A single-cell enzyme activity imaging detection system based on click chemistry and surface-enhanced Raman spectroscopy and its application

Abstract

The present invention discloses a single-cell enzyme activity imaging detection system based on click chemistry and surface-enhanced Raman spectroscopy and its application. The single-cell enzyme activity imaging detection system 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 solution flow rate and guide the introduction of the polypeptide probe and shell-isolated metal nanoparticles. The present invention utilizes the Raman signal change triggered by click chemistry, significantly enhances the Raman signal through shell-isolated metal nanoparticles, analyzes the Raman spectrum of specific groups, and realizes the imaging of enzyme activity in single cells. This method significantly improves the sensitivity and spatial resolution of Raman spectroscopy, can provide highly specific and sensitive enzyme activity detection, provides a new tool for cell metabolism and disease research, and lays a foundation for the development of future biosensing technologies and diagnostic methods.

Description

Technical Field

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

[0002] Enzymatic processes within cells play a crucial role in regulating various biochemical pathways and cellular functions. Accurately monitoring enzyme activity is of great significance for studying cell 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 has been widely used due to its high sensitivity and in vivo visualization ability. However, fluorescence imaging has some limitations, such as photobleaching, autofluorescence, and emission spectrum overlap, resulting 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 problems indicate the necessity to develop new imaging methods that can maintain high specificity and high sensitivity while not affecting spatial and temporal resolution.

[0003] As an effective alternative to fluorescence imaging, Raman imaging has advantages such as little 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. Nevertheless, 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 improvement is needed in methodology to directly correlate Raman signal changes with enzymatic processes.

[0004] In addition, in the prior art, there is a lack of devices capable of in-situ enzyme activity detection at the single-cell level, especially due to cell-to-cell heterogeneity, this problem is even 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 intracellular dynamic processes. Summary of the Invention

[0005] Object of the Invention: To solve the above technical problems, the present invention aims to provide a single-cell enzyme activity imaging detection system, which solves the problem of unable to achieve in-situ enzyme activity detection at the single-cell level.

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

[0007] Technical solution: To achieve the above object, the present invention provides a single-cell enzyme activity detection 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 guide the polypeptide probe and the shell-isolated metal nanoparticles.

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

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

[0010] Further, 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, the metal nanoparticles can be synthesized by the seed growth method, with a size of 10-80 nm; the shell coating can adopt the controlled hydrolysis of a silicon source or an aluminum source to gradually form a dense shell on the surface of the metal nanoparticles, with a thickness of 1-20 nm.

[0012] Further, the microfluidic cell culture device includes a microfluidic hose, a perforated PDMS block, and a perforated glass slide. Align the holes on the PDMS block with the holes on the perforated glass slide, and attach the PDMS block to both ends of the perforated glass slide to form a glass flow channel cover. Then use waterproof double-sided tape to bond the glass flow channel cover to the pretreated quartz wafer substrate to form a closed microfluidic channel. Insert an L-shaped steel needle into the hole of the PDMS block, and connect a micro-injection pump and a waste liquid collection bottle with the microfluidic hose.

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

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

[0015] Further, the treatment method of the pretreated quartz substrate is to soak the quartz substrate in piranha solution, take it out and rinse, and dry it with inert gas for standby.

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

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

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

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

[0020] Further, the application process is as follows: after the cells adhere to the wall, use an injection pump to introduce shell-isolated metal nanoparticles into the microfluidic culture device for endocytosis, then introduce extracellular fluid to wash away the free shell-isolated metal nanoparticles, and then place the microfluidic cell culture device under a Raman microscope, use an injection pump to introduce polypeptide probes, and perform real-time, in-situ imaging and data analysis on the alkyne peak and cyano peak through a Raman imager.

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

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

[0023] Clean the quartz substrate with piranha solution to obtain a highly hydrophilic and clean surface, bond the glass flow channel cover to the quartz substrate through waterproof double-sided tape to form a closed microfluidic channel, the thickness of the channel is determined by the thickness and number of layers of the double-sided tape, which is 50 - 500 μm, inject the cell suspension into the channel through an injection pump, and incubate it overnight in a cell culture incubator to make the cells adhere to the wall;

[0024] (2) Synthesis of Polypeptide Probe

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

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

[0027] Select gold, silver or copper as the metal core, and silica or alumina as the shell material; first obtain the metal core by the seed growth method, and then coat the shell with one or several of tetraethyl orthosilicate, 3-aminopropyltriethoxysilane, sodium silicate, and aluminum isopropoxide. The reaction temperature is 25 - 90 °C, the reaction time is 0.5 - 3 hours. After the reaction, centrifuge or filter, and wash three times with deionized water to obtain the 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 has the following method for detecting single-cell in-situ enzyme activity Raman imaging:

[0029] After the cells adhere to the wall, use an injection pump to introduce the shell-isolated metal nanoparticles into the microfluidic cell culture device for endocytosis. After incubating for 2 - 6 hours, introduce extracellular fluid to wash away the free shell-isolated metal nanoparticles, and then place the microfluidic cell culture system under a Raman microscope. Use an injection pump to introduce the polypeptide probe, and the injection flow rate is 0.1 - 1 mL / min. Perform real-time, in-situ imaging and data analysis on the alkyne peak and cyano peak through a Raman imager.

[0030] Advantages: Compared with the prior art, the present invention has the following remarkable advantages:

[0031] The present invention integrates microfluidic technology and surface-enhanced Raman spectroscopy to achieve real-time, in-situ detection of enzyme activity in a single-cell environment. Utilize the Raman signal change triggered by click chemistry reaction, significantly enhance the Raman signal through shell-isolated metal nanoparticles, analyze the Raman spectrum of specific groups, and achieve imaging of enzyme activity in a single cell. This method significantly improves the sensitivity and spatial resolution of Raman spectroscopy, and can achieve highly sensitive and highly specific in-situ detection of enzyme activity at the single-cell level. This system technology not only provides a new tool for cell metabolism and disease research, but also lays a foundation for the development of future biosensing technologies and diagnostic methods. Description of the Drawings

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

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

[0034] Figure 3 Physical diagram of the 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 the polypeptide precursor molecule Yne-CBT;

[0040] Figure 9 1H NMR spectrum of the polypeptide precursor molecule Yne-CBT;

[0041] Figure 10 13C NMR spectrum of the polypeptide precursor molecule Yne-CBT;

[0042] Figure 11 Transmission electron microscope image of Au@SiO2 nanoparticles (low magnification);

[0043] Figure 12 Transmission electron microscope image of Au@SiO2 nanoparticles (high magnification);

[0044] Figure 13 Raman spectrum of the polypeptide precursor molecule Yne-CBT incubated with different concentrations of cathepsin B (containing Au@SiO2 nanoparticles);

[0045] Figure 14 Ratio of Raman intensities of alkynyl and cyano groups (I 2120 / I 2227 ) and the quantitative relationship with cathepsin B activity;

[0046] Figure 15 Physical diagram of the microfluidic cell culture device placed under a Raman microscope;

[0047] Figure 16 Bright-field image of a single MDA-MB-231 cell;

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

[0049] Figure 18Single-cell in-situ Raman imaging (alkynyl, 2000 - 2150 cm -1 );

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

[0051] Figure 20 Raman scattering spectra at different positions of a single cell. Detailed implementation mode

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0053] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions. The experimental methods without specific conditions in the examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0054] Example 1

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

[0056] The design diagram of the single-cell enzyme activity imaging device is as Figure 1 shown, and specifically includes the following steps:

[0057] (1) Pretreatment of the quartz substrate

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

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

[0060] The glass flow channel cover is made of high-transparency optical glass, with a size of 40×22×0.15 mm. Laser drilling is used to drill holes on the glass wafer, with a hole diameter of 1 mm and a hole distance of 2 mm from the center of the short side, to obtain the drilled glass wafer 3. Use polydimethylsiloxane (PDMS, purchased from Dow Corning, product number Sylgard 184), cure it according to the instructions provided by Dow Corning, and then cut it into small cubes with a size of 2×2×2 mm. Use a punching needle to punch a round hole with a diameter of 0.5 mm on the PDMS block 2. Clean the surfaces of the PDMS block 2 and the drilled glass wafer 3 with a plasma cleaner, then align the holes on the PDMS block 2 with the holes on the drilled glass wafer 3 and fit them together, and press with fingers for 2 minutes to ensure a tight fit, thus completing the production of the glass flow channel cover (Figure 2 )。Use waterproof double-sided tape 4 to bond the glass flow channel cover to the treated quartz wafer substrate 5 to form a closed microfluidic channel. The thickness of the single-layer double-sided tape is about 50 μm, which is also the thickness of the fabricated flow 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, connect the micro-injection pump and the waste liquid collection bottle at both ends with microfluidic catheters 1 (inner diameter 0.5 mm) respectively, and finally introduce extracellular fluid to check the tightness of the system, then the construction of the microfluidic cell culture device can be completed ( Figure 3 );

[0061] (3) Cell seeding

[0062] First, culture MDA-MB-231 cells in a cell culture flask using DMEM medium (Kaiji Bio, product number KGL1206-500) containing 10% FBS (Gibco, product number 10270-106). When the cell growth density reaches 80%, add 2 mL of trypsin (Gibco, product number 25200-056), incubate at 37 °C for 5 minutes to fully digest the cells. After digestion, mix the cells with DMEM medium at a volume ratio of 1:1 and centrifuge (1000 rpm, 5 minutes). Collect the cell pellet and resuspend it in fresh DMEM medium. Use an injection pump to inject the treated cell suspension into the microfluidic cell culture device at an inoculation density of 20,000 cells / mL. Remove the microfluidic catheter 1 of the culture system and place it 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 polypeptide 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 synthesis route are as follows:

[0066]

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

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

[0069] 168.6 μL (1.3 mmol) of isobutyl chloroformate (IBCF) 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. Stir at 0 °C for 1 h, then add a solution of 2-cyano-6-aminobenzothiazole (CBT, 192.7 mg, 1.1 mmol), and continue to stir at 0 °C for 2 h. Stir the mixture overnight at room temperature. High performance liquid chromatography (HPLC) purification was carried out using a water / acetonitrile mixed solvent (volume ratio from 30:70 to 0:100) and adding 0.1% TFA as the eluent, with an elution time of 25 min, to obtain Compound A (325.8 mg, yield: 71.7%). Mass spectrometry (MS) measurement 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] 12% piperidine was added to a solution of N,N-dimethylformamide (DMF, 3 mL), and then all of the Compound A synthesized in the previous step was added to remove the Fmoc protecting group of Compound A. The reaction was carried out at 0 °C for 7 min, and then 360 μL of trifluoroacetic acid (TFA) was added to neutralize to alkaline. HPLC purification was carried out using a water / acetonitrile mixed solvent (volume ratio from 20:80 to 0:100) and adding 0.1% TFA as the eluent, with an elution time of 25 min, to obtain Compound B (130.3 mg, yield: 78.0%). Mass spectrometry measurement 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 polypeptide Fmoc-Val-Cit-Cys(StBu)-Pra-OH (C) was synthesized using solid phase peptide synthesis (SPPS, 1 g of 2-chlorotrityl chloride resin). HPLC purification was carried out using a water / acetonitrile mixed solvent (volume ratio from 30:70 to 0:100) and adding 0.1% TFA as the eluent, with an elution time of 25 min, to obtain Compound C (520 mg, yield: 66.5%). Mass spectrometry measurement results: calculated value is 783.3 [M+H]+, 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 the eluent, with an elution time of 25 minutes, HPLC purification was carried out to obtain compound D (150.5 mg, yield: 75.5%). Mass spectrometry determination result: calculated value is 997.3 [M+H]+, measured value is 997.3 (ESI-MS)( Figure 7 )

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

[0078] 12% piperidine was added to DMF (3 mL), and then all of the compound D synthesized in the previous step was added to remove the Fmoc protecting group of compound D. The reaction was carried out at 0 °C for 7 minutes, and then 360 μL of TFA was added to neutralize the alkalinity. HPLC purification was used to obtain Yne-CBT (93.8 mg, yield: 80.1%), and it was freeze-dried to obtain a solid powder, which is the polypeptide probe. Mass spectrometry determination result: calculated value is 775.3 [M+H]+, measured value is 775.3 (ESI-MS)( Figure 8 ) 1 1H NMR (600 MHz, DMSO-d6) and 13 13C NMR (151 MHz, DMSO-d6) spectra are shown in the appendix Figure 9 、 Figure 10 。

[0079] Example 3

[0080] Synthesis of shell-isolated metal nanoparticles

[0081] (1) Synthesis of Au seeds:

[0082] Au seeds with a diameter of approximately 16 nm were synthesized using the sodium citrate reduction method. The specific operation was as follows: 50 mL of an aqueous solution of chloroauric acid (0.5 mM) was heated to boiling with stirring at 1200 rpm, and then 1.5 mL of an aqueous solution of sodium citrate (38.8 mM) was rapidly 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 in the dark at 4 °C before use.

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

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

[0085] Two solutions were prepared: The first solution (solution 1) was a mixture of 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 a mixture of 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 stirring was continued at room temperature for 30 minutes. The resulting AuNPs were stored in the dark at 4 °C before use.

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

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

[0088] Through the above steps, Au@SiO2 nanoparticles with an SiO2 shell, namely shell-isolated metal nanoparticles 9, were successfully synthesized, and their concentration was approximately 2 nM. The transmission electron microscope (TEM) images are shown in Figure 11 、 Figure 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] Dissolve 1.8 mg of freeze-dried bovine spleen Cathepsin B (CTSB) solid (Sigma-Aldrich, catalog number: C6286) 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) In vitro enzymatic activity Raman quantitative detection:

[0094] Dilute the above-mentioned TSB working solution with buffer (20 mM phosphate buffer, 1 mM EDTA, 0.2 mM TCEP, pH 7.4) to make its final concentrations 0, 0.125, 0.25, 0.5, 1.0, 2.0 U / mL respectively, with a volume of 1 mL each. Add 2 μL of Yne-CBT (10.0 mM) prepared in Example 2 to each of them. After mixing evenly, incubate at 37 °C for 2 hours. For surface-enhanced Raman spectroscopy measurement, mix 20 μL of the above reaction solution with 20 μL of Au@SiO2 nanoparticles (100 pM) prepared in Example 3 respectively. Drop 10 μL of the mixed solution on a pre-cleaned silicon wafer (Lijing Silicon Co., Ltd., square single-polished 5 mm × 5 mm, thickness 650 μm), and measure its Raman spectrum with a Raman spectrometer, excited at 638 nm, integration time 1 second, and perform ratio analysis on the Raman peaks at 2120 cm -1 (alkynyl) and 2220 cm -1 (cyano) to obtain the quantitative relationship between Raman intensity and enzymatic activity. The results show that in the CTSB concentration range of 0 - 1 U / mL, the Raman intensity ratio shows good linearity with the CTSB concentration, and the correlation coefficient is 0.999. ( Figure 13 、 Figure 14 )

[0095] Example 5

[0096] Single-cell enzymatic activity Raman imaging measurement

[0097] Add the Au@SiO2 nanoparticles (50 pM, 0.1 mL) prepared in Example 3 to the microfluidic cell culture device incubated with MDA-MB-231 cells prepared in Example 1 through an injection pump, and incubate for 6 hours to ensure that the shell-insulated metal nanoparticles 8 (Au@SiO2 nanoparticles) do not enter the interior of MDA-MB-231 cells. Then, introduce extracellular fluid (Beyotime, catalog number C0216-500ml) to wash away the excess Au@SiO2 nanoparticles, and image the microfluidic cell culture device under a Raman microscope ( Figure 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. Figure 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 ( Figure 17 ), alkynyl ( Figure 18 ) and cyano ( Figure 19 ) channel and measured its Raman spectrum, and found that cyano (2220cm -1 ) signal relative to the alkynyl (2120cm -1 ) has decreased significantly ( Figure 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, wherein 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; 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; The side chain of Pra is grafted with an alkynyl group, and the end of CBT contains a cyano group; 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.

2. 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).

3. The single cell enzyme activity imaging detection system according to claim 2, characterized in that: The pre-treated quartz substrate (5) 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.

4. The single cell enzyme activity imaging detection system according to claim 3, characterized in that: The piranha wash solution is a mixture of concentrated sulfuric acid and hydrogen peroxide with a concentration ratio of 3-5:1-3, and the soaking time is 8-10 minutes.

5. An application of the single-cell enzyme activity imaging detection system according to claim 1 in detecting single-cell enzyme activity.

6. The use according to claim 5, characterized in that: The single cell enzyme is any one or more of cathepsin B, acid phosphatase, furin or aminopeptidase N.

7. The use according to claim 5, 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 the microfluidic culture device for cell endocytosis, and then extracellular fluid is introduced to wash away the free shell-isolated metal nanoparticles (8). The microfluidic cell culture device is then placed under a Raman microscope, and a peptide probe (7) is introduced using a syringe pump. The alkynyl peak and the cyano peak are imaged in real time and in situ and data analyzed using a Raman imager.

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