CN114235773A - Raman imaging method for dynamically monitoring cell membrane repairing process - Google Patents
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- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 claims abstract description 41
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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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- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention designs a Raman imaging method for dynamically monitoring a cell membrane repairing process. According to the method, a reaction product SLO-DBCO of a pore-forming protein SLO and dibenzyl cyclooctyl-sulfo-N-hydroxysuccinimide ester (DBCO-sulfo-NHS ester) is used as a pore-forming monomer, pores are formed on a living cell membrane in an aggregating manner, and then a Surface Enhanced Raman (SERS) probe is aggregated to the periphery of a membrane pore to generate SERS signals by utilizing a click reaction between the SLO-DBCO and a gold nano star probe co-modified by Raman beacon molecules (p-mercaptobenzoic acid) and azide-polyethylene glycol (PEG-N3), so that Raman imaging of the membrane pore is realized. In the process of repairing the cell membrane, the membrane pores gradually become small, the SLO and the coupled SERS probe continuously fall off from the surface of the cell, so that the SERS signal is gradually reduced until the membrane pores are completely repaired, and the dynamic monitoring of the whole repairing process is realized through the change of the SERS signal. The invention provides a simple and sensitive method for dynamic monitoring of the cell membrane repair process and research on the repair mechanism thereof, and also provides a new technical support for treatment of diseases related to the membrane pore repair.
Description
One, the technical field
The invention relates to a Raman imaging method for dynamically monitoring a cell membrane repairing process.
Second, background Art
The biofilm pores are widely present on the cell surface and are closely related to the death of cells in the immune system, inflammation or other related diseases. The pore may be formed by aggregation of endotoxin-perforin secreted by NK cells or T cells, or by aggregation of exotoxins, such as the bacterial exotoxin Streptolysin (SLO) in the cholesterol-dependent cytolysin (CDC) family, after attachment to the cell membrane. The mechanism of pore formation has been well establishedHowever, due to the shortage of the related art, it is difficult to monitor the repair process of the membrane pores on the cell membrane and study the repair mechanism. Currently, the visualization of the membrane pores is mainly achieved by means of electron microscopy and atomic force microscopy on fixed dead cells or artificial phospholipid bilayers. In addition, some are based on indicating Ca flow through the pores of the membrane2+Or the fluorescence method for monitoring the membrane pore repair by fluorescence labeling the perforation monomer, because the reading of the fluorescence signal cannot be synchronous with the actual repair process, and the sensitivity is insufficient, the real process of the membrane pore repair on the living cell cannot be indicated. Therefore, there is an urgent need in the art to develop a sensitive test method for dynamic monitoring and repairing mechanism research of the membrane pore repairing process on living cells.
The patent designs a dynamic Raman imaging method induced by pore-forming protein, and realizes dynamic imaging of living cell membrane repairing process by reducing Surface Enhanced Raman (SERS) signals in the repairing process.
Third, the invention
The purpose of the invention is: the method is characterized in that a SERS signal is generated based on aggregation of aureonano star (AuNSs), and the SERS signal generated by aggregation of pore-forming protein-SLO on a cell membrane and induction of aggregation of an SERS probe on a pore of the membrane is designed. In the cell membrane repairing process, the SLO and the coupled SERS probe continuously fall off from the cell surface, so that the Raman signal on the cell membrane is reduced, the dynamic monitoring of the cell membrane repairing process is realized, the repairing mechanism is clarified, and a new technical support is provided for the treatment of related diseases of cell membrane repairing.
The dynamic SERS imaging strategy for cell membrane repair proposed by the present invention is shown in fig. 1. First, SLO (SLO-DBCO) modified with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester (DBCO-sulfo-NHS ester) was synthesized, and accumulated on a cell membrane with SLO-DBCO to form a pore. Simultaneously, a Raman beacon molecule (p-mercaptobenzoic acid) and an azide-polyethylene glycol (PEG-N)3) And modifying the AuNSs surface to obtain the SERS probe. And the SERS probe is subjected to click chemical reaction with SLO-DBCO on the cell membrane pore to be gathered around the membrane pore to generate an SERS signal, so that Raman imaging of the membrane pore is realized. During the process of repairing cell membrane, the pore becomes smaller, SLO andthe coupled SERS probe continuously falls off from the cell surface, so that the SERS signal is gradually reduced until the membrane pore is completely repaired, and the dynamic monitoring of the whole repairing process is realized through the change of the SERS signal.
The invention is realized by the following technical scheme:
1) the pore-forming protein SLO is reacted with DBCO-sulfo-NHS ester for 2 hours at room temperature to form an SLO-DBCO conjugate with pore-forming activity.
2) Mixing Raman beacon molecule (p-mercaptobenzoic acid) and azido-polyethylene glycol (PEG-N)3) And co-modifying to obtain the SERS probe on the AuNSs surface.
3) The living cells are incubated in the culture solution containing SLO-DBCO, so that the SLO-DBCO can be gathered on cell membranes to form large membrane pores.
4) As shown in fig. 1, the SLO-DBCO composing the membrane pore causes the SERS probe to gather above the membrane pore by a click reaction with the SERS probe, thereby generating a SERS signal.
5) As shown in fig. 1, as the membrane pores of the cells are repaired, the membrane pores gradually become smaller, and the SLO and the coupled SERS probe thereof continuously fall off from the surface of the cells, so that SERS signals are gradually reduced, thereby realizing dynamic raman imaging for repairing the membrane pores of living cells.
Compared with the prior art, the invention has the following characteristics:
the monitoring method designed in the invention has the characteristics of simplicity, no damage, sensitivity and in-situ dynamic tracking. After the membrane pores are formed on living cells, the cells do not need to be fixed and then imaged, the cells and the membrane pores are not damaged by a signal generation strategy, and the signal amplification is realized based on the inherent signal enhancement effect of the gold nanoparticles so as to meet the requirement of the in-situ dynamic monitoring membrane pore repair process.
Description of the drawings
FIG. 1 is a schematic diagram of a surface enhanced Raman spectroscopy method for dynamic monitoring of cell membrane repair process
Fifth, detailed description of the invention
Example 1: synthesis and cell perforation experiment of SLO-DBCO conjugate
After activating the pore-forming protein SLO with Dithiothreitol (DTT) at 37 ℃ for 2 hours, the SLO solution (0.55. mu.M) was mixed with DBCO-sulfo-NHS ester (47. mu.M) and reacted on a vertical rotator for 2 hours. The excess DBCO-sulfo-NHS ester in the reaction system was removed by ultrafiltration (14000g, 4 ℃)5 times using a 30kDa ultrafiltration tube to obtain the SLO-DBCO conjugate having the perforation activity.
MCF-7 cells were incubated overnight in an incubator at 37 ℃ with MCF-7 breast cancer cell line as a cell model, and Hank's balanced salt buffer (HBSS, containing 2mM CaCl)2) After washing 3 times, the reaction mixture was mixed with SLO-DBCO (200U mL)-1) After incubation in an incubator at 37 ℃ for 20 minutes, a hole for SLO-DBCO to aggregate on the cell membrane was formed.
Example 2: synthesis of SERS probes
First 5mL of 1% trisodium citrate was added to 100mL of boiling HAuCl4The solution reacts for 20 minutes to prepare 15-nm gold seeds, and 8.6mL PVP (25.6g L) is dripped after the gold seeds are cooled to room temperature-1MW 10000) overnight, and 15mL of 0.12mM HAuCl4And reacted with 10mM PVP in DMF for 15 min to obtain gold nanostars (AuNSs). After AuNSs was dispersed in 500. mu.L of water, 10. mu.L of 10mM Raman reporter MBA in ethanol and 490. mu.L of freshly prepared HS-PEG-N were added3(2mM) solution, shaking overnight at room temperature. Centrifugally washing twice at 8000rpm to obtain SERS probe (AuNSs-MBA/PEG-N)3) And suspended in 500. mu.L of water for use.
Example 3: in conjunction with FIG. 1, the accumulation of SERS probes on cell membranes generates a SERS signal
The cells obtained in example 1 were incubated with SERS probes (0.2nM) for 30 min at 37 ℃ in an incubator by-PEG-N3Through the click reaction with-DBCO, the SERS probe can be gathered on a hole formed by the SLO on a cell membrane to generate an SERS signal, and the Raman imaging of the cell membrane hole is realized.
Example 4: dynamic Raman imaging monitoring of Living cell Membrane repair Process in conjunction with FIG. 1
The cells aggregated by SERS probe in example 3 were transferred to 1640 culture medium containing 10% fetal bovine serum and cultured at 37 ℃ to initiate the repair process of the cells. And taking out the cells repaired at different time every 10 minutes, and lightly washing the cells for 3 times by using 1640 culture solution without fetal calf serum until the whole repairing behavior is completed, so that the dynamic monitoring of the repairing process of the living cells can be realized.
Claims (4)
1. A Surface Enhanced Raman Scattering (SERS) imaging method for dynamically tracking a cell membrane repairing process is characterized in that a SERS probe is coupled with a pore-forming protein SLO on the cell surface, corresponding SERS signals are generated by gathering on the cell surface, and the reduction of the SERS signals is caused by cell membrane repairing, so that the dynamic monitoring of cell membrane repairing is realized.
2. The imaging method as claimed in claim 1, wherein the amino group of the pore-forming protein SLO is reacted with dibenzylcyclooctyl-sulfo-N-hydroxysuccinimide ester (DBCO-sulfo-NHS ester) to produce a SLO-DBCO having a perforating activity.
3. The imaging method of claim 1, wherein the SLO-DBCO is formed into a pore on a cell membrane by contacting with azide-polyethylene glycol (PEG-N) on a SERS probe3) The click reaction of (3) induces the SERS probe to gather around the membrane pore to generate an SERS signal, and the Raman imaging of the membrane pore is realized.
4. The imaging method of claim 1, wherein the SLO and its coupled SERS probe are continuously detached from the cell surface following cell membrane repair, resulting in a decrease in raman signal on the cell membrane, enabling dynamic monitoring of the cell membrane repair process.
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