CN114544721A - Flexible micro-nano electrode sensor and preparation method thereof - Google Patents

Abstract

The invention discloses a flexible micro-nano electrode sensor and a preparation method thereof. The sensor includes a flexible substrate with a nano-porous membrane and a nano-needle electrode array. The nano-needle electrode array includes nano-needles, electrodes with preset shapes and a serpentine shape. The electrode of the preset shape is connected to the nano-needle and the serpentine circuit, the serpentine circuit is used to connect an external circuit, and the electrode of the preset shape is covered on the surface of the flexible substrate, so The nanoneedles penetrate the nanopores of the flexible substrate. The embodiments of the present invention can realize high-throughput and long-term detection of electrical signals in somatic cells, and can be widely used in the field of sensor technology.

Description

Flexible micro-nano electrode sensor and preparation method thereof

technical field

The invention relates to the technical field of sensors, in particular to a flexible micro-nano electrode sensor and a preparation method thereof.

Background technique

Bioelectric signal detection focuses on the heart and brain, and its detection range includes cells, tissues, organs, and living bodies. By detecting the signals of electrophysiological characteristics, different disease mechanisms can be studied, which has extremely high guiding significance for disease mechanisms and treatment methods. In addition, the detection and recording of cell electrical signals is also a key technology for drug development and screening: studying the changes in the action potential peak spectrum of drugs on cells can analyze the regulatory mechanism of drug molecules on the opening and closing of cell ion channels, and then understand the relationship between drug treatment effects and Effective information such as toxic effects.

At present, the conventional method of intracellular electrical signal recording is the patch clamp technique. The current-clamp technique of patch-clamp technology, which can acquire standard action potential signals, and the voltage-clamp technique, which can acquire and record the response of ion channels, have become the basis for deciphering the behavior of electrically excitable cells. The patch clamp technique generally uses a pointed glass tube containing a metal wire as a recording electrode, which is attached to the cell surface, and at the same time, a negative pressure is applied to rupture the cell membrane to record the action potential in the cytoplasm. However, due to the low throughput of manual patch clamp, complicated and time-consuming operation, and damage to cells, it is impossible to record electrophysiological signals of cells for a long time. Currently, the duration of signal acquisition using manual patch clamp technology does not exceed 2 hours. . In addition, the prior art cannot detect electrical signals in somatic cells.

SUMMARY OF THE INVENTION

In view of this, the purpose of the embodiments of the present invention is to provide a flexible micro-nano electrode sensor and a preparation method thereof, so as to realize high-throughput and long-term detection of electrical signals in somatic cells.

In a first aspect, an embodiment of the present invention provides a flexible micro-nano electrode sensor, comprising a flexible substrate with a nanoporous membrane and a nanoneedle electrode array, wherein the nanoneedle electrode array includes nanoneedles, electrodes with preset shapes and snakes the electrode of the preset shape is connected to the nanoneedle and the serpentine circuit, the serpentine circuit is used to connect an external circuit, the electrode of the preset shape is covered on the surface of the flexible substrate, The nanoneedles penetrate the nanopores of the flexible substrate.

Optionally, the surface of the nanoneedles is modified with a positive ionic polymer.

Optionally, the nanopore density of the flexible substrate ranges from 0.5 pores/μm ² to 1.5 pores/μm ² .

In a second aspect, an embodiment of the present invention provides a method for preparing a flexible micro-nano electrode sensor, including:

providing a flexible substrate with a nanoporous membrane;

The nanoholes on the first surface of the flexible substrate cover a mask or photoresist with a preset shape array pattern; each preset shape pattern has a serpentine curved pattern;

After the conductive layer is plated on the mask or the photoresist, the mask or the photoresist is removed to form a predetermined-shaped electrode array and a serpentine circuit;

Based on the preset shape electrode array, a metal solid column structure is formed on the inner wall of the nanopore by electrodeposition;

An insulating layer is coated on the first surface of the flexible substrate to encapsulate the device, and a part of the substrate is etched on the second surface of the flexible substrate to form exposed metal nanoneedles.

Optionally, the method further includes:

Hydroxylation is carried out on the surface of the metal nanoneedles through a thiol reaction;

Modification of positive ionic polymers on the surface of hydroxylated metal nanoneedles;

Block unreacted groups.

Optionally, the method further includes:

The structural characteristics and distribution characteristics of the metal nanoneedles were evaluated by scanning electron microscopy.

Optionally, the method further includes:

Whether the metal nanoneedles are well connected to the predetermined shape electrode array is detected by cyclic voltammetry.

Optionally, the method further includes:

The electrical properties of the metal nanoneedles were tested by electrochemical impedance spectroscopy.

Optionally, the method further includes:

The blank background noise of the flexible micro-nano electrode sensor was evaluated by standard instrumentation of the micro-electrode array.

The implementation of the embodiment of the present invention includes the following beneficial effects: In this embodiment, the flexible micro-nano electrode sensor includes a flexible substrate with a nanoporous membrane and a nano-needle electrode array, and the nano-needle electrode array includes nano-needles, electrodes with a preset shape and a serpentine shape The circuit, the electrodes of the preset shape are connected to the nano-needle and the serpentine circuit, and the serpentine circuit is used to connect the external circuit; the detection of the electrical signal in the somatic cell is realized through the flexible substrate, and the high-level detection of the electrical signal in the cell is realized through the nano-needle electrode array. throughput and long-term detection.

Description of drawings

1 is a schematic structural diagram of a flexible micro-nano electrode sensor provided by an embodiment of the present invention;

FIG. 2 is a schematic flowchart of steps of a method for preparing a flexible micro-nano electrode sensor provided by an embodiment of the present invention;

3 is a flow chart of preparing a flexible micro-nano electrode sensor provided by an embodiment of the present invention;

Fig. 4 is the flow chart of a kind of nano-needle that is provided in the embodiment of the present invention shows the modification of positive ionic polymer;

FIG. 5 is a diagram of a flexible micro-nano electrode sensor for detecting intracellular action potential according to an embodiment of the present invention;

6 is a schematic diagram of a single intracellular action potential peak being evenly divided into 25 segments by time according to an embodiment of the present invention;

FIG. 7 is a heat map of voltage values corresponding to 24 feature points of a multi-channel provided by an embodiment of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The numbers of the steps in the following embodiments are only set for the convenience of description, and the sequence between the steps is not limited in any way, and the execution sequence of each step in the embodiments can be adapted according to the understanding of those skilled in the art Sexual adjustment.

As shown in FIG. 1, an embodiment of the present invention provides a flexible micro-nano electrode sensor, wherein FIG. 1(a) represents a side view of the flexible micro-nano electrode sensor, and FIG. 1(b) represents a top view of the flexible micro-nano electrode sensor , comprising a flexible substrate 1-1 with a nanoporous membrane and a nanoneedle electrode array, the nanoneedle electrode array comprising nanoneedles 1-2, electrodes 1-3 with preset shapes and a serpentine circuit 1-4, the Electrodes 1-3 with a preset shape are connected to the nanoneedles 1-2 and the serpentine circuit 1-4, and the serpentine circuit 1-4 is used to connect an external circuit, and the electrodes 1-3 with a preset shape Covered on the surface of the flexible substrate 1-1, the nanoneedles 1-2 penetrate the nanopores of the flexible substrate 1-1.

It should be noted that the material of the flexible substrate includes polyimide PI, polycarbonate, silk protein or SU8 and the like. The flexible substrate can be attached to the curved and uneven organ surface to form a good coupling interface with the organ surface, so as to record the physiological electrical signals of cells in different regions of different organs.

Optionally, the surface of the nanoneedles is modified with a positive ionic polymer.

It should be noted that the positive ionic polymer was modified on the surface of the nanoneedle electrode to optimize the conditions for maintaining the nanoelectrode to penetrate the cell membrane for a long time, and to reveal the cell membrane penetration mechanism and cell safety.

Optionally, the nanopore density of the flexible substrate ranges from 0.5 pores/μm ² to 1.5 pores/μm ² .

It should be noted that the nanopore density range of the flexible substrate is determined according to the diameter of the cell, so that one cell can contact multiple nanoneedles.

The implementation of the embodiment of the present invention includes the following beneficial effects: In this embodiment, the flexible micro-nano electrode sensor includes a flexible substrate with a nanoporous membrane and a nano-needle electrode array, and the nano-needle electrode array includes nano-needles, electrodes with a preset shape and a serpentine shape The circuit, the electrodes of the preset shape are connected to the nano-needle and the serpentine circuit, and the serpentine circuit is used to connect the external circuit; the detection of the electrical signal in the somatic cell is realized through the flexible substrate, and the high-level detection of the electrical signal in the cell is realized through the nano-needle electrode array. throughput and long-term detection.

As shown in FIG. 2 , an embodiment of the present invention provides a method for preparing a flexible micro-nano electrode sensor, including:

S100, providing a flexible substrate with a nanoporous membrane;

S200, the nano-holes on the first surface of the flexible substrate cover a mask or photoresist with a preset shape array pattern; each preset shape pattern has a serpentine curved pattern;

S300, after plating a conductive layer on the mask or the photoresist, remove the mask or the photoresist to form a preset-shaped electrode array and a serpentine circuit;

S400. Based on the preset shape electrode array, a metal solid column structure is formed on the inner wall of the nanopore by electrodeposition;

S500 , coating an insulating layer on the first surface of the flexible substrate to encapsulate the device, and etching part of the substrate on the second surface of the flexible substrate to form exposed metal nanoneedles.

It should be noted that the preset shape array pattern is determined according to practical applications, and is not specifically limited in this embodiment, such as a square or a circle.

It should be noted that the conductive layer includes, but is not limited to, a metal layer, such as a gold layer or a platinum layer.

Referring to FIG. 3, in a specific embodiment, gold nanoneedle arrays and flexible circuit patterns are constructed on a polycarbonate nanoporous membrane flexible substrate. The substrate film contains nanopores with a pore diameter of 100 nm (pore density is 0.1 pore/μm ² ), and gold nanoneedle arrays can be prepared by electrodeposition method and plasma etching process. In order to realize the patterned array of gold nanoneedles, firstly, by designing a mask of 6×6 square (10 μm×10 μm) array pattern, or exposing 6×6 squares on the backside of the porous membrane by UV lithography (dimensions 10 μm x 10 μm) photoresist pattern of the array. Depending on the spacing of the nanopores on the porous membrane, the fabricated square array will have an average of 10 nanoneedles per square array (each cardiomyocyte is >10 μm in diameter, so each cell will be in contact with an average of 10 nanoneedles). The spacing design of the nanoneedles can effectively ensure that a large number of cells have enough nanoneedles for contact. The square array pattern has a serpentine curved pattern that independently extends the square array to the outside block by block. The entire gold nanoneedle sensing device is mainly composed of two parts: a 6×6 electrode array with gold nanoneedles as the sensing module and a flexible circuit pattern for electrical signal recording. To build flexible circuits on the backside to connect each individual sensing module for subsequent electrical signal recording. Design a serpentine circuit pattern as a lead to connect the nanoneedle sensing module and the external circuit contact module. Then, magnetron sputtering is used to coat a layer of Au with a thickness of about 40 nm, and then the mask is directly removed, or the photoresist at the unexposed part is removed by degumming, so as to obtain a 6×6 Au layer arranged Square (10 μm×10 μm in size) pattern. The square array pattern also has a serpentine curved Au circuit pattern, which independently extends the square array to the outside block by block. The serpentine-bending Au circuit is flexible and bendable, preventing rigid circuit designs from destroying conductivity during the bending process. These square squares of the Au layer can provide good conductive contacts for the electrodeposition of the metal nanoneedles, so that the nanoneedle structure can be deposited only in the pores of the Au pattern. Then, Ag/AgCl was used as the reference electrode, Pt was used as the counter electrode, and the gold layer was used as the working electrode at the bottom of the porous film _. , potassium chloride (1mM/L) electroplating solution for electrodeposition. In the constant pressure working mode, gold was deposited on the inner wall of the porous membrane to form a solid columnar structure. After gold electroplating, photolithography is used to process a serpentine flexible structure circuit (line width 1 μm) to connect the square electrode, and prepare a counter electrode to form a circuit. Finally, a SU-8 insulating layer was spin-coated on the backside to encapsulate the device. Subsequently, a small amount of excess gold film on the front side was removed by mechanical polishing, and the upper surface of the polycarbonate film was etched with O ₂ plasma to expose gold nanoneedle structures with a diameter of 100 nm and a length of 2 μm. The electrical signal was recorded using an Ag/AgCl electrode as a reference electrode. The serpentine circuit is guided out through metal leads for easy connection to an external power source. Encapsulate the device to prevent electrical or liquid leakage.

The nanopore template method, micro-nano processing etching, electrodeposition and other multi-process synergistic new strategies proposed in this example realize the large-area preparation of high-precision vertical nano-needle electrodes on flexible devices.

Optionally, the method further includes:

Hydroxylation is carried out on the surface of the metal nanoneedles through a thiol reaction;

Modification of positive ionic polymers on the surface of hydroxylated metal nanoneedles;

Block unreacted groups.

Referring to FIG. 4 , in a specific embodiment, the surface of the gold nanoneedle electrode is hydroxylated by thiol reaction. The device was first cleaned with 95% ethanol, and then an ethanol solution of 11-mercapto-undecanoic acid (7-mercaptoheptanol, 5 mM) was added to react for 24 hours. The gold nanoneedle electrodes were then cleaned with ethanol. Next, 5% 3-glycidoxypropyl trimethoxysilane (3-GPS) was dissolved in 99% ethanol solution, then added to the gold nanoneedle electrode and heated to 60 °C for 24 hours. After the reaction, wash with ethanol. Then the PEI polymer was dissolved in carbonate buffer (pH 9.5) at a concentration of 10%, and then added to the gold nanoneedle electrode to react for 24 hours. After the reaction, it was washed several times with 1M NaCl solution and purified water. Then 1M ethanolamine solution was added to react for 6 hours to block unreacted epoxy resin groups. The whole gold nanoneedle array sample was subsequently washed several times with 1M NaCl solution and purified water.

In order to optimize the cell membrane penetration efficiency and safety of PEI-modified nanoneedles, branched PEI (800, 2K, 10k, 50k) polymer-modified nanoneedles with different molecular weights are planned to be tried. After the gold nanoneedle array of PEI was modified, X-ray electron spectroscopy (XPS) was used to characterize the elements and contents on the surface of the material, so as to test whether the PEI was successfully modified to the surface of the nanoneedles. When the molecular weight of PEI is 10k and the concentration is 1%, the modification effect is better.

Optionally, the method further includes:

The structural characteristics and distribution characteristics of the metal nanoneedles were evaluated by scanning electron microscopy.

After the patterned gold nanoneedle array was prepared, the patterned distribution of the gold nanoneedle array was observed by scanning electron microscope (SEM) to evaluate the uniformity of the local distribution of the nanoneedle and the microstructure of the gold nanoneedle. The geometric parameters of the gold nanoneedle array, including diameter, height, and density, can be controllably adjusted to optimize the morphology of the metal nanoneedle, which is suitable for subsequent intracellular electrical signal recording. The diameter and density of gold nanoneedles are determined by the pore size and pore density of the polycarbonate porous membrane. The diameter and density of gold nanoneedles can be adjusted by selecting polycarbonate membranes with different specification parameters, while the height can be adjusted by changing the _O2 plasma. The length of etching time can be adjusted. After the technology is mature, the array modules arranged in 6×6 can be designed to be increased to 8×8 or even 10×10 array modules according to the recording requirements to increase the throughput of parallel recording.

Optionally, the method further includes:

Whether the metal nanoneedles are well connected to the predetermined shape electrode array is detected by cyclic voltammetry.

Optionally, the method further includes:

The electrical properties of the metal nanoneedles were tested by electrochemical impedance spectroscopy.

By testing the basic electrical performance of the sensor, its ability to record high-quality signals is evaluated. Based on the fabrication of the nanoneedle array and the electrode leads on the backside, there are two process steps. In order to ensure the reliability of the electrical signal recording function of the nanoneedle sensor, electrochemical tests are first required. Cyclic voltammetry (CV) was used to scan the CV curve of the nanoneedle sensor in K ₃ Fe(CN) ₆ solution, and it was judged by the experimental current whether the connection between the nanoneedle and the back electrode of the substrate was good. At the same time, the electrochemical impedance spectroscopy test method was used to scan the impedance spectrum of the nanoneedle sensor in phosphate buffer solution (PBS) between 100Hz and 1 MHz, and compared with the impedance spectrum of a typical microelectrode array and nanosensor, so as to further determine Whether the basic performance of the developed nanoneedle sensor is good. Through impedance spectrum scanning, it is studied whether the impedance performance of the nanoneedle sensor changes greatly under different curvature conditions, so as to determine the stability of the sensor electrical performance of the flexible substrate.

Optionally, the method further includes:

The blank background noise of the flexible micro-nano electrode sensor was evaluated by standard instrumentation of the micro-electrode array.

After basic electrical performance testing, the blank background noise level of the nanoneedle sensor needs to be evaluated using standard instrumentation of the microelectrode array. The nanoneedle sensor was used to record the blank background noise of electrically excited cells cultured in vitro to evaluate the electrical signal noise level in vitro. At the same time, the blank background noise of the animal brain and heart organ tissue was recorded using the nanoneedle sensor to evaluate the in vivo noise level. electrical signal noise level under conditions. By analyzing the impedance spectrum and noise level, the coefficient of variation (CV) was calculated, allowing the evaluation of the consistency among the multi-channel nanoneedle sensors. Through the above performance tests, this research will further optimize and improve the processing technology of the nano-needle sensor, and develop a nano-needle sensor with good electrical signal recording performance.

In addition, the sensor in this application is not only used for the electrical signal recording of electrically excitable cells in vitro, but also for the electrical signal recording of the in vivo brain and heart. In order to achieve good bio-device interface coupling, flexible materials need to be used as sensors. base. The stability and robustness of the electrical properties of the micro-nano sensors based on flexible material substrates were evaluated for their stability and robustness under the tensile-distortion state of the brain and heart applications. The anti-fatigue test of the flexible sensor is carried out repeatedly for tensile and torsional deformation, and its robustness is determined by the impedance spectrum characteristics before and after the test.

The stability of the nanoneedle sensor is the basis and guarantee of high-performance long-term recording of bioelectrical signals. The actual application environment of bioelectric recording of nanoneedle sensors is usually 37°C, high humidity, in vitro cell culture medium rich in organic matter and inorganic salts or in body fluid environment. Therefore, it is necessary to adopt a variety of stability tests that simulate the actual environment. . For isolated cell and tissue culture environmental conditions, it is necessary to explore the quality change of the long-term electrical signal recorded by the nanoneedle sensor. For different electrically excitable cells (cardiomyocytes and nerve cells), based on the data of each time period in a long period (one week or several weeks), compare the data recorded by the traditional microelectrode array sensor synchronously, and grasp the evolution law of the bioelectric signal itself. Thus, the evaluation criteria of signal quality are established, which is helpful to select conductive materials with better biocompatibility, stronger wear resistance and corrosion resistance to process nanoneedles.

The optimal working conditions and service life of the flexible sensor are finally determined through stability and robustness tests.

The use of the sensor of the present application will be described below with a specific embodiment.

First, the isolated cardiomyocytes and myocardial tissue of the experimental mouse were cultured, and the isolated heart was perfused to prepare the living heart tissue of the experimental mouse.

Then, in order to detect intracellular electrical signals, excitable cardiomyocytes in vivo or in vitro are used as research, test and application objects. The cells were cultured on a multi-well plate, and during the experiment, the nanoneedle electrode device modified by the cationic polymer was adjusted in height through a micro-operating table, and gradually pressed onto the cells. If the nanoneedles penetrate the cell membrane and the electrodes come into contact with the intracellular environment, electrical signals within the cell can be recorded. Often the intracellular electrical signal resembles a standard action potential shape. Through data analysis, the amplitude of the electrical signal recorded by the nanoneedle sensor can be calculated. In order to evaluate the sensitivity of the sensor's electrical signal recording, the gold standard patch-clamp technique was used to simultaneously record the action potential of the same cell, and then the ratio of the intracellular potential amplitude to the standard action potential amplitude was calculated to determine the ability of the nanoneedle sensor to record the intracellular signal. At the same time, the signal-to-noise ratio can be calculated according to the noise level of the two recording methods, so as to comprehensively evaluate the basic performance of the nanoneedle sensor combined with the photoelectric permeable membrane technology to record the intracellular electrical signal.

Evaluation of the continuity of the intracellular electrical signal recording: After the intracellular electrical signal is recorded by the nanoneedle sensor, the quality of the intracellular electrical signal will decrease or even disappear quickly due to the fusion of the nanocracks after the cell membrane is perforated. The continuity of the electrical signal recording is evaluated, and the changes in the nature and quality of the electrical signal are analyzed through the evaluation of the signal amplitude and shape. This project will explore the optimization of various PEI modification conditions, different nanoneedle geometries (diameter, length, spacing), and nanoneedle pressing conditions on cells, and the effects and effects on prolonging the continuous recording time of intracellular electrical signals of nanosensor devices.

Evaluation of the recording accuracy of intracellular electrical signals: The accuracy of intracellular electrical signals is also an important part of evaluating the recording function of nanosensors. The intracellular signal of the same electrically excited cell was recorded synchronously with the nanoneedle sensor and patch clamp, and the signal analysis method of signal proportional compression was used to directly compare whether the shapes of the two recorded signals were similar, and intuitively evaluate the similarity between the two. At the same time, the data correlation calculation method is used to analyze whether the correlation coefficient between the two is close to 1, and the similarity of the two signals is further evaluated theoretically. In addition, according to the intracellular electrical signal characteristics (such as time course, each characteristic period) of nerve cells, cardiac pacemaker cells, atrial myocytes, and cardiomyocytes recorded by the nanoneedle sensor, they were compared with the standard action potential characteristics recorded by patch clamp. , to assess the accuracy of ion channel activity-related signals recorded by nanoneedle sensors.

The experimental data are as follows: refer to Figure 5, Figure 5(a) shows the typical intracellular action potential recorded by the device after electroporation, and Figure 5(b) shows the multi-channel intracellular action potential recorded by the device. Referring to Figure 6 and Figure 7, Figure 6 shows that the action potential peak in a single cell is evenly divided into 25 segments according to time, and 24 feature points are taken; Figure 7 shows the heat map of the voltage values corresponding to the 24 feature points of the multi-channel. 7 The voltage values of the 9th to 15th feature points of the recorded intracellular signals are higher, and the voltage values of the remaining feature points are lower, which are similar to the intracellular signals recorded by the gold standard patch-clamped micro-nano sensor.

The above is a specific description of the preferred implementation of the present invention, but the present invention is not limited to the described embodiments, and those skilled in the art can make various equivalent deformations or replacements without departing from the spirit of the present invention. , these equivalent modifications or substitutions are all included within the scope defined by the claims of the present application.

Claims (9)

1. The utility model provides a flexible electrode sensor that receives a little, its characterized in that, including flexible substrate and the nanometer needle electrode array that has nanometer porous membrane, nanometer needle electrode array includes nanometer needle, the electrode and the serpentine circuit of predetermineeing the shape, the electrode connection of predetermineeing the shape the nanometer needle with serpentine circuit, serpentine circuit is used for connecting external circuit, the electrode cover of predetermineeing the shape is in the surface of flexible substrate, the nanometer needle pierces through the nanopore of flexible substrate.

2. The flexible micro-nano electrode sensor according to claim 1, wherein the surface of the nano needle is modified with a positive ion polymer.

3. The method of claim 1, wherein the flexible substrate has a nanopore density in a range of 0.5 pores/μ ι η²1.5 pores/. mu.m²。

4. A preparation method of a flexible micro-nano electrode sensor is characterized by comprising the following steps:

providing a flexible substrate having a nanoporous membrane;

covering a mask plate or photoresist with a preset shape array pattern on the nano holes on the first surface of the flexible substrate; each preset shape pattern is provided with a snake-shaped bending pattern;

after plating a conducting layer on the mask plate or the photoresist, removing the mask plate or the photoresist to form an electrode array with a preset shape and a snake-shaped circuit;

based on the preset-shaped electrode array, forming a metal solid columnar structure on the inner wall of the nanopore by adopting electrodeposition;

and coating an insulating layer on the first surface of the flexible substrate to package the device, and etching part of the substrate on the second surface of the flexible substrate to form the exposed metal nano-needle.

5. The method of manufacturing according to claim 4, further comprising:

hydroxylating the surface of the metal nano needle by a sulfydryl reaction;

modifying a positive ion polymer on the surface of the hydroxylated metal nano needle;

blocking unreacted groups.

6. The method of manufacturing according to claim 4, further comprising:

and evaluating the structural characteristics and the distribution characteristics of the metal nano-needle by a scanning electron microscope.

7. The method of manufacturing according to claim 4, further comprising:

and detecting whether the metal nano needle is well connected with the preset-shaped electrode array or not by a cyclic voltammetry method.

8. The method of manufacturing according to claim 4, further comprising:

and testing the electrical property of the metal nano needle by electrochemical impedance spectroscopy.

9. The method of manufacturing according to claim 4, further comprising:

and evaluating blank background noise of the flexible micro-nano electrode sensor by a standard instrument of the microelectrode array.

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