A copper death dynamic sensor and a preparation method and application thereof

Abstract

This invention discloses a copper death dynamic sensor, its fabrication method, and its application. The sensor comprises a theta-shaped dual-channel nanopipette, with the inner walls of the two channels respectively modified with Cu. + Probes Cul and Cu 2+ The probe GHK alters the surface charge density and ion current rectification ratio after the target ion binds to the probe, achieving highly sensitive ratio detection with detection limits as low as 3.55 × 10⁻⁶. ‑16 M and 2.88×10 ‑16 M. This sensor features reversible regeneration, interference resistance, and long-term stability. It can be used for real-time monitoring of Cu⁺ / Cu at the single-cell level. 2+ Dynamically assessing cellular reductive metabolic capacity and performing spatially resolved electrochemical imaging in three-dimensional tumor spheres reveals metabolic resistance mechanisms in the tumor core region, providing analytical tools for evaluating the efficacy of copper death therapy and optimizing combination therapy.

Description

Technical Field

[0001] This invention relates to a copper death dynamic sensor based on an asymmetric functionalized dual-channel nanopipette, its preparation method, and its application in in-situ detection of copper ion valence state in single cells and three-dimensional tumor spheres. Background Technology

[0002] Copper is an essential micronutrient element for the human body, and its different valence states (Cu) + and Cu 2+ The transformation of copper (Cu) is closely related to cellular redox homeostasis and disease progression. Copper death, as a novel form of cell death, depends on Cu. 2+ Reduced to cytotoxic Cu + Therefore Cu + / Cu 2+ Concentration ratio is a key indicator for assessing the efficacy of copper apoptosis treatment. However, existing copper detection methods generally suffer from insufficient valence state discrimination, low spatial resolution, and inability to perform in-situ dynamic monitoring. While inductively coupled plasma mass spectrometry (ICP-MS) offers high sensitivity, it cannot distinguish copper valence states, and sample pretreatment is complex and the equipment is expensive. Fluorescent probes, colorimetric methods, and traditional electrochemical sensors can distinguish valence states, but they suffer from poor stability in complex physiological environments, are susceptible to signal interference, and struggle to achieve high spatial resolution imaging of three-dimensional biological samples. These limitations severely restrict the accurate analysis of the dynamic changes of copper ions during copper apoptosis.

[0003] Solid-state nanochannel sensors have demonstrated advantages in biosensing in recent years, including rapid response, label-free detection, and good biocompatibility. Nanochannels with asymmetric structures, in particular, can amplify detection signals through ion current rectification. However, most reported solid-state nanochannel sensors are single-channel designs, making it difficult to simultaneously and independently detect the concentrations of copper ions in two valence states at the same cell or tissue site. Furthermore, existing sensors are mostly limited to transient detection at the single-cell level, lacking in-situ imaging capabilities for the spatial distribution of copper ions in complex tissue models such as three-dimensional tumor spheroids, and thus failing to reveal drug resistance mechanisms caused by tumor metabolic heterogeneity. Therefore, developing a sensor capable of in-situ, real-time, and valence-state-specific detection of Cu is crucial. + and Cu 2+ It also possesses dynamic sensors capable of single-cell and three-dimensional tissue spatial imaging, which are of great significance for a deeper understanding of copper death mechanisms and optimization of combination therapy strategies. Summary of the Invention

[0004] Objective of the Invention: One objective of this invention is to provide a dual-channel nanopipette-mediated dynamic sensor for copper death, which can simultaneously, independently, and with high sensitivity detect Cu. + and Cu 2+The concentration of Cu. Another object of the present invention is to provide a method for preparing the sensor, which is simple to operate, has mild conditions, and good repeatability. A final object of the present invention is to provide the sensor for detecting Cu in single cells and three-dimensional tumor spheroids. + / Cu 2+ Application of concentration ratios in assessing the efficacy of copper-based treatments.

[0005] Technical Solution: The present invention discloses a dual-channel nanopipette-mediated copper death dynamic sensor. The sensor comprises a dual-channel nanopipette, the tip of which has two physically isolated and independently operating nanochannels. The inner walls of the two channels are independently modified with Cu. + Specific probes and Cu 2+ Specific probes.

[0006] Among them, Cu + The specific probe is bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine (Cul), Cu 2+ The specific probe is the GHK peptide (Gly-His-Lys); Cu + Specific probes and Cu 2+ The specific probes are all activated by EDC / NHS and then covalently linked to the carboxyl groups on the inner wall of the channel. The dual-channel nanopipette is made of theta-type quartz material, with a tip channel pore size of 100–150 nm and a diaphragm thickness of 15–25 nm between the two channels.

[0007] The present invention discloses a method for fabricating a dual-channel nanopipette-mediated copper death dynamic sensor, comprising the following steps:

[0008] (1) A dual-channel nanopipette was prepared by laser drawing method;

[0009] (2) Introduce amino groups by APTES silanization treatment of nanopipettes;

[0010] (3) It introduces a carboxyl group by reacting with succinic anhydride;

[0011] (4) Selective modification of the two channels: one channel is modified with Cu + Specific probe, another channel modified Cu 2+ Specific probes;

[0012] (5) Seal the unreacted sites, wash, and you will get the product.

[0013] Step (1) includes the following steps:

[0014] Quartz capillary tubes (outer diameter 1.2 mm, inner diameter 0.9 mm, Sutter Instrument Co.) were used as raw materials. Theta-type dual-channel nanopipettes were prepared using a Sutter P-2000 laser drawing instrument and a two-stage drawing process was adopted: the parameters of the first stage were Heat 870, Fil 4, Vel 30, Del 190, Pul 50; the parameters of the second stage were Heat 900, Fil 3, Vel 20, Del 140, Pul 150.

[0015] In step (2), the introduction of amino groups into the nanopipette through APTES silanization includes the following steps:

[0016] The tip of the dual-channel nanopipette prepared in step (1) was immersed in a mixed solution containing 3-aminopropyltriethoxysilane (APTES) and ethanol to react with the silanol groups on the inner and outer walls of the nanopipette to generate amino groups, thus obtaining an amino-modified nanopipette. The reaction was carried out at room temperature for more than 2 hours, followed by washing with anhydrous ethanol more than three times, and then vacuum drying at 100-120℃ for more than 1 hour.

[0017] Step (3) includes the following steps:

[0018] The nanopipette tip obtained in step (2) was immersed in a solution containing succinic anhydride to react with the amino group to generate a carboxyl group, followed by washing. The reaction was carried out at room temperature for more than 12 hours. After the reaction, the nanopipette tip was washed with anhydrous ethanol more than three times, and then with deionized water more than three times.

[0019] Step (4) includes the following steps:

[0020] (A1) Carboxyl activation: The tip of the nanopipette obtained in step (3) is immersed in a mixed aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group and convert it into an active ester. Then it is washed.

[0021] (A2) Selective modification of Cu 2+ Specific probe: Using a microfluidic perfusion method, PBS buffer containing GHK peptide (Gly-His-Lys) was injected into one channel of the nanopipette activated in step (A1) for reaction, while blank PBS buffer was injected into the other channel as a protection, so that the amino group of GHK peptide covalently binds to the active ester on the inner wall of the channel, thus obtaining a GHK peptide modified nanopipette.

[0022] (A3) Selective modification of Cu +Specific probe: The channel of the nanopipette obtained in step (A2) that has been modified with GHK is injected into PBS buffer for protection, and another channel is injected into PBS buffer containing Cul probe (bis(2-(2-(ethylthio)ethyl)thio)ethyl)amine) so that the amino group of Cul probe is covalently bound to the active ester on the inner wall of the channel.

[0023] In step (A1), the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the mixed aqueous solution is at least 0.1 M, the concentration of N-hydroxysuccinimide is at least 0.2 M, and the solution is activated at room temperature for more than 30 min.

[0024] In step (A2), the concentration of each GHK peptide in the PBS buffer containing GHK peptide is 5-10 mM, and the reaction is carried out at room temperature for more than 4 hours. Then, the mixture is washed three times or more with PBS buffer and deionized water.

[0025] In step (A3), the concentration of Cul probe in the PBS buffer containing Cul probe is 5-10 mM, and the reaction is carried out at room temperature for more than 4 hours. The sample is then washed three times each with PBS buffer and deionized water.

[0026] The dual-channel nanopipette-mediated copper death dynamic sensor described in this invention can detect Cu in single cells and three-dimensional tumor spheroids. + and Cu 2+ Applications.

[0027] In the application process, the sensor tip is inserted into the sample to be tested (single cell or three-dimensional tumor spheroid), the IV curve is measured, and the ion current rectification ratio of the two channels is calculated separately, using the formula ICR = I +1V / I -1V Through Cu + and Cu 2+ By observing the change in the rectification ratio before and after binding with a specific probe within the corresponding channel, independent quantitative detection of two ions is achieved. For single-cell detection, the sensor tip is gently inserted into the cell membrane to record intracellular Cu under different treatment conditions in real time. + and Cu 2+ The concentration change was observed. For a three-dimensional tumor sphere, the sensor was inserted into the sphere layer by layer at progressively deeper depths (e.g., 10 μm), and the rectification ratio signal at different depths was recorded to plot the Cu concentration. + and Cu 2+ Spatial distribution map.

[0028] Mechanism: such as Figure 2 As shown, the sensor detects Cu + and Cu 2+ The detection is based on the ion current rectification effect and surface charge modulation mechanism. The Cul probe specifically binds to Cu. +Subsequently, the surface charge on the inner wall of the induced channel changes from negative to positive, leading to a reversal of the rectified polarity of the ion current; the GHK peptide binds to Cu 2+ Subsequently, the positive charge density on the inner wall of the channel was further increased, thereby increasing the rectification ratio. This enabled high-fidelity detection of the valence state of copper ions. In the detection of single cells and tumor spheroids, a three-dimensional electrochemical fingerprint of the valence state of copper ions was constructed through multi-point measurements, revealing the cell's reducing metabolic capacity and tissue heterogeneity.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0030] (1) The dual-channel nanopipette sensor of the present invention adopts a θ-type dual-channel structure, with the two channels physically isolated and operating independently, and modified with Cu respectively. + and Cu 2+ This specific probe enables, for the first time, independent, in-situ, and ratiometric detection of two copper ions in the same detection site, avoiding cross-interference, with detection limits as low as 3.55 × 10⁻⁶. -16 M(Cu) + ) and 2.88×10 -16 M(Cu) 2+ Its sensitivity is significantly better than existing methods.

[0031] (2) The sensor of the present invention uses the ion current rectification ratio as the signal readout method, which does not require marking or external probes and is easy to operate; at the same time, through EDTA chelation regeneration treatment, the sensor can be reused more than 5 times and its performance remains stable, which greatly reduces the detection cost.

[0032] (3) The sensor of the present invention has excellent spatial resolution and can realize the detection of Cu in a single living cell. + / Cu 2+ Real-time monitoring of dynamic changes revealed for the first time that decreased cellular reductive metabolism under hypoxic conditions leads to Cu + / Cu 2+ The mechanism of the ratio reduction; at the same time, it can perform depth-resolved electrochemical imaging of three-dimensional tumor spheres, clearly present the copper ion valence gradient from the periphery to the core of the sphere, and accurately locate the metabolic drug resistance region. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the fabrication process of the dual-channel nanopipette-mediated copper death dynamic sensor of the present invention.

[0034] Figure 2 This is a schematic diagram illustrating the principle of the dual-channel nanopipette-mediated copper death dynamic sensor of the present invention for the detection of Cu⁺ and Cu²⁺.

[0035] Figure 3The image shows a scanning electron microscope (SEM) image of the dual-channel nanopipette prepared in Example 1; where A is a planar SEM image and B is a cross-sectional SEM image.

[0036] Figure 4 This is a graph showing the ion current-voltage characteristics during the stepwise functionalization process of the Cul probe sensor in Example 2.

[0037] Figure 5 This is a graph showing the ion current-voltage characteristics during the stepwise functionalization modification process of the GHK probe sensor in Example 2.

[0038] Figure 6 The CDS sensor in Example 3 of this invention measures different concentrations of Cu. + The detection results are shown in the figure; where A represents the addition of different concentrations of Cu. + (10) -5 M to 10 -15 M) is the IV characteristic curve, and B is the ΔI-Log C correction curve;

[0039] Figure 7 The CDS sensor in Example 3 measures different concentrations of Cu. 2+ The detection results are shown in the figure; where A represents the addition of different concentrations of Cu. 2+ (10) -5 M to 10 -15 M) is the IV characteristic curve, and B is the ΔI-Log C correction curve;

[0040] Figure 8 This is a bright-field micrograph of the CDS sensor penetrating a single cell in Example 4;

[0041] Figure 9 In Example 4, the CDS sensor was used to detect Cu in single cells under incubation with different concentrations of CuCl2. + and Cu 2+ Concentration results graph;

[0042] Figure 10 In Example 4, the CDS sensor was used to detect Cu at different depths of a three-dimensional tumor sphere under incubation with different concentrations of CuCl2. + and Cu 2+ Concentration results graph;

[0043] Figure 11 This is a comparison chart of the detection limit performance of the CDS sensor in Example 5 and existing copper ion detection methods;

[0044] Figure 12The graph shows the time optimization of the CDS sensor GHK and Cul probe modification in Example 6; where A is a bar chart of the rectification ratio of the GHK modified channel changing over time, and B is a bar chart of the rectification ratio of the Cul modified channel changing over time.

[0045] Figure 13 The CDS sensor and Cu in Example 7 + and Cu 2+ The incubation time optimization diagram. Detailed Implementation

[0046] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0048] Example 1: Preparation and Characterization of Dual-Channel Nanopipettes

[0049] Quartz capillary tubes (outer diameter 1.2 mm, inner diameter 0.9 mm, Sutter Instrument Co.) were used as raw materials. Before drawing, the capillary tubes were sequentially treated with acetone, ethanol, and ultrapure water (resistivity 18.2 MΩ·cm). -1 Ultrasonic cleaning for 10 minutes was performed to remove surface contaminants. Theta-shaped dual-channel nanopipettes were fabricated using a Sutter P-2000 laser drawing instrument with a two-stage drawing procedure: the first stage parameters were Heat 870, Fil 4, Vel 30, Del 190, Pul 50; the second stage parameters were Heat 900, Fil 3, Vel 20, Del 140, Pul 150. The drawing time fluctuation was controlled within 0.1 s to ensure consistency in the tip aperture geometry. The nanopipette tip morphology was characterized using scanning electron microscopy (Zeiss Sigma), with the aperture of each channel defined as the length of the major axis of an approximately semi-elliptical opening. Ten nanopipettes fabricated independently from multiple drawing batches were measured, with each of the two channels of each nanopipette measured separately, and each channel measured three times independently by different researchers. The fabricated nanopipette tip channel has a pore size of 115 ± 10 nm, and the quartz diaphragm between the two channels has a thickness of 18 ± 3 nm. The dual-channel structure of the θ-type nanoinjector can be seen from the planar and cross-sectional images obtained by scanning electron microscopy. Figure 3 (A in middle school and B in middle school 3).

[0050] Example 2: Functional Modification of the Sensor

[0051] (1) Surface amination modification: The tip of the dual-channel nanopipette prepared in Example 1 was immersed in a mixed solution containing 3-aminopropyltriethoxysilane (APTES) and ethanol (volume ratio 1:9) and reacted at room temperature for 2 h. After the reaction, it was washed three times with anhydrous ethanol and then dried under vacuum at 120 °C for 1 h to obtain the amination-modified nanopipette.

[0052] IV curve analysis was performed on the amino-modified nanopipettes, and the results are as follows: Figure 4 and Figure 5 As shown, before modification ( Figure 4 and Figure 5 The inner wall of the quartz (BARE) is negatively charged due to the presence of silanol groups; after modification, amino groups are introduced onto the surface. Figure 4 and Figure 5 APTES (in this context) undergoes protonation in solution and becomes positively charged. Through... Figure 4 The IV curve test showed that the ion current rectification direction of the nanopores changed significantly, proving that the amination modification was successful.

[0053] (2) Surface carboxylation modification: The tip of the above amino-modified nanopipette was immersed in an anhydrous ethanol solution containing 10 mM succinic anhydride and reacted at room temperature for 12 h. It was washed three times with anhydrous ethanol and then three times with deionized water to obtain the carboxyl-modified nanopipette.

[0054] IV curve analysis was performed on the carboxyl-modified nanopipettes, and the results are as follows: Figure 4 As shown, succinic anhydride reacts with surface amino groups via an amidation reaction, converting the terminal functional groups into carboxyl groups. At this point, the inner wall surface becomes negatively charged again. Figure 4 The IV curve shows a reversal in the rectification ratio ( Figure 4 and Figure 5 The presence of COOH in the middle of the charge indicates that the negative charge characteristic has been restored, proving that the carboxylation modification was successful.

[0055] (3) Carboxyl activation: The tip of the carboxyl-modified nanopipette was immersed in a mixed aqueous solution containing 0.1 M EDC and 0.2 M NHS and activated at room temperature for 30 min to convert the carboxyl group into an active ester. After activation, it was washed three times with deionized water.

[0056] (4) Selective modification of Cu 2+ Specific probe: Using a microfluidic perfusion method, one channel of the activated nanopipette from step (3) was filled with PBS buffer containing 10 mM GHK peptide (Gly-His-Lys), and the other channel was filled with blank PBS buffer for protection. The reaction was carried out at room temperature for 4 h. After the reaction, the nanopipette was washed three times with PBS buffer and deionized water, respectively, to obtain the GHK peptide-modified nanopipette.

[0057] IV curve analysis was performed on the carboxyl-modified nanopipettes, and the results are as follows: Figure 5 As shown, the GHK peptide binds to the surfactant ester via its amino group. After modification ( Figure 5 (GHK), due to the influence of the charge properties of the GHK peptide, such as Figure 5 The negative charge density on the channel surface decreases, and the corresponding rectification ratio of the channel before activation decreases.

[0058] (5) Selective modification of Cu + Specific probe: The GHK-modified channel of the GHK peptide-modified nanopipette was injected with PBS buffer for protection, while the other channel was filled with PBS buffer containing 10 mM Cul probe (bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine). The reaction was carried out at room temperature for 4 h. The nanopipette was washed three times with PBS buffer and deionized water, respectively, to obtain the Cul-modified nanopipette. A dual-channel nanopipette-mediated copper death dynamic sensor, denoted as the CDS sensor, was thus prepared. The entire preparation process is as follows: Figure 1 As shown.

[0059] IV curve tests were performed on the Cul-modified nanopipettes, and the results are as follows: Figure 4 As shown, combined with Figure 4 A comparison of the IV curves before and after modification shows that the negative charge density on the pore surface significantly decreases after modification with the Cu⁺ probe. This is because the probe molecules occupy the original carboxyl sites through covalent coupling, altering the charge distribution on the inner wall. The change in this electrical characteristic signal is consistent with theoretical expectations, fully demonstrating that the Cu⁺-specific probe has been successfully grafted onto the inner wall of the nanopipette.

[0060] Example 3 Sensor for Cu + and Cu 2+ Quantitative detection and performance evaluation

[0061] The tip of the CDS sensor prepared in Example 2 was immersed in a 1 mM KCl solution containing different concentrations of Cu⁺ or Cu²⁺. Using an Ag / AgCl electrode as a reference electrode, IV curves within a range of ±1.0 V were recorded using an electrochemical workstation, and the ion current rectification ratio ICR was calculated. +1V / I -1V , among which, I +1V I represents the current at a voltage of +1 V. -1V This indicates the current at a voltage of -1V. For example... Figure 6 As shown, for Cu⁺ detection, the ICR increases in a stepwise manner with increasing Cu⁺ concentration, and the linear correction equation is ΔICR = 5.576 + 0.779 Log C, R 2=0.997, where ΔICR is the difference between the rectification ratio induced by the target analyte and the blank signal, and C is the Cu⁺ concentration (detection limit is 3.55 × 10⁻¹). 6 M). For example Figure 7 As shown, for Cu 2⁺ Detection, with Cu 2⁺ With increasing concentration, the ICR also showed an increasing trend, and the linear correction equation was ΔICR = 13.72 + 1.903Log C, R 2 =0.997, where ΔICR is the difference between the rectification ratio induced by the target and the blank signal, and C is Cu 2⁺ Concentration, (detection limit is 2.88 × 10⁻¹) 6 This sensor utilizes a dual-channel asymmetric functionalized interface to amplify the ion current rectification signal through the surface charge density change induced by the target ion, achieving a high-sensitivity ratio detection of copper ions in two valence states.

[0062] Example 4: Cu in single cells and three-dimensional tumor spheroids + and Cu 2+ Detection applications

[0063] The CDS sensor prepared in Example 2 was used for in-situ detection of Cu⁺ and Cu²⁺ in A549 single cells and A549 three-dimensional tumor spheres cultured using the liquid covering culture method.

[0064] like Figure 8 As shown, during single-cell detection, the sensor tip was gently inserted into the A549 cell membrane under an inverted microscope, and the IV curve was recorded in real time. The Cu⁺ and Cu²⁺ concentrations were calculated from the curve obtained in Example 3 based on the change in the ion current rectification ratio. Repeated measurements were performed on multiple independent cells (n=10), and the results are as follows: Figure 9 The results showed that with increasing copper chloride concentration, Cu in single cells... 2+ and Cu + The concentrations all increased, and Cu 2+ The rate of increase in concentration was greater than that of Cu. + Concentration. During three-dimensional tumor spheroid detection, punctures were performed layer by layer from the edge of the spheroid towards the center in 10 μm increments, and the ion distribution at different depths was recorded. Results are as follows: Figure 10 The results show that in the CuCl2 treatment group, Cu 2+ and Cu + The concentration peaks all occurred at a depth of 35 μm from the surface, and the concentration decreased significantly beyond 60 μm. This radial gradient distribution reveals the diffusion-restricted characteristics of copper chloride in solid tumor tissue, confirming that the CDS sensor possesses three-dimensional spatial resolution imaging capabilities and can be used to quantitatively assess drug penetration depth and heterogeneous distribution within tumor tissue.

[0065] Example 5: Performance comparison of the sensor of the present invention with publicly reported copper ion detection methods

[0066] The dual-channel nanopipette sensor (CDS) prepared in Example 2 was used to test Cu. + and Cu 2+ The detection performance was compared with several reported copper ion detection methods, including real-time detection based on a single SERS probe (see J. Liu et al., Angew. Chem. 2021, 133, 21521), detection based on carbon nanotubes (see J. Chen et al., ACS Nano 2025, 19, 28743), detection based on fluorescence (see T. Gao et al., Adv. Opt. Mater. 2024, 12, 2400458), detection based on electrochemiluminescence (see H. Yang et al., Anal. Chim. Acta 2025, 344505), and detection based on near-infrared fluorescent probes (see X. Qian et al., Dyes Pigm. 2021, 194, 109561). The comparison results are as follows: Figure 11 As shown in the figure. The comparison results show that the CDS sensor prepared in Example 2 has a detection limit for Cu+ as low as 3.55 × 10⁻⁶. -16 M, for Cu 2+ The detection limit is as low as 2.88 × 10⁻⁶. -16 M, the linear dynamic range is 1×10 -15 M to 1×10 -5 M exhibits significantly superior detection performance compared to most reported copper ion detection methods, particularly demonstrating irreplaceable advantages in valence state differentiation, in-situ ratio detection, and three-dimensional spatial imaging.

[0067] Example 6: Optimization of Modification Time for Sensor GHK and Cul Probes

[0068] A carboxyl-modified dual-channel nanopipette sensor was prepared according to the method described in Example 2. The sensor tip was immersed in a buffer solution containing either a GHK probe or a Cul probe. Using Ag / AgCl as the reference electrode, IV curves (scan range -1.0 V to +1.0 V) were recorded at different modification time points (0 h, 1 h, 2 h, 4 h, 6 h, 8 h) using an electrochemical workstation. The ion current rectification ratio (ICR) of the corresponding channel at each time point was calculated as I₊1V / I₋1V. Figure 12As shown, when modifying GHK or Cul probes, the rectification ratio is low and has not yet stabilized when the modification time is too short (e.g., 1 h). With prolonged modification time, the rectification ratio gradually increases. When the modification time reaches 4 h, the rectification ratios of both the GHK and Cul channels reach a plateau and tend to stabilize. Further extending the modification time to 6-8 h does not significantly change the rectification ratio. These results indicate that the covalent modification of GHK and Cul probes on the channel inner wall reaches saturation within 4 h, and further extending the modification time does not significantly improve the modification density or rectification response. Therefore, to ensure sensor modification efficiency and batch-to-batch reproducibility, this invention selects 4 h as the optimal modification time for GHK and Cul probes. Sensors prepared under these conditions exhibit high probe density and stable signals, meeting the application requirements for subsequent copper ion detection and cell imaging.

[0069] Example 7 Sensor for Cu + and Cu 2+ Response time optimization

[0070] The CDS sensor was prepared according to the method described in Example 2. The sensor tip was filled with a test solution containing 1 μM Cu⁺ or 1 mM Cu²⁺, respectively. Using Ag / AgCl as the reference electrode, IV curves (scan range -1.0 V to +1.0 V) were continuously recorded at different time points (0, 15, 30, 45, 60 min) using an electrochemical workstation. The ion current rectification ratio (ICR) of the corresponding channel at each time point was calculated as I₊1V / I₋1V, and the rectification ratio was used as the response signal. Three sensors were tested independently at each time point, and each sensor was measured three times, with the average value taken. Figure 13 As shown, when the incubation time is too short (e.g., 15 min), the rectification ratio has not yet reached stability, resulting in insufficient response signal. With the incubation time extended to 40 min, the rectification ratios of both the Cu⁺ and Cu²⁺ channels increase rapidly and tend to stabilize. Further extending the incubation time to 60 min does not significantly change the rectification ratio. These results indicate that the binding of the sensor to the target ion reaches equilibrium within 40 min, and further extending the incubation time does not significantly improve the response signal. Therefore, to ensure detection sensitivity and repeatability, while also considering operational efficiency in single-cell and tissue imaging, this invention selects 40 min as the optimal incubation time between the sensor and the sample (or solution). Under these conditions, the sensor's response signals to Cu⁺ and Cu²⁺ are stable and repeatable, meeting the requirements for real-time dynamic monitoring.

Claims

1. A dual-channel nanopipette-mediated copper death dynamic sensor, characterized in that, The copper death dynamic sensor includes a dual-channel nanopipette, the inner walls of which are independently modified with Cu in each of the two channels. + Specific probes and Cu 2+ Specific probes.

2. The dual-channel nanopipette-mediated copper death dynamic sensor according to claim 1, characterized in that, Cu + The specific probe is bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine, Cu 2+ The specific probe is the GHK peptide.

3. The dual-channel nanopipette-mediated copper death dynamic sensor according to claim 1, characterized in that, The dual-channel nanopipette is a theta-type quartz nanopipette with pore sizes of 100–150 nm for both channels and a diaphragm thickness of 15–25 nm between the channels.

4. The dual-channel nanopipette-mediated copper death dynamic sensor according to claim 1, characterized in that, Cu + Specific probes and Cu 2+ The specific probes were all activated by EDC / NHS and then covalently linked to the carboxyl groups on the inner wall of the channel.

5. A method for fabricating the dual-channel nanopipette-mediated copper death dynamic sensor according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) A dual-channel nanopipette was prepared by laser drawing method; (2) The nanopipette was subjected to APTES silanization treatment to introduce amino groups; (3) It introduces a carboxyl group by reacting with succinic anhydride; (4) Selective modification of the two channels: one channel is modified with Cu + Specific probe, another channel modified Cu 2+ Specific probes; (5) Seal the unreacted sites, wash, and you will get the product.

6. The preparation method according to claim 5, characterized in that, Step (1) includes the following steps: Quartz capillary tubes were used as raw material to prepare θ-type dual-channel nanopipettes using a Sutter P-2000 laser drawing instrument. A two-stage drawing process was adopted: the parameters for the first stage were Heat 870, Fil 4, Vel 30, Del 190, Pul 50; and the parameters for the second stage were Heat 900, Fil 3, Vel 20, Del 140, Pul 150. In step (2), the APTES silanization treatment of the nanopipette to introduce amino groups includes the following steps: immersing the tip of the dual-channel nanopipette prepared in step (1) into a mixed solution containing 3-aminopropyltriethoxysilane (APTES) and ethanol to react, so that the silanol groups on the inner and outer walls of the nanopipette react with APTES to generate amino groups.

7. The preparation method according to claim 5, characterized in that, Step (3) includes the following steps: immersing the tip of the nanopipette obtained in step (2) into a solution containing succinic anhydride, reacting the amino group with the succinic anhydride to generate a carboxyl group, and then cleaning. Step (4) includes the following steps: (A1) Carboxyl activation: The tip of the nanopipette obtained in step (3) is immersed in a mixed aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group into an active ester, and then cleaned. (A2) Selective modification of Cu 2+ Specific probe: Using microfluidic perfusion, one channel of the activated nanopipette in step (A1) was injected with PBS buffer containing GHK peptide for reaction, while the other channel was injected with blank PBS buffer as protection, so that the amino group of GHK peptide covalently binds to the active ester on the inner wall of the channel, thus obtaining a GHK peptide-modified nanopipette. (A3) Selective modification of Cu + Specific probe: The channel of the nanopipette obtained in step (A2) that has been modified with GHK is injected into PBS buffer for protection, and another channel is injected into PBS buffer containing bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine, so that the amino group of bis(2-((2-(ethylthio)ethyl)thio)ethyl)amine is covalently bound to the active ester on the inner wall of the channel.

8. The use of the dual-channel nanopipette-mediated copper death dynamic sensor according to any one of claims 1-4 in the preparation of products for copper death efficacy assessment, single-cell copper ion detection, or three-dimensional tumor spherical spatial imaging.

9. The application according to claim 8, characterized in that, In application, the sensor tip is inserted into the sample to be tested, the IV curve is measured, and the ion current rectification ratio of the two channels is calculated separately. The formula is ICR = I +1V / I -1V Through Cu + and Cu 2+ The change in rectification ratio before and after binding with the specific probe in the corresponding channel enables Cu + Cu 2+ Independent quantitative detection of two ions.

10. The application according to claim 8, characterized in that, For single-cell detection, the sensor tip is gently inserted into the cell membrane to record intracellular Cu under different treatment conditions in real time. + and Cu 2+ The concentration change; for a three-dimensional tumor sphere, the sensor is used to penetrate the sphere layer by layer, recording the rectification ratio signal at different depths, and plotting Cu. + and Cu 2+ Spatial distribution map.

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