hot spot region 2 of HepG2 cell activity assay is 269[ M-H]-、255[M-H]-And 283[ M-H]-The correlation coefficients of the peak height data Array (Array1) of their excimer ion peaks and the cell proliferation inhibition rate data Array (Array2) of the corresponding Array units are 0.560, 0.454 and 0.305, respectively; further, the correlation of the middle row of the array was examined and their correlation coefficients were 0.600, 0.334 and-0.376, respectively, and the corresponding results are shown in Table 3.
TABLE 3
Figure RE-GDA0001635586440000201
d) From the calculated correlation coefficient, 327[ M-H]-And 269[ M-H]-The selected quasi-molecular ion is the quasi-molecular ion of the biological activity attribution compound which is the interdependence of the mass spectrum quasi-molecular ion peak intensity and the biological activity intensity and is coupled with the chromatographic behavior.
The activity attributed to the same molecular ion peak in array units of the same array region is taken into consideration as the activity contribution normalization of the molecule.
In this embodiment, according to the bio-activity hotspot graph, array units with significant cell activity on a two-dimensional thin-layer chromatography microarray sample and adjacent array units thereof are purposefully selected for ESI-MS analysis, and a correspondence relationship between a bio-activity hotspot array unit region and a corresponding array unit mass spectrogram is established. Based on the corresponding relation, the correlation analysis such as the interdependence of the biological activity intensity and the mass spectrum excimer ion peak intensity, the chromatographic behavior coupling and the like can be carried out on a computer, the digitization of the planar chromatographic component characteristics with irregular chromatographic spot shape and position distribution and the computer processing are realized, the efficiency is high, the simplicity is realized, and the screening efficiency is greatly improved.
The traditional method is to develop or develop the planar chromatogram, take an image to obtain an image file, then scan the image, identify the peak, and convert the image into a number for data processing. For the biological activity reaction and chemical analysis which can not be carried out in situ in the planar chromatography, the chromatographic layer is taken out according to the color development pattern, and the chromatographic components are eluted out for carrying out the experiment. This is done on the one hand because of the difficulties and enormous efforts brought by the irregular distribution of the shape and position of the chromatographic spots; on the other hand, no universal and nondestructive in-situ detection method for the plane chromatography exists so far, the sensitivity of different detection methods is different from the applicable compounds, the obtained graphs are different, and more importantly, the graphs obtained by chemical analysis detection and biological activity reaction detection are different and difficult to correspond; therefore, the method of screening for bioactive ingredients, which first locates the thin layer spots chemically, may result in screen leakage! Also, the integrity of the planar chromatography is destroyed by taking the chromatographic layer irregularly, wherein a large amount of information useful for screening bioactive components is destroyed. When the chemical components of the bioactive array units are not single compounds, the biological activity attribution cannot be judged simply by the content of the compounds, and scientific basis is needed for screening and selecting the compounds. The experiment takes the dependence relationship between the bioactivity intensity on the area of the obvious bioactivity array unit and the mass spectrum excimer ion peak intensity of the corresponding array unit and the chromatographic behavior coupling degree of the dependence relationship as indications, provides screening bases through correlation analysis, indicates bioactive chemical components from coexisting chemical components, and shows the advantages of screening and screening the bioactive components through the combination of chromatographic separation, bioactivity detection and mass spectrum analysis.
The biological activity of the individual array elements in the hot spot region shown in fig. 8d1 and fig. 8d2 is not too high, but the biological activity of the array elements in the same array region can be integrally exhibited by taking the activity attributed to the same molecular ion peak in the array elements as the activity contribution normalization of the molecule into consideration in the screening. This is beneficial for screening of bioactive components where the planar chromatographic spot does not fall exactly into a single array element.
2) High-flux screening of bioactive components from large and complex Chinese medicine and natural medicine
Peak of quasi-molecular ion 327[ M-H ] in Alpinia officinarum extract]-And 269[ M-H]-Identification of belonging Compound (Arisaema, Shaoguiwu, ancient exercise right, Zhangmin. chemical component research of Alpinia galanga. Chinese medicinal materials 2000,23(2):84-87), speculation 327[ M-H]-The compound is diphenyl heptane A [ 1-phenyl-7- (3 '-methoxy-4' -hydroxy) phenyl-5-ol-3-heptanone]，269[M-H]-The compound is galangin.
3) Evaluation of biological Activity of selected Components
The pharmacodynamic activity of the two selected compounds was evaluated by the same method as in example 6 using 5-fluorouracil as a positive control. The data of the cell growth inhibition curves are substituted into SPSS18.0 software to obtain that the IC50 of diphenyl heptane A, galangin and 5-fluorouracil for lung cancer cell A549 is respectively 0.247, 0.089 and 0.023mol.L-1, and the IC50 for liver cancer cell HepG2 is respectively 0.259, 0.085 and 0.092 mol.L-1.
Example 9 screening of G-quadruplex ligands on thin layer chromatography component microarray samples
1) Principle of experiment
The G-quadruplex (G-quadruplex) is a four-strand DNA helical structure formed by connecting four guanines, and the formation of the G-quadruplex can effectively inhibit the activity of telomerase and the extension of telomeres, thereby inhibiting the mass proliferation of tumor cells. The method takes the G-quadruplex as a target spot, screens a ligand which can be specifically identified and combined with the G-quadruplex, has a stable G-quadruplex structure or promotes the formation of the G-quadruplex so as to inhibit the proliferation of cancer cells, and is an effective way for finding potential anticancer drugs.
In the experiment, nanogold (GNPs) are used as colorimetric probes, and G-quadruplex ligands are screened by a colorimetric method based on the difference between single-stranded DNA and G-quadruplex and GNPs. Specifically, single-stranded G-DNA can be adsorbed on the surfaces of colloidal Gold Nanoparticles (GNPs), so that colloidal gold can still be in a red dispersion state at a certain salt (such as NaCl) concentration; however, the ligand can induce the GDNA to form a G-quadruplex structure so as to be detached from the surfaces of the GNPs, and the colloidal gold is in a blue aggregation state. The G-quadruplex ligand can be rapidly screened through the change of the color of the GNPs solution before and after the addition of the medicament.
2) G-quadruplex ligand screening experiments
On the galangal two-dimensional thin-layer chromatography component microarray sample prepared as in example 5, the test solution was dispensed according to the array coordinates into the corresponding reaction cell of a 384-well round-mouth microplate for the G-quadruplex ligand screening test.
GNPs (prepared by sodium citrate reduction chloroauric acid method, particle size of 12nM, concentration of 9.45nM) and 1.0 μm G-DNA (sequence of 5'-TTAGGGTTAGGGTTAGGGTTA GGG-3', synthesized by Shanghai bioengineering Co., Ltd., order No. 300064096) were added at a molar ratio of 1: 150, and reacted at 25 deg.C for 16h to prepare GNPs-GDNA probes; adding GNPs-GDNA probes to the thin-layer chromatography component microarray sample at 60 μ L/well, and reacting at 25 deg.C for 3 h; adding 0.106M NaCl, and reacting at 25 ℃ for 30 min; after the liquid is added in the steps, the liquid is treated by centrifugation for removing bubbles (1400rpm, 1 min).
Detecting the absorption spectrum (400-850 nm) of each hole of the thin-layer chromatographic component microarray sample on a microplate reader, and calculating the ratio lambda of the absorbance at 670nm to the absorbance at 520nm670/520Observing color change and imaging, and inspecting the aggregation capability change of each array unit component of the thin-layer chromatography component microarray sample on the GNPs-GDNA probe caused by NaCl resistance.
The image of the G-quadruplex ligand screening experiment of the thin layer chromatography component microarray sample is shown in FIG. 10 a.
3) Combined screening and screening of bioactive components by chromatographic separation, bioactivity detection and ESI-MS analysis
a) The G-quadruplex ligand screening experiment is carried out on the lambda of each array unit of a thin-layer chromatography component microarray sample670/520As the ligand activity value, it was subjected to EXCEL to construct a bio-activity heat map, and the bio-activity of the corresponding region of the planar chromatography was digitally expressed in an array format, as shown in FIG. 10 b.
b) According to the biological activity heat dot diagram, array units with significant ligand activity on a two-dimensional thin-layer chromatography microarray sample and adjacent array units thereof are selected in a targeted mode to carry out ESI-MS analysis. The ligand active hot spot array elements and their adjacent regions are enclosed by boxes in figure 10 b.
c) ESI-MS analysis shows that the main excimer ion peak related to the active hot spot array unit is 269[ M-H]‐、255[M‐H]‐And 283[ M-H]‐. FIG. 11 shows the ESI-MS mass spectrum of the bioactive hot spot array unit.
d) Taking the peak intensity of each main excimer ion peak coexisting in the ESI-MS mass spectrum of the hot spot Array unit as Array unit data Array1, lambda reflecting the ligand activity of the corresponding Array unit670/520As Array cell data Array2, Array correlation analysis was performed using the statistical function CORREL (Array1, Array2) of Excel.
e) Table 1 lists the ligand activity values for the hot spot array elements and an array of peak intensity data for each of the main excimer ion peaks coexisting in the corresponding ESI-MS mass spectrum. Correlation analysis results show that the correlation coefficients of the peak height data Array (Array1) of the main excimer peaks 269[ M-H ] -, 255[ M-H ] -and 283[ M-H ] -associated with active hot spot Array elements and the ligand activity data Array (Array2) of the corresponding Array elements are 0.758, 0.411 and 0.209, respectively, and the calculation results are shown in Table 4.
TABLE 4
Figure RE-GDA0001635586440000221
Based on the calculated correlation coefficient, 269[ M-H]‐The selected mass spectrum quasi-molecular ion is the quasi-molecular ion with the interdependence of the peak intensity and the biological activity intensity and the coupling of the chromatographic behavior. The activity attributed to the same molecular ion peak in array units of the same array region is taken into consideration as the activity contribution normalization of the molecule.
The present experiment shows an example in which, in the case where a plurality of compounds coexist in a bioactive array unit and the excimer ion peak having bioactivity is not the highest among them, a correlation between the intensity of bioactivity in a region having a significant bioactive array unit region and the intensity of mass spectrum excimer ion peaks of the corresponding array unit and the degree of chromatographic behavior coupling thereof are used as indicators, a screening basis is given by correlation analysis, and a bioactive chemical component is indicated from coexisting chemical components.
The experiment also shows that the activity of the ion peaks belonging to the same molecule in the array units of the same array area is taken as the activity contribution normalization of the molecule into screening consideration, so that the biological activity of the molecule can be integrally displayed, and the biological active components of which the plane chromatographic spots do not exactly fall into a single array unit are prevented from being leaked.
4) High-flux screening of bioactive components from large and complex Chinese medicine and natural medicine.
The selected excimer ion 269[ M-H ] -, referring to the cell experiment results and reference literature of example 8 (Chaozhenxian, Xiaoguiwu, Guzhuang, Zhangyi. galangal chemical composition research. Chinese medicinal material 2000,23(2):84-87), was presumed to belong to the compound galangin.
The results of the screening experiments of G-quadruplex ligands and the results of the cell survival experiments in example 8 are combined to speculate that the induction of the formation of G-quadruplex and the inhibition of the proliferation of cancer cells may be one of the action mechanisms of galangin in anti-cancer activity.
Example 10 construction of "Spectrum-Effect" related traditional Chinese medicine and Natural product thin-layer chromatography finger-prints
The galangal thin-layer chromatography fingerprint obtained in the above example, the growth inhibition rate experimental results of HepG2 hepatoma cell lines on the thin-layer chromatography component microarray sample, and the activity screening compounds are corresponded, the array format of the thin-layer chromatography component microarray sample is X, Y two-dimensional coordinates, and the mapped to the corresponding thin-layer chromatography fingerprint array unit area, a bar chart is established on the array unit to mark the biological activity value of the array unit and the structural formula of the corresponding compound components galangin and diphenylheptane a, and the traditional Chinese medicine and natural product thin-layer chromatography fingerprint related to "spectrum-effect" is established, as shown in fig. 12.

Claims (35)

1. The device for preparing the planar chromatography microarray is characterized by comprising a planar chromatography, a grid plate and a microporous plate which correspond to each other in an array format one by one,
the grid plate is provided with a plurality of channel-shaped spaces defined by grid frames, and each channel-shaped space is provided with a first end and a second end which are open;
the array format of the grid plate corresponds to the array format of the micropore pool; on the open plane of the first end of the grid plate, the area of the grid frame for limiting each channel-type space is less than or equal to 10% of the area of the first end of the channel-type space; the open second end of the channel-shaped space is used for being butted with a micropore pool of a micropore plate; on the open plane of the second end, the thickness of the grid frame for limiting each channel-shaped space is larger than that on the open plane of the first end, and is the same as the thickness of the edge of the micropore pool of the micropore plate;
the planar chromatogram is used for constructing a planar chromatogram fingerprint of a sample to be screened;
the grid plate is used for dividing and stripping a chromatographic thin layer of the planar chromatographic fingerprint according to a micropore plate array format;
the micro-porous plate is used for positioning and receiving components of the planar chromatography and preparing a planar chromatography component microarray sample;
the device also comprises a microporous filter plate; the microporous filter plate is used for positioning and eluting the chromatographic thin layer divided and stripped by the grid interface to a microporous plate.
2. The apparatus of claim 1, wherein the area of the frame of the mesh defining each channel-shaped space in the open plane of the first end of the mesh plate is less than or equal to 5% of the area of the first end of the channel-shaped space.
3. The apparatus of claim 1, wherein the area of the frame of the mesh defining each channel-shaped space in the open plane of the first end of the mesh plate is equal to or less than 3% of the area of the first end of the channel-shaped space.
4. The apparatus of claim 1, wherein the channel-shaped space is a regular n-sided polygon in the first end plane, and satisfies n-sided polygon interior angle =180(n-2) ÷ n.
5. The apparatus of claim 4, wherein n is 4, and the channel-shaped space of the grid plate is a square pyramid.
6. The apparatus of claim 1, wherein the longitudinal cross-section of the frame of the grid defining each channel-shaped space is an isosceles trapezoid, the width of the lower base of the isosceles trapezoid being the same as the thickness of the edge of the cell of the microplate; the width of the upper bottom edge of the isosceles trapezoid is less than or equal to 0.15 mm.
7. The apparatus of claim 1 wherein the grid plate array format corresponds to a 384 microplate array format of 4.5mm x 4.5mm square grid 24 x 16 array format.
8. The apparatus of claim 1, wherein the longitudinal section of the grid frame is an isosceles trapezoid with a height of 2.5mm, and the width of the lower base edge of the isosceles trapezoid is 0.68-0.72mm, which is the same as the thickness of the cell edge of the micropore cell of the 384-square-mouth micropore plate; the width of the upper bottom edge of the isosceles trapezoid is less than or equal to 0.15 mm.
9. The apparatus of claim 1, wherein the mesh plate is prepared by 3D printing.
10. The device according to claim 1, wherein the planar chromatography is selected from the group consisting of unsupported matrix planar chromatography and/or supported matrix planar chromatography.
11. The device of claim 10, wherein the unsupported planar chromatography matrix is selected from the group consisting of gel electrophoresis, polymer membrane thin layer chromatography, and electrophoretic band-to-film polymer membranes.
12. The apparatus of claim 10, wherein the planar chromatograph having a support substrate is selected from a preparative thin silica gel chromatograph plate of a glass plate substrate or an aluminum-based thin silica gel chromatograph plate.
13. The apparatus of claim 12, wherein the planar chromatography is selected from the group consisting of aluminum based thin layer silica gel plates.
14. The apparatus according to claim 13, wherein when the planar chromatography is selected from the group consisting of aluminum-based thin-layer silica gel plates, the planar chromatography further comprises a PVDF membrane.
15. The device of claim 14, wherein the planar chromatograph further comprises a sheet of silicone rubber and a sheet of paper.
16. The apparatus according to claim 15, wherein a silicone rubber sheet, a mesh plate, a PVDF film, an aluminum-based thin layer silicone plate, and a paper sheet are stacked in parallel from bottom to top in this order.
17. The apparatus of claim 1, wherein the planar chromatographic fingerprint has a chromatogram span and a channel width corresponding to a microplate array format.
18. The device of claim 17, wherein the planar chromatogram has a span and channel width that are multiples of 4.5mm, and comprises a one-dimensional planar chromatogram having a span of 9.0cm x 7.2cm and a two-dimensional planar chromatogram having a span of 7.2cm x 7.2 cm.
19. The apparatus of claim 1 wherein said microplate is a square-mouth microplate.
20. The apparatus of claim 19 wherein said square-mouth microplate is a 384-well square-mouth microplate having an array format of 4.5mm square, 24 x 16 array.
21. The apparatus of claim 19, wherein the microporous filter plate is a 384 square-mouthed microporous filter plate.
22. The apparatus as claimed in claim 21, wherein the 384 square-mouthed microporous filter plate is model number Pall No. 5072.
23. The method for using the apparatus for preparing a planar chromatography microarray of claim 1, comprising the steps of:
1) constructing a planar chromatography fingerprint of a sample to be screened by using a planar chromatography;
2) dividing, stripping, positioning and eluting a chromatographic thin layer of a planar chromatographic fingerprint image into a micropore pool of a micropore plate according to a micropore plate array format by adopting a grid plate to prepare a planar chromatographic component microarray sample;
in the step 2), the chromatographic thin layer stripped and positioned in the grid plate is firstly pushed into a micropore pool of a micropore filter plate, then the micropore filter plate is aligned with a micropore plate receiving eluent, and then a solvent is adopted to position and elute the chromatographic components on the chromatographic stationary phase into the corresponding micropore pool in the micropore plate.
24. The planar chromatography microarray prepared by the method of claim 23, wherein the planar chromatography microarray is a sample on a microplate comprising a chromatographic thin layer matrix to be screened and bioactive to be removed by separation with a planar chromatography thin layer and division with a grid plate corresponding to the microplate array format.
25. A bioactive component screening system comprising the planar chromatography microarray of claim 24, a bioactive detection unit, and a mass spectrometry analysis unit;
the mass spectrometry analysis unit is used for measuring mass spectrometry information of active ingredients in the planar chromatographic ingredient microarray sample;
the biological activity detection unit is used for detecting biological activity in the microarray sample of the planar chromatographic component.
26. The system of claim 25, wherein the mass spectrometry unit is selected from the group consisting of mass spectrometry units with soft ionizing ion sources.
27. The system of claim 26, wherein the soft ionization ion source is electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, matrix assisted laser desorption ionization, or chemical ionization in gas chromatography-mass spectrometry.
28. The system of claim 26, wherein the soft ionization ion source is electrospray ionization.
29. The system of claim 25, wherein the bioactivity detection unit comprises one or more of a microplate reader, a multifunctional microplate detection system, and a high-throughput microplate detector.
30. The system of claim 25, wherein the system comprises a data processing system.
31. The system of claim 30, wherein said data processing analysis is used to perform one or more of the operations of digital representation of planar chromatographic components in an array format, construction of a bioactivity profile, correlation of bioactivity intensity with mass spectral information, coupling, normalization analysis, active substance selection, and construction of a "spectrum-effect" related planar chromatographic fingerprint.
32. A method of using a bioactive ingredient screening system as claimed in any of claims 25 to 31, comprising the steps of:
1) constructing a planar chromatography fingerprint of a sample to be screened by using a planar chromatography;
2) dividing, stripping, positioning and eluting a chromatographic thin layer of a planar chromatographic fingerprint image into a micropore pool of a micropore plate according to a micropore plate array format by adopting a grid plate to prepare a planar chromatographic component microarray sample;
3) performing biological activity detection on the planar chromatographic component microarray sample by adopting a biological activity detection unit;
4) and carrying out mass spectrometry on the active hot spot array unit and the adjacent area array unit by adopting a mass spectrometry unit.
33. The use of claim 32, wherein in step 2) a microarray sample of planar chromatographic components is prepared and the planar chromatographic components are digitally represented in an array using a data processing system.
34. The use of claim 32, wherein in step 3), after the biological activity assay is completed, a data processing system is used to construct the biological activity profile.
35. The use of the method according to claim 32, wherein in step 4), after mass spectrometry, the data processing system is used to correlate, couple and normalize the information of the biological activity intensity and the mass spectrometry, and active substances are selected to construct a planar chromatographic fingerprint related to spectrum-effect.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6414306B1 (en) * 1999-08-07 2002-07-02 Bruker Daltonik Gmbh TLC/MALDI carrier plate and method for using same
CN204022796U (en) * 2014-05-19 2014-12-17 遵义市李龙基葡萄种植农民专业合作社 A kind of Domestic hand grape crusher
CN104792914A (en) * 2015-04-03 2015-07-22 广东医学院 Plane chromatography and micro-porous plate array mapping correlation experiment method and application thereof
CN105833932A (en) * 2016-05-17 2016-08-10 山西省交通科学研究院 Electronic waste shearing and breaking device
CN107155348A (en) * 2014-09-27 2017-09-12 特雷西-艾比利蒂有限公司 The system based on supporting plate for phenetic analysis thing
CN206764846U (en) * 2017-04-21 2017-12-19 武汉爱博泰克生物科技有限公司 Multifunctional adjustable grid knife

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101738466A (en) * 2008-11-13 2010-06-16 上海裕隆生物科技有限公司 Immunological detection integrated reaction box
CN102323429A (en) * 2011-08-17 2012-01-18 上海裕隆生物科技有限公司 Protein membrane chip
CN203350063U (en) * 2013-08-16 2013-12-18 天威新能源控股有限公司 Sampler for cross linking degree experiment of photovoltaic industry
CN104960006B (en) * 2015-06-04 2018-01-16 上海海洋大学 A kind of rubber tapping method of gas defence gel cutter
CN205889360U (en) * 2016-08-23 2017-01-18 郑州市中医院 Cutting device of traditional chinese medicinal materials

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6414306B1 (en) * 1999-08-07 2002-07-02 Bruker Daltonik Gmbh TLC/MALDI carrier plate and method for using same
CN204022796U (en) * 2014-05-19 2014-12-17 遵义市李龙基葡萄种植农民专业合作社 A kind of Domestic hand grape crusher
CN107155348A (en) * 2014-09-27 2017-09-12 特雷西-艾比利蒂有限公司 The system based on supporting plate for phenetic analysis thing
CN104792914A (en) * 2015-04-03 2015-07-22 广东医学院 Plane chromatography and micro-porous plate array mapping correlation experiment method and application thereof
CN105833932A (en) * 2016-05-17 2016-08-10 山西省交通科学研究院 Electronic waste shearing and breaking device
CN206764846U (en) * 2017-04-21 2017-12-19 武汉爱博泰克生物科技有限公司 Multifunctional adjustable grid knife

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Yuan Cheng等.A rapid, micro-scale preliminary screening method for activecomponents in Galangal with protective effect against hydrogenperoxide induced cell apoptosis through "thin layer chromatography"and "tetrazolium-based colorimetric assay" array correspondence.《Journal of Chromatography A》.2015,第1395卷第167～172页. *

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