Peptide hydrogel multifunctional platform for inducing tumor cells to form tumor stem cells as well as preparation method and application of peptide hydrogel multifunctional platform

Abstract

The invention discloses a peptide hydrogel multifunctional platform capable of reprogramming tumor cells into tumor stem cells (CSCs) as well as a preparation method and application of the peptide hydrogel multifunctional platform. The platform is formed by embedding tumor cells in 9-fluorenyl methoxy carbonyl-diphenylalanine (Fmoc-FF) self-assembled hydrogel and modifying the tumor cells on a three-dimensional electrode. When tumor cells are subjected to three-dimensional culture on the platform, Fmoc-FF self-assembled hydrogel acts on the surfaces of the cells, expression of various dry related genes and proteins of the cells is obviously up-regulated, and CSCs can be quickly obtained within 5 days; meanwhile, the platform can be used as a working electrode to monitor the change of a stemness marker acetaldehyde dehydrogenase 1 (ALDH1) in the stemness enhancement process of the cultured tumor cells in situ. The platform provided by the invention can combine rapid construction of a CSCs model with in-situ and nondestructive electrochemical monitoring of cell stemness change, and has lower time and money cost compared with the existing CSCs preparation and detection technology.

Description

Technical Field

[0001] This invention relates to the field of tumor stem cell preparation and detection technology, and in particular to a multifunctional platform that can induce tumor cells to reprogram into tumor stem cells and perform in situ monitoring of their stemness, as well as its preparation method and application. Background Technology

[0002] Cancer stem cells (CSCs) are a type of tumor cell within the tumor parenchyma that exhibits relatively inactive division, strong tumorigenic capacity, and stem cell-like characteristics. They are highly metastatic and resistant to radiotherapy and chemotherapy, making them a significant cause of cancer recurrence and treatment failure. Currently, researchers primarily use two methods to obtain CSCs for study: serum-free suspension culture and immunological sorting. However, both methods suffer from cumbersome procedures, high costs, and low efficiency. Furthermore, in CSC-related research, characterizing changes in cell stemness requires cell separation or disruption before testing, leading to the loss of biophysical information about the surrounding cellular environment, thus limiting the development of CSC-related research. Therefore, developing efficient methods for constructing CSC models is of great significance.

[0003] Studies have shown (S. Tanaka, et al. Rapid reprogramming of tumor cells into cancer stem cells on double-network hydrogels[J]. Nature Biomedical Engineering, 2021, 5(8): 914–925.) that when tumor cells are cultured on the surface of a synthetic double-crosslinked network polymer hydrogel (DN gel), the cells undergo rapid reprogramming due to stimulation from external signals such as the stiffness and charge of the gel, thus transforming into cancer stem cells (CSCs). In recent years, the role of biomechanical and chemical factors in the regulation of pluripotency of stem cells and cancer cells has attracted increasing attention from researchers. However, cell reprogramming is a highly complex and dynamic process, and the mechanisms by which physical and chemical signals in the cancer cell growth environment induce reprogramming remain unclear. In existing studies, characterizing reprogrammed cancer cell stem cells (CSCs) requires separating the cells from the scaffold material, which leads to the loss of important cellular environmental information and severely hinders further research into the biological processes and mechanisms involved in cancer cell reprogramming. While synthetic materials such as polymer gels or nanomaterials can stimulate tumor cell reprogramming, their physical and chemical properties differ significantly from the extracellular matrix, limiting their application in studying the occurrence and development of CSCs within real tumors. Therefore, developing a method to efficiently acquire CSCs and perform rapid and sensitive in-situ, real-time characterization to construct an effective CSC research model that accurately simulates the real tumor environment is of significant research importance in exploring the formation mechanism of CSCs and developing targeted therapies. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides a multifunctional peptide hydrogel platform for inducing tumor cell reprogramming into cellular stem cells (CSCs), along with its preparation method and applications. This platform enables rapid acquisition of CSCs while simultaneously allowing in-situ electrochemical monitoring of changes in cell stemness during the process.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a multifunctional peptide hydrogel platform, characterized in that: the multifunctional platform is obtained by one-step co-assembly of 9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) and tumor cells in a three-dimensional porous foam carbon electrode with gold nanoparticles deposited on it. The Fmoc-FF hydrogel has a three-dimensional nanofiber porous structure, and its morphology and properties are similar to those of the extracellular matrix, with cells uniformly distributed in the three-dimensional morphology of the hydrogel.

[0007] 1. The preparation method of the multifunctional platform proposed in this invention is as follows:

[0008] (1) Preparation of AuNPs / CF electrode: 0.5-2 wt% chloroauric acid solution was used as electrolyte solution, carbon foam was used as working electrode, platinum wire was used as counter electrode, and Ag / AgCl electrode was used as reference electrode to form a three-electrode system; gold nanoparticles were electrodeposited on carbon foam under the three-electrode system using cyclic voltammetry; the potential window was set to -0.2 to -0.9 V, the scan rate was 50 mV / s, and the number of deposition cycles was 10 to 30 to obtain AuNPs / CF electrode;

[0009] (2) Dissolve the lyophilized Fmoc-FF monomer powder in hexafluoroisopropanol (HFIP) solution, with a concentration of Fmoc-FF of 90-110 mg / mL, and sonicate until transparent;

[0010] (3) Take 10-30 μL of the Fmoc-FF HFIP solution from step (2) into the wells of a 96-well plate, place the sterilized AuNPs / CF electrode from step (1), and add 200 μL of a 1×10⁻⁶ solution. 4 ~1×10 5 A suspension of tumor cells per mL is added into the well using a pipette, and a hydrogel is formed in 5–10 seconds, thus obtaining the peptide hydrogel multifunctional platform. The final concentration of the Fmoc-FF hydrogel is 9–11 mg / mL.

[0011] 2. One application of the peptide hydrogel multifunctional platform according to claim 1, wherein the interaction between the Fmoc-FF self-assembled hydrogel and the cell surface can induce tumor cells to form tumor stem cells, and the specific application steps are as follows:

[0012] Add 100–300 μL of DMEM complete cell culture medium to the wells and place them in a cell culture incubator for cell culture and induction. The DMEM complete cell culture medium used contained 4500 mg / L glucose in DMEM high-glucose medium, accounting for 90% of the total volume; fetal bovine serum accounted for 10% of the total volume; and penicillin-streptomycin antibiotics accounted for 1% of the total volume. The culture conditions were: 37℃, saturated humidity, CO2 concentration of 5%, and culture time of 48–168 h. After this, the expression of multiple stemness-related genes and proteins in the cells was significantly upregulated.

[0013] 3. A second application of the peptide hydrogel multifunctional platform described in claim 1, which enables in-situ, non-destructive electrochemical monitoring of changes in the content of acetaldehyde dehydrogenase 1 (ALDH1), a stem cell marker, in cultured cells, for monitoring changes in cell stemness. The specific application steps are as follows:

[0014] The platform serves as the working electrode, with an Ag / AgCl electrode as the reference electrode and a platinum wire electrode as the counter electrode, using 1 μM NAD. +The Tris-HCl buffer solution (pH = 7.4) was used as the electrolyte solution, and 20 μL of 1 μM acetaldehyde was added as ALDH1.

[0015] ALDH1

[0016] Enzyme substrate; Catalytic equation: CH3CHO + NAD + →NADH+CH3COOH, in NAD + In the presence of ALDH1 enzyme, ALDH1 catalyzes the formation of NADH and acetic acid from acetaldehyde. Differential pulse voltammetry was performed in a three-electrode system with a potential range of 0.3–1.0 V. At a potential of around 0.81 V, the NADH generated by this characteristic reaction is oxidized to produce a current, the magnitude of which is proportional to the ALDH1 expression level, thus indicating the trend of changes in cell stemness.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This invention utilizes Fmoc-FF peptide hydrogel to construct a multifunctional platform for three-dimensional culture of tumor cells. Compared to other synthetic cell culture scaffolds, it exhibits better biocompatibility and possesses a three-dimensional nanofiber morphology and shear-thinning properties resulting from non-covalent polymerization. Physically and chemically, it better mimics the extracellular matrix than polymer scaffolds, providing rich biophysical and chemical signals during the investigation of the development and progression of cancer cells (CSCs). The constructed platform leverages the suitable stiffness and negative surface charge of the peptide hydrogel to rapidly reprogram cancer cells within the three-dimensional culture, quickly obtaining CSCs. Furthermore, utilizing the conductivity resulting from the π-π stacking within the peptide hydrogel fibers, this invention combines the induction of rapid cell reprogramming with electrochemical sensing, enabling in-situ, rapid monitoring of changes in tumor cell stemness without the need for cell separation or disruption. Compared to existing CSC acquisition technologies, this invention offers lower time and cost, and its combination with in-situ electrochemical characterization shows promising application prospects in exploring the mechanisms of CSC occurrence and development, as well as in the development of targeted drug screening. Attached Figure Description

[0019] Figure 1 The results of SEM and EDSMapping of the MCF-7 / Fmoc-FF / AuNPs / CF platform used to prepare peptide hydrogels in Example 1 are shown.

[0020] Figure 2 This is a 3D laser confocal image of the microsphere-shaped MCF-7 cells cultured in three dimensions five days after Example 1.

[0021] Figure 3This study compares the real-time quantitative reverse transcription PCR results of CD44, SOX2, and other stem gene expression levels in MCF-7 cells cultured in a multifunctional platform for five days with those in the two-dimensional culture control group.

[0022] Figure 4 This study compares the flow cytometry results of stem cell markers CD44 and ALDH1 in MCF-7 cells cultured for five days on a multifunctional platform with those in the two-dimensional culture control group.

[0023] Figure 5 This study compares the Western Blot results of stem cell markers CD44, ALDH1, and KMT1A in MCF-7 cells cultured for five days on a multifunctional platform with those in the two-dimensional culture control group.

[0024] Figure 6 The results of DPV analysis of the change in ALDH1 enzyme expression, a stem cell marker, five days after culture-induced reprogramming using a peptide hydrogel multifunctional platform, compared to the two-dimensional culture control group.

[0025] Figure 7 This is a comparison of the real-time quantitative reverse transcription PCR results of the expression levels of stem genes such as SOX2 in MCF-7 cells after two days of three-dimensional culture on the multifunctional platform in Example 2 and the two-dimensional culture control group MCF-7 cells (the results of biomarker genes with fluorescence signals below the detection limit were not displayed).

[0026] Figure 8 This is a comparison of real-time quantitative reverse transcription PCR results of SOX2 and other stem gene expression levels in MCF-7 cells cultured in the multifunctional platform for seven days and in the two-dimensional culture control group MCF-7 cells. (Results for biomarker genes with fluorescence signals below the detection limit were not displayed).

[0027] Figure 9 This is a comparison of the real-time quantitative reverse transcription PCR results of CD44, SOX2 and other stem gene expression levels in BIU cells cultured in the multifunctional platform for five days and BIU cells in the two-dimensional culture control group.

[0028] Figure 10 This is a comparison of the real-time quantitative reverse transcription PCR results of the expression levels of stem genes such as CD44 and SOX2 in HepG2 cells after five days of three-dimensional culture on the multifunctional platform in Example 5, and those in the two-dimensional culture control group HepG2 cells. Detailed Implementation

[0029] Example 1:

[0030] (1) Select a CF with a specification of 5mm×4mm×2mm as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. Use a chloroauric acid solution with a concentration of 1wt% as the electrolyte solution. Electrodeposit the CF using cyclic voltammetry. The potential range is selected as -0.2 to -0.9V, the scan rate is 50mV / s, and the number of deposition cycles is 20 to obtain AuNPs / CF.

[0031] (2) MCF-7 cells were resuspended in DMEM complete cell culture medium (DMEM high glucose medium with a glucose content of 4500 mg / L, accounting for 90% of the total volume; fetal bovine serum accounting for 10% of the total volume; and penicillin-streptomycin antibiotics accounting for 1% of the total volume) to prepare a breast cancer cell suspension, and the cell density was adjusted to 5 × 10⁻⁶ cells / year. 4 cells / mL;

[0032] (3) Weigh 10 mg of Fmoc-FF lyophilized powder, dissolve it in 100 μL of hexafluoroisopropanol (HFIP), and sonicate until completely dissolved.

[0033] (4) Using a pipette, pipette 20 μL of Fmoc-FF in HFIP solution and add it to the wells of a 96-well plate; place the prepared AuNPs / CF electrode into the well, and add 200 μL of the prepared MCF-7 cell suspension. After 10 s, a hydrogel forms, yielding the MCF-7 / Fmoc-FF / AuNPs / CF platform. Add 200 μL of DMEM complete culture medium and place in a cell culture incubator. The culture conditions are 37℃, 5% CO2, and saturated humidity. Change the culture medium once a day. The SEM and EDS mapping results of the MCF-7 / Fmoc-FF / AuNPs / CF platform are shown below. Figure 1 As shown.

[0034] (5) After 5 days of culture, the hydrogel was prepared for scanning electron microscopy and confocal microscopy characterization to observe the three-dimensional growth state of cells in the hydrogel. Figure 2 As shown, the cells became spherical after five days of growth in the hydrogel and were evenly distributed within the hydrogel.

[0035] (6) After diluting the gel with phosphate buffer to obtain three-dimensionally cultured cells, real-time quantitative reverse transcription PCR, Western blotting, and flow cytometry were performed. The results are as follows: Figure 3 , Figure 4 , Figure 5As shown in the figure. PCR results showed that the expression of stem cell genes such as CD44, SOX2, and OCT4 in MCF-7 cells cultured in the platform for 5 days was significantly upregulated compared to cells cultured in two-dimensional culture dishes. Flow cytometry results showed that the proportion of CD44+ MCF-7 cells increased from 18.2% to 27.5% and the proportion of ALDH1+ cells increased from 3.44% to 31.0% after 5 days of culture in the platform. Western blotting tests on important biomarkers of breast cancer CSCs, CD44, ALDH1, and KMT1A, showed that the bands of these three proteins were significantly thicker after 5 days of culture compared to the control group, indicating increased protein expression. All characterization results showed that the genes and proteins related to various stem cell markers in MCF-7 cells cultured in the platform for 5 days were significantly upregulated, demonstrating that this multifunctional platform rapidly produced breast cancer CSCs within 5 days.

[0036] (7) The MCF-7 / Fmoc-FF / AuNPs / CF platform, which had been placed in the cell culture incubator for different days, was removed and used as the working electrode, the Ag / AgCl electrode as the reference electrode, and the titanium wire electrode as the counter electrode. The platform was then placed in an NAD system. + The DPV was measured in a three-electrode system by adding 20 μL of 1 μM acetaldehyde to a Tris-HCl buffer solution, and the results were as follows. Figure 6 As shown, the peak current generated by MCF-7 cells after five days of culture in the platform significantly increased, indicating that the expression of ALDH1 enzyme on the surface of MCF-7 cells increased and stemness was enhanced after five days of culture in the platform. The trend of the results is consistent with the biological characterization in step (6), indicating that the prepared multifunctional platform can not only rapidly obtain CSCs, but also realize in-situ and rapid electrochemical monitoring of changes in stemness of cancer cells.

[0037] Example 2:

[0038] (1) Select a CF with a specification of 5mm×4mm×2mm as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. Use a chloroauric acid solution with a concentration of 1wt% as the electrolyte solution. Electrodeposit the CF using cyclic voltammetry. The potential range is selected as -0.2 to -0.9V, the scan rate is 50mV / s, and the number of deposition cycles is 30 to obtain AuNPs / CF.

[0039] (2) Human liver cancer MCF-7 cell line was digested with trypsin, and resuspended in DMEM complete medium (DMEM high glucose medium with a glucose content of 4500 mg / L, accounting for 90% of the total volume; fetal bovine serum accounting for 10% of the total volume; penicillin and streptomycin antibiotics accounting for 1% of the total volume) to prepare an MCF-7 cell suspension. The cell density was adjusted to 1×10⁶ cells / year using the above-mentioned complete medium.5 cells / mL;

[0040] (3) Weigh 10 mg of Fmoc-FF lyophilized powder, dissolve it in 100 μL of HFIP, and sonicate until completely dissolved;

[0041] (4) Using a pipette, 20 μL of Fmoc-FF HFIP solution was added to the wells of a 96-well plate; the prepared AuNPs / CF electrode was placed in the well, and 200 μL of the prepared MCF-7 cell suspension was added. After 10 seconds, a hydrogel was formed, resulting in the MCF-7 / Fmoc-FF / AuNPs / CF platform. 100 μL of DMEM complete medium was added, and the plate was placed in a cell culture incubator. The culture conditions were 37°C, 5% CO2, and saturated humidity, with the medium changed daily. After 2 days of culture in the platform, the changes in various stem genes of MCF-7 cells were as follows: Figure 10 As shown, the expression of stemness genes such as SOX2 and OCT4 in MCF-7 cells cultured in the platform for 2 days was significantly upregulated compared to cells cultured in two-dimensional culture dishes. This indicates that the multifunctional platform can reprogram MCF-7 cells, significantly improving cell stemness within 2 days.

[0042] Example 3:

[0043] (1) Select a CF with a specification of 5mm×4mm×2mm as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. Use a chloroauric acid solution with a concentration of 1wt% as the electrolyte solution. Electrodeposit the CF using cyclic voltammetry. The potential range is selected as -0.2 to -0.9V, the scan rate is 50mV / s, and the number of deposition cycles is 10 to obtain AuNPs / CF.

[0044] (2) Human liver cancer MCF-7 cell line was digested with trypsin, and resuspended in DMEM complete medium (DMEM high glucose medium with a glucose content of 4500 mg / L, accounting for 90% of the total volume; fetal bovine serum accounting for 10% of the total volume; penicillin and streptomycin antibiotics accounting for 1% of the total volume) to prepare an MCF-7 cell suspension. The cell density was adjusted to 1×10⁶ cells / year using the above-mentioned complete medium. 4 cells / mL;

[0045] (3) Weigh 10 mg of Fmoc-FF lyophilized powder, dissolve it in 100 μL of HFIP, and sonicate until completely dissolved;

[0046] (4) Using a pipette, 20 μL of Fmoc-FF HFIP solution was added to the wells of a 96-well plate; the prepared AuNPs / CF electrode was placed in the well, and 200 μL of the prepared MCF-7 cell suspension was added. After 10 seconds, a hydrogel was formed, resulting in the MCF-7 / Fmoc-FF / AuNPs / CF platform. 300 μL of DMEM complete medium was added, and the plate was placed in a cell culture incubator. The culture conditions were 37°C, 5% CO2, and saturated humidity, with the medium changed daily. After 7 days of culture in the platform, the changes in various stem genes of MCF-7 cells were as follows: Figure 10 As shown, the expression of stemness genes such as SOX2 and KMT1A in MCF-7 cells cultured in the platform for 7 days was significantly upregulated compared to cells cultured in two-dimensional culture dishes. This indicates that the multifunctional platform can reprogram MCF-7 cells, significantly improving cell stemness within 7 days.

[0047] Example 4:

[0048] (1) A CF with a specification of 5mm×4mm×2mm was selected as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. A chloroauric acid solution with a concentration of 0.5wt% was used as the electrolyte solution. The electrodeposition was carried out by cyclic voltammetry with a potential range of -0.2 to -0.9V, a scan rate of 50mV / s, and 20 deposition cycles to obtain AuNPs / CF.

[0049] (2) Human bladder cancer BIU cell line was digested with trypsin, and resuspended in DMEM complete medium (DMEM high glucose medium with a glucose content of 4500 mg / L, accounting for 90% of the total volume; fetal bovine serum accounting for 10% of the total volume; penicillin and streptomycin accounting for 1% of the total volume) to prepare BIU cell suspension. The cell density was adjusted to 5 × 10⁶ cells / year using the above-mentioned complete medium. 4 cells / mL;

[0050] (3) Weigh 9 mg of Fmoc-FF lyophilized powder and dissolve it in 100 μL of HFIP. Sonicate until completely dissolved.

[0051] (4) Using a pipette, 20 μL of Fmoc-FF HFIP solution was added to the wells of a 96-well plate; the prepared AuNPs / CF electrode was placed in the well, and 200 μL of the prepared BIU cell suspension was added. After 10 seconds, a hydrogel was formed, resulting in the BIU / Fmoc-FF / AuNPs / CF platform. 200 μL of DMEM complete medium was added, and the plate was placed in a cell culture incubator. The culture conditions were 37°C, 5% CO2, and saturated humidity, with the medium changed daily. After 5 days of culture in the platform, the changes in various stem genes of BIU cells were as follows: Figure 9As shown, the expression of stemness genes such as CD44, SOX2, and OCT4 in BIU cells cultured in the platform for 5 days was significantly upregulated compared to cells cultured in two-dimensional culture dishes. This indicates that the multifunctional platform can reprogram BIU cells, significantly improving cell stemness within 5 days.

[0052] Example 5:

[0053] (1) Select a CF with a specification of 5mm×4mm×2mm as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. Use a chloroauric acid solution with a concentration of 2wt% as the electrolyte solution. Electrodeposit the CF using cyclic voltammetry. The potential range is selected as -0.2 to -0.9V, the scan rate is 50mV / s, and the number of deposition cycles is 20 to obtain AuNPs / CF.

[0054] (2) Human hepatocellular carcinoma HepG2 cell line was digested with trypsin, and resuspended in DMEM complete medium (DMEM high glucose medium with a glucose content of 4500 mg / L, accounting for 90% of the total volume; fetal bovine serum accounting for 10% of the total volume; penicillin and streptomycin antibiotics accounting for 1% of the total volume) to prepare HepG2 cell suspension. The cell density was adjusted to 5 × 10⁶ cells / year using the above-mentioned complete medium. 4 cells / mL;

[0055] (3) Weigh 11 mg of Fmoc-FF lyophilized powder, dissolve it in 100 μL of HFIP, and sonicate until completely dissolved;

[0056] (4) Using a pipette, 20 μL of Fmoc-FF HFIP solution was added to the wells of a 96-well plate; the prepared AuNPs / CF electrode was placed in the well, and 200 μL of the prepared HepG2 cell suspension was added. After 10 seconds, a hydrogel was formed, resulting in the HepG2 / Fmoc-FF / AuNPs / CF platform. 200 μL of DMEM complete medium was added, and the plate was placed in a cell culture incubator. The culture conditions were 37°C, 5% CO2, and saturated humidity, with the medium changed daily. After 5 days of culture in the platform, the changes in various stem genes of HepG2 cells were as follows: Figure 10 As shown, the expression of stemness genes such as CD44, SOX2, and OCT4 in BIU cells cultured in the platform for 5 days was significantly upregulated compared to cells cultured in two-dimensional culture dishes. This indicates that the multifunctional platform can reprogram HepG2 cells, significantly improving cell stemness within 5 days.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A multifunctional peptide hydrogel platform for reprogramming tumor cells into tumor stem cells, characterized in that, The tumor cells were encapsulated in a self-assembled hydrogel of 9-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) and modified onto a three-dimensional electrode.

2. The peptide hydrogel multifunctional platform according to claim 1, characterized in that, The three-dimensional electrode is a three-dimensional porous carbon foam (AuNPs / CF) electrode modified with gold nanoparticles.

3. A method for preparing the peptide hydrogel multifunctional platform according to claim 1, characterized in that, Includes the following steps: (1) Preparation of AuNPs / CF electrode: 0.5-2 wt% chloroauric acid solution was used as electrolyte solution, carbon foam was used as working electrode, platinum wire was used as counter electrode, and Ag / AgCl electrode was used as reference electrode to form a three-electrode system; gold nanoparticles were electrodeposited on carbon foam under the three-electrode system using cyclic voltammetry; the potential window was set to -0.2 to -0.9 V, the scan rate was 50 mV / s, and the number of deposition cycles was 10 to 30 to obtain AuNPs / CF electrode; (2) Dissolve the lyophilized Fmoc-FF monomer powder in hexafluoroisopropanol (HFIP) solution, with a concentration of 90-110 mg / mL, and sonicate until transparent; (3) Take 10-30 μL of the Fmoc-FF HFIP solution from step (2) into the wells of a 96-well plate, place the sterilized AuNPs / CF electrode from step (1) inside, and add 100-300 μL of a 1×10⁻⁶ solution. 4 ~1×10 5 A suspension of tumor cells per mL is added into the well using a pipette, and a hydrogel is formed in 5–10 seconds, thus obtaining the peptide hydrogel multifunctional platform. The final concentration of the Fmoc-FF hydrogel is 9–11 mg / mL.

4. One application of the peptide hydrogel multifunctional platform according to claim 1, wherein the interaction between the Fmoc-FF self-assembled hydrogel and the cell surface can induce tumor cells to form tumor stem cells, and the specific application steps are as follows: Tumor cells embedded in the platform were cultured in three dimensions. 100–300 μL of DMEM complete cell culture medium was added to each well, and the cells were placed in a cell culture incubator for culture and induction. The DMEM complete cell culture medium used contained 4500 mg / L glucose (90% of the total volume), fetal bovine serum (10%), and penicillin-streptomycin antibiotics (1%). Culture conditions were 37°C, saturated humidity, and 5% CO2 concentration. After 48–168 hours of culture, the expression of multiple stemness-related genes and proteins in the cells was significantly upregulated.

5. A second application of the peptide hydrogel multifunctional platform according to claim 1, which enables in-situ, non-destructive electrochemical monitoring of changes in the content of acetaldehyde dehydrogenase 1 (ALDH1), a stem cell marker, in cultured cells, for monitoring changes in cell stemness. The specific application steps are as follows: The platform serves as the working electrode, with an Ag / AgCl electrode as the reference electrode and a platinum wire electrode as the counter electrode, using 1 μM NAD. + The Tris-HCl buffer solution (pH = 7.4) was used as the electrolyte solution, and 20 μL of 1 μM acetaldehyde was added as ALDH1. Enzyme substrate. Catalytic equation: CH3CHO + NAD+ + →NADH+CH3COOH, in NAD + In the presence of ALDH1, the enzyme catalyzes the formation of NADH and acetic acid from acetaldehyde. Differential pulse voltammetry was performed in a three-electrode system with a potential range of 0.3–1.0 V. At approximately 0.81 V, the NADH generated in this characteristic reaction is oxidized, producing a current whose magnitude is directly proportional to the ALDH1 expression level, thus indicating the trend of changes in cell stemness.

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