A method for analyzing protein kinase activity by AuNPs conjugated single molecule fluorescence imaging

Abstract

The application relates to the field of biotechnological analysis and detection, in particular to a method for analyzing protein kinase activity by using AuNPs coupled monomolecular fluorescence imaging; the method for realizing super-sensitive detection of protein kinase based on plasmonic nanoparticle coupled monomolecular dynamics fingerprinting is to use AuNPs as a substrate, fix other components for convenient observation, meanwhile, due to the addition of AuNPs, the surface plasmon effect of gold particles is used to enhance molecular fluorescence, improve detection signal and signal-to-noise ratio, break the limitation of imaging chain concentration, and accelerate the imaging speed; compared with a traditional amplification method, the background signal is greatly reduced, zero-background detection is basically realized, non-specific amplification is prevented, and false positive signals in the amplification process are avoided.

Description

Technical Field

[0001] This invention relates to the field of biotechnology analysis and detection, and in particular to a method for analyzing protein kinase activity using AuNPs coupled with single-molecule fluorescence imaging. Background Technology

[0002] Protein kinase (PKA) phosphorylation is closely related to important biological processes such as cell differentiation, gene expression, metabolism, immune response, and apoptosis. PKA can serve as a biomarker for disease diagnosis and as a target for drug discovery. Abnormal PKA activity is closely associated with many important diseases, such as diabetes, cancer, Alzheimer's disease, immunodeficiency, and cardiac dysfunction. Therefore, developing a rapid and sensitive PKA detection method is of great significance for biomedical research and clinical diagnosis.

[0003] Among the existing methods for determining PKA activity, radioisotope techniques are commonly used. These techniques rely on radioactive phosphate groups [γ-] 32 Monitoring phosphorylation reactions by transferring P-ATP to substrate proteins / peptides is a method that can characterize PKA activity, but it has low sensitivity and is time-consuming and labor-intensive. Furthermore, radioactive labeling can cause some harm to the human body and the environment due to radiation. To overcome this limitation, researchers have tried colorimetric methods, electrochemical methods, and mass spectrometry to detect PKA activity. However, these methods have poor detection sensitivity and cannot show sensitive responses to low concentrations of the target. Therefore, they have high requirements for the concentration of the analyte. Moreover, equipment such as mass spectrometry is expensive and complex to operate, making it unsuitable for widespread laboratory applications.

[0004] Common fluorescence amplification detection methods are widely used in the field of biotechnology analysis and detection. For example, patent WOUS04000480 discloses an analytical method for detecting protein kinase activity using fluorescence assay. This method uses fluorescent groups to modify the phosphate sites of protein kinases to determine their activity. Through numerous experiments and condition optimizations, fluorescence detection methods have shown high biosafety and high throughput in PKA activity detection. However, their shortcomings are also significant and cannot be ignored. Fluorescence-based detection often suffers from photobleaching and low signal output, while amplification-based detection methods typically lead to false positive signals. These problems have a significant impact on PKA activity detection results and urgently need to be addressed.

[0005] Given these technical challenges, there is an urgent need to develop an accurate, background-free protein kinase detection method. Improving signal output intensity and overcoming the effects of photobleaching and false positive signals on PKA activity detection results are currently top priorities in research.

[0006] Single-molecule fluorescence imaging (TIRFM) has become a powerful tool for the sensitive and accurate detection of biomolecules. TIRFM refers to the transient hybridization of fluorescently labeled short DNA oligonucleotides (imaging strand 8–12 nt) with their complementary strands (i.e., docking strands) at room temperature. Once the fluorescently labeled imaging strand binds to its docking strand, it is specifically illuminated by TIRFM, producing a typical "flickering event," where binding (dark to light) and dissociation (light to dark) events occur at the same location (N). b+d This phenomenon is defined as the fluorescence "on" state. However, if the imaging chain is not briefly locked to the imaging surface without hybridizing with the docking chain, the fluorophore will not be excited due to the exponential decay of the evanescent field. Even if it is excited, it diffuses rapidly in solution, and the fluorescence signal is difficult to capture on the measurement timescale. b+d The event will not occur; this is defined as the fluorescence "off" state. Repeated dynamic hybridization of the labeled imaging strand with the docking strand produces a unique "kinetic fingerprint" pattern distinct from the control group, allowing for specific identification of background signals. Applying this feature to PKA activity detection analysis establishes a method that enables single-molecule identification of PKA, breaking the limitations of traditional fluorescence imaging methods on imaging strand concentration, avoiding photobleaching and false positives. This method is then applied to the detection of biomarker enzyme activity and inhibitor screening. Furthermore, the main direction of this invention is to expand the toolbox of biodetection by designing different target-corresponding substrate sequences to detect other biomolecules. Summary of the Invention

[0007] The purpose of this invention is to establish a method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging.

[0008] Another object of the present invention is the application of a method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging.

[0009] The technical solution adopted to achieve the purpose of this invention is:

[0010] To overcome the aforementioned technical problems, a method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging (PNP-SMKF) was developed to achieve ultrasensitive detection of protein kinase (PKA). The steps are as follows:

[0011] Step 1: Establishing the flow-through pool:

[0012] The slides were first cleaned with Piranha solution, and then functionalized with (3-aminopropyl)triethoxysilane (APTES) in ethanol for 100 min. During sample preparation, an 18×18 mm coverslip and a 22×22 mm slide were sandwiched together with epoxy adhesive to form a channel, also known as a flow cell, with a volume of approximately 20 μL.

[0013] Step 2: Immobilize AuNPs on the surface of an aminated glass slide, and then chemically assemble the base peptides (SPs) onto the AuNPs surface using gold thiol:

[0014] AuNPs coated with an appropriate concentration of sodium citrate were injected into the flow cell and incubated for 0.5 h. After incubation, free gold particles were washed away with deionized water. To prevent nonspecific adsorption of peptide substrates (SPs) and fluorescent probes onto the slide surface, excess APTES sites were quenched with 0.5 mg / mL mPEG-succinovalerate (mPEG-SVA) for 2 h.

[0015] The AuNPs have a particle size of 50.0 ± 2.4 nm.

[0016] Then, 1 μM SPs were injected into the flow cell and assembled on the surface of AuNPs via Au-S bonds. The reaction was carried out at 37 °C for 12 h. After the reaction was completed, excess unreacted SPs were rinsed with deionized water.

[0017] Step 3: Specific phosphorylation of serine residues on the polypeptide matrix:

[0018] Different concentrations of PKA, 500 μM ATP, and 10×PKA reaction buffer (MgCl2 20 mM, Tris-HCl 50 mM, pH 7.5) were introduced into the flow cell for phosphorylation. The mixture was incubated at 30 °C for 1 h in the flow cell to obtain phosphorylated SPs. The reaction was then terminated by washing three times with 1×PKA reaction buffer, thus completing the phosphorylation reaction of PKA.

[0019] Step 4: Fix the 5'-phosphorylated docking chain onto the imaging surface:

[0020] After the phosphorylation reaction is complete, 500 μM Zr is added. 4+ The solution was injected into the flow channel and incubated at 30°C for 1 hour, then passed through Zr. 4+ Chelating with phosphate, Zr 4+ Finally, it binds to the phosphorylation sites of SPs; the flow channel is rinsed three times with deionized water. A final concentration of 50 nM containing the 5'-phosphorylated terminal of the docking chain is added to the flow cell, along with Zr. 4+ Chelation, thereby fixing the docking chain to the imaging surface.

[0021] Step 5: At room temperature, Cy5-labeled imaging strands are introduced. Dynamic hybridization occurs between the imaging and docking strands in a salt-containing buffer solution. Transient hybridization between the imaging and docking strands is detected under 635 nm laser irradiation. Fluorescence signals of the samples are acquired using a 100×TIRF objective lens and recorded by an EMCCD.

[0022] The salt-containing buffer solution was a 900mM NaCl solution. The EMCCD pixel size used in this experiment was 16×16μm, the sample exposure time was set to 300ms, the gain was 40, and the total acquisition time was 10 minutes (2000 frames).

[0023] Step 6: Analyze the original image.

[0024] Candidate regions of interest (ROIs) in the image with inter-frame intensity fluctuations greater than those of surrounding pixels are identified. Then, the trajectory of fluorescence intensity changes over time for each candidate molecule is plotted. Based on the trajectory plot, a Hidden Markov Model (HMM) is used to determine the number of binding and dissociation events and the dwell time of the binding and dissociation states, generating kinetic fingerprint data. When the imaging chain specifically binds to the target docking chain, it typically leads to more binding (dark to light) and dissociation (light to dark) events (N) at the same location. b+d Non-specific binding results in relatively less N b+d Therefore, by evaluating N b+d The number of molecules, signal-to-noise ratio (SNR), and average dwell time of the imaging probe can be used to determine the threshold for dynamic filtering based on single-molecule fluorescence intensity trajectory statistics, accurately identifying the true target. This screens out non-specific molecules and removes false positive signals.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. This is a single-molecule dynamic fingerprint based on plasmonic nanoparticle coupling for the analysis of single-cell enzyme activity. By adding AuNPs, the detection signal can be effectively enhanced without further amplification. The plasmonic effect on the surface of gold particles enhances molecular fluorescence, while reducing the limitation of imaging chain concentration on detection, improving the detection signal and signal-to-noise ratio, and accelerating the imaging speed. Compared with traditional amplification methods, it greatly reduces the background signal, basically achieving zero background detection, preventing non-specific amplification, and avoiding the occurrence of false positive signals during the amplification process.

[0027] 2. Gold particles of about 50 nm are selected as the fluorescence enhancement substrate. Multiple peptide substrates can be modified on the surface of each gold particle. Therefore, due to the surface enrichment effect, more targets will be captured by the probe on the imaging surface. This will increase the efficiency of target capture by the specific substrate, improve the detection sensitivity, and reduce the detection limit. Target substances can be detected at the level of a single molecule.

[0028] 3. By setting up a flow cell, the imaging probe can be continuously replenished, overcoming the problem of photobleaching caused by prolonged imaging in previous experiments, which affected the experimental results and reduced the detection sensitivity. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of a single-molecule fluorescence imaging method involving AuNPs for the analysis of protein kinase activity.

[0030] Figure 2 For PNP-SMKF detection of PKA activity: (A) Transient hybridization of the Cy5-labeled imaging strand with the target docking strand detected by TIRFM; (B) Representative single-frame field-of-view (FOV) image from TIRF microscopy; (C) Intensity fluctuation plot of the FOV shown in the figure; (D) Representative fluorescence intensity trajectory over time; (E) Further depth analysis of Figure D using the HMM model to obtain subsequent data; (F) Setting threshold filtering to separate true target molecules from false positive molecules;

[0031] Figure 3 (A) and (D) are schematic diagrams of phosphorylation processes with and without PKA, respectively; (B) and (E) are imaging chains τ with and without PKA, respectively. off The distribution of the imaging chain τ; (C) and (F) represent the imaging chain τ in the cases without and with PKA, respectively. on Distribution;

[0032] Figure 4 Selectivity analysis chart for PNP-SMKF method for PKA detection;

[0033] Figure 5 (A) The change in the actual target molecule count under different conditions with PKA concentration; (B) Linear relationship between the count of target molecules and PKA concentration;

[0034] Figure 6 (A) Chemical structure of inhibitor H-89; (B) Relationship between relative activity of PKA and concentration of inhibitor H-89;

[0035] Figure 7 (A) and (B) show the changes in the count of real target molecules obtained under different concentrations of Fsk / IBMX stimulation and the presence of inhibitors;

[0036] Figure 8 (A) The actual target count obtained changes with the number of cells; (B) The actual target count obtained is linearly related to the PKA concentration within a certain range.

[0037] Figure 9 The effect of NaCl concentration on the actual target molecule count;

[0038] Figure 10 The change in the actual number of target molecules with exposure time. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0041] Example 1: PKA Detection Imaging Surface Preparation and Single-Molecular Imaging Experiment

[0042] The slides were first cleaned with Piranha solution, and then functionalized with (3-aminopropyl)triethoxysilane (APTES) in ethanol for 100 minutes. For sample preparation, an 18×18 mm coverslip and a 22×22 mm slide were sandwiched together with epoxy adhesive to form a flow cell with a channel volume of approximately 20 μL.

[0043] like Figure 2 As shown in Figure A, AuNPs coated with an appropriate concentration of sodium citrate were injected into the flow cell and incubated for 0.5 h. Free gold particles were then washed away with deionized water. To prevent non-specific adsorption of SPs and fluorescent probes onto the slide surface, excess APTES sites were quenched with 0.5 mg / mL mPEG-succinovalerate (mPEG-SVA) for 2 h.

[0044] Different concentrations of PKA, 500 μM ATP, and 10×PKA reaction buffer (MgCl2 20 mM, Tris-HCl 50 mM, pH 7.5) were introduced into the flow cell for phosphorylation. The mixture was incubated at 30°C for 1 h in the flow channel to obtain phosphorylated SPs. The reaction was then terminated by washing three times with 1×PKA reaction buffer. After phosphorylation, 500 μM Zr was added... 4+ The solution was injected into the flow channel and incubated at 30°C for 1 hour, then passed through Zr. 4+Chelating with phosphate groups, Zr 4+ Finally, the molecules were bound to the phosphorylation sites of SPs, and the flow channel was rinsed three times with deionized water. A final concentration of 50 nM docking strand with 5'-phosphorylated ends was added to the flow cell. Finally, single-molecule fluorescence imaging experiments were performed on the TIRFM imaging system. Before imaging, 10 nM of Cy5-labeled fluorescent probe was introduced into the imaging buffer containing 900 mM NaCl, i.e., the imaging strand was injected into the flow channel.

[0045] The transient binding of the imaging chain and docking chain was detected under 635nm laser illumination. The fluorescence signal of the sample was acquired by a 100×TIRF objective lens and recorded by an EMCCD. The EMCCD used in this experiment had a pixel size of 16×16μm, an exposure time of 300ms, a gain of 40, and a total acquisition time of 10 minutes (2000 frames).

[0046] The acquired images are analyzed, such as Figure 2 BC identifies candidate regions of interest (ROIs) in an image where inter-frame intensity fluctuations are greater than those of surrounding pixels. White circles in the image indicate the locations of local maxima in the fluctuation graph, from which candidate ROIs can be identified for further analysis. Then, based on the trajectory of fluorescence intensity changes over time for each candidate molecule... Figure 2 D; Then, using a Hidden Markov Model (HMM), the number of binding and dissociation events and the dwell time of the binding and dissociation states are determined, generating dynamic fingerprint data such as... Figure 2 E; By setting an appropriate threshold (dashed line), specific and non-specific molecules can be distinguished, such as... Figure 2 F.

[0047] Furthermore, by using AuNPs with a particle size of 50 nm as substrates, the fluorescence of target binding events can be precisely enhanced, improving the signal-to-noise ratio and increasing fluorescence by nearly 12 times. At the same time, it avoids the concentration limitation of the imaging chain for detection. Compared with the previously reported SMKF method, the use of plasmonic nanoparticles further shields the non-specific interactions close to the nanoparticles.

[0048] Example 2: Feasibility Verification

[0049] To verify that the PNP-SMKF method can be used to analyze PKA activity, the imaging results before and after PKA addition were compared, such as... Figure 3 A and 3D mainly observe the changes in the average binding (on) and dissociation (off) time of the probe.

[0050] like Figure 3 As shown in B and 3E, when PKA is added, τ off Approximately 4 times lower than without PKA; τ offThe decrease is mainly due to the phosphorylation reaction mediated by the addition of PKA, which anchors the docking chains to the surface. When imaging chains are added, more binding and dissociation events occur at the same site, increasing the number of "on" states and the total residence time in the "on" state.

[0051] For τ on By calculating hundreds of trajectories, the following conclusion can be drawn: when PKA does not exist, τ on The calculated value is 19.9s. Figure 3 C, longer than 1.5s when PKA exists, is as follows. Figure 3 F.

[0052] Since there are significant differences in the dynamic hybridization fingerprint patterns between the experimental group (with PKA) and the control group (without PKA), the PNP-SMKF method can be used to analyze PKA activity.

[0053] Example 3: Selective Experiment

[0054] Unrelated proteins glucose oxidase (GOX) and glutathione (GSH) were selected as negative controls, such as... Figure 4 As shown, when 4 U / mL PKA was added, a large number of real target molecules could be counted after kinetic filtration. However, when PKA was replaced with the same concentrations of GOX and GSH, i.e., the concentrations of GOX and GSH added were 4 U / mL and 24 ng / mL, respectively, the molecule count obtained was negligible, similar to the results of the control group without the addition of PKA.

[0055] Example 4: Sensitivity Detection (LOD)

[0056] Because this method has good specificity, its sensitivity is calculated by statistically analyzing the number of actual targets after dynamic filtering at different PKA concentrations.

[0057] from Figure 5 As shown in Figure A, the number of true targets obtained after dynamic filtration increases monotonically with increasing PKA concentration. Within the range of 0.001 to 4 U / mL, there is a linear relationship between the true target count and the logarithm of the PKA concentration. Figure 5 B. The regression equation is y = 57.10 + 17.31log 10 x, with a regression coefficient of 0.990, where y is the true target molecule count and x is the PKA concentration (U / mL). The LOD was calculated to be 0.0005 U / mL by adding three times the standard deviation to the mean response value of the control group.

[0058] Example 5: Application in Inhibitor Screening

[0059] Since PKA overexpression is closely related to a variety of diseases, screening for corresponding inhibitors is of great significance for the discovery of PKA-targeting drugs.

[0060] In this experiment, H-89 was selected as the model inhibitor for PKA activity detection. The chemical structure of H-89 is as follows: Figure 6 A,H-89 is a competitive inhibitor of PKA that binds to the ATP site of the catalytic subunit, thereby blocking PKA activity.

[0061] In this experiment, different concentrations of H-89 were mixed with 4 U / mL PKA and 500 μM ATP, and phosphorylated at 30 °C for 1 hour. The relative activity (RA) of PKA was then measured.

[0062] The formula for calculating RA is as follows: RA = (N i -N0) / (N t -N0)×100%, where N0, N t N i The molecular counts were calculated for extractions in the absence of PKA, the presence of 4 U / mL PKA, the presence of 4 U / mL PKA, and H-89, respectively. Finally, the half-maximum inhibitory concentration (IC50) of H-89 was obtained by fitting a curve between RA and H-89 concentrations. 50 value.

[0063] like Figure 6 As shown in Figure B, the activity of PKA gradually decreases with increasing inhibitor concentration. The IC50 of H-89 was calculated. 50 The value was 34.61 nM, which is basically consistent with the previously reported results. This indicates that the analytical method can be used to detect which substances have inhibitory effects on PKA, and can be used for screening PKA inhibitors.

[0064] Example 6: Cell Culture and Preparation of Cell Extracts

[0065] The cell model used in this experiment was HeLa cells, cultured in DMEM. Before stimulation with Forskolin (Fsk) and 3-isobutyl-1-methylxanthine (IBMX), the previous culture medium was replaced with 3 mL of serum-free medium, and then incubated for 4 h. Then, different concentrations of Fsk and IBMX were added to the above culture medium to activate intracellular PKA, and the reaction was carried out for 30 min.

[0066] For comparison, we defined an equal volume of DMSO medium as a negative control. In the inhibitor assay, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-89), FSK, and IBMX were simultaneously added to the cells and reacted for 30 min. Afterward, the cells were washed three times with PBS buffer (pH 7.4), and cells were collected using trypsin. To prevent trypsin digestion of the cells and to remove the medium, the cells were centrifuged at 1000 rpm for 5 min.

[0067] For cell extracts, the already counted 10 6 Cells were resuspended in 100 μL of lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.25 mM sodium deoxycholate, 1.0% glycerol, 0.1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride). The mixture was then incubated on ice for 30 min, vortexing for 30 s every 5 min. After lysis, the cell lysate was centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was collected in a clean tube at -20 °C for further use.

[0068] The concentration of extracted protein was determined according to the Bradford method. The concentration of cell lysate was diluted to 50 μg / mL before PKA activity detection and analysis.

[0069] Cellular experiment results as follows Figure 7 As shown in A and 7B, the count was very low in the unstimulated cell lysate (No. 1), indicating that the proposed method is unaffected by other matrices in the cell lysate. However, with the addition of the stimulating drug, the unimolecular count gradually increased accordingly. A stable level was reached when the concentrations of Fsk and IBMX were 25 μM and 50 μM, respectively. Conversely, when 4 μM of H-89 was added to the drug-stimulated cell lysate, the count almost dropped to a level comparable to that of the drug-free cell lysate. This further demonstrates that H-89 has an inhibitory effect on PKA activity. Furthermore, we further measured the PKA activity in the cell lysate stimulated with 25 μM Fsk / 50 μM IBMX, from... Figure 8 As shown in Figure A, the molecular count gradually increases with the increase in the number of HeLa cells. Within the range of 1–10,000 cells, the molecular count is linearly correlated with the logarithm of the cell number. Figure 8 B, the resulting regression equation is y = 5.51 + 21.67log 10 x, with a regression coefficient of 0.994, where y represents the true number of target molecules and x represents the number of HeLa cells. The calculated LOD is 1 cell.

[0070] Comparative Example 1

[0071] Due to the negative charge on the DNA strand, monovalent cations (such as Na) + and K + NaCl is typically used to shield against electrostatic repulsion to stabilize DNA strands. This experiment uses NaCl to promote DNA hybridization.

[0072] The experimental conditions were the same as in Example 1, except that the NaCl concentration was changed. When the NaCl concentration varied between 50 and 900 mM, N b+d As the number of molecules gradually increases, the target molecules for counting also increase. When the NaCl concentration exceeds 900 mM, N... b+d The number of targets no longer increases, and the number of actual targets obtained gradually decreases, such as... Figure 9 As shown.

[0073] Therefore, the optimal NaCl concentration is 900 mM.

[0074] Comparative Example 2

[0075] The experimental conditions were the same as in Example 1, except for the exposure time, i.e., the time for acquiring each frame of image. For a 9-bp DNA strand, such as Figure 10 As shown, the number of target molecules increases as the exposure time increases from 100 to 300 ms, and then gradually decreases after 300 ms. Therefore, the optimal exposure time is 300 ms.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging, comprising the following steps: (1) substrate immobilization; (2) phosphorylation of PKA with ATP assistance; (3) after phosphorylation, Zr 4+ As a bridge, the 5'-phosphorylated docking chain is fixed on the imaging surface; (4) the imaging chain is introduced, and the imaging chain and the docking chain can undergo a dynamic hybridization reaction in a salt-containing buffer solution; (5) the transient binding of the imaging chain and the docking chain is captured, and the obtained raw image is analyzed, characterized in that: In step (1), AuNPs are introduced as a substrate, and the AuNPs particle size is 50.0 ± 2.4 nm; The immobilization of the substrate specifically includes the following steps: a. Establishment of a flow cell; b. Immobilizing AuNPs on an aminated glass slide surface, and then chemically assembling the peptide substrate onto the AuNPs surface using gold thiol.

2. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 1, characterized in that: In step (1)a, before the reaction, the glass slide is first cleaned with Piranha solution, then functionalized with (3-aminopropyl)triethoxysilane in ethanol for 100 min, and then a 18 × 18 mm coverslip and a 22 × 22 mm glass slide are sandwiched together with epoxy adhesive to form the flow cell, which has a volume of 20. μ L.

3. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 1, characterized in that: In step (1)b, after incubating the negatively charged AuNPs coated with sodium citrate in a flow cell for 0.5 h, the excess free gold particles were washed away with deionized water, and the excess APTES sites were shielded with 0.5 mg / mL mPEG-succinyl valerate for 2 h. The peptide substrate was then injected into the flow cell and reacted at 37 °C for 12 h. After the reaction was completed, the excess unreacted peptide substrate was rinsed with deionized water.

4. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 1, characterized in that: In step (2), different concentrations of PKA, ATP, and 10 × PKA reaction buffer are introduced into the flow cell. The flow cell is incubated at 30 °C for 1 h. Then, the reaction is terminated by rinsing three times with 1 × PKA reaction buffer, thus completing the phosphorylation reaction involving PKA.

5. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 1, characterized in that: In step (3), Zr 4+ The solution was injected into the flow cell and incubated at 30 °C for 1 h. The flow channel was then rinsed three times with deionized water. The docking chain containing the 5'-phosphorylated terminus was added to the flow cell and reacted with Zr. 4+ A chelation reaction occurs, thereby fixing the docking chain to the imaging surface.

6. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 1, characterized in that: Step (4): At room temperature, a fluorescent probe labeled with Cy5 on the imaging chain is introduced, and a dynamic hybridization reaction occurs between the imaging chain and the docking chain in a salt-containing buffer.

7. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 6, characterized in that: The salt-containing buffer solution is a 900 mM NaCl solution.

8. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 1, characterized in that: Step (5) involves detecting the transient binding of the imaging chain and the docking chain under 635 nm laser irradiation. The fluorescence signal of the sample is acquired by a 100 × TIRF objective lens and recorded by an EMCCD to evaluate N. b+d The number of samples, signal-to-noise ratio, and average dwell time are used to determine the filtering threshold and screen out false positive signals.

9. The method for analyzing protein kinase activity using AuNPs-coupled single-molecule fluorescence imaging as described in claim 8, characterized in that: The EMCCD pixel size is 16 × 16 µ m, the exposure time of the sample was set to 300 ms, the gain was 40, the total acquisition time was 10 minutes, and a total of 2000 frames were collected.

Images