CN113960011A - Method for detecting local biochemical environment of regulated cells based on Raman spectrum - Google Patents
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Abstract
The invention discloses a method for detecting a local biochemical environment of a regulated cell based on Raman spectroscopy, 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, and a template for integrating single cell regulation and detection is obtained; assembling gold nanorods of the template and modifying; taking the modified template as a substrate for cell growth, and sowing adherent cells on the substrate 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 the specific single cell or the 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 a sample, and has important significance for revealing the internal physiological change of cell regulation on line in real time.
Description
Technical Field
The invention relates to a method for detecting regulated cells, in particular to a micro-nano structure combined with self-assembled gold nanorods, a method for regulating the growth of cells through the micro-nano structure and simultaneously carrying out Raman detection on specific sites of the regulated cells.
Background
The micro-nano structures with different shapes can affect the behavior and the state of the cell. For example, cells are more likely to adhere to rougher micro-nano structured surfaces, and anisotropic structural features on the micro-nano scale can direct cell growth, alter the skeletal structure of cells and induce cell differentiation. The regulation of the growth and behavior of cells through micro-nano structures plays an important role in tissue engineering and clinical application, and with the development of micro-nano manufacturing technology, the regulation of cells based on micro-nano structures has gradually become one of the hot fields. In the process that the cells are regulated and controlled, the biochemical microenvironment around the cells can be changed, and the research on the functions of the cells and the prediction on the fate of the cells can be realized by detecting the local biochemical environment of the cells, such as the pH value. For example, tumor cells have a lower extracellular pH than normal cells, and a lower pH favors cell migration and invasion behavior. However, there is often a lack of extensive research into the changes in the localized biochemical microenvironment of the cells being regulated.
The means for detecting the regulated cells is to observe the change of the external appearance of the cells by means of microscope imaging, or to perform fluorescence imaging by using biomolecules such as fluorescently labeled specific proteins, or to extract the internal components of the cells by puncturing the cells with microneedles or the like. The methods have many limitations in quantitatively detecting the local biochemical environment of the regulated cells, for example, the phenomenon of spectral overlap is easily generated due to a wider fluorescence spectrum, so that two molecules with overlapped fluorescence spectra are difficult to distinguish, the phenomena of photobleaching, photoquenching and the like also influence the accuracy of quantitative detection based on fluorescence intensity, and meanwhile, because many fluorescent substances are difficult to mark living cells, the cells need to be fixed during fluorescent dyeing, so that the fluorescence intensity characterization is not favorable for dynamically detecting the local biochemical microenvironment of the living cells in real time. The methods of puncturing cells to extract internal components of the cells have 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 damaged cells can be changed due to stress reaction when the cells are damaged, so that the accuracy of measurement is influenced. Therefore, although many documents report cell regulation based on micro-nano structures, it is difficult to realize real-time dynamic detection of local biochemical microenvironment of the regulated cells after the micro-nano structures regulate the cells.
In order to realize real-time dynamic detection of the regulated single-cell local biochemical microenvironment, a detection method which is free of cell damage, convenient to operate and high in sensitivity is particularly important. The detection method based on the Raman spectrum has small damage to the sample, needs small amount of samples and can provide rich chemical composition information, but the application of the Raman detection is limited due to weak signals and low detection sensitivity. However, when laser is irradiated on a rough metal surface, due to the enhancement of a local electromagnetic field, charge transfer and other phenomena, a Raman scattering signal is enhanced by ten thousands or even hundred million times, surface-enhanced Raman scattering is realized, and the sensitivity and the signal-to-noise ratio of Raman detection are greatly improved. Because the components inside and outside the cell are complex and the content is low, the surface enhanced Raman scattering with high sensitivity, small damage to the sample and rich contained information becomes an effective means for realizing the detection of the living cells.
In order to obtain a surface enhanced raman scattering substrate having high sensitivity and high reproducibility, i.e., a roughened metal surface having a uniform structure, there are generally a top-down method and a bottom-up method. The top-down method is to obtain a micro-nano structure 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 approach favors the formation of ordered patterns on the micrometer scale, but presents difficulties in the construction of nanoscale or arbitrary three-dimensional structures. The bottom-up method is to assemble molecules or atomic components into a micro-nano structure, such as self-assembly, chemical vapor deposition, atomic layer deposition, and the like. Among them, the self-assembly method is not required to have a complicated process flow and expensive equipment, and has low raw material consumption, cost-effectiveness, and has received wide attention. In order to realize self-assembly of gold nanorods, common methods include solvent evaporation self-assembly, organic solvent assisted self-assembly, template assisted self-assembly, and the like. Because the evaporation speed of the solvent at the three-phase interface of the substrate, the air and the water is higher than that of the liquid drop center and the concentration of the gold nanorods is higher, the self-assembly of the gold nanorods obtained by the solvent evaporation method has a coffee ring effect, and the uniformity and the repeatability of the gold nanorods serving as the cell culture and Raman detection substrate are limited. By controlling the conditions of the solvent evaporation, such as temperature, humidity, hydrophilicity and hydrophobicity of the substrate and the like, the evaporation speed of the solvent is reduced, the concentrations of the gold nanorods at different positions of the liquid drop are kept close, and the assembly effect can be improved.
Disclosure of Invention
Aiming at the prior art, the invention provides a method for detecting the local biochemical environment of the regulated cells based on Raman spectroscopy, which realizes the integrated single cell detection with high flux, high sensitivity and low damage to samples and has important significance for revealing the internal physiological change of cell regulation on line and in real time.
In order to solve the technical problems, the invention provides a method for detecting a local biochemical environment of a regulated cell based on Raman spectroscopy, 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 the gold nanorods to be self-assembled in the micro-nano structure to prepare a template for integrating single cell regulation and detection;
step 2) according to different detected indexes, modifying the assembly of the gold nanorods of the template to obtain a modified template, wherein the modification of the assembly of the gold nanorods comprises the following steps of:
a) modifying 4-mercaptopyridine on the gold nanorod assembly for performing single cell local pH detection or single cell specific part pH detection;
b) modifying 1-thio decane on the gold nanorod assembly for detecting single-cell glucose;
c) modifying a dopamine aptamer on the gold nanorod assembly for detecting the single-cell dopamine;
d) modifying a DNA single chain on the gold nanorod assembly for RNA detection matched with the DNA single chain base;
step 3) taking the modified template obtained in the step 2) as a substrate for cell growth, sowing 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 morphological structure of the substrate surface, 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 a 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.
Further, in the method for detecting the local biochemical environment of the regulated cells based on the raman spectroscopy, in the step 1), the preparation of the micro-nano structure comprises one of the following methods:
a) a silicon template having a micron-scale structure obtained by photolithography;
b) obtaining a silicon template with a micron-scale structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template;
c) firstly, obtaining a silicon template with a micron-scale structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template; and then, the flexible template is attached to the surface of a glass slide, so that polydimethylsiloxane is bonded with the glass, and after the flexible template is peeled off, a polydimethylsiloxane structure with a nano-scale thickness is remained on the surface of the glass slide, and hydrophilic and hydrophobic alternate micro-nano structures are obtained.
Compared with the prior art, the invention has the beneficial effects that:
the invention integrates cell growth regulation and cell local part detection, and realizes integrated single cell detection with high flux, high sensitivity and low damage to samples. The method realizes the controllable self-assembly of the gold nanorods by constructing an interface micro-nano structure, enables cells to be specifically combined with the surfaces of the gold nanorods, and realizes the regulation and control of the 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 directionally assembling gold nanorods. Meanwhile, the Raman spectrum of the cell is found to contain a characteristic peak reflecting the change of the pH value, 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 online real-time revealing of the internal physiological change of cell regulation.
Drawings
Fig. 1 is a schematic diagram of preparing a micron-scale structure template for integrating single cell regulation and detection, wherein (a) is a schematic diagram of obtaining a flexible template by utilizing a Polydimethylsiloxane (PDMS) reverse mold, and (b) is a schematic diagram of bonding and then peeling the PDMS and glass to obtain a hydrophilic-hydrophobic alternate micro-nano structure on the surface of the glass.
Figure 2 is a schematic of template functionalization by modified gold nanorod assembly and raman indicator factor.
FIG. 3 is a scanning electron microscope image of the integrated platform for cell control and detection, 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) under different pH environments.
FIG. 5 is a scanning electron microscope image of the use of the integrated platform to regulate cell growth.
FIG. 6 is a schematic diagram of the use of the integrated platform for localized pH detection at specific locations of different cells.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, which are not intended to limit the invention in any way.
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 topographic factors on adherent cells can be realized by designing cell culture substrates with different morphological structures. The cell culture substrate has the effects of regulating and controlling the adhesion of cells, the directional growth of the cells and the differentiation of the cells by the hardness, the roughness and the anisotropy of the surface topography. The study of the intrinsic physiological changes of the regulated cells, such as changes in pH, protein, and DNA, can reveal the intrinsic mechanisms of changes in cell growth. According to the invention, by designing a platform combining the gold nanorods and the micro-nano structure, the integration of cell regulation and cell detection is realized. The assembled gold nanorods generate a plasma resonance effect on the surface under laser irradiation, so that Raman signals are improved by tens of thousands or even hundreds of millions of times, and the obtained surface-enhanced Raman spectrum has high sensitivity and signal-to-noise ratio. By modifying the organic 4-mercaptopyridine with Raman characteristic peaks changing with pH, the cell regulation and detection platform can realize the detection of the pH value of the cell. Because the diameter of the laser spot is only 0.9 micron when the Raman spectrum detection is carried out, the local pH value of a specific cell can be detected by aligning the laser spot with the regulated cell, and the local pH difference of different regulated cells and different parts of the cell can also be compared.
The design idea of the method for detecting the local biochemical environment of the regulated cells based on the Raman spectrum is as follows: the gold nanorods are modified on templates with 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 cells with regulated growth, the pH detection of a specific part of a specific cell is realized by utilizing the Raman enhancement effect. The detection method comprises the following specific steps:
step 1) preparing a template for single cell regulation and detection integration, wherein the process comprises the following steps:
firstly, preparing a micro-nano structure according to one of the following methods:
a) a silicon template having a micron-scale structure obtained by photolithography;
b) obtaining a silicon template with a micron-scale structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template;
c) firstly, obtaining a silicon template with a micron-scale structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template; and then, the flexible template is attached to the surface of a glass slide, so that polydimethylsiloxane is bonded with the glass, and after the flexible template is peeled off, a polydimethylsiloxane structure with a nano-scale thickness is remained on the surface of the glass slide, and hydrophilic and hydrophobic alternate micro-nano structures are obtained.
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 nanorods to be self-assembled in the micro-nano structure to prepare a template for integrating single cell regulation and detection;
step 2) according to different detected indexes, modifying the assembly of the gold nanorods of the template to obtain a modified template, wherein the modification of the assembly of the gold nanorods comprises the following steps of:
a) modifying 4-mercaptopyridine on the gold nanorod assembly for performing single cell local pH detection or single cell specific part pH detection;
b) modifying 1-thio decane on the gold nanorod assembly for detecting single-cell glucose;
c) modifying a dopamine aptamer on the gold nanorod assembly for detecting the single-cell dopamine;
d) modifying a DNA single chain on the gold nanorod assembly for RNA detection matched with the DNA single chain base;
step 3) taking the modified template obtained in the step 2) as a substrate for cell growth, sowing 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 morphological structure of the substrate surface, 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 a 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.
Example (b):
and manufacturing the template with the micron-sized morphology structure. And photoetching is utilized to obtain the silicon template with the micron-scale morphology structure. And (3) obtaining the flexible template with elasticity and easy deformation by back-molding Polydimethylsiloxane (PDMS). Bonding PDMS with glass, and peeling PDMS to leave nano-structure with nano-scale thickness on the surface of the glass. As shown in fig. 1, wherein (a) is a schematic diagram of obtaining a flexible template by using a Polydimethylsiloxane (PDMS) reverse mold, and (b) is a schematic diagram of bonding and peeling off the PDMS and the glass to obtain a micro-nano structure with hydrophilic and hydrophobic phases on the surface of the glass. 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 was functionalized by modifying gold nanorods and raman indicator. Dropwise adding the gold nanorod solution on a template with a micrometer structure, utilizing a structure with alternate hydrophilic and hydrophobic template interfaces, and directionally assembling a layer of gold nanorods on the surface of the micrometer structure by adjusting conditions such as temperature, humidity and the like during solvent evaporation to obtain a substrate with a Raman enhancement effect. And then soaking the substrate in a pH-sensitive 4-mercaptopyridine (4-MPy) ethanol solution, and modifying 4-MPy on the gold nanorods through gold-sulfur bonds, so that the Raman-enhanced substrate has a pH detection function. FIG. 2 is a schematic diagram of template functionalization by modified gold nanorod assembly and Raman indicator, wherein 4 is a PDMS flexible template, 5 is a glass template with a surface modified with hydrophilic and hydrophobic alternate micro-nano structure, 6 is a gold nanorod solution, 7 is a gold nanorod self-assembly array, and 8 is pH-sensitive Raman indicator 4-mercaptopyridine (4-MPy).
Regulating the growth process of the cell. The functionalized different templates are used as cell culture substrates, cells are sown on the surfaces of the templates, and after the cells grow in an adherent manner, the extension and migration of the cells are influenced by the structure of the growth substrates, so that the cells have different morphological characteristics and directional growth characteristics. FIG. 3 is a scanning electron microscope image of a template integrated with cell conditioning and detection, wherein (a) is a grid structure and (b) is a circular array structure. Different masks are designed for photoetching to obtain a silicon template, and then the manufacturing method in the figure 1 and the figure 2 can be used for obtaining various platforms integrating cell regulation and detection with different shapes and sizes.
Localized pH detection of specific cells. Through the surface enhanced Raman scattering effect of the gold nanorod self-assembly, the Raman spectrum of the cell growing on the gold nanorod assembly is greatly enhanced, so that the Raman spectrum of the cell growing on the assembly can be enhanced by tens of thousands of times. After the gold nanorod assembly is modified with 4-mercaptopyridine, the difference of Raman characteristic peaks is related to the change of pH value, and a Raman spectrum of a cell has a characteristic peak reflecting the change of the pH value, so that the change of the pH value of a peripheral local area can be reflected by detecting the Raman spectrum, namely the change of the pH value of the cell growing on the gold nanorod assembly. Because the diameter of a laser spot in Raman detection is only 0.9 micron, the local pH detection of a specific cell can be realized by aligning the laser spot to the specific cell. For a particular cell whose growth is regulated by the template structure, detection of differences in raman spectra at particular locations (e.g., nucleus, cytoplasm, cellular pseudopodia, junctions between multiple cells) may reflect localized pH changes at different locations in the regulated cell.
The process of detecting the specific part of the 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 position of the cell is realized.
FIG. 4 is a surface enhanced Raman spectrum of 4-mercaptopyridine (4-MPy) under different pH environments. 4-MPy pyridine protein ring at 1006cm-1The characteristic peak and C ═ S bond at 1100cm-1The characteristic peak at (A) is related to the pH value of the surrounding environment, and is at 1006cm-1After the Raman spectrum is normalized by the intensity of the characteristic peak, 1100cm can be seen along with the increase of the pH value-1The intensity of the characteristic peak at (A) gradually increases. Therefore, the template with the micro structure assembled by the gold nanorods is modified by 4-MPy, 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 the regulation of cell growth using a template integrated with single cell regulation and detection. The diameter of the round assembly of gold nanorods in the figure is 20 μm, and the distance between two circles is also 20 μm. HeLa cells are sown on the micro-nano structure, and because the adherent growth of the cells is regulated and controlled by the shape of the substrate, the cells are in a stretching state spanning a plurality of small circles and a circular state growing on only one small circle. And the cells have the tendency of directional growth and tend to stretch to small circles of the gold nanorods rather than to the surrounding gaps.
FIG. 6 is a schematic diagram of the detection of localized biochemical environments of a conditioned cell based on surface enhanced Raman scattering spectroscopy.
In the figure, 9 is a PDMS film with a micron structure, 10 is a gold nanorod circular assembly, 11 is a cell growing on the gold nanorod assembly, and under the regulation and control action of a growth substrate structure, the cell presents a circular or extended shape, 12 is a cell nucleus, and 13 is laser with the wavelength of 785 nm. The laser 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 through a Raman spectrum. By comparing the local pH values at the cytoplasm and nucleus of the round cells and the stretched cells, the internal physiological environment change of the regulated cells can be revealed.
While the present invention has been described with reference to the accompanying drawings, the present invention is not limited to the above-described embodiments, which are illustrative only and not restrictive, and various modifications which do not depart from the spirit of the present invention and which are intended to be covered by the claims of the present invention may be made by those skilled in the art.
Claims (2)
1. A method for detecting the local biochemical environment of a regulated cell based on Raman spectroscopy 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 the gold nanorods to be self-assembled in the micro-nano structure to prepare a template for integrating single cell regulation and detection;
step 2) according to different detected indexes, modifying the assembly of the gold nanorods of the template to obtain a modified template, wherein the modification of the assembly of the gold nanorods comprises the following steps of:
a) modifying 4-mercaptopyridine on the gold nanorod assembly for performing single cell local pH detection or single cell specific part pH detection;
b) modifying 1-thio decane on the gold nanorod assembly for detecting single-cell glucose;
c) modifying a dopamine aptamer on the gold nanorod assembly for detecting the single-cell dopamine;
d) modifying a DNA single chain on the gold nanorod assembly for RNA detection matched with the DNA single chain base;
step 3) taking the modified template obtained in the step 2) as a substrate for cell growth, sowing 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 morphological structure of the substrate surface, 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 a 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 Raman spectroscopy according to claim 1, wherein: in the step 1), the preparation of the micro-nano structure comprises one of the following methods:
a) a silicon template having a micron-scale structure obtained by photolithography;
b) obtaining a silicon template with a micron-scale structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template;
c) firstly, obtaining a silicon template with a micron-scale structure through photoetching; then, carrying out reverse molding on the silicon template through Polydimethylsiloxane (PDMS) to obtain a flexible template; and then, the flexible template is attached to the surface of a glass slide, so that polydimethylsiloxane is bonded with the glass, and after the flexible template is peeled off, a polydimethylsiloxane structure with a nano-scale thickness is remained on the surface of the glass slide, and hydrophilic and hydrophobic alternate micro-nano structures are obtained.
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