Calpain activity detection method based on fluorescence sensor and its application

Abstract

The present invention discloses a method and application of detecting calpain activity based on a fluorescence sensor. The present invention belongs to the fields of biotechnology, bioanalysis and nanomaterial application. The present invention constructs a fluorescence sensor based on FRET of a fluorescently labeled polypeptide and nanographene oxide, adds it to a tissue or cell sample, realizes rapid and sensitive detection of calpain activity, and further optimizes the real-time monitoring method of calpain activity. In addition, the present invention proves through experiments that the cell imaging effect of NGO‑FSP is good, and the NGO‑FSP fluorescence sensing system can be applied to cell imaging.

Description

Technical Field

[0001] The invention belongs to the field of biological analysis, and in particular relates to a calpain activity detection method based on a fluorescence sensor and an application thereof. Background Art

[0002] Nanographene oxide (NGO) is a good biomaterial obtained by strongly oxidizing and then peeling off nanographene materials. It is widely used in various biosensors. The hydrophilic polar oxygen-containing groups on its surface, such as hydroxyl, alkoxy, carboxyl and other active groups, make NGO have good dispersibility in solvents, especially water, and enable NGO to be stably distributed on different matrices. Therefore, NGO is hydrophilic and biocompatible, and can interact with a variety of biological molecules. At the same time, NGO can serve as an excellent energy receptor in the energy transfer system. In addition, the efficient energy absorption and storage function of nanographene oxide has made it the focus of research by scholars at home and abroad.

[0003] Fluorescence resonance energy transfer (FRET) is a homogeneous analytical technique and a powerful tool for detecting nanoscale distances and changes in nanoscale distances of biological macromolecules in living organisms. It has the advantages of high sensitivity, good selectivity, simplicity and rapidity. FRET is a non-radiative energy transfer. When two different fluorescent groups, one of which is an energy donor (Donor) and the other is an energy acceptor (Acceptor), the emission spectrum of the energy donor and the excitation spectrum of the energy acceptor can effectively overlap, and the distance between the two is between 1.0 and 10.0 nm. When excited by the excitation light of the energy donor, the energy transfer from the donor to the acceptor can be observed. Due to the efficient energy transfer and absorption of nano-graphene oxide, when the fluorescent molecule and NGO are close to each other at the nanoscale distance, NGO hijacks the fluorescence energy of the fluorescent molecule and a rapid fluorescence quenching effect occurs; when the two dissociate, the fluorescence energy transfer effect does not occur, and the fluorescence can be revived. Therefore, NGO is usually used as a sensitive fluorescent sensor material to design and construct a FRET-based detection technology system.

[0004] Calpain is a conserved calcium-dependent neutral cysteine ​​enzyme in cells that requires calcium ions to activate. It can be divided into micromolar µ-calpain (calpain1) and millimolar m-calpain (calpain2) according to the required calcium ion concentration. Calpain can not only degrade proteins in the body, but also participate in many major physiological processes such as cell proliferation, apoptosis, and signal transduction. It has the characteristics of subtype diversity, wide distribution, and substrate specificity. Its activity regulation mechanism is very complex and has always been a hot spot and difficulty in the field of biochemistry. A large number of studies have shown that calpain is involved in the occurrence and development of various diseases such as neurodegenerative diseases, Alzheimer's disease, muscular dystrophy, atherosclerosis, and tumors. In recent years, scientists have not only studied the structure and activity regulation mechanism of calpain, but also further explored its biological effects. However, the calpain activity detection kits currently on the market are generally expensive and the detection steps are cumbersome and time-consuming, and there is a lack of real-time monitoring technology. Therefore, it is urgent to build a convenient and fast calpain activity detection method. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for detecting calpain activity based on a fluorescent sensor.

[0006] Another object of the present invention is to provide an application of the above-mentioned method for detecting calpain activity based on a fluorescent sensor.

[0007] The purpose of the present invention is achieved by the following technical solution: A method for detecting calpain activity based on a fluorescent sensor comprises the following steps:

[0008] (1) Mixing nanographene oxide (NGO) and fluorescent labeled polypeptide (FSP) to obtain NGO-FSP fluorescent sensor;

[0009] (2) The sample to be tested is mixed with the NGO-FSP fluorescent sensor of step (1), and the fluorescence intensity is detected after incubation to determine the activity of calpain in the sample to be tested.

[0010] The NGO-FSP fluorescent sensor described in step (1) is obtained by adsorption and binding of nanographene oxide (NGO) and fluorescent labeled polypeptide (FSP).

[0011] The fluorescently labeled polypeptide described in step (1) is obtained by labeling fluorescein isothiocyanate (FITC) on the N-terminus of the CAPN-specific enzyme-cleaved substrate polypeptide (SP).

[0012] The polypeptide is preferably a sequence containing a calpain2 restriction site at the N-terminus of selenoprotein K, and its amino acid sequence is: RRMGRISHLRGPSPPPMAGG, and its molecular weight is 2130.49 Da.

[0013] The excitation light wavelength of the fluorescein isothiocyanate is 488 nm, and the emission light wavelength is 520 nm.

[0014] The nano-graphene oxide described in step (1) has a sheet size of 50 to 200 nm and a specific surface area of ​​50 to 100 m 2 / g; the preferred flake size is 86 nm and the specific surface area is 90 m 2 / g.

[0015] The concentration of the nano-graphene oxide described in step (1) in the system is 0.75-200 μg / mL, preferably 1 μg / mL.

[0016] The concentration of the fluorescently labeled polypeptide in step (1) in the system is 0.1-40 μM, preferably 0.2 μM.

[0017] The mixing described in step (1) is to add nano-graphene oxide to the fluorescent labeled polypeptide solution and mix by blowing.

[0018] The fluorescently labeled polypeptide solution preferably uses water as a solvent, more preferably uses ultrapure water as a solvent, and even more preferably uses milli-Q water as a solvent.

[0019] The sample to be tested in step (2) includes at least one of cells, tissues, or supernatant obtained after the cell / tissue undergoes a total protein extraction step.

[0020] The above-mentioned system of the sample to be tested does not contain Ca 2+ When calpain is added, calpain buffer should be added to meet the environmental conditions for enzyme cleavage.

[0021] The formula of the above calpain buffer is 500 mM Tris-HCl (pH 7.5), 100 mM CaCl2, 300 mM NaCl, 50 mM DTT.

[0022] The incubation time in step (2) is 40 to 60 minutes, preferably 50 minutes.

[0023] The incubation temperature in step (2) is 35-40°C, preferably 37°C.

[0024] The fluorescence intensity in step (2) is detected by at least one of an ELISA reader, a fluorescence spectrophotometer or a laser confocal microscope.

[0025] The above steps (1) and (2) are all carried out under light-proof conditions.

[0026] The application of the above-mentioned calpain activity detection method based on fluorescence sensor in cell imaging.

[0027] The application of the above-mentioned calpain activity detection method based on fluorescent sensor in detecting calpain activity in samples.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] The present invention uses nano-graphene oxide and fluorescent labeled polypeptide to prepare a FRET-based fluorescent sensor. In the constructed NGO-FSP fluorescent sensing system, NGO has a stable adsorption and sensitive fluorescence quenching effect on FSP. It is added to the sample to be tested and incubated. The active calpain contained in the sample to be tested cleaves the fluorescent polypeptide, causing the fluorescein FITC end to be released, so that the fluorescence can be restored ( Figure 1 ). Therefore, the activity of calpain can be detected according to the fluorescence intensity. The method of the present invention requires lower cost and has the advantages of being convenient and fast. The present invention can quickly and sensitively detect the activity of calpain in cell and muscle tissue samples, and further optimize the real-time monitoring method of calpain activity. In addition, the present invention proves through experiments that the cell imaging effect of NGO-FSP is good, and the NGO-FSP fluorescent sensing system can be applied to cell imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the principle of the present invention.

[0031] Figure 2 It is a fluorescence intensity result diagram of the doubly diluted fluorescent polypeptide FSP in Example 1; wherein A is a fluorescence spectrum diagram of the doubly diluted FSP, and B is a fluorescence intensity curve diagram.

[0032] Figure 3 It is a graph showing the detection results of the fluorescence quenching effect of NGO at different concentrations on FSP in Example 1; A is a fluorescence spectrum graph showing the fluorescence quenching effect of NGO at different concentrations on FSP, and B is a fluorescence intensity curve graph.

[0033] Figure 4 This is a result diagram of the effect of different reaction systems on the fluorescence quenching effect of NGO-FSP in Example 1.

[0034] Figure 5 This is a graph showing the fluorescence intensity stability results of the NGO-FSP reaction system (NGO concentration is 1 μg / mL, FSP concentration is 0.2 μM) and the fluorescent polypeptide FSP within 5 minutes in Example 1.

[0035] Figure 6 It is a graph showing the fluorescence regeneration results of the NGO-FSP system after adding calpains of different activities in Example 2; wherein A is a fluorescence spectrum graph after adding calpain; B is a fluorescence curve graph; and C is a linear fitting graph of relative fluorescence intensity to calpain activity.

[0036] Figure 7 It is the result diagram of the anti-interference analysis of the NGO-FSP system and the calpain selection specificity in Example 2; wherein A is the result diagram of the effect of different interferents on NGO-FSP; and B is the result diagram of verifying that the calpain activity leads to fluorescence recovery.

[0037] Figure 8 It is a graph showing the detection results of calpain activity in tissue and cell samples by the NGO-FSP system in Example 3; wherein A is a fluorescence spectrum graph of a muscle tissue sample; B is a fluorescence curve graph of a muscle tissue sample; C is a fluorescence spectrum graph of a MAEC cell sample; D is a fluorescence curve graph of a MAEC cell sample; E is a fluorescence spectrum graph of a Raw264.7 cell sample; and F is a fluorescence curve graph of a Raw264.7 cell sample.

[0038] Fig. 9 This is a cell imaging result diagram of the cell model after the NGO-FSP system treated with calpain inhibitors in Example 4; the CK group is the control group cells, which are not treated with calpain inhibitors; the Z-LLY-FMK group is the cells treated with calpain 2 inhibitors.

[0039] Fig.10 The laser confocal microscopy imaging results of different cell models constructed after the NGO-FSP system stimulated or inhibited the intracellular calcium ion flow in the cells in Example 4; the CK group was the control group cells; the Ionomycin group was the cells treated with ionomycin; and the BAPTA-AM group was the cells treated with a calcium ion chelator. DETAILED DESCRIPTION

[0040] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and the accompanying drawings, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If specific conditions are not specified, the conditions are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0041] Example 1 Construction of a fluorescence resonance energy transfer sensor

[0042] (1) Customized fluorescent labeled peptides

[0043] A sequence containing a Calpain2 restriction site at the N-terminus of selenoprotein K (SP, amino acid sequence: RRMGRISHLRGPSPPPMAGG, molecular weight: 2130.49Da) was customized by chemical synthesis (Shanghai Xinhao Biotechnology Co., Ltd.). During the solid phase peptide synthesis process, fluorescein isothiocyanate (FITC, excitation wavelength: 488 nm; emission wavelength: 520 nm) was labeled at the N-terminus of the peptide sequence to obtain 2 mg of fluorescent labeled peptide (FSP).

[0044] (2) Building an NGO-FSP system

[0045] Under light-proof conditions, ultrapure water was used to set a series of concentration gradients of FSP obtained in step (1), namely 0.4 μM, 0.2 μM, 0.1 μM, and 0.05 μM FSP, and the fluorescence intensity of FSP at different concentrations was detected by a fluorescence spectrophotometer ( Figure 2 ), the optimal concentration of FSP in the system was selected as 0.2 μM within a reasonable linear range.

[0046] Under light-shielding conditions, the FSP obtained in step (1) was set to a concentration of 0.2 μM in the system, and a series of amounts of NGO (purchased from Nanjing Xianfeng Nanomaterial Technology Co., Ltd., with a flake size range of 50-200 nm, an average flake size of 86 nm, a thickness of 0.8-1.6 nm, a monolayer ratio of 96%, a purity of 98%, and a specific surface area (BET): 90 m2 / g. It is a brown-yellow suspension at a concentration of 0.5 mg / mL in pure water) were added. The concentrations of NGO in the system were 0 μg / mL, 0.015 μg / mL, 0.03 μg / mL, 0.06 μg / mL, 0.12 μg / mL, 0.24 μg / mL, 0.48 μg / mL, 0.96μg / mL, and 1.92 μg / mL, respectively. The mixture was pipetted and mixed, and the fluorescence intensity and fluorescence spectrum were detected by fluorescence spectrophotometer ( Figure 3 ), the results showed that the fluorescence intensity decreased with the increase of NGO concentration. When the NGO concentration reached 1 μg / mL, the fluorescence quenching effect could reach 98%. After that, the fluorescence intensity would not decrease much when the NGO concentration was increased.

[0047] (3) In order to explore the effects of different reaction systems on the NGO-FSP fluorescence quenching system, the fluorescence intensity under different reaction systems was detected using a fluorescence spectrophotometer ( Figure 4). In addition to ultrapure water, different reaction systems also include phosphate buffered saline (PBS), cell lysate, complete culture medium, calcein buffer (CRB, calcein reaction buffer 10×CRB: 500 mM Tris-HCl, pH7.5; 100 mM CaCl2, 300 mM NaCl, 50 mM DTT), and normal saline. The results showed that the fluorescence quenching values ​​in different reaction systems were relatively close to the blank values, and the fluorescence quenching had a good effect relative to the initial fluorescence value, indicating that NGO-FSP has relative stability in different reaction systems.

[0048] In addition, in order to explore the dynamic effect of the NGO-FSP fluorescence quenching system within a certain period of time, the fluorescence intensity of the system was detected every 30 s using a fluorescence spectrophotometer ( Figure 5 ). The concentration of NGO in the system is 1 μg / mL, and the concentration of FSP is 0.2 μM. The fluorescent peptide FSP itself has a certain stability. According to the results, the fluorescent sensor NGO-FSP system produced after the fluorescent peptide FSP is adsorbed onto NGO has strong stability.

[0049] Example 2 Analysis of the reactivity and specificity of NGO-FSP to calpain 2 activity

[0050] (1) Using the NGO-FSP prepared in Example 1 as the system, first pre-mix FSP and NGO in a calpain buffer (CRB), the concentrations of FSP and NGO in the system were 0.2 μM and 1 μg / mL, respectively, and then different active calpain 2 (purchased from Abcam (Shanghai) Trading Co., Ltd.) were added to the NGO-FSP mixed system. The calpain 2 with a concentration of 0 to 20 μg / mL was measured by the Calpain Activity Assay Kit (purchased from abcam) to obtain an RFU range of 0 to 2500, and the calpain activity was expressed as relative fluorescence units (RFU) per milligram of protein. After incubation at 37°C in the dark for 50 min, the fluorescence intensity and fluorescence spectrum were detected using a fluorescence spectrophotometer, and the fluorescence intensity curve of calpain 2 with different activity sizes and the linear fitting diagram of the relative fluorescence intensity were obtained ( Figure 6 , the relative fluorescence intensity (F / F0) refers to the ratio of the fluorescence intensity after adding calpain 2 to the initial fluorescence intensity without adding calpain 2. ).

[0051] The results showed that the fluorescence intensity increased with the increase of calpain 2 activity. The fluorescence recovery effect was optimal when the RFU of calpain 2 was 2500, and the fluorescence recovery effect could reach 90%. In addition, the relative fluorescence unit had a good linear relationship with the relative fluorescence intensity in the range of 0 to 2500 (R 2 =0.9911).

[0052] (2) At the same time, the inventors also conducted interference experiments and specificity analysis, adding potassium chloride (2-150 mM), sodium chloride (2-150 mM), glycine (0.1-0.4 mM), glutamine (1-4 mM), glucose (5-35 mM), bovine serum albumin (20 μg / mL), calpain 1 (20 μg / mL) and calpain 2 (20 μg / mL) in the physiological medium range as interfering substances to the composite system NGO-FSP prepared in Example 1. The results showed that ( Figure 7 A) The fluorescence intensity barely changes after adding interfering substances, but the fluorescence is significantly enhanced after adding calpain 2 with an RFU of 2500 (calpain 2 at a concentration of 20 μg / mL obtained by the Calpain Activity Assay Kit).

[0053] In addition, a calpain 2 inhibitor (Z-LLY-FMK) was added to the NGO-FSP system to confirm that the enhanced fluorescence of the system was due to the activity of calpain ( Figure 7 B). It can be seen that the fluorescence recovery caused by calpain 2 is almost eliminated in the presence of calpain 2 inhibitor, and the calpain 2 inhibitor has no direct effect on the fluorescence intensity of the NGO-FSP system. Therefore, it is shown that the fluorescence recovery of the NGO-FSP system is caused by the cleavage of the fluorescent peptide by activated calpain 2.

[0054] The above conclusions all indicate that the constructed NGO-FSP has good selective specificity and anti-interference ability for active calpain 2.

[0055] Example 3 Detection of Calpain Activity in Actual Samples by NGO-FSP

[0056] (1) NGO-FSP detection of active calpain in muscle tissue samples

[0057] 8-week-old C57B / L mice were killed by dislocation, and the mouse legs were separated to obtain mouse skeletal muscle. To extract total protein from mouse skeletal muscle tissue: 0.1g-0.2g muscle was weighed into an EP tube, 200 μL of RIPA lysis buffer containing protease inhibitor (Cocktail Protease Inhibitor, 1000×) was added, ice-bath homogenization was performed, and ultrasonic disruption was performed. After thorough vortexing, the supernatant was centrifuged at 10000×g and 4°C for 10 minutes, and the supernatant was collected into a new EP tube. The protein concentration in the supernatant was determined by Bradford method.

[0058] Taking the NGO-FSP prepared in Example 1 as the system, first, FSP and NGO were pre-mixed in advance in a calpain buffer (CRB), and the concentrations of FSP and NGO in the system were 0.2 μM and 1 μg / mL, respectively. Then, mouse skeletal muscle protein extracts containing calpains with different RFU activities (total protein concentration ranged from 0 to 3.2 μg / mL, and RFU measured by the Calpain Activity Assay kit was 0 to 180) were added to the NGO-FSP mixed system, and the mixture was incubated in the dark at 37° C. for 50 min, and the fluorescence spectrum was detected using a fluorescence spectrophotometer. The fluorescence spectra and fluorescence intensity curves of the samples containing calpains with different activities were obtained ( Figure 8 A~B). The results showed that within the range of RFU of 0~180, when the activity of calpain in muscle samples was higher, the fluorescence regeneration phenomenon was more significant after incubation with NGO-FSP sensor, so the content of active calpain in muscle samples could be determined by fluorescence intensity.

[0059] (2) Detection of calpain activity in cells using NGO-FSP

[0060] Two types of cells were selected here, namely mouse aortic endothelial cells MAEC (from the American Model Culture Collection ATCC) and mouse macrophages Raw264.7 (from the Guangdong Academy of Agricultural Sciences) as actual cell samples to detect their intracellular calpain activity. First, the total cell protein was extracted, and RIPA lysis buffer containing protease inhibitor (Cocktail Protease Inhibitor, 1000×) was added to the culture dish where the cells were cultured, and incubated on ice for 30 minutes. Use a cell scraper to evenly scrape the bottom of the culture dish, collect the lysate in an EP tube, vortex thoroughly, centrifuge at 10000×g, 4℃ for 10 minutes, collect the supernatant in a new EP tube, and determine the protein concentration in the supernatant by Bradford method to obtain the total cell protein extract.

[0061] Taking the NGO-FSP prepared in Example 1 as the system, FSP and NGO were pre-mixed in advance in a calpain buffer, and total cell protein extracts containing calpains with different RFU activities were added to the NGO-FSP mixed system (the total protein concentration gradients of MAEC and Raw264.7 were both 0-16 μg / mL, and the RFU of calpain activity in MAEC was measured by the Calpain Activity Assay kit to be 0-3800; the RFU of calpain activity in Raw264.7 was 0-4200), and the fluorescence intensity was detected using a fluorescence spectrophotometer after incubation at 37°C in the dark for 50 min. The fluorescence spectra and fluorescence intensity curves of cell samples containing calpains with different activities can be obtained ( Figure 8 C~F). The results show that when the calpain activity of the cell sample is higher, the fluorescence regeneration effect is more significant after incubation, so the active calpain content in the cell sample can be determined by the fluorescence intensity.

[0062] Example 4 Cellular imaging of active calpains in different cell types using NGO-FSP

[0063] (1) Different cell models were constructed by inhibiting the enzymatic activity of intracellular calpain 2 using a calpain 2 inhibitor (Z-LLY-FMK, purchased from Merck). Mouse macrophages (Raw264.7) were cultured in complete culture medium (containing 10% fetal bovine serum, 100 U / mL penicillin, and 50 U / mL streptomycin). The cells were set up into two groups, each containing three replicate wells (2.5×10 5 cells / well). One group of cells was not added with inhibitors as the control group, and the other group was added with calpain 2 inhibitor (Z-LLY-FMK) at a concentration of 20 μM in advance. All cells were incubated in an incubator at 37°C for 12 hours. After removing the culture medium, the cells were washed twice with PBS buffer. The NGO-FSP system was prepared with DMEM stock solution (without 10% fetal bovine serum, 100 U / mL penicillin and 50 U / mL streptomycin). The final concentration of the fluorescent polypeptide FSP in the system was 20μM, and the final concentration of nano-graphene oxide was 100μg / mL. After mixing, the cells were added to the cells that had been washed twice with PBS and incubated at 37°C in the dark for 2 hours. All cells were washed twice with PBS buffer and then imaged using a cell imaging system ( Fig. 9 ).

[0064] The results showed that in the presence of calpain 2 inhibitors, the activity of intracellular calpain 2 was weakened, so there was almost no fluorescence in the cells after NGO-FSP treatment. However, there was a certain amount of calpain activity in the cells without the addition of calpain 2 inhibitors, which resulted in obvious fluorescence after NGO-FSP treatment. The results showed that NGO-FSP released the FITC fluorescent end under the cleavage of calpain 2, and the intensity of fluorescence could be used to simply, quickly and sensitively detect the size of intracellular calpain activity.

[0065] (2) By stimulating or inhibiting the changes in intracellular calcium flow-induced changes in calpain activity, cell models with different calpain activity were constructed. Mouse aortic endothelial cells (MAEC) were cultured in complete culture medium ( , and the cells were set up into three groups, each group of cells contained three wells (10 5 Cells / well) were cultured in an incubator at 37°C for 12 hours. The first group of cells was used as a blank control group, and the second and third groups of cells were added with 5 μM ionomycin (purchased from Invitrogen) and 20 μM calcium ion chelator BAPTA-AM (purchased from Selleck) to stimulate or inhibit calcium ion flow in the cells, respectively, and incubated in a 37°C incubator for 1 hour. After removing the culture medium from all cells, they were washed twice with PBS buffer, and the NGO-FSP system was prepared with DMEM stock solution (without 10% fetal bovine serum, 100 U / mL penicillin and 50 U / mL streptomycin). The final concentration of the fluorescent polypeptide FSP in the system was 20 μM, and the final concentration of nanographene oxide was 100 μg / mL. After mixing, it was added to the cells washed twice with PBS and incubated at 37°C in the dark for 2 hours. All cells were washed twice with PBS buffer, fixed with 4% (m / v) paraformaldehyde at room temperature for 15 min, washed three times with PBS, stained with PI (ex / em: 492 / 635 nm), and finally sealed with PVP anti-fluorescence quenching sealing solution. Cell imaging was performed using a confocal microscope ( Fig.10 ).

[0066] The results showed that the first group of cells had no stimulation or inhibition of calcium flow, and the cells themselves had a certain amount of active calpain 2, which had a certain fluorescence recovery after incubation with NGO-FSP; the second group of cells were stimulated by ionomycin, which mobilized the intracellular calcium ion flow to cause the activation of calpain, and the enhanced calpain activity made NGO-FSP have a strong fluorescence recovery; and the third group of cells were treated with the calcium ion chelator BAPTA-AM, and the intracellular calcium ion concentration decreased, causing the calpain activity to weaken, so NGO-FSP showed weaker fluorescence, which was consistent with the previous conclusion. In addition, it can be seen from the results that the cell imaging effect of NGO-FSP is good. Therefore, the above method can be used to detect the activity of calpain in cells and apply it to cell imaging.

[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for detecting calpain activity based on a fluorescent sensor, characterized in that The steps include: (1) Mixing nanographene oxide and fluorescent labeled peptide to obtain NGO-FSP fluorescent sensor; (2) mixing the sample to be tested with the NGO-FSP fluorescent sensor of step (1), and detecting the fluorescence intensity after incubation to determine the activity of calpain in the sample to be tested; The fluorescently labeled polypeptide described in step (1) is obtained by labeling fluorescein isothiocyanate on the N-terminus of the CAPN-specific enzyme-cleaved substrate polypeptide; The nano-graphene oxide described in step (1) has a sheet size of 50 to 200 nm and a specific surface area of ​​50 to 100 m 2 / g; The polypeptide is a sequence containing a calpain2 restriction site at the N-terminus of selenoprotein K, and its amino acid sequence is: RRMGRISHLRGPSPPPMAGG, with a molecular weight of 2130.49 Da; The excitation wavelength of the fluorescein isothiocyanate is 488 nm, and the emission wavelength is 520 nm; The concentration of the nano-graphene oxide described in step (1) in the system is 1 μg / mL; The concentration of the fluorescently labeled polypeptide in step (1) is 0.2 μM; The mixing described in step (1) is to add nano-graphene oxide to the fluorescent labeled polypeptide solution and mix by blowing; The fluorescent labeled polypeptide solution uses water as solvent; The sample to be tested in step (2) includes at least one of cells, tissues, or supernatant obtained after a total protein extraction step of cells / tissues; The system of the sample to be tested in step (2) does not contain Ca 2+ When calpain is added, calpain buffer is also needed to meet the environmental conditions for calpain2 enzyme cleavage; The incubation time in step (2) is 40 to 60 minutes; The incubation temperature in step (2) is 35-40°C; The fluorescence intensity in step (2) is detected by at least one of an ELISA reader, a fluorescence spectrophotometer or a laser confocal microscope; The steps (1) and (2) are all carried out under light-proof conditions.

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