CN114660148A - Monoatomic cobalt nanoenzyme material, flexible electrochemical chip sensor and application thereof - Google Patents
Monoatomic cobalt nanoenzyme material, flexible electrochemical chip sensor and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the technical field of biosensing, and relates to a monoatomic cobalt nanoenzyme material, a flexible electrochemical chip sensor and application thereof, wherein the monoatomic cobalt nanoenzyme material is synthesized by taking a cobalt-containing compound as a cobalt precursor and a carbon nanomaterial as a substrate through chemical bonding; the chemical bonding includes pi-pi interaction, amide bond cross-linking, or covalent bond bonding. The monoatomic cobalt nanoenzyme material prepared by the invention can be used for constructing a flexible electrochemical chip sensor and detecting NO released by living cells and mouse living bodies and organs in situ in real time; in addition, the sensor has wide linear range, low detection limit, quick sensitive response, good reproducibility, stability and selectivity, and can realize high-sensitive detection of NO in vivo and in vitro.
Description
Technical Field
The invention belongs to the technical field of biosensing, and relates to a monoatomic cobalt nanoenzyme material, a flexible electrochemical chip sensor and application thereof, which can be specifically used for in-situ detection of nitric oxide released by cells and mice.
Background
Nitric Oxide (NO) is a key biomolecule with redox activity in nature, which was awarded the nobel prize in 1998. It plays a key role in almost all life forms, from bacteria to humans. NO is produced by the oxidation of L-arginine to L-citrulline by a series of enzymes called Nitric Oxide Synthases (NOS). NO performs different functions depending on the site and concentration of production and physiological environment differences. For example, in the brain, NO modulates synaptic transmission and neuronal activity at normal concentrations (nanomolar). However, at higher concentrations (up to micromolar), NO can cause neuronal damage caused by oxidative stress, acting as a double-edged sword. Therefore, accurate detection of NO is of great significance to the concentration threshold for cytotoxicity. However, accurate monitoring of nitric oxide in complex biological systems remains a major challenge due to its short half-life (6-10s), low concentration (nM- μ M), and susceptibility to oxidation by oxygen and metalloproteins in biological systems. A variety of techniques for capturing NO have been proposed, including fluorescence sensors, electron spin resonance spectroscopy, magnetic resonance imaging, etc., but these techniques have limited ability to achieve non-invasive, high spatial and temporal resolution detection of deep tissues and in vivo. Compared with the prior art, the electrochemical sensor has the characteristics of high sensitivity, quick response, wide linear range and low detection limit, and can ensure the real-time sensing of NO. At present, the flexible printed electrochemical sensor has good monitoring performance and biocompatibility, and is a promising tool for monitoring cell or histochemical signals in real time.
Inkjet printing is considered to be an optimal method for selectively printing various materials on a large scale due to the characteristics of high material utilization rate, low cost, good compatibility with flexible substrates, multiple solution parameters and the like. Screen printing can realize large-scale, simple-process electrode printing, and has been widely used in various fields. The monatomic nanoenzymes (SAEs) have been the leading edge of research because they have abundant metal atom active centers, adjustable electronic structures, and can further regulate and control the adsorption energy of reactants and reaction intermediates. Among transition metal catalysts, cobalt-based catalysts have attracted much attention because of their excellent biocompatibility and high-efficiency biomimetic catalytic activity. Therefore, the Co-based monatin nanoenzyme has great potential in manufacturing flexible printed electrochemical sensors, and can be used for in vivo and in vitro biological research and clinical diagnosis.
Disclosure of Invention
The invention aims to provide a monoatomic cobalt nanoenzyme material and a flexible electrochemical chip sensor, which can be used for in-situ detection of NO at the level of cells and animal organs and can also be used for in-vivo detection of mice.
According to the technical scheme of the invention, the monatomic cobalt nanoenzyme material is synthesized by taking a cobalt-containing compound as a cobalt precursor and a carbon nanomaterial as a substrate through chemical bonding; the chemical bonding includes pi-pi interaction, amide bond cross-linking, or covalent bond bonding.
Further, the cobalt-containing compound is cobalt phthalocyanine, cobalt acetate or cobalt chloride.
Further, the carbon nano material is carbon nano tube, graphene, graphite alkyne or carbon tri-nitrogen tetra.
Further, the mass ratio of the cobalt-containing compound to the carbon nanomaterial is 1: 5-20, preferably 1: 10.
further, the synthesis using pi-pi interactions operates as follows: dispersing a carbon nano material in organic, adding a cobalt-containing compound, and reacting to obtain the monoatomic cobalt nanoenzyme material;
the synthesis by amide bond crosslinking is carried out as follows: mixing carbon nano material with SOCl2Reacting to obtain a carboxyl acyl chlorinated carbon material, adding a cobalt-containing compound, and reacting to obtain the monoatomic cobalt nanoenzyme material;
the covalent bond synthesis was performed as follows: dispersing a cobalt-containing compound in an organic solvent, fully reacting under the action of acidic sodium nitrite, adding a carbon nano material, adjusting the pH value to 9-11, and reacting to obtain the monatomic cobalt nano enzyme material.
Further, the organic solvent is selected from common organic solvents such as ethanol, DMF (N, N-dimethylformamide), THF (tetrahydrofuran) and the like.
Further, carbon nano-material and SOCl2Reaction(s) ofThe temperature of (A) is 65-80 ℃.
Specifically, the operation of synthesis using pi-pi interaction may be as follows: taking 300-500mg of carbon nano-substrate material, 30mL of DMF, carrying out ultrasonic treatment for 2h, fully and uniformly mixing, adding 30-50mg of cobalt compounds such as cobalt phthalocyanine and the like serving as raw materials into the carbon material solution, carrying out ultrasonic treatment for 30-60min, and stirring at room temperature for 23-25 h; centrifuging, washing with DMF (dimethyl formamide), washing with ethanol, and freeze-drying to obtain a monatomic cobalt nanoenzyme material (CNT @ Co-PP);
the procedure for cross-linking synthesis using amide bonds can be as follows: 1g of carbon nanomatrix material was added with 50mL of SOCl2And 8mL DMF, heated to 70 ℃ for 24h, centrifuged, washed with THF, and dried under vacuum to give the carboxylic acid chlorinated carbon material. Adding 300-500mg of the material and 30-50mg of cobalt compounds such as cobalt phthalocyanine and the like into 30mL of DMF (dimethyl formamide), performing ultrasonic treatment for 30min, fully and uniformly mixing, refluxing at 70 ℃ for 96h, centrifuging, washing with ethanol, and performing freeze drying to obtain a monoatomic cobalt nanoenzyme material (CNT @ Co-CONH);
the operation of the covalent bond synthesis can be as follows: dispersing 30-50mg cobalt compounds such as cobalt phthalocyanine in 30mL ethanol, adding 6mL DMMF and 12mL HCl, stirring in ice bath for 2h, adding sodium nitrite solution (120mg sodium nitrite, 3mL water), stirring for 2h, and reacting completely. Then, adding 10mL of 30-50 mg/mL carbon nano substrate material into the solution, dropwise adding 25 wt% of sodium hydroxide until the pH value is equal to 10, stirring for 2h, and removing the ice bath; centrifuging, washing with water and ethanol, and freeze-drying to obtain the monatomic cobalt nanoenzyme material (CNT @ Co-COV).
The second aspect of the invention provides a biochip electrode comprising the monatomic cobalt nanoenzyme material.
Specifically, a monatomic cobalt nanoenzyme material can be added into a conductive paste (such as carbon paste), and a high-resolution direct current body or screen printing is adopted to obtain the biochip electrode; or
The preparation method comprises the steps of taking conductive slurry (such as carbon slurry) as a raw material, adopting high-resolution direct current body or screen printing to print to obtain an electrode, and modifying the monatomic cobalt nanoenzyme material on the electrode to obtain the biochip electrode.
Further, the viscosity of the conductive paste is 14000-.
Further, the concentration of the monatomic cobalt nanoenzyme material is 5-20 mg/mL; preferably at a concentration of 10mg/mL, and in a volume of 10. mu.L.
The third aspect of the invention provides a flexible electrochemical chip sensor, which comprises a flexible substrate and a three-electrode or two-electrode system loaded on the flexible substrate, wherein the working electrode of the three-electrode or two-electrode system is the biochip electrode.
Further, the flexible substrate is selected from a polyester film, a polyvinyl alcohol film, a polyimide, polyethylene naphthalate, a film, or a flexible fabric.
The third aspect of the invention provides the use of the monatomic cobalt nanoenzyme material or the flexible electrochemical chip sensor in NO detection.
Further, the flexible electrochemical chip sensor is placed in detection liquid containing NO or NO release source to detect the electrochemical response of NO.
Further, the NO release source is cells or mouse living bodies and organs.
Furthermore, the electrochemical response of NO is detected by electrochemical technologies such as cyclic voltammetry, differential pulse voltammetry, chronoamperometry and the like, so that qualitative and quantitative detection of NO is achieved.
Further, the pH value of the detection solution is 6-9, and preferably 7.4.
Specifically, the method for detecting NO by using the flexible electrochemical chip sensor (sensing chip) comprises the following steps: dripping a solution to be detected into PBS base solution in which the sensing chip is placed, detecting an electrochemical signal of the solution to be detected, and further evaluating the selective response performance of the sensor to NO, wherein NO is dripped into the PBS solution as a detection object in the detection process, and the concentration of NO can be 1.8 mM;
a method for detecting NO release from cells using the sensor chip, comprising the steps of: culturing cells to a certain density (single cell or multiple cells), taking out cell culture solution, adding PBS (phosphate buffer solution) with pH of 7.4, stimulating the cells to release NO by adopting a medicament (such as acetylcholine), and detecting a corresponding electrochemical signal of NO by using the sensing chip;
a method for detecting NO release of a mouse living body and organs by using the sensor chip comprises the following steps: dripping PBS (phosphate buffer solution) and a stimulating medicament (such as acetylcholine) at the wound of the mouse, and detecting an electrochemical signal corresponding to NO by using the sensing chip; or
The organs of the mouse including heart, liver, spleen, lung, kidney, brain and endothelial tissue are put into PBS, the organs are stimulated by the medicine, and the electrochemical signal corresponding to NO is detected by the sensor chip.
Compared with the prior art, the technical scheme of the invention has the following advantages: the monoatomic cobalt nanoenzyme material prepared by the invention can be used for constructing a flexible electrochemical chip sensor and detecting NO released by living cells and mouse living bodies and organs in situ in real time; in addition, the sensor has wide linear range, low detection limit, quick sensitive response, good reproducibility, stability and selectivity, and can realize high-sensitive detection of NO in vivo and in vitro.
Drawings
FIG. 1 is a schematic diagram of flexible electrochemical chip sensor printing and mouse and living cell sensing.
FIG. 2 is a (a) scanning electron microscope image and (b) high resolution transmission electron microscope image of a CNT; (c) CNT @ Co-PP, (d) CNT @ Co-CONH, (e) CNT @ Co-COV, a high resolution transmission electron microscopy image, (f) a spherical aberration electron microscopy image of Co @ CNT-PP, and (g) an element distribution map of Co @ CNT-PP.
FIG. 3 is an X photoelectron spectroscopy elemental analysis diagram of a monatomic cobalt nanoenzyme material: (a) nitrogen element, (b) cobalt element.
FIG. 4 shows the optimization of the performance and conditions of the monatomic cobalt nanoenzyme material for the electrochemical oxidation of NO: (a) cyclic voltammograms recorded with Co @ CNT-PP nanoenzyme in 0.01M PBS (pH 7.4) solution with different concentrations of NO; (b) DPV response of different catalysts to 580 μ MNO in 0.01MPBS (pH 7.4) solution, scan rate 0.05V/s; and (3) testing the regulating current value of the pH value (c) or the potential (d) of the base solution on the electrochemical response of the monatomic cobalt nanoenzyme material to NO.
FIG. 5 is a plot of (a) CNT @ Co-PP, CNT @ Co-CONH, CNT @ Co-COV versus NO for a test potential of 0.75V; (b) response time of CNT @ Co-PP to NO; (c) a linear regression curve corresponding to the NO chronoamperometric curve; (c) and (4) selective testing of the sensor.
FIG. 6 is a time-series current response of the CNT @ Co-PP biosensor chip at different cell densities with 0.5mM acetylcholine (ACh) (a) or 1.0mM ACh (b) stimulating NO release from human hepatoma cells (HepG-2); (c) timed current response to NO release from human normal hepatocytes (L02) stimulated by different drug concentrations; (d) HepG-2 and L02 cells released concentrations of NO under different conditions.
FIG. 7 is a graph showing the in vitro real-time detection of NO produced by various organs such as brain, spleen, liver, heart, kidney, endothelial tissue, lung, etc. (a) to (g); the inset is a picture of different organ detection processes; (h) concentration of NO released by the corresponding organ. (i) In situ real-time detection of NO release from live mice.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
EXAMPLE 1 preparation of monatomic cobalt nanoenzyme materials
Synthesizing the monoatomic cobalt nanoenzyme material by adopting three connection models of p-p interaction, acylation reaction and covalent crosslinking
In the synthesis of a pi-pi interaction monatomic cobalt nanoenzyme material (Co @ CNT-PP), 30mg of cobalt phthalocyanine was mixed with 300mg of carbon material such as Carbon Nanotubes (CNTs) in 30mL of DMF and stirred continuously for 24h at room temperature with sonication. Finally, the Co @ CNT-PP can be obtained by washing and freeze drying.
In the synthesis of covalently cross-linked monatomic cobalt nanoenzyme material (Co @ CNT-COV), 30mg of cobalt phthalocyanine was dispersed in a solution consisting of 30mL of ethanol, 12mL of HCl, and 6mL of DMF. After stirring in an ice bath for 2h, 3mL of NaNO was added to the mixture2(40mg/mL) and stirred for an additional 2 h. Next, 10mL of a suspension of carbon material such as CNTs (30mg/mL) was added with constant stirring, and stirring was continued for 0.5 h. Subsequently, the pH of the mixture was adjusted to 10 with 25% by weight of sodium hydroxide and stirred for 2 h. Final product water and alcoholWashed three times and then freeze dried to obtain the final product CNT @ CNT-COV.
To synthesize an acylation reaction monatomic cobalt nanoenzyme material (Co @ CNT-CONH), 1g of a carbon material such as CNTs was added to a solution containing 50mL of SOCl2And 8mL DMF, stirred at 70 ℃ for 2h, then washed with Tetrahydrofuran (THF), centrifuged and dried under vacuum. Then, 300mg of the treated CNTs and 30mg of cobalt phthalocyanine were added to 30mL of DMF, and stirred at 70 ℃ for 96 hours. And finally, washing with ethanol for multiple times, centrifuging, collecting, and freeze-drying to obtain a final product Co @ CNT-CONH.
Example 2 preparation of Flexible electrochemical chip Sensors (monoatomic cobalt Sensors) and electrochemical testing
A high-resolution direct-current printer is adopted, a flexible film such as a film is used as a chip substrate, and the viscosity of the conductive carbon paste and the prepared monoatomic cobalt nanoenzyme material is controlled to be about 16000 cp. The two-electrode or three-electrode system is designed by CAD software and is input into a control computer of a high-resolution direct-current printer, so that printing can be performed. And selecting parameters such as a square wave mode and a duty ratio of 20%, and the like to obtain the flexible electrochemical chip sensor.
Or preparing a flexible electrochemical chip sensor by using a screen printer, taking a flexible film such as a film as a chip substrate, taking the conductive carbon paste and the prepared monoatomic cobalt nanoenzyme material as ink, and controlling the viscosity to be about 16000 cp. And designing a proper two-electrode or three-electrode system screen printing plate, and printing. The obtained flexible electrochemical chip sensor is further used for in-situ real-time detection of NO released by cells and mice, as shown in figure 1.
Test example
1. Structural morphology characterization of monatomic cobalt nanoenzyme material
As shown in fig. 2, the morphology of the carbon nanotubes can be observed by scanning and transmission electron microscopy (fig. 2a and b). Indicating that the CNTs have a hollow tubular structure. In addition, the tube wall has a distinct lattice structure. After the monoatomic cobalt nanoenzyme material is modified, a layer of cobalt metal atoms is obviously deposited on the tube wall of the carbon nanotube. FIGS. 2c to e are Co @ CNT-PP, Co @ CNT-COV and Co @ CNT-CONH in this order, with the thickness of the cobalt single atoms being 4.17nm, 0.625nm and 1.62nm in this order. FIG. 2f is a spherical aberration electron microscope of Co @ CNT-PP, from which many bright spots of single cobalt atoms on the carbon nanotubes can be observed, demonstrating that cobalt is indeed present as a single atom. FIG. 2g is the elemental distribution diagram of Co @ CNT-PP, from which it can be observed that C, Co, N elements are uniformly distributed in the material.
FIG. 3 is a high resolution X-ray photoelectron spectrum of the material. FIG. 3a shows the high resolution XPS spectra of N1s in the single atom Co @ CNT-PP, Co @ CNT-COV and Co @ CNT-CONH materials, which exist primarily as pyridine N (398.78 eV), pyrrole N (399.65 eV) and quaternary ammonium N (400.76 eV). Furthermore, Co 2p3/2(FIG. 3b) the binding energy in the monatomic material is 780.6eV, which is closer to Co (II) in cobalt phthalocyanine (CoPc), Co 2P1/2The bond energy of (2) is mainly 796.18eV, and the valence state of Co in the monatomic material can be deduced to be +2 valence.
2. Electrochemical Performance of the sensor
The activity of the cobalt monoatomic oxidase-like enzyme is further verified by researching the electrochemical catalytic oxidation of the printed flexible electrochemical chip sensor on NO. The Cyclic Voltammogram (CV) in fig. 4a shows that Co @ CNT-PP has no significant response in 0.01M PBS (pH 7.4). A distinct oxidation peak was seen after 160. mu.M NO in PBS, with an oxidation peak potential of 0.73V, due to the oxidation of NO by Co @ CNT-PP. As the NO concentration was further increased to 580. mu.M, the oxidation current increased significantly, indicating that Co @ CNT-PP has good oxidase-like activity. During the reaction process, NO as a high-activity free radical molecule is firstly adsorbed on Co atom and then loses an electron to generate NO+(Nitro ion), which is a strong Lewis acid, readily reacts with OH-to form NO2 -. Therefore, the cobalt monoatomic material can be used as a catalyst for detecting NO.
To further investigate the catalytic performance of cobalt monatomic materials, different catalysts were used such as: carbon Nanotubes (CNT), cobalt phthalocyanine (Co (Phen)3) Physical mixture Co (Phen)3And @ CNT, Co @ CNT-COV, Co @ CNT-CONH, Co @ CNT-PP and the like are used for sensing and detecting 580 mu MNO. Fig. 4b gives the measurement curve of Differential Pulse Voltammetry (DPV).The results show that Co (Phen)3And physical mixture Co (Phen)3@ CNT is weakly reactive to NO and has NO distinct oxidation peak. CNT, Co @ CNT-COV, Co @ CNT-CONH and Co @ CNT-PP all have catalytic activity for NO, wherein the intrinsic enzyme activity of the Co @ CNT-PP is the best, and the CNT-PP shows the minimum anode peak potential and the maximum current response. Furthermore, the experimental sequence for the NO-demonstrated oxidase activity of several catalysts was Co @ CNT-PP>Co@CNT-CONH>Co@CNT-COV>CNT>Co(Phen)3@CNT>Co(Phen)3The inherent advantages of the monatomic nanoenzyme are shown.
3. Regulation and control of pH value and test potential value of buffer solution to Co @ CNT-PP (carbon nanotube-polypropylene) determined NO signal
As shown in FIG. 4c, the effect of pH on the electrochemical performance of Co @ CNT-PP for oxidizing NO was investigated using DPV technology. The results show that the proposed Co @ CNT-PP sensor responds best at pH 7.4. In addition, the pH value of 7.4 is close to the pH value of the biological environment, and the method is suitable for monitoring NO in vivo and in vitro. The influence of the applied potential on the catalytic NO oxidation of the Co @ CNT-PP sensor was investigated by using a chronoamperometric I-t response method, as shown in FIG. 4 d. As the applied potential was increased from 0.50V to 0.70V, the current response of Co @ CNT-PP to NO also increased, and after further increasing the potential, the current response tended to level off. Therefore, 0.70V (Ag/AgCl as reference electrode) potential was chosen as the optimum for subsequent I-t measurements.
4. Detection limit and sensitivity of sensor
Co @ CNT-COV, Co @ CNT-CONH and Co @ CNT-PP are adopted to respectively prepare corresponding sensors, a current i-t curve is recorded, and the electrocatalytic behavior of Co-SAEs nanoenzyme on NO oxidation is further researched, as shown in figure 5 a. After continuous addition of NO to 0.01MpH 7.4.4 PBS, a fast step current was obtained from FIG. 4a due to the electrocatalytic effect of Co-SAEs nanoenzyme on NO oxidation. Notably, Co @ CNT-PP quickly reached 95% of the steady state current in 1.7 seconds (FIG. 5 b). The half-life of NO is 3-6 seconds, so that the quick response performance of the sensor provides guarantee for in-situ in-vivo and in-vitro detection of NO. FIG. 5c shows calibration curves of different Co-SAEs nanoenzymes for NO sensing, respectively. Co @ CNT-PP achieves a wide linear range from 36nM to 405500nM with a linear equation of I (. mu.A). + -. 2.75+0.63CNO(nM) (R ═ 0.9993, n ═ 38), and the sensitivity of the calibration curve was 8917.19 μ a μ M-1cm-2. The low detection limit calculated from the expression LOD 3S/K, where S is the standard deviation of the blank signal (nB 20) and K is the sensitivity of the calibration curve slope calculation, is 12 nM. The linear range of the Co @ CNT-CONH sensor is 396-21028 nm and 24537-313404 nm, and the equation is that I (mu A) is 4.78+1.23 multiplied by 10-4CNO(nM) (R ═ 0.9976, n ═ 14) and I (μ a) ═ 0.89+3.04 × 10-4CNO(nM) (R ═ 0.9997, n ═ 20). The sensitivity was 1.74 and 4.31. mu.A. mu.M, respectively-1cm-2The detection limit was 132 nm. The Co @ CNT-COV sensor shows linear relation in the ranges of 756-7207 nm, 7207-21028 nm and 21028-313404 nm, and the equation is that I (mu A) is 0.80+3.43 multiplied by 10-5CNO(nM)(R＝0.9964,n＝8),I(μA)＝0.27+1.03×10-4CNO(nM)(R＝0.9959,n＝6)and I(μA)＝-1.84+1.80×10-4CNO(nM) (R ═ 0.9997, n ═ 21) sensitivities were 0.48, 1.46 and 2.55 μ Α μ M respectively-1cm-2The detection limit was 252 nm.
5. Selectivity and stability studies of sensors
The interference of a flexible electrochemical chip sensor on the electrochemical oxidation of common interfering species coexisting with NO in a biological system is researched by adopting a timing current i-t curve. As shown in FIG. 5d, the monoatomic cobalt sensor showed a significant current response to 4.3. mu.M NO at a potential of 0.7V, however, when a series of physiological interferents such as glucose (4mM), AA (0.125mM), DA (1. mu.M) and 100. mu.M K were added+,Na+,Ca+,Cl-SO4 2-The sensor did not exhibit a significant response. Experimental results show that the constructed flexible electrochemical chip sensor has good selectivity and anti-interference capability on NO.
In addition, the monoatomic cobalt nanoenzyme material can reach over 90.6 percent of the original activity after being stored for 30 days, and has a long service life. When 54 μ M NO was measured using 5 sets of monoatomic cobalt sensors prepared under the same experimental conditions, the calculated RSD was 5.38%, indicating good reproducibility. The repeatability of the monatomic cobalt sensor was studied by six tests on the same sensor. The results were satisfactory, with calculated RSD as low as 3.63%. Therefore, the prepared sensor has good stability.
6. Research application in living cell detection
By accurately controlling the cell density and the concentration of the stimulation drug, the Co @ CNT-PP sensor can detect NO molecules released by living cells in real time. HepG-2 and L02 cells were selected as model cell lines. As shown in FIGS. 6a-c, the prepared monoatomic cobalt sensor has excellent NO sensing performance released by cells, and shows higher catalytic activity and excellent selectivity. In a typical chronoamperometric i-t curve, the sensor generates a significant oxidation current as 0.5 or 1.0mM ACh (a drug commonly used to stimulate the release of NO from cells) is injected into the cell culture medium, which is caused by the NO released by the cells. However, addition of a mixture of ACh and Hb (a drug that can consume NO) to the cultured cells did not observe a significant galvanic reaction, indicating that NO released by the cells was effectively consumed by Hb. The NO concentration can be calculated from the calibration curve equation of fig. 5 c. Figure 6d summarizes the effect of cell density and drug concentration on the release of NO molecules by living cells. Density of 5X 104And 1X 105The concentration of NO released by HepG-2 cells per ml stimulated by 0.5mM or 1.0mM MACh was approximately 1.1 and 2.1. mu.M (FIG. 6a) or 1.9 and 4.1. mu.M (FIG. 6b), respectively. Indicating that the release of NO shows cell density and drug dependent behavior. In addition, normal L02 cells were studied as a control experiment. As shown in FIG. 6c, when the ACh concentration was changed from 0.5mM to 1.0mM, it was 1X 105The concentration of NO released by L02 cells per ml was 1.2. mu.M and 2.5. mu.M, respectively. The results showed that HepG-2 cancer cells (1X 10) were present at the same concentration of ACh (1.0mM)5One/ml) released about 1.6 times higher concentration of NO than normal L02 cells. These results indicate that the monoatomic cobalt sensor can quantitatively detect NO molecules in living cells in real time under a complex biological environment.
7. Research application in mouse organ and living body detection
Real-time sensing of NO release from living organs is crucial to exploring the role of NO in neural signal transduction and inflammatory processes. The monatomic sensor for high-resolution direct-current body printing has good electrochemical response and biocompatibility, and can be applied to a nude mouse model to detect NO released by a living organ in real time. As shown in fig. 7a to g, 0.2MACh was injected into PBS containing different organs such as brain, spleen, liver, heart, kidney, lung and endothelial tissue, and the monatomic cobalt sensor produced an acute galvanic response. Fig. 7a to g are insets illustrating the sensing process for monitoring NO using the print chip. The results show that under ACh stimulation, the above organs all produce NO and the corresponding concentrations are summarized in FIG. 7h, with 1.29 μ M for heart, 0.98 μ M for brain, 2.23 μ M for lung, 0.20 μ M for liver, 0.55 μ M for spleen, 0.74 μ M for kidney, and 2.03 μ M for endothelial tissue, respectively, with the highest concentration of NO produced by lung. The printed monoatomic cobalt sensor has excellent NO sensing capability and can play an important role in researching the function of NO in the processes of triggering organ physiology and pathology.
To evaluate the utility of printed monoatomic cobalt sensors for in vivo monitoring of NO, one nude mouse with a wound on the skin was used as a study model. A monoatomic cobalt sensor was placed on the wound of a nude mouse, and ACh was then added on the wound to stimulate NO production. As shown in fig. 7i, an increase in current was observed upon the addition of ACh, indicating that the monoatomic cobalt sensor successfully captured and catalyzed NO. From the current responses in FIG. 7i, the concentration of NO released from the wound surface of the nude mouse in vivo was calculated to be 19.99. mu.M. Experimental results prove that the constructed monoatomic cobalt sensor can realize in-situ real-time detection of NO released by a wound of a living nude mouse, can help research the relation between NO concentration and wound inflammatory reaction, and can provide some related information for further wound healing treatment.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Various other modifications and alterations will occur to those skilled in the art upon reading the foregoing description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
Claims (10)
1. The monatomic cobalt nanoenzyme material is characterized in that the monatomic cobalt nanoenzyme material is synthesized by taking a cobalt-containing compound as a cobalt precursor and a carbon nanomaterial as a substrate through chemical bonding; the chemical bonding includes pi-pi interaction, amide bond cross-linking, or covalent bond bonding.
2. The monatomic cobalt nanoenzyme material of claim 1, wherein the cobalt-containing compound is cobalt phthalocyanine, cobalt acetate, or cobalt chloride.
3. The monatomic cobalt nanoenzyme material of claim 1, wherein the carbon nanomaterial is a carbon nanotube, graphene, a graphdyne, or a carbon triazo.
4. The monatomic cobalt nanoenzyme material of any of claims 1-3, wherein the mass ratio of the cobalt-containing compound to the carbon nanomaterial is 1: 5-20.
5. A biochip electrode comprising the monatomic cobalt nanoenzyme material of any one of claims 1 to 4.
6. A flexible electrochemical chip sensor, comprising a flexible substrate and a three-electrode or two-electrode system loaded on the flexible substrate, wherein the working electrode of the three-electrode or two-electrode system is the biochip electrode of claim 5.
7. The flexible electrochemical chip sensor according to claim 6, wherein said flexible substrate is selected from the group consisting of polyester film, polyvinyl alcohol film, polyimide, polyethylene naphthalate, film, or flexible fabric.
8. Use of a monatomic cobalt nanoenzyme material of any of claims 1-4, or a flexible electrochemical chip sensor of claim 6 or 7, for the detection of NO.
9. The use according to claim 8, wherein the flexible electrochemical chip sensor according to claim 6 or 7 is placed in a detection solution containing NO or a source of NO release to detect the electrochemical response of NO.
10. The use of claim 9, wherein the pH of the detection solution is 6-9.
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