CN113237929A

CN113237929A - Construction method of flexible sensing electrode modified by nano particles

Info

Publication number: CN113237929A
Application number: CN202110472683.0A
Authority: CN
Inventors: 包静; 邱晓沛; 杨慧思; 高铭萱; 周洋; 侯长军; 陈鸣
Original assignee: First Affiliated Hospital of PLA Military Medical University
Current assignee: Nanfang Hospital; First Affiliated Hospital of PLA Military Medical University
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2021-08-10

Abstract

The invention relates to the technical field of biosensors and discloses a construction method of a flexible sensing electrode modified by nano particles; also discloses a CC/GWs/AuPt sensing electrode and application thereof. The CC/GWs/AuPt sensing electrode prepared by the method has the advantages of excellent performance, low price, simple preparation steps, strong repeatability, high detection efficiency and high accuracy, is closer to the real three-dimensional physiological environment of cell culture, can be applied to the field of real-time in-situ detection of more biomolecules and can be applied to the field of continuous dynamic monitoring of cell activities and metabolites thereof; the electrode of the invention can be used for the direct growth of living cells and is beneficial to the sensing electrode pair H₂O₂The cell can grow freely on any dimension (three-dimensional skeleton or wall) of the electrode, the number of adhered cells is large, the growth state is good, and the electrode pair H prepared by the invention₂O₂Has wide linear range, fast response speed and high response speedLow detection limit and good anti-interference capability.

Description

Construction method of flexible sensing electrode modified by nano particles

Technical Field

The invention relates to the technical field of biosensors, in particular to a construction method of a nanoparticle modified flexible sensing electrode.

Background

Reactive Oxygen Species (ROS), which are the general term for the products of the one-electron reduction of oxygen in organisms, play a very important role in cell signaling and maintenance of homeostasis, and examples include superoxide anion (O)₂H), hydrogen peroxide (H)₂O₂) Hydroxyl radical (OH-), ozone (O-)₃) And the like, because the compounds contain unpaired electrons, the half-life period is short, the chemical reaction activity is high, the diffusion of the compounds in a biological system is fast, the concentration of the compounds is low, and the rapid quantitative detection of the ROS is still an international problem. Wherein, hydrogen peroxide (H)₂O₂) As important signal transduction molecules and second messengers in ROS, the protein has important significance for the research of cell physiological metabolism and disease pathology. H₂O₂Excessive accumulation in cells generates oxidative stress, damages lipid, protein and DNA, and is related to the occurrence and development of neuronal fading diseases such as aging, cancer, diabetes and Alzheimer's disease.

Therefore, the method has the advantages of quick response, strong specificity, high sensitivity and capability of realizing in-situ real-time quantitative detection of H released by cells under physiological conditions₂O₂The method for analyzing the concentration of (A) has important significance for the early diagnosis of diseases and the research of related pathogenic mechanisms. At present, the analysis methods adopted for the quantitative detection of ROS inside and outside cells mainly comprise a chemical reaction method, a chemiluminescence method, a fluorescence method, a spectrophotometry method, an electrochemical method and the like. Among them, fluorescence analysis and electrochemical methods are the most widely studied methods that can achieve direct quantitative detection of ROS. However, the fluorescence analysis method has reduced sensitivity due to pH sensitivity and instability thereof, and limits the application of the fluorescence analysis method in the detection of ROS with ultra-low concentration.The electrochemical method has the advantages of rapidness, sensitivity, simple and convenient operation, high sensitivity, easy construction of a specific sensing interface and the like, and is widely applied to the in-situ real-time detection of ROS released by living cells. An ideal electrochemical biosensing detection platform needs to have good catalytic performance and excellent selectivity, and needs to have good biocompatibility and a growth environment close to real physiological conditions. Therefore, the inventor develops a construction method of the nanoparticle modified flexible sensing electrode.

Disclosure of Invention

Based on the problems, the CC/GWs/AuPt sensing electrode prepared by the method has the advantages of excellent performance, low price, simple preparation steps and strong repeatability, can be applied to the field of real-time in-situ detection of more biomolecules, and can be applied to the field of continuous dynamic monitoring of cell activities and metabolites thereof.

In order to solve the technical problems, the invention provides the following technical scheme:

a construction method of a nanoparticle modified flexible sensing electrode comprises the following steps:

s1 growing GWs on 5 × 5cm by RF-PECVD technique²Obtaining a CC/GWs electrode, and treating the CC/GWs electrode for 20s under oxygen by using a plasma cleaner;

s2: the CC/GWs electrode processed in the step S1 is cut into 1X 1cm²The edges of the cut CC/GWs electrode were encapsulated with epoxy resin so that the exposed active area of the CC/GWs electrode was 0.9X 0.9cm²Soaking CC/GWs electrode in 5mL H₂PtCl₆And HAuCl₄In the mixed solution of (1), H₂PtCl₆And HAuCl₄The concentration of the mixed solution of (1 mM), H in the mixed solution₂PtCl₆And HAuCl₄The concentration ratio of the metal oxide is 1:1, and then electrodeposition is carried out under the working potential of-0.2V by adopting a chronoamperometry, wherein the deposition time is 200 s; and after the deposition is finished, washing the electrode for a plurality of times by using deionized water to obtain the CC/GWs/AuPt sensing electrode.

Further, step S2 is H₂PtCl₆And HAuCl₄The mixed solution of (A) is 0.01M H₂SO₄And 0.01M Na₂SO₄And (4) diluting.

In order to solve the technical problems, the invention also provides a CC/GWs/AuPt sensing electrode.

In order to solve the technical problems, the invention also provides application of the CC/GWs/AuPt sensing electrode in preparation of products for detecting or monitoring biomolecules.

Further, the product can be used for in-situ real-time analysis and detection and dynamic monitoring of biomolecules.

Further, the biomolecule is H₂O₂。

Compared with the prior art, the invention has the beneficial effects that: the CC/GWs/AuPt sensing electrode prepared by the method has the advantages of excellent performance, low price, simple preparation steps, strong repeatability, high detection efficiency and high accuracy, is closer to the real three-dimensional physiological environment of cell culture, can be applied to the field of real-time in-situ detection of more biomolecules and can be applied to the field of continuous dynamic monitoring of cell activities and metabolites thereof; the electrode of the invention can be used for the direct growth of living cells and is beneficial to the sensing electrode pair H₂O₂The cell can grow freely on any dimension (three-dimensional skeleton or wall) of the electrode, the number of adhered cells is large, the growth state is good, and the electrode pair H prepared by the invention₂O₂The response detection has wide linear range, fast response speed, low detection limit and good anti-interference capability.

Drawings

FIG. 1 is a topographical view of CC/GWs prepared in accordance with an embodiment of the present invention;

FIG. 2 is a microscopic topography of bare CC, CC/GWs and CC/GWs/AuPt electrodes studied using SEM in accordance with an embodiment of the present invention;

FIG. 3 is an XPS spectrum of an example of the present invention;

FIG. 4 is a graph of current response results for an embodiment of the present invention;

FIG. 5 shows a CC/GWs/AuPt sense electrode pair H according to an embodiment of the present invention₂O₂The detection result graph of (2);

FIG. 6 is a graph of CV curves and a linear relationship between peak current and scan rate for an embodiment of the present invention;

FIG. 7 shows a CC/GWs/AuPt sense electrode pair H according to an embodiment of the present invention₂O₂The response curve of the chronoamperometric current (i-t) and the fitting scatter diagram;

FIG. 8 is a graph showing the measurement of the flexible CC/GWs/AuPt sensing electrode pair H at an operating potential of-0.32V by using i-t in an embodiment of the invention₂O₂A linear dependence result graph of the response;

FIG. 9 is a graph showing the results of selectivity and reproducibility studies on CC/GWs/AuPt sensing electrodes according to an embodiment of the present invention;

FIG. 10 is a photomicrograph, fluorescence micrograph, and SEM of A549 cells of an embodiment of the invention;

FIG. 11 is a diagram of a CC/GWs/AuPt sensing electrode of the embodiment of the invention culturing A549 cells, a photograph of a testing device under a traditional three-electrode system and H released by cells on the CC/GWs/AuPt sensing electrode under drug stimulation₂O₂In-situ monitoring;

FIG. 12 shows the direct cell growth CC/GWs/AuPt sensor electrode of the present invention for real-time detection of H released by living cells₂O₂The construction schematic diagram of (1).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example (b):

the reagents and instrumentation used in this example are shown in the following table:

primary reagent

Main equipment

In this example, a CC/GWs/AuPt sensing electrode is first prepared, which includes the following steps:

s1: before Graphene (GWs) growth, a flexible conductive carbon cloth (CC, 5X 5 cm)²) Sequentially soaking in acetone and ethanol, performing ultrasonic treatment for 10min to remove organic impurities on the surface, washing with deionized water for several times, and drying for later use; directly and vertically growing graphene sheets on smooth carbon fibers by a radio frequency enhanced plasma chemical vapor deposition (RF-PECVD) technology to form a continuous Graphene Wall (GWs) which is stacked layer by layer; the cleaned and dried CC was placed in the center of a vacuum tube furnace in hydrogen (H)₂) Raising the temperature of the tube furnace to 750 ℃ under constant flow, then bombarding the surface of the CFP by using hydrogen plasma for 10min to remove oxygen and impurities on the surface of the CC substrate, and then treating the surface of the CC substrate by using a plasma jet method according to the flow ratio of 3: 2 ratio of methane (CH)₄) And hydrogen (H)₂) The system pressure is 40Pa, the power is 200W, the growth time is 30min, 45min and 60min, and then, when the temperature of the tube furnace is H₂After the protection is carried out and the temperature is reduced to the room temperature, the prepared CC/GWs is taken out, and the CC/GWs electrode is treated for 20s by a plasma cleaning machine under the power of 50 percent and the oxygen flow of 80 percent;

s2: the CC/GWs electrode processed in the step S1 is cut into 1X 1cm²The edges of the cut CC/GWs electrode were encapsulated with epoxy resin so that the exposed active area of the CC/GWs electrode was 0.9X 0.9cm²The CC/GWs was clamped by a platinum sheet electrode holder, and the CC/GWs electrode was immersed in 5mL of H₂PtCl₆And HAuCl₄In the mixed solution of (1), H₂PtCl₆And HAuCl₄The concentration of the mixed solution of (1 mM, H)₂PtCl₆And HAuCl₄The mixed solution of (A) is 0.01M H₂SO₄And 0.01M Na₂SO₄Diluted, mixed solution of H₂PtCl₆And HAuCl₄The concentration ratio of (A) to (B) is 1: 1; then, carrying out electrodeposition at a working potential of-0.2V by adopting a chronoamperometry (i-t), wherein the deposition time is 200 s; and after the deposition is finished, washing the electrode for a plurality of times by using deionized water to obtain the CC/GWs/AuPt sensing electrode.

As shown in the attached drawings 1A and 1B, the prepared black flexible CC/GWs has very good flexibility, can bear very large physical deformation, can still recover the original appearance after being folded, and the prepared CC/GWs has the properties of easy carrying, easy processing and cutting and the like besides the flexibility. FIG. 1B shows CC/GWs being processed into a square shape of 1cm by 1cm and edge-sealed with epoxy, and further electrochemical deposition and testing experiments were carried out using a platinum sheet electrode clamp to clamp CC/GWs to provide an effective area of about 0.9 by 0.9cm²。

The physical characterization and related electrochemical performance of the CC/GWs/AuPt sensing electrode prepared by the inventor are tested, and the test method is shown as follows.

The prepared CC/GWs/AuPt sensing electrode adopts a field emission electron microscope (FESEM) to analyze the surface appearance characteristics and the size and surface distribution state of AuPt nano particles, and uses an X-ray photoelectron spectroscopy (XPS) to analyze the surface element chemical state of the prepared sensing electrode. All electrochemical tests such as Cyclic Voltammetry (CV), alternating impedance (EIS) and chronoamperometry (i-t) used a CHI 760E electrochemical workstation using a three-electrode system consisting of a platinum wire electrode (Pt) counter electrode, a silver/silver chloride (Ag/AgCl) reference electrode, and a CC/GWs/AuPt working electrode. CC/GWs/AuPt electrode pair H₂O₂In the electrocatalytic performance test, CV and i-t experiments were performed in 0.01M PBS buffer at pH 7.4. Test parameters of CV: the working voltage range is-1.0V, and the scanning rate is 50mV s^-1(ii) a i-t detection electrode pair H₂O₂The linear response of (A) is carried out at an optimum operating voltage of-0.32V.

Referring to fig. 2A and 2B, the Carbon Cloth (CC) is formed by interweaving a plurality of carbon fibers with smooth surfaces, and fig. 2C and 2D show that a large number of folded graphene nano sheets continuously and vertically grow on the smooth carbon fibers to form a Graphene Wall (GWs) stacked layer by layer, and the unique three-dimensional structure provides a large specific surface area, which is beneficial to the mass deposition of AuPt, and also provides a favorable environment for the adhesion and growth of cells. As can be seen from the high power SEM of fig. 2E, a large number of AuPt nanoparticles were uniformly distributed on any side of the framework and inner wall of GWs by electrodeposition, wherein the size distribution of the nanoparticles was 20-40 nm; the EDS face scan results of FIG. 2F show that the elements contained in the CC/GWs/AuPt electrode are C, Au and Pt, and that the three elements are uniformly distributed over the surface of the electrode.

The XPS survey of fig. 3A shows that the electrode contains C, Au and Pt elements, where C is from carbon fiber and graphene. From the C1s high resolution XPS spectrum of FIG. 3B, it can be observed that the main peak exhibits a characteristic C-C peak at 284.78eV, whereas the binding energy is 286.28eV for the C-O functionality, possibly due to the oxygen plasma treatment of the surface. FIG. 3C shows that the binding energies of 84.13eV and 87.78eV in the Au 4f high resolution XPS spectrum correspond to those of Au 4f_7/2And Au 4f_5/2The chemical binding state, Pt 4f absorption peak is further divided into peaks, and the binding energy of 71.09eV and 74.12eV are respectively assigned to Pt 4f_7/2And Pt 4f_5/2A chemically bound state. The results of FIG. 3 show that the inventors have successfully fabricated a flexible CC/GWs/AuPt sensing electrode.

This example investigated bare CC, CC/Gws, CC/AuPt and CC/GWs/AuPt electrodes in 5mM [ Fe (CN) ] containing 0.1M KCl by CV method₆]^3-/4-Current response in solution. See FIG. 4A, bare CC electrode at [ Fe (CN)₆]^3-/4-A pair of wider Fe is presented in the solution^2+/3+The redox peaks are oxidized, and with the modification of GWS, AuPt and GWs/AuPt, the oxygen bloom reduction peak current of the redox peaks is obviously increased and the peak potential difference is reduced, mainly because GWs with good conductivity and a unique three-dimensional structure loads a large amount of AuPt nanoparticles, and an electron carrier can migrate along the interlaced network framework of the electron carrier, so that the electron transfer rate is promoted. In addition, EIS methods have also been used to analyze the characteristics and properties of the electrode and electrolyte interfaces. As shown in FIG. 4B, the resistance (Rct) was gradually decreased from 120 Ω of the bare CC electrode, and the variation trend of the resistance was similar to that of CVThus, the prepared CC/GWs/AuPt electrode (red curve) can carry out more effective electron transfer and has better conductivity.

The prepared flexible CC/GWs/AuPt sensing electrode was examined for electrocatalytic process of H2O2 in 10mL of 0.01M PBS (pH 7.4) system by CV method and chronoamperometry (i-t). FIG. 5A shows bare CC, CC/Gws, CC/AuPt and CC/GWs/AuPt sense electrode pairs 5mM H₂O₂The electrocatalytic reduction CV plot of (1) can be observed for bare CC and CC/GWs sense electrode pair H₂O₂No obvious current response characteristic peak appears, but a clear reduction peak appears at-0.32V of CC/AuPt and CC/GWs/AuPt electrodes, which proves that AuPt nanoparticles have H pair₂O₂Has strong catalytic action. As is clear from the plot of the chronoamperometric response i-t of FIG. 5B, at an operating potential of-0.32V, the results obtained are consistent with the CV curve as a function of H₂O₂The current signal of the continuous addition of (2) shows a step response, and the reduction current signal of the CC/GWs/AuPt electrode (curve d) is obviously higher than that of the CC/AuPt electrode (curve c). And, the CC/GWs/AuPt electrode was accompanied by H added₂O₂The CV curves were shown to have increasing current signals at the reduction peaks with increasing concentrations (0, 1, 2, and 5mM), and the potential at the reduction peaks was slightly shifted in the negative direction (FIG. 5C).

To explore H₂O₂The dynamic process of the catalyst on a flexible CC/GWs/AuPt sensing electrode is tested by a CV method under different scanning rates on 2mM H₂O₂The catalytic current signal of (a). As can be seen from the CV curve of FIG. 6A, the scan rate is increased by 20mV s^-1Increased to 300mV s^-1In the process, H is produced₂O₂The reduction current signal increases and its reduction peak position moves slightly to a negative potential. FIG. 6B shows that the reduction peak current is well linear with the square root of the scan rate (R)²0.996), indicating H₂O₂The catalytic reduction reaction that occurs at the surface of the CC/GWs/AuPt electrode is a diffusion-controlled, irreversible process.

The inventor researches a CC/GWs/AuPt sensing electrode pair H under different working potentials by adopting a chronoamperometry (i-t)₂O₂Influence of catalytic properties. Referring to FIG. 7, FIG. 7A shows the sensor at different operating potentials for successive additions of 150 μ M H₂O₂I-t current response curves (5. mu.L per 100s, 7 additions). When H is present₂O₂After the sensor electrode is added, the sensing electrode rapidly generates a reduction current response. FIG. 7B, which corresponds to the fitted scatter plot, clearly shows that the CC/GWs/AuPt sense electrode pair H increases with the operating potential from-0.28V to-0.32V₂O₂The current response of (a) gradually increases and reaches a maximum at-0.32V, and the catalytic current signal decreases as the operating potential further increases to-0.38V. It is evident that the flexible CC/GWs/AuPt sense electrode pair H is at-0.32V₂O₂The best catalytic performance is shown, so-0.32V is selected as the working potential of the experiment.

Similarly, the flexible CC/GWs/AuPt sensing electrode is measured for H under the working potential of-0.32V by using i-t₂O₂The linear relationship of the response. Referring to FIG. 8, FIG. 8A shows that increasing concentrations of H were added continuously (5. mu.L per 100 s) to 10mL of 0.01M PBS test base₂O₂Solution of H added thereto₂O₂Time, concentration of solution and corresponding H in detection system₂O₂The final concentration related parameters are shown in the following table:

in 10mL of 0.01M PBS aqueous solution, 5 mu L of H with increasing concentration is continuously dropped into the solution at intervals of 100s₂O₂Test System after solution H₂O₂Concentration, corresponding to FIGS. 8A and B

The reduction current of the CC/GWs/AuPt sensing electrode rapidly presents step-type response and reaches an equilibrium state within 5s, which shows that the response speed of the electrode is high, and H can be realized₂O₂Rapid catalysis of. In addition, the first and second substrates are,FIG. 8C is the i-t curve of the low concentration region (1.5-98 μ M) of FIG. 8A, where the detectable concentration of the electrode is as low as 1.5 μ MH₂O₂The response current of (2). FIG. 8B and FIG. 8D show the current signal and H, respectively₂O₂The linear relationship curve of concentration (corresponding to fig. 8A and 8C, respectively) has a linear range divided into two segments, 1.5-98 μ M and 98-7148 μ M, and the corresponding linear fitting equation I (μ a) is 0.56(μ M) +0.54 (R)²0.991 and 0.54(μ M) +0.69 (R) ═ I (μ a) (-)²0.993), the lowest detection was calculated to be 0.084 μ M (signal-to-noise ratio S/N-3). CC/GWs/AuPt flexible sensing electrode pair H prepared by the research₂O₂Compared with other sensors of the same type, the detection shows excellent performance no matter in a linear range or a detection limit, and specifically the following table is shown:

prepared sensors and other electrochemistry H₂O₂Performance comparison of sensors

The inventors then investigated the selectivity and reproducibility of CC/GWs/AuPt sensing electrodes. In actual sample detection, the selectivity and reproducibility of the sensor are two very important factors. The inventor records the H detection of several interference substances (glucose (Glu), cysteine (Cys), Glutathione (GSH), tryptophan (Trp), Ascorbic Acid (AA), Dopamine (DA) and Uric Acid (UA)) on a CC/GWs/AuPt sensing electrode by using an i-t curve₂O₂The interference situation of (1). See FIG. 9A, at-0.32V operating voltage, when 150 μ M H is added₂O₂The reduction current is obviously increased and shows a step-like response, but the reduction current has no obvious current response to other 1mM interferent, and the addition of 150 mu M H is continued after the interferent is added₂O₂Can still rapidly appear almost the same current response as the initial one, fully shows that the sensor has good performanceSelectivity of (2). On the other hand, we measured 5 CC/GWs/AuPt sense electrode pairs 100. mu. M H₂O₂And each electrode was tested 3 times in duplicate. The result is shown in fig. 9B, and the Relative Standard Deviation (RSD) between the electrodes was obtained to be 3.19%, indicating that the sensor had good reproducibility.

The inventor also carries out cell culture on a CC/GWs/AuPt electrode and detects H released by living cells in real time₂O₂To study the growth status and characteristics of living cells on the CC/GWs/AuPt sensing electrode. First, the CC/GWs electrode was plasma surface treated under oxygen for 20s prior to electrodeposition of AuPt to make the electrode more conducive to cell adhesion and growth. The CC/GWs/AuPt electrode was then sterilized with 75% ethanol and UV light. The pretreated CC/GWs/AuPt electrode was placed in a six-well plate, followed by 1X 10⁵cells/mL were inoculated with human lung cancer cells (A549, from ATCC cell bank) at a density and in a 37 ℃ incubator (95% air and 5% CO)₂) Culturing in RPMI-1640 medium containing 10% fetal calf serum and 1% penicillin/streptomycin double antibody solution for 24 hr, and placing CC/GWs/AuPt electrode for growing A549 cells in electrochemical detection system for releasing H₂O₂Real-time detection of. Wherein injection of 4 μ M fMLP stimulating agent into the assay stimulates cells to release H₂O₂And recording the released H of the CC/GWs/AuPt electrode pair at-0.32V working potential by a chronoamperometry (i-t)₂O₂The response current of (2). Under the same experimental conditions, 10. mu.L of 200. mu.g mL were added simultaneously^-1The catalase is added into the detection system together to eliminate H released by the cells₂O₂As an experimental control group.

In addition, in order to better observe the growth state of A549 cells on a CC/GWs/AuPt sensing electrode, the inventor uses a Calcein-AM kit to perform fluorescence staining (living cell staining and green fluorescence) on the A549 cells grown on the CC/GWs/AuPt electrode, and 5 mu L of 4mM Calcein-AM and 10 mu L of 2mM EthD-1 are added into 5mL of D-PBS so that the final concentration of Calcein-AM is 4 mu M. After culturing the cells for 24h, sucking out the culture medium in a six-well plate, washing the electrodes for 3 times by using a sterilized PBS solution, then adding 200 mu L of the prepared Calcein-AM/EthD-1 staining solution, incubating for 40min in an incubator at 37 ℃ in a dark place, taking out the cells, washing the cells for 3 times by using the sterilized PBS buffer solution, and placing the cells under a fluorescence microscope to observe the growth state of the cells on the electrodes. In addition, a549 cells grown on the CC/GWs/AuPt sensor electrode were fixed with a cell fixing solution (4% paraformaldehyde), fixed for 30min, washed 3 times with 0.01M PBS, and then dehydrated with 10%, 30%, 50%, 70%, 90%, and 100% absolute ethanol in a gradient, and the growth state was further observed with SEM.

The attachment and growth of cells on the sensing matrix is crucial for the in situ detection of target biomolecules released by living cells. The inventor explores that the cell culture is directly carried out on a flexible CC/GWs/AuPt sensing electrode, so that the sensor can capture and detect H released by cells in time₂O₂And the instant in-situ detection of signals is realized. Referring to FIG. 10A, A549 cells were grown in a culture flask adherently, and after 24h of culture, their cell morphology was observed to be spindle or polygonal. Similarly, the inventor cultures the cells on the CC/GWs/AuPt sensing electrode, after 24 hours, the growing cells on the CC/GWs/AuPt sensing electrode are subjected to fluorescent staining by using a Calcein-AM/EthD-1 double staining kit, and then the growth condition of the cells is observed by using a fluorescent microscope. Calcein-AM is a cell staining reagent that fluorescently labels living cells, producing green fluorescence, while EthD-1 intercalates into the DNA double helix of dead cells, producing red fluorescence. In FIG. 10B, a large number of A549 cells (green fluorescence) can be observed to grow on the unique three-dimensional interweaving space structure of the CC/GWs/AuPt sensing paper-based electrode (only can be focused on the same plane when observed), and in the enlarged view 10C, the cell growth state is observed to be good, and the cells (the diameter is about 10-20 μm) can freely adhere to the surface and any internal surface of the three-dimensional framework of the CC/GWs/AuPt electrode. Only a small number of cells that appeared spherical dead (red fluorescence) were observed from fig. 10D. In addition, the inventors further characterized the cell adhesion growth using SEM, after fixing the cells and dehydrating with ethanol gradient and vacuum drying for SEM observation. FIG. 10E shows that a large number of cells grew on the CC/GWs/AuPt electrodeThe long, enlarged 10F shows more clearly that the cells are in a better growing state, and that their morphology is also spindle or polygonal, and that many cell tentacles extend to attach firmly to the CC/GWs/AuPt electrode. All the results show that the flexible CC/GWs/AuPt sensing electrode prepared in the embodiment provides an adhesion interface beneficial to the growth of cells and an environment similar to that in an organism under the condition of not modifying any extracellular matrix protein or amino acid, so that the cells can grow well, and the sensing electrode has better biocompatibility and cell adhesion.

After A549 cells are cultured on a flexible CC/GWs/AuPt sensing electrode for 24 hours, H is released to the cells by adopting a chronoamperometry (i-t)₂O₂And carrying out in-situ detection. FIG. 11A is a photograph of a real object of A549 cells cultured on a CC/GWs/AuPt sensor electrode, followed by detection using a test set-up under a conventional three-electrode system (FIG. 11B). FIG. 11C shows the CC/GWs/AuPt sensor electrode real-time detection of H released from cells under drug stimulation in a 10mL 0.01M PBS detection system at-0.32V operating voltage₂O₂The chronoamperometric response curve of (a). As can be seen from the figure, the addition of fMLP stimulation to the CC/GWs/AuPt sense electrode without cell growth (FIG. 11C, black curve) and the simultaneous addition of 2. mu.M fMLP and 200. mu.g mL of fMLP to the CC/GWs/AuPt sense electrode with cell growth (FIG. 11C, black curve)^-1No significant current response signal was generated by peroxidase (catalase) (FIG. 11C, blue curve), but a significant current response appeared after 2 μ M fMLP stimulation was applied to the CC/GWs/AuPt sensing electrode for cell growth (FIG. 11C, red curve). The results show that the CC/GWs/AuPt sensing electrode can sensitively capture H released by cells₂O₂The current response signal is that the activity of active micromolecules such as ROS and the like released by the cells is higher, the cells are directly cultured and grown on the electrode, and H released by the cells can be greatly shortened₂O₂Diffusion distance to sensing electrode surface in solution, thereby for H₂O₂And the in-situ real-time detection which is faster and more sensitive is realized.

This example constructs a nanoparticle modified CC/GWs around GWs with a unique three-dimensional maze structureAuPt flexible sensing electrode for releasing H from cells₂O₂Real-time monitoring. The 3D framework of the prepared sensing electrode for direct growth of living cells is beneficial to the electron transfer rate, loads a large number of AuPt nanoparticles with specific surface binding sites and is beneficial to the H electrode pair of the sensing electrode₂O₂Detection of (3). Electrode pair H prepared in this example₂O₂The response detection has wide linear range (1.5-7148 mu M) and faster response speed (<5s), lower detection limit (0.084 mu M) and good anti-interference capability.

Due to the good biocompatibility of graphene and AuPt nanoparticles, cells can grow freely on any dimension (three-dimensional framework or wall) of the electrode, the number of adhered cells is large, and the growth state is good. Under the stimulation of drugs, the CC/GWs/AuPt/A549 of cells after direct culture and growth realizes the release of H from living cells₂O₂The reduction current signal is rapidly captured, and the in-situ real-time detection is realized. The CC/GWs/AuPt flexible sensing electrode prepared by the embodiment has the advantages of excellent performance, low price, simple preparation steps and strong repeatability, can be applied to more fields of real-time in-situ detection of biomolecules, and has the potential of being further applied to continuous dynamic monitoring of cell activities and metabolites thereof as cells can continuously grow on the 3D CC/GWs/AuPt electrode.

Therefore, the CC/GWs/AuPt sensing electrode constructed in the embodiment can be applied to the preparation of products for detecting or monitoring biomolecules, the products can be used for in-situ real-time analysis detection and dynamic monitoring of the biomolecules, and the products can be instruments or other forms of products.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the verification process of the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all equivalent structural changes made by using the contents of the specification and the drawings of the present invention should be covered by the scope of the present invention.

Claims

1. A construction method of a flexible sensing electrode modified by nanoparticles is characterized by comprising the following steps:

2. The method for constructing a nanoparticle-modified flexible sensing electrode as claimed in claim 1, wherein the H in step S2₂PtCl₆And HAuCl₄The mixed solution of (A) is 0.01M H₂SO₄And 0.01M Na₂SO₄And (4) diluting.

3. A CC/GWs/AuPt sensing electrode constructed according to the method of construction set forth in any one of claims 1-2.

4. Use of the CC/GWs/AuPt sensing electrode of claim 3 in the manufacture of a product for detecting or monitoring biomolecules.

5. The use according to claim 4, wherein the product is useful for in situ real-time analytical detection and dynamic monitoring of biomolecules.

6. According to claim4, wherein the biomolecule is H₂O₂。