CN113960011B - Detection method for local biochemical environment of regulated cell based on Raman spectrum - Google Patents
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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Abstract
The invention discloses a detection method for a local biochemical environment of a regulated cell based on Raman spectrum, which comprises the steps of dropwise adding a gold nanorod solution on the surface of a micro-nano structure, so that the gold nanorod is self-assembled in the micro-nano structure to obtain a template for single cell regulation and detection integration; modifying the gold nanorod assembly of the template; taking the modified template as a substrate for cell growth, and sowing the adherent cells on the substrate, so as to obtain single cells with specific cell morphology and growth direction; and detecting the Raman spectrum of the obtained single cell to obtain data of a local biochemical microenvironment, and aligning the light spot of the incident laser to a specific single cell or a specific part of the single cell when detecting the Raman spectrum of the detected cell. The detection method integrates cell growth regulation and cell local part detection, realizes high-flux, high-sensitivity and low-damage integrated single-cell detection on the sample, and has important significance for revealing the inherent physiological change of cell regulation in real time on line.
Description
Technical Field
The invention relates to a method for detecting regulated cells, in particular to a method for regulating the growth of cells and carrying out Raman detection on specific sites of regulated cells through a micro-nano structure combined with a self-assembled gold nanorod.
Background
Micro-nano structures with different morphologies can affect the behavior and state of cells. For example, cells are more likely to adhere to and grow on a rougher micro-nano structured surface, while micro-nano scale anisotropic structural features can orient cells to grow, alter the cytoskeletal structure of cells and induce cell differentiation. The growth and behavior of the cells regulated by the micro-nano structure play an important role in tissue engineering and clinical application, and with the development of micro-nano manufacturing technology, the regulation of the cells based on the micro-nano structure has gradually become one of the hot fields. In the process of regulating and controlling the cells, the surrounding biochemical microenvironment can be changed, and the exploration of the cell functions and the prediction of the cell fate can be realized by detecting the local biochemical environment of the cells, such as the pH value. For example, tumor cells have lower extracellular pH than normal cells, and lower pH favors migration and invasion behavior of the cells. However, there is generally no in-depth study of the change in the local biochemical microenvironment of the cells being modulated.
The means for detecting the regulated cells is usually to observe the change of the external appearance of the cells by microscopic imaging, or to perform fluorescent imaging by using biomolecules such as fluorescent labeled specific proteins, or to puncture the cells by using microneedles or the like to extract the internal components thereof. The methods have a plurality of limitations in quantitative detection of the local biochemical environment of the regulated cells, such as the fact that the fluorescence spectrum is wider and the spectrum overlapping phenomenon is easy to generate, so that two molecules with overlapped fluorescence spectrums are difficult to distinguish, and the phenomena of photobleaching, light quenching and the like also influence the accuracy of quantitative detection based on the fluorescence intensity. The method of puncturing the cells to extract the internal components of the cells has great destructiveness on the cells, so that the real-time dynamic detection of the cells is difficult to realize, and the local biochemical environment of the cells can be changed due to stress response when the cells are damaged, thereby influencing the accuracy of measurement. Thus, although many documents have reported cell regulation based on micro-nano structures, it is difficult to realize real-time dynamic detection of the local biochemical microenvironment of the regulated cells after the micro-nano structures regulate the cells.
In order to realize the real-time dynamic detection of the regulated single-cell local biochemical microenvironment, a detection method which is nondestructive to cells, convenient to operate and high in sensitivity is particularly important. The detection method based on Raman spectrum has small damage to the sample and small required sample size, and can provide abundant chemical component information, but the application of Raman detection is limited due to weak signal and low detection sensitivity. However, when laser irradiates on a rough metal surface, the raman scattering signal is enhanced by tens of thousands or even hundreds of millions times due to the enhancement of a local electromagnetic field, the generation of charge transfer and other phenomena, the surface enhanced raman scattering is realized, and the sensitivity and the signal-to-noise ratio of raman detection are greatly improved. Because of the complex components inside and outside the cell and low content, the surface enhanced Raman scattering with high sensitivity, small damage to the sample and rich information is an effective means for realizing living cell detection.
In order to obtain a surface enhanced raman scattering substrate with high sensitivity and high reproducibility, i.e. a roughened metal surface with a uniform structure, there are generally top-down and bottom-up methods. The top-down method refers to that the micro-nano structure is obtained by removing part of materials to reduce the larger size to the required size, and comprises photoetching, wet etching, electron beam etching, nano-imprint etching and the like. The top-down method is advantageous for forming ordered patterns on the micrometer scale, but has difficulty in constructing nano-scale or arbitrary three-dimensional structures. The bottom-up method refers to the assembly of molecular or atomic components into micro-nanostructures, such as self-assembly, chemical vapor deposition, atomic layer deposition, and the like. Among them, the self-assembly method has received a lot of attention because it does not require complicated process flow and expensive equipment, and it is cost-effective with low costs for raw materials. In order to realize self-assembly of the cash nanorods, common methods include solvent evaporation self-assembly, organic solvent assisted self-assembly, template assisted self-assembly, and the like. The solvent evaporation speed at the three-phase interface of the substrate, air and water is faster than that at the center of the liquid drop, and the concentration of the gold nanorods is higher, so that the gold nanorod self-assembly obtained by the solvent evaporation method has a coffee ring effect, and the uniformity and the repeatability of the gold nanorod self-assembly serving as a cell culture and Raman detection substrate are limited. The solvent evaporation speed is reduced by controlling the conditions of solvent evaporation, such as temperature, humidity, substrate hydrophilicity and hydrophobicity, so that the gold nanorod concentration at different positions of the liquid drop is kept similar, and the assembly effect can be improved.
Disclosure of Invention
Aiming at the prior art, the invention provides a detection method for the local biochemical environment of the regulated cells based on Raman spectrum, realizes high-flux and high-sensitivity integrated single-cell detection for low damage of samples, and has important significance for revealing the internal physiological change of cell regulation in real time on line.
In order to solve the technical problems, the invention provides a detection method for a local biochemical environment of a regulated cell based on Raman spectrum, which comprises the following steps:
step 1) dropwise adding a gold nanorod solution on the surface of a micro-nano structure, and controlling the temperature and humidity conditions during solvent evaporation to enable self-assembly of the gold nanorod in the micro-nano structure to prepare a template for single-cell regulation and detection integration;
and 2) modifying the gold nanorod assembly of the template according to different detected indexes to obtain a modified template, wherein the modification comprises modification of one of the following organic matters on the gold nanorod assembly:
a) The gold nanorod assembly is modified with 4-mercaptopyridine for single-cell local pH detection or single-cell specific part pH detection;
b) Modifying 1-thiodecane on the gold nanorod assembly for detecting single-cell glucose;
c) Modifying dopamine aptamer on the gold nanorod assembly for single-cell dopamine detection;
d) Modifying a DNA single strand on the gold nanorod assembly for RNA detection in base pairing with the DNA single strand;
step 3) taking the modified template obtained in the step 2) as a substrate for cell growth, sowing the adherent cells on the substrate, utilizing the hydrophilicity of the gold nanorods to enable the cells to specifically grow on the micro-nano structure, and regulating and controlling the growth of the adherent cells through the morphology structure on the surface of the substrate so as to obtain single cells with specific cell morphology and growth direction;
and 4) detecting the Raman spectrum of the single cell obtained in the step 3) to obtain data of a local biochemical microenvironment, and aligning the light spot of the incident laser to a specific single cell or a specific part of the single cell when detecting the Raman spectrum of the detected cell.
Furthermore, in the detection method for the local biochemical environment of the regulated cell based on the Raman spectrum, in the step 1), the preparation of the micro-nano structure comprises one of the following methods:
a) A silicon template having a micron-sized structure obtained by photolithography;
b) Obtaining a silicon template with a micron-sized structure through photoetching; then, the flexible template is obtained after the silicon template is subjected to reverse molding through Polydimethylsiloxane (PDMS);
c) Firstly, obtaining a silicon template with a micron-sized structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template; and then attaching the flexible template to the surface of a glass slide to bond the polydimethylsiloxane and the glass, and after the flexible template is peeled off, the polydimethylsiloxane structure with the nanoscale thickness is remained on the surface of the glass slide to obtain the hydrophilic-hydrophobic interphase micro-nano structure.
Compared with the prior art, the invention has the beneficial effects that:
the invention integrates cell growth regulation and control with cell local part detection, and realizes high-flux, high-sensitivity and low-damage integrated single cell detection on samples. The invention firstly realizes controllable self-assembly of the gold nanorods by constructing an interface micro-nano structure, ensures that cells are specifically combined on the surfaces of the gold nanorods, and realizes regulation and control of directional growth of the cells by utilizing the micro-nano structure. And then, the integration of cell regulation and online detection is realized by utilizing the Surface Enhanced Raman Scattering (SERS) effect of gold nanorod directional assembly. Meanwhile, the Raman spectrum of the cell contains characteristic peaks reflecting the pH value change, so that the detection of the pH value of the specific position of the regulated single cell is realized through the prepared platform. The invention has important significance for revealing the internal physiological change of cell regulation in real time on line.
Drawings
Fig. 1 is a schematic diagram of a micro-scale structure template for single-cell regulation and detection integration, wherein (a) is a schematic diagram of a flexible template obtained by using Polydimethylsiloxane (PDMS) to perform reverse molding, and (b) is a schematic diagram of a micro-nano structure in which PDMS is bonded with glass and then peeled off, so that the surface of the glass is hydrophilic-hydrophobic phase.
FIG. 2 is a schematic illustration of assembly of gold nanorods by modification and functionalization of templates with Raman indicators.
Fig. 3 is a scanning electron microscope image of the cell control and detection integrated platform, wherein (a) is a grid structure and (b) is a circular array structure.
FIG. 4 is a surface enhanced Raman spectrum of 4-mercaptopyridine (4-MPy) at different pH.
Fig. 5 is a scanning electron microscope image of cell growth regulated using the integrated platform.
FIG. 6 is a schematic diagram of a local pH assay for different cell specific locations using the integrated platform.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and specific examples, which are in no way limiting.
The growth, differentiation, apoptosis, canceration and other processes of the cells can be influenced by the surrounding biochemical environment and physical environment, wherein the influence of the topographic factors on the adherent cells can be realized by designing cell culture substrates with different morphological structures. The hardness, roughness and surface morphology anisotropy of the cell culture substrate have regulatory effects on cell adhesion, cell directional growth and cell differentiation. The study of the intrinsic physiological changes of the regulated cells, such as changes in pH, proteins and DNA, reveals the intrinsic mechanisms of the change in the growth conditions of the cells. According to the invention, the integration of cell regulation and control and cell detection is realized by designing a platform combining the gold nanorods and the micro-nano structure. The assembled gold nanorods generate a plasma resonance effect on the surface under laser irradiation, so that the Raman signal is improved by tens of thousands or even hundreds of millions, and the obtained surface enhanced Raman spectrum has high sensitivity and signal-to-noise ratio. The cell regulation and detection platform can realize the detection of the cell pH value by modifying the organic 4-mercaptopyridine with the Raman characteristic peak changing along with the pH. Because the diameter of the laser spot is only 0.9 micron when Raman spectrum detection is carried out, the laser spot is aligned to the regulated cell, so that the local pH value of the specific cell can be detected, and the regulated different cells and the local pH difference of different parts of the cell can be compared.
The design idea of the detection method of the local biochemical environment of the regulated cells based on the Raman spectrum is as follows: the gold nanorods are modified on templates of different micro-nano structures, and the hydrophilicity of the gold nanorods is utilized to enable cells to specifically grow on the micro-nano structures, so that the growth of adherent cells can be regulated and controlled through the morphological structure difference of the substrate surface. Meanwhile, for the cells with regulated growth, the pH detection of specific parts of specific cells is realized by utilizing the Raman enhancement effect. The specific steps of the detection are as follows:
step 1) preparing a template for single cell regulation and detection integration, wherein the process is as follows:
first, a micro-nanostructure is prepared according to one of the following methods:
a) A silicon template having a micron-sized structure obtained by photolithography;
b) Obtaining a silicon template with a micron-sized structure through photoetching; then, the flexible template is obtained after the silicon template is subjected to reverse molding through Polydimethylsiloxane (PDMS);
c) Firstly, obtaining a silicon template with a micron-sized structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template; and then attaching the flexible template to the surface of a glass slide to bond the polydimethylsiloxane and the glass, and after the flexible template is peeled off, the polydimethylsiloxane structure with the nanoscale thickness is remained on the surface of the glass slide to obtain the hydrophilic-hydrophobic interphase micro-nano structure.
Then, dropwise adding a gold nanorod solution on the surface of the micro-nano structure, and controlling the temperature and humidity conditions during solvent evaporation to enable the gold nanorod to self-assemble in the micro-nano structure so as to prepare a template for single-cell regulation and detection integration;
and 2) modifying the gold nanorod assembly of the template according to different detected indexes to obtain a modified template, wherein the modification comprises modification of one of the following organic matters on the gold nanorod assembly:
a) The gold nanorod assembly is modified with 4-mercaptopyridine for single-cell local pH detection or single-cell specific part pH detection;
b) Modifying 1-thiodecane on the gold nanorod assembly for detecting single-cell glucose;
c) Modifying dopamine aptamer on the gold nanorod assembly for single-cell dopamine detection;
d) Modifying a DNA single strand on the gold nanorod assembly for RNA detection in base pairing with the DNA single strand;
step 3) taking the modified template obtained in the step 2) as a substrate for cell growth, sowing the adherent cells on the substrate, utilizing the hydrophilicity of the gold nanorods to enable the cells to specifically grow on the micro-nano structure, and regulating and controlling the growth of the adherent cells through the morphology structure on the surface of the substrate so as to obtain single cells with specific cell morphology and growth direction;
and 4) detecting the Raman spectrum of the single cell obtained in the step 3) to obtain data of a local biochemical microenvironment, and aligning the light spot of the incident laser to a specific single cell or a specific part of the single cell when detecting the Raman spectrum of the detected cell.
Examples:
and manufacturing a template with a micron-sized morphology structure. And obtaining the silicon template with the micron-sized morphology structure by utilizing photoetching. A flexible template with elasticity and easy deformation is obtained by a Polydimethylsiloxane (PDMS) reverse mould. And bonding the PDMS with the glass, and then peeling the PDMS, so that a nano-scale micro-nano structure can be remained on the surface of the glass. As shown in fig. 1, wherein (a) is a schematic diagram of obtaining a flexible template by using Polydimethylsiloxane (PDMS) inverse mold, and (b) is a schematic diagram of bonding PDMS to glass and then peeling the PDMS off, so that the surface of the glass obtains a hydrophilic-hydrophobic phase-to-phase micro-nano structure. In the figure, 1 is a silicon template having a micro-nano structure obtained by photolithography, 2 is Polydimethylsiloxane (PDMS), and 3 is glass.
The template is functionalized by modifying gold nanorods and raman-indicating factors. And (3) dropwise adding the gold nanorod solution onto a template with a micrometer structure, utilizing a structure with hydrophilic and hydrophobic phases at the interface of the template, and directionally assembling a layer of gold nanorod on the surface of the micrometer structure by adjusting conditions such as temperature, humidity and the like when a solvent is evaporated, so as to obtain a substrate with a Raman enhancement effect. Then the substrate is soaked in a pH sensitive 4-mercaptopyridine (4-MPy) ethanol solution, and 4-MPy is modified on the gold nanorods through gold sulfide bonds, so that the Raman enhanced substrate has a pH detection function. Fig. 2 is a schematic diagram of assembly of gold nanorods and functionalization of a template by modifying a raman indicator, wherein 4 is a PDMS flexible template, 5 is a glass template with a surface modified with a micro-nano structure with hydrophilic and hydrophobic phases, 6 is a gold nanorod solution, 7 is a gold nanorod self-assembled array, and 8 is a pH-sensitive raman indicator molecule 4-mercaptopyridine (4-MPy).
Regulating the growth process of the cells. The functionalized different templates are used as cell culture substrates, cells are sown on the surfaces of the templates, and after the cells are grown by adherence, the stretching and migration of the cells can be influenced by the structure of the growth substrates, so that the cell culture substrates have the characteristics of different morphological characteristics and directional growth. FIG. 3 is a scanning electron microscope image of a template integrating cell control and detection, wherein (a) is a grid structure and (b) is a circular array structure. The silicon template is obtained by photoetching through designing different masks, and then various cell regulation and detection integrated platforms with different shapes and sizes can be obtained through the manufacturing methods in fig. 1 and 2.
Local pH detection of specific cells. The Raman spectrum of the cells growing on the gold nanorod assembly is greatly enhanced by the surface enhanced Raman scattering effect of the gold nanorod self-assembly, so that the Raman spectrum of the cells growing on the assembly can be enhanced by tens of thousands times. After the gold nanorod assembly is modified with 4-mercaptopyridine, the difference of the Raman characteristic peaks is related to the change of the pH value, and the Raman spectrum of the cell has the characteristic peaks reflecting the change of the pH value, so that the change of the surrounding local pH value, namely the change of the pH value of the cell growing on the gold nanorod assembly, can be reflected by detecting the Raman spectrum. Because the diameter of the laser spot is only 0.9 micron during Raman detection, the laser spot is aligned to a specific cell, and the local pH detection of the specific cell can be realized. For specific cells whose growth is regulated by the template structure, detecting raman spectrum differences at specific locations (e.g., nuclei, cytoplasm, cell pseudopodia, junctions between multicellular) can reflect local pH changes at different locations of the regulated cells.
The process of detecting a specific part of a cell is as follows: when the Raman spectrum of the regulated cell is detected, the light spot of the incident laser is aligned to a specific cell or a specific position of the cell, so that the detection of a specific site of the cell is realized.
FIG. 4 is a surface enhanced Raman spectrum of 4-mercaptopyridine (4-MPy) at different pH. 4-MPy pyridine protein ring at 1006cm -1 Characteristic peaks at 1100cm with c=s bonds -1 The characteristic peak at the point is related to the pH value of the surrounding environment, and is 1006cm -1 The characteristic peak intensity at the site attributes the raman spectrumAfter normalization, it can be seen that with increasing pH, 1100cm -1 The characteristic peak intensity at the point gradually increases. Therefore, the 4-MPy is modified on the microstructure template with gold nanorod assembly, so that the template has the capability of detecting the pH value of the surrounding environment.
FIG. 5 is a scanning electron microscope image of a template-mediated cell growth using single cell modulation and detection integration. The round assembly diameter of the gold nanorods in the figure is 20 μm, and the distance between the two circles is also 20 μm. HeLa cells are sown on the micro-nano structure, and the cells are in an extending state which spans a plurality of small circles and a circular state which only grows on one small circle as the adherent growth of the cells is regulated and controlled by the shape of the substrate. And the cells have a tendency to grow directionally, more toward the small circles of gold nanorods than the surrounding gaps.
FIG. 6 is a schematic diagram of detection of the local biochemical environment of a regulated cell based on surface enhanced Raman scattering spectroscopy.
In the figure, 9 is a PDMS film with a micrometer structure, 10 is a gold nanorod circular assembly, 11 is a cell growing on the gold nanorod assembly, the cell is subjected to the regulation and control action of a growth substrate structure, 12 is a cell nucleus, and 13 is a laser with the wavelength of 785 nm. The laser light spot is aligned to a specific part of a specific cell, so that the Raman detection of the position can be realized, and the local pH value of the position is reflected by Raman spectrum. By comparing the local pH values at the cytoplasm, nucleus of round cells with expanded cells, changes in the internal physiological environment of the cell being regulated can be revealed.
Although the invention has been described above with reference to the accompanying drawings, the invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many modifications may be made by those of ordinary skill in the art without departing from the spirit of the invention, which fall within the protection of the invention.
Claims (2)
1. A detection method for the local biochemical environment of a regulated cell based on Raman spectrum is characterized in that: the method comprises the following steps:
step 1) dropwise adding a gold nanorod solution on the surface of a micro-nano structure, and controlling the temperature and humidity conditions during solvent evaporation to enable self-assembly of the gold nanorod in the micro-nano structure to prepare a template for single-cell regulation and detection integration;
and 2) modifying the gold nanorod assembly of the template according to different detected indexes to obtain a modified template, wherein the modification comprises modification of one of the following organic matters on the gold nanorod assembly:
a) The gold nanorod assembly is modified with 4-mercaptopyridine for single-cell local pH detection or single-cell specific part pH detection;
b) Modifying 1-thiodecane on the gold nanorod assembly for detecting single-cell glucose;
c) Modifying dopamine aptamer on the gold nanorod assembly for single-cell dopamine detection;
d) Modifying a DNA single strand on the gold nanorod assembly for RNA detection in base pairing with the DNA single strand;
step 3) taking the modified template obtained in the step 2) as a substrate for cell growth, sowing the adherent cells on the substrate, utilizing the hydrophilicity of the gold nanorods to enable the cells to specifically grow on the micro-nano structure, and regulating and controlling the growth of the adherent cells through the morphology structure on the surface of the substrate so as to obtain single cells with specific cell morphology and growth direction;
and 4) detecting the Raman spectrum of the single cell obtained in the step 3) to obtain data of a local biochemical microenvironment, and aligning the light spot of the incident laser to a specific single cell or a specific part of the single cell when detecting the Raman spectrum of the detected cell.
2. The method for detecting the local biochemical environment of the regulated cells based on the Raman spectrum according to claim 1, wherein the method comprises the following steps: in the step 1), the preparation of the micro-nano structure comprises one of the following methods:
a) A silicon template having a micron-sized structure obtained by photolithography;
b) Obtaining a silicon template with a micron-sized structure through photoetching; then, the flexible template is obtained after the silicon template is subjected to reverse molding through Polydimethylsiloxane (PDMS);
c) Firstly, obtaining a silicon template with a micron-sized structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template; and then attaching the flexible template to the surface of a glass slide to bond the polydimethylsiloxane and the glass, and after the flexible template is peeled off, the polydimethylsiloxane structure with the nanoscale thickness is remained on the surface of the glass slide to obtain the hydrophilic-hydrophobic interphase micro-nano structure.
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