Construction of enzyme-activated ratio type pH sensor and application of enzyme-activated ratio type pH sensor in ischemic stroke accurate imaging

Abstract

The invention discloses construction of an enzyme-activated ratio type pH sensor and application of the enzyme-activated ratio type pH sensor in ischemic stroke accurate imaging, and belongs to the technical field of biomedical sensing and nanotechnology. The sensor is formed by encapsulating a double-stranded DNA probe (PF) by lipidosome; the PF probe comprises a Cy3 / Cy5 double-labeled F chain capable of forming an i-motif and a complementary P chain containing an AP site. The sensor realizes high specificity through a dual activation mechanism: in normal tissues, the P chain inhibits the F chain function; in a cerebral arterial thrombosis focus, an up-regulated purine-free / pyrimidine-free endonuclease 1 (APE1) cuts an AP site on a P chain, then an acidic microenvironment triggers an F chain to be folded to form an i-motif, the distance between Cy3 and Cy5 is shortened, fluorescence resonance energy transfer (FRET) is activated, and a ratio type pH response signal is generated. Liposome encapsulation enhances the delivery efficiency and stability of the probe. The invention provides a new tool for solving the problems of specificity and reliability of pH dynamic imaging in stroke research.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the construction of an enzyme-activated ratiometric pH sensor and its application in precise imaging of ischemic stroke. Background Technology

[0002] Ischemic stroke (IS) is a common neurological disorder caused by interruption of blood flow to the brain, characterized by high incidence, high disability rate, and high mortality. Its pathological process is complex, with the dynamic changes in the ischemic core and the surrounding ischemic penumbra being crucial in determining clinical prognosis. Although functionally impaired, the penumbra retains structural integrity and is a key target for acute intervention and salvage. Studies have shown that oxygen-glucose deprivation following cerebral ischemia rapidly triggers anaerobic metabolism, leading to a large accumulation of lactic acid and intracellular acidosis. This decrease in pH is not only a core marker of neuronal damage, but its spatial distribution and dynamic gradient are also closely related to the delimitation of the penumbra and the expansion of the infarct foci. Therefore, achieving high-resolution, dynamic spatiotemporal imaging of pH levels within ischemic brain regions is of vital clinical significance for early diagnosis, assessment of tissue viability, and guidance of personalized treatment (such as thrombolysis or neuroprotection).

[0003] Currently, the technologies used clinically for stroke diagnosis and pH monitoring have significant limitations. For example, while magnetic resonance imaging (MRI) can provide anatomical details, it is difficult to achieve specific and dynamic imaging of pH; arterial blood gas analysis is an invasive procedure and reflects the pH of the whole body rather than local brain tissue; brain microdialysis technology has low spatial resolution and is invasive. None of these methods can meet the need for in vivo, real-time, high-resolution monitoring of dynamic changes in brain pH.

[0004] In recent years, the development of fluorescence imaging technology and molecular probes has provided new avenues for solving this challenge. A series of fluorescence sensors based on small molecule dyes, gene-encoded proteins, and functional nanomaterials have been developed to achieve quantitative pH detection by measuring fluorescence intensity, ratio signals, or fluorescence lifetime. Among these, DNA-based nanosensors have shown great potential due to their excellent biocompatibility, ease of programming and design, and precise molecular recognition capabilities. In particular, molecular switches constructed using pH-sensitive structures such as DNA i-motifs or triplexes can generate readable fluorescence signals through conformational changes, enabling high-resolution pH imaging in complex biological environments.

[0005] However, existing DNA-based pH sensing technologies still face two key challenges and shortcomings when applied to ischemic stroke research: (1) Poor disease specificity: Most probes only respond to pH changes and cannot distinguish between ischemic acidosis and acidic environments caused by other pathological processes (such as inflammation) or normal physiological fluctuations. This non-specific activation leads to severe off-target signals, making it difficult to accurately distinguish between the infarct core and salvageable penumbra tissue; (2) Low reliability in in vivo application: First, naked DNA probes are difficult to effectively penetrate the blood-brain barrier (BBB) ​​and accumulate in ischemic neurons, resulting in low delivery efficiency; second, probes are easily degraded by nucleases in vivo, producing false positive signals, and the detection mode based on single fluorescence intensity is easily affected by environmental factors such as probe concentration and instrument efficiency, affecting the accuracy and reliability of quantitative measurement.

[0006] Apurinic / apyrimidinic endonuclease 1 (APE1) is a key enzyme that plays a central role in cellular responses to oxidative stress. Under oxidative stress and inflammation following ischemic stroke, APE1 expression is significantly upregulated and undergoes cytoplasmic translocation, and its activity is closely related to the severity of ischemic injury. This characteristic makes it a highly promising endogenous triggering target for disease-specific activation.

[0007] In summary, there is an urgent need in the field to develop a novel sensing strategy that integrates disease-specific activation, efficient targeted delivery, and interference-resistant ratiometric measurement to achieve accurate and reliable imaging of intracellular pH dynamics during ischemic stroke. This is precisely the core technical problem that this invention aims to solve. Summary of the Invention

[0008] In view of the shortcomings of the prior art, the purpose of this invention is to provide an enzyme-activated ratiometric pH sensor, its construction method and application, so as to solve the technical problems of poor disease specificity, low delivery efficiency and insufficient detection reliability of pH fluorescent probes in the application of ischemic stroke.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an enzyme-activated ratiometric pH nanosensor, wherein the sensor is a liposome-encapsulated double-stranded DNA probe (Liposome-encapsulated PF, LPF). The double-stranded DNA probe (PF) is formed by hybridization of one F strand and one P strand; The F chain is a cytosine-rich sequence that can remain in an extended state at neutral pH and fold to form an i-motif structure at acidic pH, with a donor fluorophore (Cy3) and an acceptor fluorophore (Cy5) labeled at its two ends, respectively. The P chain is a partially complementary sequence to the F chain, and contains a purine-free / pyrimidine-free (AP) site for specific recognition and cleavage by purine-free / pyrimidine endonuclease 1 (APE1). When the P chain is not cleaved, it stably hybridizes with the F chain, inhibits F chain folding, and causes the distance between Cy3 and Cy5 to exceed the effective distance of fluorescence resonance energy transfer (FRET), and the sensor is in the "off" state. After the P chain is cleaved by APE1, its dissociation ability from the F chain is significantly reduced. Under acidic conditions, the F chain can fold to form an i-motif, bringing Cy3 and Cy5 closer together, triggering efficient FRET, generating a ratiometric fluorescence signal, and the sensor is "activated". The liposomes encapsulate the PF probe to enhance its delivery efficiency and stability.

[0010] Preferably, the F chain is 35-40 nucleotides long and the P chain is 28-32 nucleotides long.

[0011] Preferably, the liposomes comprise egg yolk lecithin, cationic cholesterol, DSPC, and PEG2000 in a molar ratio of 50:38.5:10:1.5.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned enzyme-activated ratiometric pH nanosensor, comprising the following steps: Preparation of PF complex: P chain and F chain solutions were mixed, heated to denature, and then slowly annealed to hybridize and form PF double-stranded complex; Lipid film preparation: Egg yolk lecithin, cationic cholesterol, DSPC and PEG2000 were dissolved in an organic solvent, and the solvent was removed by rotary evaporation to form a uniform lipid film; Liposome hydration and encapsulation: Phosphate buffer and PF complex solution were added to the lipid film to carry out the hydration reaction. During the process, liposome formation and PF probe encapsulation were promoted by physical oscillation or aspiration. Post-processing: The hydrated mixture is sonicated until it becomes clear, and then filtered to remove bacteria, thus obtaining the LPF nanosensor.

[0013] Thirdly, the present invention provides the use of the above-described enzyme-activated ratiometric pH nanosensor in the preparation of reagents or compositions for imaging or detection.

[0014] Preferably, the imaging or detection is for samples with an acidic microenvironment and / or without purine / pyrimidine endonuclease 1 (APE1) expression.

[0015] Preferably, the sample is derived from the ischemic stroke region.

[0016] Preferably, the use includes distinguishing between the ischemic penumbra and the infarct core area.

[0017] Fourthly, the present invention provides a method for detecting pH changes in a sample, including the use of the enzyme-activated ratiometric pH nanosensor described above.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1) High disease specificity: This invention innovatively utilizes two key pathological features—the acidic microenvironment and APE1 enzyme upregulation—that coexist in ischemic stroke lesions as a cascade activation switch; the sensor is only activated in the presence of both APE1 and acidic pH, which greatly avoids off-target activation in non-target tissues or other acidic pathological environments, achieving specific identification of ischemic lesions and providing a possibility for distinguishing the penumbra from the core area.

[0019] 2) Reliable ratiometric detection: This invention uses Cy3 / Cy5 as the FRET pair, and monitors its fluorescence intensity ratio (IL). Cy5 / I Cy3 This ratiometric detection mode effectively compensates for errors caused by environmental factors such as probe concentration differences, instrument fluctuations, and photobleaching, significantly improving the accuracy, stability, and quantification of the detection.

[0020] 3) Highly efficient targeted delivery and stability: Encapsulation with liposomes (especially formulations containing cationic lipids) significantly enhances the ability of DNA probes to penetrate the blood-brain barrier and cell membranes, enabling them to accumulate effectively within ischemic neurons. Simultaneously, liposomes protect the internal DNA probes from nuclease degradation, reducing false positive signals and improving probe stability in complex in vivo environments.

[0021] 4) Dynamic and precise imaging: This sensor can perform dynamic, high-resolution spatiotemporal imaging of intracellular pH gradients. This can be used not only for monitoring stroke pathological processes in basic research, but also provides a powerful tool platform for early diagnosis, assessment of tissue viability, and guidance of personalized treatment interventions at the clinical level.

[0022] In summary, this invention, through a synergistic design of "enzyme activation-ratio sensing-efficient delivery," successfully overcomes the core defects of existing technologies and provides a high-performance nanosensor that can be used for precise imaging of ischemic stroke. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the construction of the LPF nanosensor and the stroke-specific monitoring principle in an embodiment of the present invention.

[0024] Figure 2 This is a comparison of the fluorescence intensity of PF probes with different P chain base lengths at different pH values ​​(pH 5.0 and pH 7.4).

[0025] Figure 3 This is a comparison of the fluorescence intensity of the PF probe and the nPF (without AP site modification) control probe at different pH values ​​(pH 5.0 and pH 7.4) in the embodiments of this invention.

[0026] Figure 4 This is a heatmap analysis of the stability of the PF probe in an embodiment of the present invention.

[0027] Figure 5 These are the fluorescence response curves of the PF probe under different pH conditions in the presence or absence of APE1 enzyme in the embodiments of the present invention.

[0028] Figure 6 This is a transmission electron microscope (TEM) image of the LPF nanosensor prepared according to an embodiment of the present invention.

[0029] Figure 7 This is the ZETA potential and DLS characterization of the liposomes and LPF nanosensors in the embodiments of the present invention.

[0030] Figure 8 These are laser confocal microscopy (CLSM) images of OD cells and cells cultured under oxygen-glucose deprivation (OGD) conditions under different pH conditions (5.0-7.0) in embodiments of the present invention.

[0031] Figure 9 This is a fluorescence image of an LPF sensor used for in vivo imaging of a mouse model of middle cerebral artery occlusion (MCAO) in an embodiment of the present invention.

[0032] Figure 10 These are verification images of successful MCAO model mouse modeling in this embodiment of the invention; (a) Coronary brain slices after TTC staining, with the white area representing the infarct foci; (b) Representative laser speckle blood flow imaging image; (c) Quantitative statistical analysis results of cerebral blood flow corresponding to image (b). Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0034] Example 1: Preparation of an enzyme-activated ratio pH sensor (LPF) 1.1 Assembly of the PF double-stranded complex Prepare 100 μM stock solutions of a cytosine-rich F strand (36 nt long) labeled with Cy3 at the 5' end and Cy5 at the 3' end, and a complementary P strand (30 nt long) containing a single AP site, using ultrapure water. Mix 20 μL of the P strand stock solution and 20 μL of the F strand stock solution in a 1.5 mL centrifuge tube, add 160 μL of PBS buffer (pH 7.4), and obtain a final concentration of 10 μM. Place the mixture in a PCR instrument and heat at 95°C for 5 minutes to completely denature the DNA. Then slowly cool to 4°C and incubate for 1 hour to allow for complete hybridization and formation of the PF double-stranded complex. Store at 4°C for later use.

[0035] 1.2 Preparation of blank liposomes Weigh egg yolk lecithin, cationic cholesterol, distearate phosphatidylcholine (DSPC), and DSPE-PEG2000 precisely in a molar ratio of 50:38.5:10:1.5, dissolve in 5 mL of anhydrous ethanol, and vortex until fully dissolved. Take 1 mL of this mixed lipid solution into a round-bottom flask and rotary evaporate the organic solvent in a 35°C water bath, forming a uniform lipid film on the flask wall.

[0036] 1.3 Preparation of PF probe encapsulated by liposomes (LPF) Add 800 μL of sterile PBS (pH 7.4) and 200 μL of the PF complex (10 μM) prepared in step 1.1 to the formed lipid film, and gently shake to allow the droplets to wet the entire bottle wall. Hydrate at 35°C in the dark for 2 hours, repeatedly aspirating the mixture with a syringe (at least 20 times) during this period to promote liposome formation and encapsulation of the PF probe. Then transfer the mixture to a 15 mL centrifuge tube, seal with sealing film, and sonicate in an ice-water bath until the solution becomes clear and transparent. Finally, filter sterilize using a 0.22 μm sterile filter membrane, purge with nitrogen for protection, wrap with aluminum foil to protect from light, and store at 4°C. The resulting product is the LPF nanosensor described in this invention.

[0037] 1.4 Preparation of control probe nPF The preparation process is the same as in 1.1 and 1.3, the only difference being that the P chain used is a normal sequence without AP sites, and the rest of the steps are exactly the same.

[0038] 1.5 Selection and Optimization of P-Chain Length To screen for the optimal protective chain length that can effectively protect the F chain (36 nt) and efficiently dissociate after activation, we designed and synthesized P chains of different lengths (e.g., 18 nt, 24 nt, 30 nt, 36 nt) for testing.

[0039] Take 10 μL of a PF probe (10 μM) assembled from an F chain and a P chain of a specific length, add it to 990 μL of PBS buffer at pH 5.0 or pH 7.4, and incubate at 37°C for 30 minutes. Measure its fluorescence spectrum using a fluorescence spectrometer.

[0040] The results are as follows Figure 2 As shown: When using long P chains (such as 36 nt or 30 nt), the resulting double-stranded structure is highly stable due to their complete complementarity with the F chains. Even under acidic conditions at pH 5.0, without enzymes to cleave the AP sites, the double strands cannot dissociate, resulting in almost no observable changes in FRET signal (increased Cy3 fluorescence and decreased Cy5 fluorescence). This demonstrates the excellent protective (shielding) effect of long P chains.

[0041] When using a short P chain (e.g., 18 nt, denoted as P), 18 When the P chain is partially complementary to the F chain, the double helix itself is unstable. At pH 5.0, even without enzymatic cleavage, the acidic environment is sufficient to cause partial dissociation of the double helix, resulting in a noticeable FRET signal response (i.e., "activation"). This indicates that the short P chain cannot effectively protect the F chain and is prone to generating false positive signals.

[0042] Based on the above results, we ultimately selected the 30 nt P chain (P 30 It acts as a protective chain. Its length is sufficient to stably bind the F chain under normal physiological conditions (pH 7.4) and subacidic conditions (pH 5.0), ensuring that the probe is in the "off" state.

[0043] The behavior of the 18 nt P-chain (P18) simulates the state after the guard chain has been cut. When the long guard chain (P... 30 When the AP site is cleaved by a specific enzyme, it breaks into two segments. The short segment that binds to the F chain is similar to P18, and its binding force to the F chain weakens, causing it to dissociate from the F chain and "open" the probe. The sequences of each nucleic acid chain are shown in Table 1.

[0044] Table 1. Base sequence of DNA Example 2: Structural Characterization of the LPF Sensor 2.1 Transmission Electron Microscopy (TEM) Characterization Take 10 μL of the LPF sample prepared in Case 1, dilute it 10-fold with ultrapure water, drop it onto a copper grid, blot off excess liquid with filter paper, and allow it to air dry. Observe the sample using a transmission electron microscope (JEM-1400Flash, JEOL) at an accelerating voltage of 120 kV. The results are as follows: Figure 6 As shown, the probe has a regular and uniform overall morphology and a size of less than 40 nm, making it more suitable for penetrating the blood-brain barrier to achieve stroke-specific imaging.

[0045] 2.2 Dynamic Light Scattering (DLS) and Zeta Potential Analysis Take 200 μL of LPF sample, dilute to 1 mL with PBS, and measure using a nanoparticle size and Zeta potential analyzer (Malvern Zetasizer Nano ZS90). Figure 7 As shown, the increase in DLS (from 193.2 ± 4.53 nm to 204.9 ± 2.62 nm) and the decrease in Zeta potential (from 41.7 ± 1.47 mV to -23.43 ± 0.87 mV) both demonstrate the successful preparation of the probe.

[0046] Example 3: Performance Evaluation of LPF Sensor 3.1 Verification of APE1 enzyme activation specificity Take 10 μL of PF or nPF probe (10 μM), add 1 μL of APE1 enzyme (10.0 U / mL) or an equal volume of enzyme storage buffer, and incubate at 37°C for 30 minutes. Then, add the mixture to 989 μL of PBS buffer (pH 5.0 or pH 7.4), and incubate at 37°C for another 30 minutes. Measure the fluorescence spectrum using a fluorescence spectrometer (F-7000, Hitachi), with an excitation wavelength of 515 nm (Cy3), and collect the emission spectrum from 560 to 750 nm. Results are as follows: Figure 3 As shown, a strong fluorescence signal at 670 nm (Cy5 emission peak) could only be observed under the conditions of the simultaneous presence of the PF probe, APE1 enzyme, and acidic pH (5.0), proving that its activation is highly enzyme- and pH-dependent. Furthermore, the selectivity of the probe was investigated. The experimental procedures were the same, only the reaction conditions were changed, and the results are as follows. Figure 4 As shown, under the influence of other enzymes and ions, the probe only generates a signal response when the APE1 enzyme is present in the same acidic condition (pH 5.0). This result fully demonstrates the superior performance of this probe in resisting ion and enzyme interference.

[0047] 3.2 pH response performance test The PF probes pretreated with APE1 enzyme were placed in PBS buffers at different pH values ​​(5.0, 5.5, 6.0, 6.5, 7.0, 7.4) and incubated at 37°C for 30 minutes before measuring fluorescence spectra. The fluorescence intensity ratio at each pH value was calculated (F in the figure). A / F D =I Cy5 / I Cy3 The result is as follows: Figure 5 As shown, the fluorescence intensity ratio increases regularly and significantly as the pH value decreases from 7.0 to 5.0, indicating that the activated probe has good pH response performance and can be used for quantitative detection.

[0048] 3.3 In vitro cell imaging experiments Mouse neuroma cells were plated in confocal culture dishes and divided into a normal control group and an oxygen-glucose deprivation (OGD) model group. Cells in the OGD group were replaced with glucose-free medium and cultured in a hypoxic chamber (95% N2, 5% CO2) at 37°C for 4 hours to simulate ischemia; cells in the control group were cultured in glucose-containing medium under normoxic conditions throughout. Both groups of cells were then co-incubated with medium containing LPF (2 μM) in the dark for 4 hours. Cells were washed three times with pre-chilled HBSS buffer to remove uninternalized probes. Imaging was then performed using a laser confocal microscope (LSM 880, Zeiss), with fluorescence signals collected from the Cy3 (560-590 nm) and Cy5 (650-680 nm) channels using 552 nm laser excitation. Results are shown below. Figure 8 As shown, cells in the OGD group exhibited strong Cy5 red fluorescence, while cells in the control group showed weak fluorescence signals. Furthermore, by using the ionophora rootstock nigrain to maintain the extracellular pH of OD cells at 5.0–7.0, LPF clearly distinguished the fluorescence signal changes at different pH levels, demonstrating its potential for monitoring fine pH changes within living cells.

[0049] 3.5 In vivo animal imaging experiments Healthy male C57BL / 6J mice aged 8-10 weeks were used to establish an acute ischemic stroke model by occluding the middle cerebral artery (MCAO) for 60 minutes using the suture occlusion method followed by reperfusion. In the sham-operated group, only the blood vessel was dissected without inserting the suture occluder. Twenty-four hours after successful model establishment, 200 μL of an LPF probe (10 μM) was injected into the mice via the tail vein. Whole-body fluorescence imaging was performed at 1, 2, 4, 8, and 12 hours post-injection using a small animal in vivo imaging system (IVIS Lumina II). The excitation filter was 540 nm, and the emission filter was 670 nm. After imaging, semi-quantitative analysis of the fluorescence intensity in the ischemic brain region was performed using Living Image software. Results are as follows: Figure 9As shown, stroke group mice exhibited significant fluorescent signals in the ischemic brain region 4 hours after injection, reaching their peak at 8 hours and beginning to weaken after 12 hours; while no significant signal was observed in the brain of sham-operated group mice. These results indicate that LPF can effectively penetrate the blood-brain barrier and specifically enrich and activate at ischemic lesions, enabling precise imaging of stroke sites.

[0050] 3.5 Validation of successful MACO model mouse establishment (1) TTC staining Mice were euthanized by cervical dislocation, and their brains were harvested intact. Bloodstains were rinsed with physiological saline, and the brains were pre-frozen at -20°C for 20 minutes. Five 2mm thick brain slices were cut along the coronal plane and immersed in 1% TTC staining solution (prepared with PBS, stored protected from light) for 30 minutes at 37°C in the dark (turning over once during incubation). After discarding the staining solution, the slices were fixed with 4% paraformaldehyde at 4°C for 24 hours. The slices were then blotted dry and arranged in anatomical order, with photographs taken from both sides. The infarct area (unstained white area) was quantified using ImageJ software; the infarct volume percentage was calculated as (total infarct area / total brain slice area) × 100%. Throughout the procedure, care was taken to protect the brains from light, ensure they were laid flat and not folded, and guarantee uniform staining. The TTC staining results are shown below. Figure 10 As shown in Figure a, the model group had a larger area of ​​cerebral infarction compared to the control group, proving the successful construction of the model.

[0051] (2) Laser speckle Mice in the experimental group underwent transient middle cerebral artery occlusion modeling using the standard tMCAO method, while the control group did not undergo modeling. After anesthesia with isoflurane, the scalp hair was shaved and any remaining hair was thoroughly removed with depilatory cream to ensure smooth scalp skin. Subsequently, the scalp skin of the mice was cut open with ophthalmic scissors to expose the subcutaneous cerebral cortex, and petroleum jelly was applied to the exposed area for moisturizing. The mice were fixed under a camera, and the laser speckle blood flow monitoring system and imaging software were turned on. The image size was set to 1.5 cm (width) × 1.7 cm (height), and the sampling frequency was 10 images / s. The right cerebral infarction area and a symmetrical left cerebral area of ​​equal size were circled in the imaging software, and the blood perfusion of the cerebral cortex of the mice was observed and recorded for 15 seconds. Finally, the percentage of blood perfusion in the right cerebral infarction area to the perfusion in the left cerebral (healthy side) was calculated, and the changes in blood perfusion in the experimental group and the control group at different time points were compared. The laser speckle imaging results are shown below. Figure 10 As shown in b and 10c, the successful establishment of the ischemic stroke model was demonstrated by confirming the reduction in cerebral blood flow.

[0052] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An enzyme-activated ratiometric pH nanosensor, characterized in that, The sensor is a double-stranded DNA probe encapsulated in liposomes, which is formed by hybridization of P and F strands; The F chain is a cytosine-rich sequence capable of forming an i-motif structure, and is labeled with Cy3 and Cy5 fluorophores at both ends, respectively; The P chain is a partially complementary sequence to the F chain, and includes a purine-free / pyrimidine-free site. The P chain hybridizes with the F chain when it is not cleaved, inhibiting the formation of i-motifs by the F chain and fluorescence resonance energy transfer between Cy3 and Cy5; The liposomes contain egg yolk lecithin, cationic cholesterol, DSPC, and PEG2000.

2. The sensor as described in claim 1, characterized in that, The F chain has a sequence length of 35-40 nucleotides, and the P chain has a sequence length of 28-32 nucleotides.

3. The sensor as described in claim 1 or 2, characterized in that, The molar ratio of each component in the liposome is egg yolk lecithin: cationic cholesterol: DSPC: PEG2000 = 50:38.5:10:1.

5.

4. A method for preparing an enzyme-activated ratiometric pH nanosensor as described in claim 1, characterized in that, Includes the following steps: (a) Provide a solution of P chain and F chain, mix the two, heat to 90-95°C and keep warm for 3-10 minutes, then slowly cool to 2-8°C and keep warm for at least 1 hour to form a PF double-chain complex; (b) Egg yolk lecithin, cationic cholesterol, DSPC and PEG2000 were dissolved in an organic solvent, and the organic solvent was removed by rotary evaporation to form a lipid film; (c) Add phosphate buffer and the PF double-chain complex solution obtained in step (a) to the lipid membrane and perform a hydration reaction for 1-3 hours, during which the membrane is repeatedly aspirated to promote liposome formation. (d) The hydrated mixture is sonicated until the solution is clear, and then sterilized by filtration to obtain the enzyme-activated ratio pH nanosensor.

5. The method as described in claim 4, characterized in that, The concentrations of the P chain and F chain in step (a) are each 5-20 μM independently.

6. The method as described in claim 4, characterized in that, The organic solvent mentioned in step (b) is anhydrous ethanol.

7. The method as described in claim 4, characterized in that, The volume ratio of the phosphate buffer to the PF double-stranded complex solution in step (c) is 3:1 to 5:1, and the final concentration of the PF double-stranded complex in the hydration system is 1-5 μM.

8. The use of a sensor as described in any one of claims 1-3 in the preparation of an imaging reagent for the diagnosis of ischemic stroke.

9. The application as described in claim 8, characterized in that, The imaging reagent is used to distinguish between the ischemic penumbra and the infarct core area.

10. The application of a sensor as described in any one of claims 1-3 in the detection of purine-free / pyrimidine-free endonuclease 1 activity.

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