CN118067667A - Molecular tension probe micropattern and preparation method and application thereof - Google Patents
Molecular tension probe micropattern and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular tension probe micropattern, a preparation method and application thereof, discloses a method for synchronously controlling cell morphology and detecting cell mechanics in batches, and belongs to the field of biological materials and technical methods. The method comprises the combination of two technologies, wherein the micropattern printing technology realizes cell confinement, and the other molecular tension fluorescence microscopy technology realizes measurement of cell mechanical force. The method comprises the following specific steps: 1) Ultraviolet lithography to prepare a mold to obtain soft template-Polydimethylsiloxane (PDMS); 2) PDMS amination modification gold nanoparticles; 3) And after the passivation substrate is contacted with the PDMS micro-contact to transfer the gold pattern, modifying SH-X-M molecules. The invention aims to solve the problem that the existing patterned cell mechanics measurement technology lacks molecular level information. The gold pattern in the invention has simple preparation flow and low cost; can also be coupled and combined with various molecular probes. The method researches the relation between mechanics and cell geometry from the view angle of molecular mechanical force for the first time, and can also realize the mechanical measurement of single cells by utilizing the high flux advantage of the pattern array.
Description
Technical Field
The invention relates to a method capable of synchronously controlling cell morphology and detecting cell mechanics in batches, belonging to the field of biological materials and technical methods.
Background
The in situ cellular microenvironment within an organ or tissue in the human body is complex in structure and plays a key role in determining cellular structure, mechanical properties, polarity and function. Among them, extracellular matrix (ECM) acts as a microenvironment directly acting on cells, whose physical information regulates various physiological functions of cells. For example, the geometric information of ECM is an important factor that determines the size and shape of cells and further regulates cell survival, proliferation, stem cell differentiation, and gene expression. However, in traditional cell culture environments, such as common petri dishes, cells develop randomly in an indeterminate geometry, which presents challenges in exploring the interactions between geometry and cell behavior.
To overcome this dilemma, researchers have used various single cell ECM micropatterning methods to control cell geometry and have studied in depth different aspects of cell physiology, including cell adhesion, assembly of the skeletal network of myosin, cell migration and differentiation. Given that cell geometry is largely affected by the scaffold, which in turn is regulated by the mechanical interactions mediated by integrins between cells and ECM, there has been great interest in combining cell mechanics measurement techniques with single cell ECM micropatterning techniques.
The existing micropattern mechanical measurement technology develops a cell traction measurement method of an integrated micropattern on the basis of protein microcontact printing, and the method is mainly three, namely a first elastic contractible surface technology (Fluorescent Labelled Elastomeric Sensor Surface,FLECS)(Pushkarsky I et al,Nat Biomed Eng,2018,2,124-137); based on membrane deformation and a second micropillar array technology based on micropillar deformation (Han SJ et al, biophys J,2012,103,640-648); a third traction microscopy technique based on bead displacement (Oakes et al, biophys J,2014,107,825-833). These techniques, while demonstrating the correlation between cell-ECM traction and ECM geometry and cell shape and size, are limited by optical resolution and microcolumn geometry, which can only achieve nanon/subnanon-level sensitivity and micron-level spatial resolution, and are difficult to provide on a molecular scale, the interaction between integrin-ligand forces and ECM geometry.
Therefore, development of a cell-patterned molecular mechanical measurement technique is needed to provide more molecular layer information.
Disclosure of Invention
The invention aims to solve the technical problem of realizing mechanical measurement of cell patterning on a molecular scale and provides a molecular tension probe micropattern and preparation and application thereof. The invention prepares the patterned molecular tension probe substrate for molecular mechanics research of the finite cell with the advantages of simplicity, convenience and high efficiency.
The technical scheme provided by the invention is as follows:
In a first aspect, the present invention provides a method for preparing a molecular tension probe micropattern, comprising the steps of:
(1) Preparing a silicon template with patterns;
(2) A soft template with patterns is obtained after PDMS is used for pouring;
(3) The soft template is connected with gold nano particles through amination modification to obtain a PDMS gold pattern;
(4) Preparing a PEG-passivated glass substrate;
(5) Carrying out micro-contact transfer on the PEG passivated glass substrate and the PDMS gold pattern to obtain a glass substrate with gold nanoparticle patterns;
(6) Modifying SH-X-M molecules on the gold pattern on the glass substrate; wherein SH is a sulfhydryl group, X is a molecular tensioning probe, and M is a ligand for interaction with cells to form a linkage.
Further, in the step (1), the silicon template with the pattern is prepared by an ultraviolet photoetching machine, and the pattern is a plane pattern with any shape.
Further, in the step (2), the patterned soft template includes a substrate and a cell structure arrayed thereon, wherein the patterned region is formed by relatively convex cell structures having a height difference.
Further, the unit structure is a columnar structure.
Further, in the step (3), the preparation method of the PDMS gold pattern is as follows: the soft template is firstly subjected to hydroxylation modification by plasma, amino is modified by chemical reaction of aminopropyl triethoxy silane on the basis of hydroxylation, and gold nanoparticles are adsorbed on an aminated PDMS pattern by non-covalent bonding of gold-nitrogen bonds.
Further, in the step (3), the plasma hydroxylation modification method comprises the following steps: the pattern face was placed face up in a plasma cleaner for hydroxylation. Preferably, the plasma cleaning agent comprises air and oxygen.
Further, in the step (3), the method for modifying the hydroxylated PDMS surface by amination comprises the following steps: and (3) immediately soaking the PDMS subjected to the plasma treatment into an ethanol solution provided with aminopropyl triethoxysilane for reaction, and cleaning and annealing to obtain the polyurethane.
Further, in the step (3), the method for modifying gold particles on the surface of the aminated PDMS comprises the following steps: the PDMS pattern is soaked in the gold nano solution face down and adsorbed overnight. Since the PDMS is modified with amino groups, gold particles can be adsorbed onto the pattern through gold-nitrogen bonds.
Further, in the step (4), the method for preparing the PEG-passivated glass substrate is as follows: the glass substrate is modified with hydroxyl groups by plasma, amino groups are modified with aminopropyl triethoxysilane, and then PEG molecules are attached to the amino groups.
Still further, the PEG molecules include mPEG-NHS and Lipoic acid PEG-NHS; wherein mPEG-NHS is used for resisting protein adhesion and Lipoic acid PEG-NHS is used for transferring gold nanoparticles by introducing dithiol.
Further, the method for modifying hydroxyl and amino groups of the glass substrate is consistent with the modification of the soft template.
Further, the molecular tensioning probes include at least one of reversible shear DNA molecular tensioning probes (Reversible SHEARING DNA-based Tension Probe, RSDTP), DNA hairpin structures (DNA HAIRPIN), molecular tether tensioning probes (Tension gauge tether, TGT), polyethylene glycol (Polyethylene Glycol, PEG) molecular tensioning probes; the ligand comprises one or a combination of several amino acids, polypeptide molecules and protein molecules which can form a connection with the cell. The ligand can be labeled correspondingly according to the target protein studied by a specific cell system. The sulfhydryl group in SH-X-M molecule is used for connecting gold nanoparticles.
Still further, in the ligand, the amino acid includes cysteine, lysine, and the like.
Further, among the ligands, proteins include fibronectin, collagen, antibodies, and the like.
Still further, in the ligand, the polypeptide includes Arg-Gly-Asn (RGD), pro-His-Ser-Arg-Asn (PHSRN), and the like.
Further, in the step (6), SH-X-M is prepared by the existing method:
when X is RSDTP, DNA HAIRPIN and TGT, the synthetic route is as follows:
Modifying the ligand M by click chemistry reaction of the molecular tension probe X with sulfhydryl, separating and purifying the final product by gel running, and annealing to obtain the final product;
Preferably, the preparation is carried out by the method referred to as Liu Zheng(Li,H.;Zhang,C.;Hu,Y.;Liu,P.;Sun,F.;Chen,W.;Zhang,X.;Ma,J.;Wang,W.;Wang,L.;Wu,P.;Liu,Z.Areversible shearing DNA probe for visualizing mechanically strong receptors in living cells.Nat.Cell Biol.2021,23(6),642-651.);
when X is PEG, the preparation is carried out by Liu(Liu,Yang et al.Tension sensing nanoparticles for mechano-imaging at the living/nonliving interface.JACS,2013,135,5320-5323.).
Further, in the step (5), the PDMS gold pattern is performed in a liquid environment during the micro-contact process with the glass substrate, so as to ensure that the micro-contact surface is wet. The gold particles are transferred to the glass by gold-sulfur covalent bonds that are more robust than gold-nitrogen bonds.
Still further, the liquid environment is ultrapure water.
In a second aspect, the present invention provides a molecular tensioning probe micropattern prepared by the method of the first aspect.
In a third aspect, the present invention provides an application of the molecular tension probe micropattern according to the second aspect in simultaneous batch cell morphology control and cell mechanics detection, the application method comprising: the cell receptor and the ligand on SH-X-M molecule are interacted to limit the cell to a pattern area in a specific shape, and then the mechanical detection is carried out through the fluorescent signal of the molecular tension probe.
Further, the applications include at least one of the following:
(1) Mechanical study between cell mechanical force and geometry;
(2) Providing a measurement platform for unifying cell molecular mechanical force;
(3) The high-flux screen looks up the pathological cells with abnormal mechanical force;
(4) Development of molecular mechanical biomaterials in the field of tissue engineering.
The invention provides a simple and convenient high-flux preparation method in the research of geometric shapes and cell mechanics. Firstly, the conventional gold pattern production needs to involve the combination of a plurality of ultraviolet lithography, thermal evaporation and other instruments, so that each sample is high in cost, and the sample preparation failure can be caused by errors in any production process, which is time-consuming and labor-consuming. In the invention, only chemical modification of PDMS is utilized for transfer, and the preparation of the flakes is relatively simple. After gold pattern transfer is successful, SH-X-M molecules can be patterned. The scientific research on patterning can be realized by designing a corresponding system according to the research content. Second, the invention can help researchers to understand the mechanism of action of geometry and cell mechanical force. The connection between mechanical mechanics and pattern shape is explored from the external and internal channels through the changes of the shape, the size, the curvature and other factors of the micropattern, the change of the cell membrane tension and the mechanical sensitive protein knockout. Finally, the high-throughput preparation advantage and unified measurement means provide a detection means and a guidance platform for molecular mechanics visual angles for detecting abnormal forces in disease cells and screening medicines. These examples demonstrate the good application potential of the present invention.
The method has the beneficial effects that:
1) The PDMS soft template used in the invention successfully realizes the preparation of gold nano patterns by a two-step modification method of hydroxyl and amino, and has simple and convenient operation and lower cost.
2) The micro-contact printing technology used in the invention transfers gold nano patterns, ensures the pattern domain limiting capability, provides probe connection sites for detecting cell mechanics, is easy to combine with various technologies, and has wider application prospect.
3) The molecular tension probes which can be modified by the gold pattern substrate are wide in types.
4) The molecular probe patterning technology prepared by the invention has the advantages that the mechanical measurement sensitivity is at the level of the Piniu, and the spatial sensitivity is at the micro-nano resolution.
Drawings
FIG. 1 is a schematic flow chart and method verification of pattern preparation in example 1 of the present invention. (a) specific experimental procedure for molecular tension probe patterning. (b) topography of an atomic force microscope of gold patterns. (c) Schematic of cell spreading on molecular tensioning probe pattern. (d) The fixed staining pattern of cells on the pattern spread is an image of 10, 20, 100 times objective lens in sequence from left to right. (e) Mechanical signal display of three cells 3T3, MEF, CAF on glass and pattern. (f) 17pN mean fluorescence signal intensity statistics of three cells 3T3, MEF, CAF on glass and pattern.
FIG. 2 shows the results of 56pN mechanical signals of MEF patterns with different shapes in different areas in example 2 of the present invention. (a, d, e) cell representative mechanical plots of circular, triangular, square micropatterns with areas 625, 900, 1225 and 1600 μm 2, wherein the first column is an actin fluorescent signal, the second column is a 56pN signal, the third column is a superposition of actin and 56pN, and the fourth column is an enlargement in the superposition. (b) Mean fluorescence signal intensity statistics for MEF at different areas and different shape patterns. (c) A statistical plot of the area of the plaque in different areas and different shaped patterns of MEF.
FIG. 3 is a graph showing the effect of pattern curvature on cell mechanics in example 3 of the present invention. (a) a mechanical signal result of the MEF at 56pN on an asymmetric pattern. The first column is RICM (reflection interference contrast microscope, reflection interference contrast microscopy, RICM), the second column is 56pN signal, and the third column is a superimposed plot of RICM and 56pN signal. (b) The MEF was based on 56pN mechanical signal results at the same area (1400 μm 2) for different curvature patterns. Wherein, the first column of the actin fluorescent signals, the second column of the actin fluorescent signals and 56pN signals, the third column of the actin fluorescent signals and 56pN fluorescent signals are overlapped, and the fourth column of the actin fluorescent signals and 56pN fluorescent signals are amplified in the overlapped graph. (c) the plaque area statistics of panel b. (d) a statistical plot of mean fluorescence signal intensity for plot b. (e) And dividing the e-graph representative mechanical signal into upper, middle and lower three equal division areas. (f) The ratio of the fluorescent signal intensity in the upper or lower region to the middle region is used to quantify the degree of signal non-uniformity.
FIG. 4 is a mechanical force source of the invention for exploring cell response pattern geometry in example 4. (a) Mechanical signal results after hypertonic treatment of MEF on patterns of different shapes of the same area (1225 μm 2). The first column is the GFP-tagged actin, the second column is the 56pN signal, and the third column is a superimposed plot of actin and 56 pN. (b) Mechanical signal results after hypotonic treatment of MEF on different shaped patterns of the same area (1225 μm 2). (c) statistics of the area of the plaque in a and b and control. (d) a and b, and a control group. (e) Mechanical signal results of MEF after Y27632, blebbistain, cyto D drug treatment. (f) After the MEF is subjected to hypertonic and hypotonic treatment for 30min, mechanical signal results after Y27632, blebbistain and Cyto D medicaments are added in situ.
FIG. 5 is a graph showing the effect of integrin subtypes α v and β 1 on the geometry of the pattern of cellular mechanical response in example 5 of the present invention. Schematic representation of the integrin subtype in modulating mechanical transduction. (b) Mechanical signal plot of WT MEF, alpha v -KO MEF and beta 1 -KO MEF after spreading for 1h on 56pN glass substrate. Statistical plot of cell spreading area in (c) b. A statistical plot of the mean fluorescence intensity of the cells in (d) b. (e) Immobilization of WT MEF, alpha v -KO MEF and beta 1 -KO MEF after 1h spreading on 56pN patterns of different shapes of the same area (1225 μm 2). (f) And e, a ratio graph of single cell confluence patterns. (g) WT MEF, alpha v -KO MEF and beta 1 -KO MEF 56pN mechanical signal plot on triangle pattern of large area (625 μm 2 and 1225 μm 2). (h) mean fluorescence signal intensity statistics of g plots. (i) WT MEF and beta 1 -KO MEF RICM on glass substrate. (j) WT MEF RICM before and after CK666 dosing. (k) Fixation results of CK666 treated WT MEFs on different shapes of 56pN patterns in the same area (1225 μm 2). (l) The duty cycle plot of the single cell confluence pattern in the k plot. (m) CK666 treated WT MEF 56pN mechanical signal plot on triangular patterns of different areas (625 μm 2 and 1225 μm 2).
FIG. 6 is a Western blot diagram of knock-out integrin subtypes α v and β 1 and the corresponding immobilization results in example 5 of the present invention.
FIG. 7 is a graph showing the effect of different types of probes on the pattern of mechanical response of cells in example 6 of the present invention. (a) Schematic of two different modes of probes 56pN RSDTP and 56pN TGT to regulate cell mechanical conduction. (b) The WT MEF was immobilized on patterns of different shapes over the same area (1225 μm 2) as modified by RSDTP and TGT probes. (c) a statistical plot of the duty cycle of the single cell confluent pattern in plot b.
FIG. 8 shows the structure of the probe used in examples 1 to 6 of the present invention.
Detailed Description
The following describes the specific implementation steps of the present invention with reference to the accompanying drawings, and the present invention is not limited thereto at all.
Example 1 preparation of DNA molecular tensioning Probe micropattern specific procedure and cytomechanical test
The manufacture of the silicon template needs to use ultraviolet lithography technology, and the specific flow can refer to the method Michel Bornens(Thery,M.;Pepin,A.;Dressaire,E.;Chen,Y.;Bornens,M.,Cell distribution of stress fibres in response to the geometry of the adhesive environment.Cell Motil.Cytoskeleton 2006,63(6),341-355.).
1. The specific process of ultraviolet lithography is as follows:
(1) Taking out the four-inch silicon wafer in an ultra-clean room, drying the silicon wafer in a hot plate at 100 ℃ for about 15 minutes, cooling the silicon wafer, and placing the silicon wafer in a spin coater for standby. The method aims at avoiding the consequences of glue stripping, poor adhesion and the like caused by direct gluing due to the moist environment for placing the silicon wafer.
(2) And placing the photoresist stored at the temperature of 4 ℃ at room temperature in advance, and preventing bubbles from being generated in the photoresist coating due to abrupt temperature change when the photoresist is just taken out in the process of coating. After a turntable slightly smaller than the silicon wafer is placed, spreading preservative films and the like in the spin coater in advance to prevent glue from adhering to the machine, and after the silicon wafer is placed on the turntable, firstly modulating the rotating speed of the spin coater after being sucked by a vacuum pump, and pre-running for several times, wherein the pre-rotating speed is 500rpm and 10s; the post rotation speed was 2000rpm,30s. The aim of the preset rotating speed is to uniformly spread the glue on the whole silicon wafer, and then the thickness of the glue is determined by the rotating speed. Here 2000rpm would produce a glue thickness of 40. Mu.m. Then 3mL of SU-2025 was pipetted carefully to drop from the center of the wafer, never touching the wafer. Because 2025 glue is extremely viscous, the suction effect of a dropper is hardly influenced by bubbles, and it is recommended that a bottle filled with glue is directly and slowly poured, and the dropper is used for assisting in recycling the residual glue.
(3) The pre-baking is carried out at 65 ℃ for 3min and at 95 ℃ for 6min. Cooled to room temperature. The purpose of this step is to reduce the solvent content in the glue and improve the stability of the adhesion of the glue to the silicon wafer.
(4) And (5) exposing. The first step is to first calibrate the positions of the reticle and wafer, and if necessary, align them with overlay marks. The contact ultraviolet lithography machine is provided by the micro-nano processing laboratory of the university of Wuhan university, the wavelength is 365nm, the light intensity is 4mW/cm 2, and the exposure time of 30s is adopted in the invention.
(5) Post-baking at 65deg.C for 1min and 95deg.C for 6min. The purpose is to fully complete the photochemical reaction of the photoresist by heating, and to make up the problem of insufficient exposure intensity. The micrometer pattern is visually observable during post-baking.
(6) And (5) developing. The SU8 special developer is PGMEA (propylene glycol monomethyl ether acetate), the cooled silicon wafer is immersed in the developer, a method of a small number of times of development, for example, 3×1min, is suggested, the development by immersing in isopropanol is terminated after 1min is finished, and then the development result is observed by a microscope after drying with nitrogen. In general, the fully developed film remains colorless and transparent when the isopropanol is terminated, but becomes white when the development is insufficient, and the film should be returned to development.
(7) Hardening at 155 ℃ for 20min. The purpose is to reduce the solvent in the photoresist and further improve the adhesion of the photoresist to the silicon wafer. The hardening can reduce lines generated by standing wave effect in development. Generally, the photoresist after hardening is not removed any more and is very firm.
2. The specific flow of the PDMS micro-contact printing transfer gold pattern is as follows:
(1) Preparing a soft template: mixing PDMS prepolymer with a curing agent according to the weight ratio of 10: mixing, stirring and degassing according to the mass ratio of 1, and finally pouring into the silicon template. Curing at 80 ℃ for more than 2 hours. The preferred PDMS is the dow corning DC184 brand.
(2) The soft template processing flow is as follows:
a. And (5) cleaning. The cured PDMS was carefully peeled from the silicon plate and the pattern cut to the appropriate dimensions, e.g., 25mm diameter glass as used in this experiment, typically cut to 20mm square size. Alternately cleaning with ethanol and pure water for more than 3 times, and drying and cooling.
B. Surface hydroxylation modification. The pattern is placed right side up in a plasma cleaning machine. Parameter modulation 30s.c.c.m,300mttor, time set to 3-5min. Preferably, the plasma cleaning agent is air or oxygen.
C. Surface amination modification. The plasma-treated PDMS was immediately immersed in an ethanol solution containing 3% aminopropyl triethoxysilane at room temperature for 1h. After three times of ethanol cleaning, annealing is carried out at 80 ℃ for 1h. Taking out and cooling to room temperature.
D. Gold particles adsorb. The PDMS pattern was immersed down in 10nM 13nm Au or 20nM 5nm gold nano-solution overnight at 4 ℃. Since the PDMS is modified with amino groups, gold particles can be adsorbed onto the pattern through gold-nitrogen bonds.
The size of gold is selected based on the selection of the experimental probe, and the need for a probe as a fluorescent indicator would require consideration of the NSET effect of gold. Considering that the common RSDTP probe length is in the range of 22-30nm, the NSET range of 5nm Au is in the range of 0-20 nm. For RSDTP probes, the signal-to-noise ratio of 5nm Au is higher.
(3) Preparation of PEG glass: the procedure for modifying the glass substrate to amino groups was the same as that described above for the PDMS procedure, and after the glass was removed from the oven and cooled to room temperature, a PEG solution (1% w/v mPEG 2K-NHS (MW 2000) &0.1% w/v Lipoic acid 3.4.4K-NHS (MW 3400), dissolved in 0.1M NaHCO 3) was prepared and 200. Mu.L was added dropwise to the glass at the midpoint. After overnight at 4 ℃ or 4 hours at room temperature, the product is cleaned by ultrapure water, and N 2 is dried and placed at 4 ℃ for subsequent experiments for standby.
(4) Pattern transfer: PDMS adsorbed with 13nm Au was dark red, while 5nm Au was pink. Excess gold particles were rinsed with ultrapure water, PDMS was attached to PEG glass and the interface was maintained in a liquid environment at 4 ℃ overnight. Since gold particles are attached to the glass by gold-sulfur covalent bonds that are more robust than gold-nitrogen bonds.
The patterned soft master includes a substrate and a cell structure arrayed thereon, wherein the patterned region is formed by a convex cell structure having a height difference. Preferably, the unit structure is a columnar structure.
Illustratively, the PDMS pattern is transferred by providing a protruding cell structure with a height difference of 40 μm. Furthermore, it is notable that although SU8-2007 series can produce smaller patterns due to the thin glue, the height difference of only 10 μm in film thickness can lead to gold pattern transfer failure in the subsequent step. Gold particles in the PDMS recesses in the liquid environment are also transferred to the glass substrate, so the thickness selection of SU8 photoresist is critical, and the photoresist thickness in this example is 40 μm; in addition, the liquid environment in imprint transfer is also critical. The water remaining in the PDMS, about 10 μl, provides not only capillary action in microcontacts, but also a liquid environment for gold particle transfer. In the experiment, we tried to blow-dry PDMS for gold transfer, and as a result, the experiment failed, further confirming the necessity of the existence of a liquid environment.
(5) And (5) incubating the probe. The next day patterns are gently taken, after pure water is cleaned, 5nM SH-RSDTP-RGD is dripped into the right center of the glass, and the specific structure of the probe is shown in figure 8. After 1h, the excess probe was washed with PBS and the cells were added for subsequent microscopic imaging experiments.
Specific schemes for the synthesis of the SH-RSDTP-RGD molecules can be found in the method of Liu Zheng(Li,H.;Zhang,C.;Hu,Y.;Liu,P.;Sun,F.;Chen,W.;Zhang,X.;Ma,J.;Wang,W.;Wang,L.;Wu,P.;Liu,Z.A reversible shearing DNA probe for visualizing mechanically strong receptors in living cells.Nat.Cell Biol.2021,23(6),642-651.). RGD is RGD sequence peptide.
3. And carrying out appearance characterization on the transferred gold pattern by an atomic force microscope.
The gold patterns of circles, triangles and squares can be clearly seen in fig. 1 (b). The measurement result of the cross-sectional height map was 5nm, which corresponds to the diameter of gold particles.
4. The method was tested for its domain limiting ability.
FIG. 1 (d) shows that most of the patterns are fully covered with single cells under a low power objective lens, the shapes of the cells are completely overlapped with the patterns, and the cell morphology can be clearly observed as a result of fixation staining (FIG. 1 (d)). The 100-fold objective acquisition is a mechanical signal plot of the MEF on a circular pattern.
5. The pattern was used to compare the mechanical differences between cells.
Fig. 1 (e) and 1 (f) can be seen: the three different types of cells, namely 3T3, MEF and CAF, are spread on the glass substrate without limitation, and the range of the difference of the average fluorescence intensity is large, so that comparison cannot be performed. The cells were uniformly seeded onto a circular pattern of equal area, with significant differences in cell mechanical force results. Wherein the average force of CAF is greater than MEF,3T3 minimum.
Example 2 molecular tensioning Probe micropatterning techniques for studying the correlation between cytokinin ligand forces and geometry
Gold patterns of different shapes and areas (625, 900, 1225 and 1600 μm 2) were prepared according to example 1, washed with ultrapure water, and after about 50. Mu.L (5 nM) of 56pN reversible shearing DNA molecular tension probe (Reversible SHEARING DNA-based Tension Probe, RSDTP) was dropped in the center of the glass substrate and incubated for about 1 hour, washed with PBS and immersed for use. GFP-Actin MEF was centrifuged by pancreatin digestion, resuspended in DMEM containing 10% fetal calf serum and 100U/mL penicillin-streptomycin to give a suspension containing about 1X 10 6 cells per mL, PBS was removed from the patterned substrate and replaced with 1mL of cell suspension, and the suspension was incubated in a 5% CO 2 incubator at 37℃for 1 hour and then collected by total internal reflection fluorescence microscopy (TIRF). As shown in FIG. 2, the 56pN signal density decreases as the micropattern area increases. Compared with a circular pattern, the cell force signals are isotropic, and the triangular and square patterns are distributed by vertexes, which shows that the cells can sense the geometric shape and the area so as to regulate the size and the distribution of the mechanical force.
Example 3 molecular tension probe micropatterning techniques for studying the modulation of the local curvature of micropatterns on the magnitude and distribution of cytokinin forces
An asymmetric gold pattern and gold patterns with different curvatures of the same area (1400 μm 2) were prepared as described in reference to example 1, and were washed with ultrapure water, and after incubation for about 1 hour with about 50 μl (5 nM) of 56p N RSDTP dropwise at the center of the glass substrate, washed with PBS, and immersed in PBS for use. Next, GFP-Actin MEF was centrifuged by pancreatin digestion, resuspended in DMEM containing 10% fetal calf serum and 100U/mL penicillin-streptomycin to prepare a suspension containing about 1X 10 6 cells per mL, PBS in the standby pattern substrate was removed and replaced with 1mL of cell suspension, and the suspension was incubated in a 5% CO 2 incubator at 37℃for 1 hour and used for TIRF imaging.
As can be seen from fig. 3: cells still present a typical distribution of vertices on an asymmetric pattern, and it is seen that cells can recognize the curvature of the pattern, which process is locally responsive. Comparing the results on the patterns of different curvatures of the same area, it was found that the mechanical force of the cells was more concentrated at the areas of large curvature, and that the results of both the focal adhesion and the average fluorescence signal revealed a complex series of responses of the cells to local curvatures, thereby adjusting the spatial distribution and magnitude of the mechanical force.
Example 4 molecular tensioning Probe micropatterning techniques were used to study the effects of membrane tensioning and integrin-mediated molecular clutches on cell response geometry
Gold patterns of the same area (1225 μm 2) of different shapes were prepared in accordance with example 1, washed with ultrapure water, incubated for about 1 hour by dropping about 50. Mu.L (5 nM) 56pN RSDTP in the center of the glass substrate, washed with PBS, and immersed in PBS for use. Next, GFP-actin MEF was centrifuged by pancreatin digestion, resuspended in DMEM containing 10% fetal calf serum and 100U/mL penicillin-streptomycin to prepare a suspension containing about 1X 10 6 cells per mL, PBS in the standby pattern substrate was removed and replaced with 1mL of cell suspension, and the suspension was cultured in a 5% CO 2 incubator at 37℃for 1 hour and used for TIRF imaging.
Wherein the hypotonic treatment step comprises: after GFP-actin MEF was planted on the micropatterned substrate for 1h, the culture broth was removed by pipetting and replaced with 0.5xPBS solution, and after 30min incubation, was used for TIRF imaging.
Wherein the hypertonic treatment step comprises: after GFP-actin MEF was grown on the micropatterned substrate for 1h, the culture broth was removed by pipetting and replaced with 100mM sucrose solution and incubated for 30min for TIRF imaging.
Wherein the drug treatment step: after GFP-actin MEF was grown on the micropatterned substrate for 1h, 2. Mu.M Y27632, or 2. Mu.M Blebtistatin, or 0.2. Mu.M Cyto D was added to the medium.
As shown in FIG. 4, the method finds that when hypotonic treatment is carried out, the cell membrane tension is increased, but the mechanical signal distribution is not changed obviously; when the tension of the cell membrane is reduced by the hypertonic treatment, the mechanical signal distribution is obviously changed, which indicates that the shape of the pattern of the cell mechanical force recognition response is regulated by the membrane tension. Furthermore, since there are two sources of integrin-mediated mechanical forces, the first is membrane tension and the other is myosin-regulated tension. The addition of Y27632, blebb statin and Cyto D can inhibit the tension regulated by myosin, and the cells on the pattern are treated by the medicines, so that the strength of the force signal is greatly weakened and distributed along the edge of the pattern, similar to the ring signal on the glass substrate. It was demonstrated that the shape of the pattern of cell mechanical force recognition was also affected by myosin-mediated mechanical forces. To investigate if there is a correlation between the effects of both mechanical forces, the above drugs inhibiting myosin were added in situ in hypertonic/hypotonic treated cells. The results show that under hypertonic conditions, the decrease in mysoin activity results in a reduced unevenly distributed force signal, but the spatial distribution is not affected; under hypotonic conditions, a decrease in mysoin activity will cause the peak distribution force signal to be greatly diminished or even completely diminished. This result suggests that membrane tension and myosin mediated mechanical forces are mechanical forces and spatial distributions that affect cell recognition pattern shape through independent channels.
Example 5 molecular tension probe micropatterning techniques for studying the role of two subtypes α v and β 1 integrins in identifying micropattern geometry information
1. A knockout cell line was constructed. Both alpha v -KO MEF and beta 1 -KO MEF cell lines were knocked out and constructed using CRISPR/Cas9 technology. Wherein the DNA sequence is (a v used sequence: 5'-tggagtttaagtcccaccag-3'; a 1 used sequence: 5'-aatgtcaccaatcgcagcaa-3'). The Western blot results are shown in FIG. 6.
2. Gold patterns of different shapes and two areas (625 and 1225 μm 2) were prepared according to example 1, cleaned with ultrapure water, and after incubation for about 1 hour with about 50. Mu.L (5 nM) 56pN RSDTP dropwise to the midpoint of the glass substrate, the glass substrate was rinsed with PBS and immersed in the solution for use. Then, after subjecting to respective pancreatin digestion and centrifugation of α v -KO MEF and β 1 -KO MEF, the cells were resuspended in DMEM containing 10% fetal bovine serum and 100U/mL penicillin-streptomycin to prepare a suspension containing about 1×10 6 cells per mL, PBS was removed from the patterned substrate and replaced with 1mL of cell suspension, and the suspension was cultured in a 5% co 2 incubator at 37 ℃ for 1 hour and then used for TIRF imaging.
As shown in fig. 5, α v and β 1 are important two integrins involved in mechanical force, and when knocked out, a significant decrease in force signal was emitted from the glass substrate, confirming that both integrins are involved in mechanical force transmission. Second, when α v and β 1 are knocked out, from the fixed result of the large field of view, about 53% of the α v -KO MEFs can span the entire shape, slightly below the WT MEFs (75%). However, only 11% of the β 1 -KO MEFs are pattern-able, with the vast majority of cells maintaining a smaller spread. This is in clear contrast to the results on planar substrates, and it can be seen that integrin subtypes play an important role in pattern shape recognition, with the knockdown of β 1 having a greater impact on pattern recognition than α v. In a few cells capable of confluent patterns, β 1 -KO MEF exhibited a significantly different distribution than WT MEF and α v -KO MEF. The mechanical hot spot distribution not only presents vertex distribution, but also distributes a large amount of force signals in other areas such as the center and the edge of the pattern, which is rarely found in WT MEFs and αv-KO MEFs. Second, mechanical statistics of 56pN showed that fluorescence signal intensity after β 1 knockout was positively correlated with spreading area, which is quite opposite to WT MEF phenomenon. The knock-out of beta 1 not only changes the mechanical space distribution, but also changes the relation between the force signal and the spreading area. It has been demonstrated that Arp2/3 plays a critical role in β 1 integrin-mediated mechanical transduction. CK666 is an inhibitor of the Arp2/3 complex and can block the activity of Arp2/3 to inhibit actin branch formation. After CK666 drug treatment of WT MEFs, it was found that there was a significant reduction in plate-like pseudopodia, consistent with the small plate-like pseudopodia per se in beta 1 -KO MEFs cells. The pattern fixation results and statistical graphs of the large field CK666 treated WT MEFs revealed that inhibition of Arp2/3 did affect cell recognition of the pattern. Furthermore, the uneven distribution of force signals further demonstrates that Arp2/3 belongs to the downstream key protein of β 1.
The invention demonstrates that beta 1 plays a more critical role in coordinating mechanical signal recognition micropatterns than alpha v by knockout of critical proteins inside cells.
Example 6 action of mechanical hotspots in cell recognition patterns
The gold pattern of the present invention can be used in theory with a variety of molecular tension probes. The results for different types of probes from TGT and RSDTP are shown here, as shown in FIG. 7.
The probe will break when the TGT reaches a threshold force and the force transmission will terminate. RSDTP due to the hairpin structure, the probe will not break and the signal will be transmitted continuously. To this end, the present invention compares the response of these two probes on the cell recognition pattern. From the fixed staining large field results, the proportion of cells in the TGT pattern to be spread out over the whole pattern was only 25.7%, whereas RSDTP% of cells in the pattern could be spread out over a single pattern. . This shows that the mechanical hot spot plays an important role in cell recognition patterns, and when the force signal is terminated, the adhesion behavior of the cells is broken, and no stable mechanical anchor point exists, so that the recognition of the cells to the patterns is affected.
The present invention is not limited to the above-mentioned embodiments, but any modifications, equivalents, improvements and modifications within the scope of the invention will be apparent to those skilled in the art.
Claims (10)
1. A preparation method of a molecular tension probe micropattern is characterized by comprising the following steps: the method comprises the following steps:
(1) Preparing a silicon template with patterns;
(2) A soft template with patterns is obtained after PDMS is used for pouring;
(3) The soft template is connected with gold nano particles through amination modification to obtain a PDMS gold pattern;
(4) Preparing a PEG-passivated glass substrate;
(5) Carrying out micro-contact transfer on the PEG passivated glass substrate and the PDMS gold pattern to obtain a glass substrate with gold nanoparticle patterns;
(6) Modifying SH-X-M molecules on the gold pattern on the glass substrate; wherein SH is a sulfhydryl group, X is a molecular tensioning probe, and M is a ligand for interaction with cells to form a linkage.
2. The method for preparing a molecular tension probe micropattern according to claim 1, wherein: in the step (2), the patterned soft master includes a substrate and a cell structure arrayed thereon, wherein the patterned region is formed by relatively convex cell structures having a height difference.
3. The method for preparing a molecular tension probe micropattern according to claim 1, wherein: in the step (3), the preparation method of the PDMS gold pattern is as follows: the soft template is firstly subjected to hydroxylation modification by plasma, amino is modified by chemical reaction of aminopropyl triethoxysilane, and then gold nanoparticles are adsorbed on the aminated PDMS pattern by non-covalent bonding of gold-nitrogen bonds.
4. The method for preparing a molecular tension probe micropattern according to claim 1, wherein: in the step (4), the method for preparing the PEG-passivated glass substrate is as follows: the glass substrate is modified with hydroxyl groups by plasma, amino groups are modified with aminopropyl triethoxysilane, and then PEG molecules are attached to the amino groups.
5. The method for preparing a molecular tension probe micropattern according to claim 4, wherein: the PEG molecules include mPEG-NHS and Lipoic acid PEG-NHS; wherein mPEG-NHS is used for resisting protein adhesion and Lipoic acid PEG-NHS is used for transferring gold nanoparticles by introducing dithiol.
6. The method for preparing a molecular tension probe micropattern according to claim 1, wherein: the molecular tensioning probes comprise at least one of RSDTP, hairpin, TGT, PEG molecular tensioning probes; the ligand comprises one or a combination of several amino acids, polypeptide molecules and protein molecules which can form a connection with the cell.
7. The method for preparing a molecular tension probe micropattern according to claim 1, wherein: in the step (5), the PDMS gold pattern is carried out in a liquid environment in the micro-contact process with the glass substrate, so that the wetting of the micro-contact surface is ensured.
8. A molecular tension probe micropattern characterized in that: a method according to any one of claims 1 to 7.
9. Use of a molecular tension probe micropattern according to claim 8 for simultaneous batch cell morphology control and cell mechanics detection, wherein the method of use is: the cell receptor and the ligand on SH-X-M molecule are interacted to limit the cell to a pattern area in a specific shape, and then the mechanical detection is carried out through the fluorescent signal of the molecular tension probe.
10. Use of molecular tension probe micropatterns in simultaneous bulk cell morphology control and cell mechanics detection according to claim 9, wherein the use comprises at least one of the following:
(1) Mechanical study between cell mechanical force and geometry;
(2) Providing a measurement platform for unifying cell molecular mechanical force;
(3) The high-flux screen looks up the pathological cells with abnormal mechanical force;
(4) Development of molecular mechanical biomaterials in the field of tissue engineering.
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