Single-cell enzyme activity imaging detection system based on click chemical reaction and surface enhanced Raman spectroscopy and application of single-cell enzyme activity imaging detection system

Abstract

The invention discloses a single-cell enzyme activity imaging detection system based on click chemistry reaction and surface enhanced Raman spectroscopy and application of the single-cell enzyme activity imaging detection system. The single-cell enzyme activity imaging detection system comprises a microfluidic cell culture device, a polypeptide probe and shell isolation metal nanoparticles, the micro-fluidic cell culture device forms a closed micro-fluidic channel to control the flow rate of a solution and guide a polypeptide probe, and a shell layer isolates introduction of metal nanoparticles. According to the method, Raman signal change triggered by click chemical reaction is utilized, the shell layer is used for isolating the metal nanoparticles to remarkably enhance the Raman signal, the Raman spectrum of a specific group is analyzed, the activity imaging of the enzyme in the single cell is realized, the sensitivity and spatial resolution of the Raman spectrum are remarkably improved, and the detection sensitivity of the enzyme in the single cell is greatly improved. The method can provide high-specificity and high-sensitivity enzyme activity detection, provides a new tool for cell metabolism and disease research, and lays a foundation for development of a biosensing technology and a diagnostic method in the future.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme activity imaging and detection, and in particular to a single-cell enzyme activity imaging and detection system based on click chemistry reaction and surface enhanced Raman spectroscopy and its application. Background Art

[0002] Enzymatic processes within cells play a vital role in regulating a variety of biochemical pathways and cellular functions. Accurate monitoring of enzyme activity is of great significance for studying cellular metabolism, signal transduction, and disease progression. Currently, commonly used methods for monitoring enzyme activity include enzyme-linked immunosorbent assay (ELISA), bioluminescence, and fluorescence imaging techniques. Fluorescence imaging is widely used due to its high sensitivity and in vivo visualization capabilities. However, fluorescence imaging has some limitations, such as photobleaching, autofluorescence, and emission spectrum overlap, which result in limited multiplex detection capabilities. In addition, the size of fluorophores is comparable to that of enzymes, which may interfere with the normal function of enzymes. These issues indicate the need to develop new imaging methods that can maintain high specificity and high sensitivity without compromising spatial and temporal resolution.

[0003] As an effective alternative to fluorescence imaging, Raman imaging has the advantages of low photobleaching, low background noise, and no spectral overlap. In addition, the imaging groups in Raman imaging are usually specific chemical bonds (i.e., Raman tags), which are much smaller than traditional fluorophores and can minimize interference with enzyme activity. Despite this, the application of Raman imaging in enzyme activity detection is still limited. Current methods mainly detect the overall products of enzymatic reactions rather than specific enzyme-substrate reactions, indicating that further methodological improvements are needed to directly associate Raman signal changes with enzymatic processes.

[0004] In addition, the existing technology lacks devices that can detect enzyme activity in situ at the single-cell level, especially due to the heterogeneity between cells. This problem is more prominent. Current methods mostly rely on fixed-point sampling to detect the overall enzymatic reaction products, and cannot achieve real-time, in situ enzyme activity monitoring of single cells. This limitation hinders the in-depth understanding and precise analysis of dynamic processes within cells. Summary of the invention

[0005] Purpose of the invention: In order to solve the above technical problems, the present invention aims to provide an imaging system for detecting single-cell enzyme activity, which solves the problem that it is impossible to achieve in situ detection of enzyme activity at the single-cell level.

[0006] The present invention also provides a detection method for imaging single-cell enzyme activity. The microfluidic cell culture device and method of the present invention can realize real-time Raman imaging monitoring of enzyme activity in single cells under conditions of high sensitivity and high specificity.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a single-cell enzyme activity imaging system, which includes a microfluidic cell culture device, a polypeptide probe and shell-isolated metal nanoparticles. The microfluidic cell culture device forms a closed microfluidic channel to control the flow rate and the guidance of the polypeptide probe and the shell-isolated metal nanoparticles.

[0008] Furthermore, the polypeptide probe sequence is any one or more of Val-Cit-Cys(StBu)-Pra-Gly-CBT, Cit-Val-Gly-Cys(StBu)-Pra-CBT or Val-Cys(StBu)-Pra-Gly-Cit-CBT.

[0009] Furthermore, the side chain of Pra is grafted with an alkynyl group, and the end of CBT contains a cyano group.

[0010] Furthermore, the metal nanoparticles in the shell-isolated metal nanoparticles are any one of gold, silver or copper, and the shell material is any one of silicon dioxide or aluminum oxide.

[0011] Among them, metal nanoparticles can be synthesized by seed growth method, and their size is 10-80nm; shell coating can be achieved by controlled hydrolysis of silicon source or aluminum source, and a dense shell layer is gradually formed on the surface of the metal nanoparticles, and its thickness is 1-20nm.

[0012] Furthermore, the microfluidic cell culture device includes a microfluidic hose, a PDMS block with holes and a perforated glass sheet. The holes on the PDMS block are aligned with the holes on the perforated glass sheet, and the PDMS block is attached to both ends of the perforated glass sheet to form a glass flow channel cover. The glass flow channel cover is then bonded to a pretreated quartz sheet substrate using a waterproof double-sided tape to form a closed microfluidic channel. An L-shaped steel needle is inserted into the hole of the PDMS block, and a micro-injection pump and a waste liquid collection bottle are connected using a microfluidic hose.

[0013] After the polypeptide probe of the present invention enters the cell, the high concentration of glutathione in the cell can reduce the protecting group StBu on Cys to a thiol group, and the active enzyme cleaves the specific substrate, thereby releasing the nitrogen end of Cys, and then undergoes a CBT-Cys click chemistry reaction with the CBT group, consuming the cyano group on the polypeptide precursor molecule, and the alkynyl group on Pra can be used as an internal standard peak. The Raman scattering signals of the two are enhanced by the shell-isolated metal nanoparticles and then collected in situ by a Raman imager, thereby realizing enzyme activity monitoring.

[0014] The microfluidic cell culture device of the present invention is composed of the following parts: a quartz substrate, which is cleaned with piranha washing solution to ensure the hydrophilicity and cleanliness of the surface, and is used for cell adhesion while avoiding fluorescence interference; a glass flow channel cover, which is bonded to the quartz substrate by waterproof double-sided tape and is used for flow channel packaging, and its good light transmittance makes it suitable for long-term monitoring and observation under a microscope; a flow channel control system including a syringe pump, which is used to control the flow rate and guide the introduction of polypeptide probes, shell-isolated metal nanoparticles, and the introduction of extracellular fluid, stabilize the osmotic pressure, and ensure the long-term stability of cells.

[0015] Furthermore, the pre-treated quartz substrate is treated by immersing the quartz substrate in piranha washing solution, taking it out, rinsing it, and drying it with inert gas for later use.

[0016] Furthermore, the piranha wash solution is a mixed solution of concentrated sulfuric acid / hydrogen peroxide with a concentration ratio of 3-5:1-3, and the immersion time is 8-10 minutes.

[0017] Preferably, the piranha wash is a mixed solution of concentrated sulfuric acid / hydrogen peroxide with a concentration ratio of 3:1, and the immersion time is 10 minutes.

[0018] Application of the single-cell enzyme activity imaging system of the present invention in detecting single-cell enzyme activity.

[0019] Furthermore, the single-cell enzyme is any one or more of cathepsin B, acid phosphatase, furin or aminopeptidase N.

[0020] Furthermore, the application process is as follows: after the cells adhere to the wall, the shell-isolated metal nanoparticles are introduced into the microfluidic culture device by using a syringe pump for cell endocytosis, and then the extracellular fluid is introduced to wash away the free shell-isolated metal nanoparticles. Then, the microfluidic cell culture device is placed under a Raman microscope, and the polypeptide probe is introduced by using a syringe pump. The alkynyl peak and the cyano peak are imaged and analyzed in real time and in situ by a Raman imager.

[0021] The construction of a single-cell enzyme activity imaging detection system based on click chemistry reaction and surface enhanced Raman spectroscopy provided by the present invention comprises the following steps:

[0022] (1) Construction of microfluidic cell culture device and cell inoculation

[0023] The quartz substrate was cleaned with piranha lotion to obtain a highly hydrophilic and clean surface. The glass channel cover was bonded to the quartz substrate with waterproof double-sided tape to form a closed microfluidic channel. The channel thickness was determined by the thickness and number of double-sided tape layers, which was 50-500 μm. The cell suspension was injected into the channel with a syringe pump and incubated in a cell culture incubator overnight to allow the cells to adhere to the wall.

[0024] (2) Synthesis of peptide probes

[0025] Weigh a certain amount of amino acid monomers, and use a solid phase synthesis method to gradually synthesize the target polypeptide sequence on a synthetic resin; after completion, use a release reagent to cut the polypeptide from the synthetic resin, and use high performance liquid chromatography (HPLC) to purify it to obtain the desired polypeptide probe;

[0026] (3) Synthesis of Shell-Isolated Metal Nanoparticles

[0027] Gold, silver or copper is selected as the metal core, and silicon dioxide or aluminum oxide is used as the shell material; firstly, the metal core is obtained by a seed growth method, and then one or more of tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, sodium silicate, and aluminum isopropoxide are used to coat the shell, the reaction temperature is 25-90 degrees Celsius, the reaction time is 0.5-3 hours, and after the reaction is completed, centrifugation or filtration is performed, and the shell is washed three times with deionized water to obtain shell-isolated metal nanoparticles.

[0028] The single-cell enzyme activity imaging detection system based on click chemistry reaction and surface enhanced Raman spectroscopy provided by the present invention detects single-cell in situ enzyme activity Raman imaging as follows:

[0029] After the cells adhere to the wall, the shell-isolated metal nanoparticles are introduced into the microfluidic cell culture device by using a syringe pump for cell endocytosis. After incubation for 2-6 hours, the extracellular fluid is introduced to wash away the free shell-isolated metal nanoparticles. Then the microfluidic cell culture system is placed under a Raman microscope and the peptide probe is introduced by using a syringe pump at an injection flow rate of 0.1-1 mL / min. The alkynyl peak and cyano peak are imaged in real time and in situ and data analyzed by a Raman imager.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0031] The present invention integrates microfluidics and surface-enhanced Raman spectroscopy to achieve real-time, in-situ detection of enzyme activity in a single-cell environment. It utilizes the Raman signal changes triggered by click chemistry reactions, significantly enhances the Raman signal by isolating metal nanoparticles through the shell layer, analyzes the Raman spectra of specific groups, and achieves imaging of enzyme activity in a single cell. This method significantly improves the sensitivity and spatial resolution of Raman spectroscopy, and can achieve high-sensitivity and high-specificity in-situ detection of enzyme activity at the single-cell level. This system technology not only provides new tools for cell metabolism and disease research, but also lays the foundation for the development of future biosensor technology and diagnostic methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Design diagram of the single-cell enzyme activity imaging device;

[0033] Figure 2Physical picture of the glass flow channel cover;

[0034] Figure 3 Physical picture of microfluidic cell culture chip;

[0035] Figure 4 Mass spectrum of intermediate compound A;

[0036] Figure 5 Mass spectrum of intermediate compound B;

[0037] Figure 6 Mass spectrum of intermediate compound C;

[0038] Figure 7 Mass spectrum of intermediate compound D;

[0039] Figure 8 Mass spectrum of peptide precursor molecule Yne-CBT;

[0040] Fig. 9 1HNMR spectrum of peptide precursor molecule Yne-CBT;

[0041] Fig.10 13C NMR spectrum of peptide precursor molecule Yne-CBT;

[0042] Fig.11 Transmission electron microscopy image of Au@SiO2 nanoparticles (low magnification);

[0043] Fig.12 Transmission electron microscope photo of Au@SiO2 nanoparticles (high magnification);

[0044] Fig.13 Raman spectra of peptide precursor molecule Yne-CBT after incubation with different concentrations of cathepsin B (containing Au@SiO2 nanoparticles);

[0045] Fig.14 The Raman intensity ratio of alkynyl and cyano groups (I 2120 / I 2227 ) and the quantitative relationship between cathepsin B activity;

[0046] Fig.15 A photo of a microfluidic cell culture device placed under a Raman microscope;

[0047] Fig.16 Bright field photograph of a single MDA-MB-231 cell;

[0048] Fig.17 Single cell in situ Raman imaging (lipids, 2800-3000cm -1 );

[0049] Fig.18Single cell in situ Raman imaging (alkyne, 2000-2150cm -1 );

[0050] Fig.19 Single cell in situ Raman imaging (cyano, 2150-2300cm -1 );

[0051] Fig. 20 Raman scattering spectra of different locations of a single cell. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0053] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0054] Example 1

[0055] Construction of microfluidic cell culture device and cell seeding

[0056] The design diagram of the single-cell enzyme activity imaging device is shown in the figure. Figure 1 As shown, it specifically includes the following steps:

[0057] (1) Quartz substrate pretreatment

[0058] Select high-transmittance, non-fluorescent quartz (purchased from Spectronix Optics, model JGS1) as the substrate, with a size of 50×25×0.5mm and a light transmission range of 185-2500nm. Soak the quartz sheet in piranha wash solution (concentrated sulfuric acid / hydrogen peroxide = 3:1) for surface activation, remove it after 10 minutes, rinse the quartz sheet three times with deionized water, and then blow dry with high-purity argon gas to obtain quartz sheet substrate 6 for standby use;

[0059] (2) Construction of microfluidic cell culture device

[0060] The glass flow channel cover is made of high-transmittance optical glass with a size of 40×22×0.15mm. Laser drilling is used to drill holes on the glass sheet with a hole diameter of 1mm and a hole distance of 2mm from the center of the short side to obtain a perforated glass sheet 3. Polydimethylsiloxane (PDMS, purchased from Dow Corning, item number Sylgard 184) is used to solidify it according to the instructions provided by Dow Corning, and then it is cut into small squares of 2×2×2mm. A punching needle is used to punch a circular hole with a diameter of 0.5mm on the PDMS block 2. The surface of the PDMS block 2 and the perforated glass sheet 3 is cleaned with a plasma cleaner, and then the holes on the PDMS block 2 are aligned and fitted with the holes on the perforated glass sheet 3. Press with your fingers for 2 minutes to ensure a tight fit, and the glass flow channel cover is completed ( Figure 2 ). Use waterproof double-sided tape 4 to bond the glass channel cover to the treated quartz substrate 5 to form a closed microfluidic channel. The thickness of a single layer of double-sided tape is about 50 μm, which is the thickness of the produced channel. Insert two L-shaped steel needles (outer diameter 0.6 mm, inner diameter 0.5 mm) into the holes on the PDMS block 2, and connect the microinjection pump and the waste liquid collection bottle with microfluidic catheters 1 (inner diameter 0.5 mm) at both ends. Finally, pass the extracellular fluid to check the sealing of the system, and the construction of the microfluidic cell culture device is completed ( Figure 3 );

[0061] (3) Cell seeding

[0062] First, MDA-MB-231 cells were cultured in a cell culture flask using DMEM medium (Keygen Biotechnology, catalog number KGL1206-500) containing 10% FBS (Gibco, catalog number 10270-106). When the cell growth density reached 80%, 2 mL of trypsin (Gibco, catalog number 25200-056) was added and incubated at 37°C for 5 minutes to fully digest the cells. The digested cells were mixed with DMEM medium by volume at 1:1 and centrifuged (1000 rpm, 5 minutes), and the cell pellet was collected and resuspended in fresh DMEM medium. The treated cell suspension was injected into the microfluidic cell culture device using a syringe pump, with an inoculation density of 20,000 cells / ml. The microfluidic catheter 1 of the culture system was removed and placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours to ensure cell adhesion and proliferation.

[0063] Example 2

[0064] Synthesis of peptide precursor molecule Yne-CBT

[0065] The specific sequence of the polypeptide precursor molecule Yne-CBT is Val-Cit-Cys(StBu)-Pra-Gly-CBT, and its structural formula and synthetic route are as follows:

[0066]

[0067] The specific synthesis method of the Val-Cit-Cys(StBu)-Pra-Gly-CBT is as follows:

[0068] (1) Synthesis of intermediate compound A (Fmoc-Gly-CBT):

[0069] Isobutyl chloroformate (IBCF) 168.6 μL (1.3 mmol) was added to a mixture of tetrahydrofuran (THF, 5 mL) containing Fmoc-glycine (Fmoc-Gly-OH, 297.3 mg, 1 mmol) and 4-methylmorpholine (MMP) 285.8 μL (2.6 mmol), and the reaction temperature was 0°C. Stirring at 0°C for 1 hour, then adding a solution of 2-cyano-6-aminobenzothiazole (CBT, 192.7 mg, 1.1 mmol), and stirring was continued at 0°C for 2 hours. The mixture was stirred at room temperature overnight, using a water / acetonitrile mixed solvent (volume ratio from 30:70 to 0:100), and adding 0.1% TFA as an eluent, elution time 25 minutes, and high performance liquid chromatography (HPLC) purification was performed to obtain Compound A (325.8 mg, yield: 71.7%). Mass spectrometry (MS) results: calculated value is 455.1 [M+H] +, measured value is 455.0 (ESI-MS) ( Figure 4 ).

[0070] (2) Synthesis of intermediate compound B (Gly-CBT):

[0071] Add 12% piperidine to the N,N-dimethylformamide (DMF, 3mL) solution, then add all the compound A synthesized in the previous step, remove the Fmoc protecting group of compound A, react at 0°C for 7 minutes, and then add 360μL trifluoroacetic acid (TFA) to neutralize to alkalinity. Use a water / acetonitrile mixed solvent (volume ratio from 20:80 to 0:100), add 0.1% TFA as eluent, elution time 25 minutes, HPLC purification to obtain compound B (130.3mg, yield: 78.0%). Mass spectrometry results: calculated value is 233.0[M+H]+, measured value is 233.1(ESI-MS)( Figure 5 ).

[0072] (3) Synthesis of intermediate compound C (Fmoc-Val-Cit-Cys(StBu)-Pra-OH):

[0073] The peptide Fmoc-Val-Cit-Cys(StBu)-Pra-OH(C) was synthesized using solid phase peptide synthesis (SPPS, 1 g 2-chlorotrityl chloride resin). A water / acetonitrile mixed solvent (volume ratio from 30:70 to 0:100) was used, and 0.1% TFA was added as an eluent. The elution time was 25 minutes, and HPLC purification was performed to obtain compound C (520 mg, yield: 66.5%). Mass spectrometry results: calculated value is 783.3 [M+H] +, and the measured value is 783.3 (ESI-MS) ( Figure 6 ).

[0074] (4) Synthesis of intermediate compound D (Fmoc-Val-Cit-Cys(StBu)-Pra-Gly-CBT):

[0075] Compound C (156.5 mg, 0.20 mmol), compound B (58.0 mg, 0.25 mmol), HATU (114.1 mg, 0.30 mmol), HOAT (40.8 mg, 0.30 mmol) and DIPEA (104 μL, 0.60 mmol) were mixed in DMF (3 mL) and stirred at 40° C. for 1 hour.

[0076] Using a water / acetonitrile mixed solvent (volume ratio from 20:80 to 0:100), and adding 0.1% TFA as an eluent, the elution time was 25 minutes, and HPLC purification was performed to obtain compound D (150.5 mg, yield: 75.5%). Mass spectrometry results: calculated value is 997.3 [M+H] +, measured value is 997.3 (ESI-MS) ( Figure 7 ).

[0077] (5) Synthesis of peptide probe Yne-CBT (Val-Cit-Cys(StBu)-Pra-Gly-CBT):

[0078] 12% piperidine was added to DMF (3 mL), and then all the compound D synthesized in the previous step was added, the Fmoc protecting group of compound D was removed, and the reaction was carried out at 0°C for 7 minutes, and then 360 μL TFA was added to neutralize the alkalinity. Yne-CBT (93.8 mg, yield: 80.1%) was obtained by HPLC purification, and lyophilized to obtain a solid powder, i.e., a peptide probe. Mass spectrometry results: calculated value is 775.3 [M+H] +, and the measured value is 775.3 (ESI-MS) ( Figure 8 ). 1 HNMR (600 MHz, DMSO-d6) and 13 C NMR (151MHz, DMSO-d6) spectrum is attached Fig. 9 , Fig.10 .

[0079] Example 3

[0080] Synthesis of Shell-Isolated Metal Nanoparticles

[0081] (1) Synthesis of Au seeds:

[0082] Au seeds with a diameter of about 16 nm were synthesized using the sodium citrate reduction method. The specific operation was as follows: 50 mL of chloroauric acid (0.5 mM) aqueous solution was heated to boiling under stirring at 1200 rpm, and then 1.5 mL of sodium citrate (38.8 mM) aqueous solution was quickly added. The solution continued to boil for 10 minutes under moderate stirring (1000 rpm) and then cooled naturally. The resulting Au seed solution was stored at 4 ° C in the dark before use.

[0083] (2) Synthesis of Au nanoparticles (AuNPs):

[0084] Au NPs with a diameter of approximately 32 nm were synthesized via a two-step growth method.

[0085] Two solutions were prepared: the first solution (solution 1) was mixed with 27 mL of water, hydroxylamine hydrochloride (0.2 M, 0.25 mL) and chloroauric acid (25 mM, 0.3 mL); the second solution (solution 2) was mixed with 30 mL of water, hydroxylamine hydrochloride (0.2 M, 0.224 mL) and chloroauric acid (25 mM, 0.4 mL). 7.2 mL of the above Au seed solution was added to solution 1 and stirred at room temperature for 30 minutes. Then 11.2 mL of the resulting solution was added to solution 2 and continued to stir at room temperature for 30 minutes. The resulting AuNPs were stored in a dark place at 4 °C before use.

[0086] (3) Synthesis of Au@SiO2 nanoparticles:

[0087] 0.4 mL of freshly prepared 3-aminopropyltriethoxysilane aqueous solution (1 mM) was added to 30 mL of the above AuNPs solution and stirred at room temperature for 15 minutes. Then 3.2 mL of sodium silicate aqueous solution (45.3 mM) was added under stirring at 1200 rpm. After the solution was stirred at room temperature for 3 minutes, it was heated to 98 ° C and continued to stir for 2 hours. The formed Au@SiO2NPs were collected by centrifugation (15,000 rpm, 10 minutes) The supernatant was redispersed in 3 mL of water for further use. The thickness of the resulting SiO2 shell was about 3 nm.

[0088] Through the above steps, Au@SiO2 nanoparticles with SiO2 shells, i.e., shell-isolated metal nanoparticles 9, were successfully synthesized with a concentration of about 2 nM. The transmission electron microscope (TEM) image is shown in Fig.11 , Fig.12 These shell-isolated nanoparticles will be used in subsequent in situ imaging experiments of single-cell enzyme activity.

[0089] Example 4

[0090] In vitro enzyme activity quantitative detection

[0091] (1) Preparation of CTSB enzyme working solution:

[0092] 1.8 mg of freeze-dried bovine spleen Cathepsin B (CTSB) solid (Sigma-Aldrich, Catalog No.: C6286) was dissolved in 0.5 mL of 25 mM sodium acetate / 1 mM EDTA buffer (pH 5.0) to prepare a CTSB working solution with a final enzyme concentration of 10 U / mL.

[0093] (2) Raman quantitative detection of enzyme activity in vitro:

[0094] The above TSB working solution was diluted with a buffer solution (20mM phosphate buffer, 1mM EDTA, 0.2mM TCEP, pH 7.4) to a final concentration of 0, 0.125, 0.25, 0.5, 1.0, and 2.0U / mL, respectively, with a volume of 1mL. 2μL of Yne-CBT (10.0mM) prepared in Example 2 was added thereto respectively, mixed evenly, and incubated at 37°C for 2 hours. For surface enhanced Raman spectroscopy measurement, 20μL of the above reaction solution was mixed with 20μL of Au@SiO2 nanoparticles (100pM) prepared in Example 3 respectively. The mixed solution (10μL) was dripped onto a pre-cleaned silicon wafer (Lijing Silicon Co., Ltd., square single polished 5mm×5mm, thickness 650μm), and its Raman spectrum was measured using a Raman spectrometer, with an excitation of 638nm, an integration time of 1 second, and a wavelength of 2120cm -1 (Alkynyl) and 2220cm -1 The Raman peak at (cyano) was analyzed by ratio analysis to obtain the quantitative relationship between Raman intensity and enzyme activity. The results showed that within the CTSB concentration range of 0-1U / mL, the Raman intensity ratio and CTSB concentration showed good linearity, with a correlation coefficient of 0.999. Fig.13 , Fig.14 ).

[0095] Example 5

[0096] Raman imaging measurement of single cell enzyme activity

[0097] The Au@SiO2 nanoparticles (50 pM, 0.1 mL) prepared in Example 3 were added to the microfluidic cell culture device prepared in Example 1 and incubated with MDA-MB-231 cells through a syringe pump and incubated for 6 hours to ensure that the shell layer isolated the metal nanoparticles 8 (Au@SiO2 nanoparticles) from entering the interior of the MDA-MB-231 cells. Then, the extracellular fluid (Biyuntian, item number C0216-500 ml) was introduced to wash away the excess Au@SiO2 nanoparticles. The microfluidic cell culture device was imaged under a Raman microscope ( Fig.15), the microscope is equipped with a 50X objective lens (NA 0.5) and uses a 638nm laser as the excitation light source. First, find a single cell in the bright field. Fig.16 ), Yne-CBT (Val-Cit-Cys (StBu) -Pra-Gly-CBT) 7 (20 μM) prepared in Example 2 was introduced into the above-mentioned flow channel by a syringe pump, and the injection flow rate was 0.2 mL / min. After in situ incubation for 4 h, lipid ( Fig.17 ), alkynyl ( Fig.18 ) and cyano ( Fig.19 ) channel and measured its Raman spectrum, and found that cyano (2220cm -1 ) signal relative to the alkynyl (2120cm -1 ) has decreased significantly ( Fig. 20 ), demonstrating the effectiveness of this method for in situ CTSB enzyme activity detection.

Claims

1. A single cell enzyme activity imaging detection system, characterized in that: The detection system comprises a microfluidic cell culture device, a polypeptide probe and shell-isolated metal nanoparticles. The microfluidic cell culture device forms a closed microfluidic channel to control the flow rate of the solution and guide the introduction of the polypeptide probe and the shell-isolated metal nanoparticles.

2. The single cell enzyme activity imaging detection system according to claim 1, characterized in that: The polypeptide probe sequence is any one or more of Val-Cit-Cys(StBu)-Pra-Gly-CBT, Cit-Val-Gly-Cys(StBu)-Pra-CBT or Val-Cys(StBu)-Pra-Gly-Cit-CBT.

3. The single cell enzyme activity imaging detection system according to claim 2, characterized in that: The side chain of Pra is grafted with an alkynyl group, and the terminal of CBT contains a cyano group.

4. The single cell enzyme activity imaging detection system according to claim 1, characterized in that: The metal nanoparticles in the shell-isolated metal nanoparticles are any one of gold, silver or copper, and the shell is any one of silicon dioxide or aluminum oxide.

5. The single cell enzyme activity imaging detection system according to claim 1, characterized in that: The microfluidic cell culture device comprises a microfluidic hose (1), a PDMS block with holes (2) and a perforated glass sheet (3). The holes on the PDMS block (2) are aligned with the holes on the perforated glass sheet (3), and the PDMS block (2) is attached to both ends of the perforated glass sheet (3) to form a glass flow channel cover. The glass flow channel cover is then bonded to a pre-treated quartz sheet substrate (5) using a waterproof double-sided adhesive tape (4) to form a closed microfluidic channel. An L-shaped steel needle is inserted into the hole of the PDMS block (2), and a micro-injection pump and a waste liquid collection bottle are connected using the microfluidic hose (1).

6. The microfluidic cell culture device according to claim 5, characterized in that: The pre-treated quartz substrate (5) is treated by immersing the quartz substrate in piranha washing solution, taking it out and rinsing it, and drying it with inert gas for later use.

7. The microfluidic cell culture device according to claim 6, characterized in that: The piranha wash solution is a concentrated sulfuric acid / hydrogen peroxide mixture with a concentration ratio of 3-5:1-3, and the soaking time is 8-10 minutes.

8. Use of the single-cell enzyme activity imaging detection system according to claim 1 in detecting single-cell enzyme activity.

9. The use according to claim 8, characterized in that: The single cell enzyme is any one or more of cathepsin B, acid phosphatase, furin or aminopeptidase N.

10. The use according to claim 8, characterized in that: The application process is as follows: after a single cell (6) adheres to the wall, a syringe pump is used to introduce the shell-isolated metal nanoparticles (8) into a microfluidic culture device for cell endocytosis, and then an extracellular fluid is introduced to wash away the free shell-isolated metal nanoparticles (8). Then, the microfluidic cell culture device is placed under a Raman microscope, and a polypeptide probe (7) is introduced by using a syringe pump. Real-time, in-situ imaging and data analysis of the alkynyl peak and the cyano peak are performed by a Raman imager.

Images