Application method of cholinesterase based on nanogold star

Abstract

The invention provides a cholinesterase application method based on nano-gold stars. The cholinesterase application method comprises the following steps: synthesis and characterization of a nano-gold star material, morphology regulation and control of the nano-gold star material, and application of nano-gold star etching inhibition to cholinesterase activity detection. The embodiment of the invention provides a simple, stable and sensitive nano-gold star material morphology control method, the method is applied to cholinesterase activity detection and cholinesterase inhibitor and revivifier screening, and the method has the advantages of rapidness, low cost, ultrasensitivity, quantitative colorimetric reading and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomaterial preparation and application, and particularly relates to a cholinesterase application method based on nanostar. BACKGROUND

[0002] The nanostar is a star-shaped nanoparticle synthesized based on gold elements, has a multi-branch shape with multiple sharp ends, and the sharp ends generate an electronic aggregation phenomenon through a surface plasmon resonance effect. The star-shaped structure endows the material with two characteristics: enhanced light absorption efficiency: the sharp end structure can expand the absorption spectrum to the near-infrared region II with stronger biological tissue penetration through the localized surface plasmon resonance effect; and reduced treatment damage: compared with the kilowatt-level laser required by traditional photothermal materials, the nanostar can generate an equivalent treatment effect under the excitation of a milliwatt-level laser. SUMMARY

[0003] The application provides a cholinesterase application method based on nanostar, which comprises the following steps: synthesis and characterization of nanostar material, respectively adding deionized water, 4-hydroxyethyl piperazine ethanesulfonic acid solution, gold seed solution, hydroxylamine solution and magnetic rotor, dropping chloroauric acid solution, adjusting the rotating speed to 700 rpm, and obtaining the nanostar material under the condition of room temperature for 15 minutes, and characterizing the nanostar solution and nanostar material, including: determining the wavelength corresponding to the maximum absorption peak of the nanostar solution spectrum, determining the particle size, surface potential and morphology image of the nanostar particle; morphology regulation of the nanostar material, respectively adding the nanostar solution and sodium iodide solution, and determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction under the condition of room temperature for 10 minutes, determining the detection range and detection limit of the nanostar etching coloration for the detection of iodine ion concentration, wherein the range of the sodium iodide solution is 0.01-100 µM; respectively adding the nanostar solution and cysteine solution, adding sodium iodide solution after reaction for 5 minutes under the condition of room temperature for 10 minutes, determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction, and determining the detection range and detection limit of the nanostar etching inhibition coloration for the detection of cysteine concentration, wherein the range of the cysteine solution is 0.1-50 µM; nanostar etching inhibition is used for cholinesterase activity detection, acetylthiocholine chloride is mixed with acetylcholinesterase, and after reaction for 30 minutes at 37℃, the nanostar solution and sodium iodide solution are added, and after reaction for 10 minutes under the condition of room temperature, the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction are determined, and the detection range and detection limit of the nanostar etching coloration for the detection of cholinesterase concentration are determined.

[0004] Preferably, the step further comprises: nano-gold star etching inhibition for determining cholinesterase inhibitors, mixing cholinesterase inhibitor candidate solution with acetylcholinesterase, adding acetylthiocholine chloride, after reacting at 37℃ for 30 minutes, adding nano-gold star solution and sodium iodide solution, after standing at room temperature for 10 minutes, measuring the ultraviolet-visible spectrum and spectral maximum absorption peak of the solution after reaction, and comparing with the values of the blank control group, evaluating the inhibitory effect of the inhibitor candidate on cholinesterase activity.

[0005] Preferably, the step further comprises: nano-gold star etching inhibition for determining cholinesterase inhibitors, mixing cholinesterase inhibitor candidate solution with acetylcholinesterase, adding acetylthiocholine chloride, after reacting at 37℃ for 30 minutes, adding nano-gold star solution and sodium iodide solution, after standing at room temperature for 10 minutes, measuring the ultraviolet-visible spectrum and spectral maximum absorption peak of the solution after reaction, and comparing with the values of the blank control group, evaluating the inhibitory effect of the inhibitor candidate on cholinesterase activity.

[0006] Preferably, in the morphology regulation of the nano-gold star material, the nano-gold star etching color development is the redox reaction between iodine ions and gold atoms, which makes the nano-gold star change into nano-gold ball, and the solution color gradually changes from deep blue to pink.

[0007] Preferably, in the nano-gold star etching inhibition for cholinesterase activity detection, the nano-gold star etching inhibition is that the thiol molecule forms a stable gold-sulfur bond with the gold atom through the mercapto group, and forms a self-assembled monolayer film on the surface of the nano-gold star material, thereby blocking the redox reaction between iodine ions and gold atoms.

[0008] Preferably, in the nano-gold star etching inhibition for cholinesterase activity detection, acetylthiocholine chloride is hydrolyzed by acetylcholinesterase to generate thiocholine and acetic acid, and the generated thiocholine contains mercapto group to inhibit the surface etching of the nano-gold star material, so that the nano-gold star maintains the star shape and does not change into a spherical shape.

[0009] The embodiment of the present application converts the concentration of thiol molecules into the color of the solution of nano-gold star material, and uses optical signal for quantitative readout of colorimetric results, with a linear detection range of 0.1-6 µM and a detection limit of 0.0185 µM. The embodiment of the present application provides a simple, stable and sensitive morphology regulation method of nano-gold star material, and applies the method to cholinesterase activity detection and screening of cholinesterase inhibitors and reactivators, with the advantages of rapidness, low cost, ultra-sensitivity, quantitative colorimetric readout, etc. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1A flow chart of the application method of the nanostar-based cholinesterase application of the exemplary embodiment of the present application is shown.

[0011] Figure 2 A flow chart of the nanostar-based cholinesterase application method of the exemplary embodiment of the present application is shown.

[0012] Figure 3 A flow chart of the nanostar-based cholinesterase application method of the exemplary embodiment of the present application is shown.

[0013] Figure 4 A flow chart of the nanostar-based cholinesterase application method of the exemplary embodiment of the present application is shown.

[0014] Figure 5 A flow chart of the nanostar-based cholinesterase application method of the exemplary embodiment of the present application is shown.

[0015] Figure 6 A flow chart of the nanostar-based cholinesterase application method of the exemplary embodiment of the present application is shown.

[0016] Figure 7 A flow chart of the nanostar-based cholinesterase application method of the exemplary embodiment of the present application is shown. DETAILED DESCRIPTION

[0017] The present application will be further described in details by the following. Such description is for the purpose of illustration only and not intended to limit the present application. Other advantages and effects of the present application will be readily understood by those skilled in the art from the contents disclosed in the present specification. The present application can also be carried out or applied in other different embodiments. Various modifications and changes can be made thereto without departing from the spirit of the present application.

[0018] General Terms and Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the definitions provided in this document and those provided in the art, the definitions provided in this document control.

[0019] When a number, concentration or other value or parameter is given as a range, a preferred range or a range of preferred upper and lower values, it is intended to encompass all ranges of values therebetween whether specifically disclosed or not. Unless otherwise stated, when a range of values is recited, it is intended to include the end points of the range, as well as all integers and fractions within the range. The scope of the present application is not limited to the specific values recited when defining the range. For example, "10-30" encompasses 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, as well as any sub-range formed by any two values therefrom, e.g., 10-28, 13-26, 15-22, 18-20, etc.

[0020] The terms "about," "approximately," when used in connection with a numerical value, generally mean that the recited numerical value and all numerical values of the variable are within experimental error (e.g., within a 95% confidence interval for a mean value) or within ±10% of the stated numerical value, or within a broader range (e.g., within ±15%, within ±20%, within ±25%, or within ±30%).

[0021] The terms "comprising," "including," "having," "containing," or "involving," and any variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Those skilled in the art will understand that a recited term such as "comprising" encompasses the meaning of "consisting of." The expression "consisting of" excludes any element, step, or ingredient not specified. The expression "consisting essentially of" means that the range of values is limited to the specified elements, steps, or ingredients, plus optional elements, steps, or ingredients that do not materially affect the basic and novel characteristics of the claimed subject matter. It is to be understood that the expression "comprising" encompasses the expressions "consisting essentially of" and "consisting of."

[0022] The terms "one or more" or "at least one" as used herein, means one, two, three, four, five, six, seven, eight, nine, or more.

[0023] When describing methods, compositions, or steps, identified by letters or numbers, the order or sequence of such methods, compositions, or steps is not limited to that identified, but can be modified reasonably by those skilled in the art.

[0024] In addition, where the number of things go or components (or presence) is not specified in the components or components of the present invention, it means that there is no limit to the number of things (or presence) of components or components. Therefore, it should be interpreted as including one or at least one, and the singular form of the component or component also includes a plurality, unless the number is obviously singular.

[0025] Nanogold material is a multifunctional nanomaterial with a unique three-dimensional multi-branched star structure, thus having excellent optical properties, good biocompatibility and diversified surface functionalization, etc. It has great potential in biomedical applications. The central core-radiation branch (star angle) structure of nanogold material endows it with excellent localized surface plasmon resonance characteristics, and it exhibits strong light absorption ability in the near-infrared region. At the same time, the high-energy gold atoms on the star angle can undergo redox reaction with iodine ions, so that the morphology of the material changes from star-shaped to spherical, and the ultraviolet-visible absorption peak and solution color of the material are controlled. With the increase of etching degree, the solution color gradually changes from blue to purple, pink, and finally to red. In view of this phenomenon, the embodiment of the present application provides a method for controlling the surface etching of nanogold material. Thiol molecules can form stable gold-sulfur bonds (S-Au) with gold atoms through mercapto groups (-SH), and spontaneously form self-assembled monolayer films on the surface of nanogold material, thereby blocking the redox reaction between iodine ions and gold atoms. Therefore, the concentration of thiol molecules can be converted into the color of the solution of nanogold material, and the optical signal can be used for quantitative reading of the colorimetric result.

[0026] The embodiment of the present application applies the morphology control method of nanogold material to the detection of cholinesterase activity and the screening of cholinesterase inhibitors and reactivators. Cholinesterase is a class of enzymes that catalyze the hydrolysis of acetylcholine, and its activity change is closely related to liver function, nervous system function, poisoning state, etc. Cholinesterase detection is a commonly used basic test item in clinical practice, which can be used to evaluate liver synthetic function, diagnose and monitor organophosphorus pesticide poisoning, and provide important reference for the diagnosis and condition judgment of various diseases such as nervous system diseases and malnutrition. The acetylthiocholine chloride substrate can be hydrolyzed by cholinesterase to generate thiocholine and acetic acid. The thiol group contained in the generated thiocholine can inhibit the surface etching of nanogold material, so that nanogold material maintains star-shaped and does not change into spherical shape, thereby realizing visual quantitative colorimetric detection.

[0027] S102: Synthesis and characterization of nanogold material Combination Figures 1-3The synthesis of nanostar material is shown in the following. In a conical flask, 38.5 mL of deionized water, 18.75 mL of 4-hydroxyethylpiperazine ethanesulfonic acid solution (HEPES buffer, 100 mM, pH = 8.5), 0.75 mL of gold seed, 0.75 mL of hydroxylamine solution (40 mM), and a magnetic rotor were added, 22.5 mM of chloroauric acid solution was added at a speed of 1450 rpm, and after 15 minutes of reaction at room temperature at a speed of 700 rpm, the nanostar material was obtained. The chemical reagents used in the related art are completely different from those used in the embodiments of the present application.

[0028] Characterization of nanostar material: The morphology of the nanostar material was analyzed by high-resolution transmission electron microscopy. The ultraviolet-visible spectrum of the nanostar material solution was measured by a spectrometer to determine the wavelength corresponding to the maximum absorption peak. The particle size and surface potential of the nanostar material were measured by a nanoparticle size analyzer.

[0029] The synthesis and characterization results of the nanostar material are shown in Figure 3 , where a is a high-resolution transmission electron microscopy image of the gold seed; b is a high-resolution transmission electron microscopy image of the nanostar; c is an ultraviolet-visible spectrum of the gold seed and the nanostar at the same concentration; and d is the particle size of the gold seed and the nanostar.

[0030] S104: Morphology control of nanostar material In a 96-well plate, 100 μL of nanostar solution and 100 μL of different concentrations of sodium iodide (0.01-100 μM) solution were added, and after 10 minutes of reaction at room temperature, the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction were measured by a spectrometer to determine the detection range and limit of nanostar etching color development for iodine ion concentration.

[0031] The results of iodine ion-mediated surface etching of nanostar material are shown in Figure 4 , where a is a high-resolution transmission electron microscopy image of the nanostar etched by iodine ion; b is an ultraviolet-visible spectrum of the nanostar after reaction with different concentrations of iodine ion; and c is the reading interval of the colorimetric detection of iodine ion by the nanostar.

[0032] Nanostar etching color development is a redox reaction between iodine ion (I - ) and gold atom (Au), which causes the nanostar to change into nanogold sphere, and the solution color gradually changes from deep blue to pink, and the chemical reaction equation is as follows:

[0033]

[0034] In 96-well plates, 100 μΐ, of nanostar solution and 50 μΐ, of different concentrations of cysteine (0.1-50 μΜ) solution were added, and after 5 minutes at room temperature, 50 μΐ, of sodium iodide solution (10 μΜ) was added and reacted for 10 minutes. The ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction were determined by a spectrometer to determine the detection range and detection limit of the nanostar etching inhibition color development for cysteine concentration.

[0035] S106: Nanostar etching inhibition for detection of cholinesterase activity After mixing acetylthiocholine chloride (40 μΜ) with different concentrations of acetylcholinesterase (0-12000 U / L) and reacting for 30 minutes at 37°C, 50 μΐ, of the mixed solution after reaction was taken and added to a 96-well plate, and then 100 μΐ, of nanostar solution and 50 μΐ, of sodium iodide solution (10 μΜ) were added. After 10 minutes of reaction at room temperature, the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction were determined by a spectrometer to determine the detection range and detection limit of the nanostar etching color development for cholinesterase concentration. In some embodiments, the reaction time is 25-35 minutes, preferably about 30-35 minutes, and more preferably 30 minutes.

[0036] The results of the etching inhibition of nanostar material mediated by thiol molecules are shown in Figure 5 , where a is a high-resolution transmission electron microscopy image of nanostar protected by thiol molecules after etching by iodine ions; b is an ultraviolet-visible spectrum of nanostar etching under protection of different concentrations of thiol molecules; and c is the reading interval of colorimetric detection of nanostar for thiol molecules.

[0037] The principle of nanostar etching inhibition is that thiol molecules (cysteine) form a stable gold-sulfur bond (S-Au) on the surface of nanostar material through the mercapto group (-SH) to spontaneously form a self-assembled monolayer, thereby blocking the redox reaction between iodine ions and gold atoms. The chemical reaction equation is as follows:

[0038]

[0039]

[0040] The results of cholinesterase activity detection based on nanostar material are shown in Figure 6 , where a is an ultraviolet-visible spectrum of nanostar etching in the presence of different concentrations of cholinesterase; b is the reading interval of colorimetric detection of nanostar for cholinesterase activity; and c is the detection result of the recovery of the method in a serum sample.

[0041] In some embodiments, when detecting the concentration of the iodine standard solution, a standard curve is plotted with the maximum absorption peak value of the gold nanostar solution after the reaction as the abscissa and the acetylcholinesterase activity as the ordinate.

[0042] In some embodiments, when quantifying the acetylcholinesterase activity, a standard curve is plotted with the acetylcholinesterase activity as the abscissa and the ratio of the light absorption at 765 nm and 540 nm (A 765 540 ) of the ultraviolet-visible spectrum as the ordinate.

[0043] In some embodiments, the above steps further comprise: nanogold star etching inhibition for determining cholinesterase inhibitors. The cholinesterase inhibitor candidate solution is mixed with acetylcholinesterase (6000 U / L), acetylthiocholine chloride (40 µM) is added and reacted at 37°C for 30 minutes. 50 µL of the mixed solution after the reaction is taken and added to a 96-well plate, then 100 µL of the nanogold star solution and 50 µL of the sodium iodide solution (10 µM) are added, and after standing at room temperature for 10 minutes, the ultraviolet-visible spectrum and the maximum absorption peak value of the solution after the reaction are measured by a spectrometer, and compared with the values of the blank control group, to evaluate the inhibitory effect of the inhibitor candidate on the cholinesterase activity.

[0044] In some embodiments, the above steps further comprise: nanogold star etching inhibition for determining cholinesterase reactivators. Neostigmine (500 µg / mL) is mixed with acetylcholinesterase (6000 U / L) at a ratio of 1:5, which can inhibit 90% of the acetylcholinesterase activity. The acetylcholinesterase reactivator candidate solution is mixed with the acetylcholinesterase (6000 U / L) whose activity is inhibited by 90%, acetylthiocholine chloride (40 µM) is added and reacted at 37°C for 30 minutes, 50 µL of the mixed solution after the reaction is taken and added to a 96-well plate, then 100 µL of the nanogold star solution and 50 µL of the sodium iodide solution (10 µM) are added, and after standing at room temperature for 10 minutes, the ultraviolet-visible spectrum and the maximum absorption peak value of the solution after the reaction are measured by a spectrometer, and compared with the values of the blank control group, to evaluate the recovery effect of the reactivator candidate on the cholinesterase activity.

[0045] As Figure 7 ​The results of screening of cholinesterase inhibitors and reactivators based on nanostar materials of the embodiments of the present application are shown, wherein a is the UV-visible spectrum of nanostar etched in the presence of cholinesterase and different concentrations of neostigmine; b is the inhibition rate of cholinesterase activity in the presence of different concentrations of neostigmine; c is the result of the method in screening potential cholinesterase inhibitors; d is the UV-visible spectrum of nanostar etched in the presence of cholinesterase whose activity is inhibited and different concentrations of pralidoxime; b is the inhibition rate of cholinesterase activity in the presence of different concentrations of pralidoxime; c is the result of the method in screening potential cholinesterase reactivators.

[0046] The embodiments of the present application convert the concentration of thiol molecules into the color of the solution of nanostar materials, and use optical signals for quantitative readout of colorimetric results, with a linear detection range of 0.1-6 µM and a detection limit of 0.0185 µM. The embodiments of the present application provide a simple, stable and sensitive method for morphology control of nanostar materials, and apply the method to detection of cholinesterase activity and screening of cholinesterase inhibitors and reactivators, with advantages of rapidity, low cost, ultra-sensitivity, quantitative colorimetric readout, etc.

[0047] The embodiments of the present application have been described in detail above, but the content described is only the preferred embodiments of the present application, and cannot be considered as limiting the scope of the embodiments of the present application. The true scope and spirit of the present application are shown by the appended claims, and the specification and embodiments are only exemplary.

Claims

1. A method for the application of cholinesterase based on nanostar, characterized by, The method comprises the following steps: Synthesis and characterization of nanostar material, respectively adding deionized water, 4-hydroxyethyl piperazine ethanesulfonic acid solution, gold seed solution, hydroxylamine solution and magnetic rotor, dropping chloroauric acid solution, adjusting the speed to 700 rpm, and reacting for 15 minutes at room temperature to obtain nanostar material, and the nanostar solution and nanostar material are characterized, including: determining the wavelength corresponding to the maximum absorption peak of the nanostar solution spectrum, determining the particle size, surface potential and morphology image of the nanostar particles; Morphology regulation of nanostar material, respectively adding nanostar solution and sodium iodide solution, standing for 10 minutes at room temperature, and then determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction, to determine the detection range and limit of nanostar etching coloration for iodine ion concentration, wherein the sodium iodide solution ranges from 0.01 to 100 µM; respectively adding nanostar solution and cysteine solution, standing for 5 minutes at room temperature, then adding sodium iodide solution and reacting for 10 minutes, and then determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction, to determine the detection range and limit of nanostar etching inhibition coloration for cysteine concentration, wherein the cysteine solution ranges from 0.1 to 50 µM; Nanostar etching inhibition is used for detection of cholinesterase activity, acetylthiocholine chloride is mixed with acetylcholinesterase, nanostar solution and sodium iodide solution are added after reaction for 30 minutes at 37℃, and then standing for 10 minutes at room temperature, and then determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction, to determine the detection range and limit of nanostar etching coloration for cholinesterase concentration.

2. The nanogold star-based cholinesterase application method according to claim 1, wherein, The steps further comprise: Nanostar etching inhibition is used for determining cholinesterase inhibitors, cholinesterase inhibitor candidate solution is mixed with acetylcholinesterase, and acetylthiocholine chloride is added, and then standing for 30 minutes at 37℃, and then adding nanostar solution and sodium iodide solution, and then standing for 10 minutes at room temperature, and then determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction, and comparing with the values of the blank control group, to evaluate the inhibition effect of the inhibitor candidate on cholinesterase activity.

3. The nanogold star-based cholinesterase application method according to claim 2, wherein, The steps further comprise: Nanostar etching inhibition is used for determining cholinesterase reactivators, acetylcholinesterase reactivator candidate solution is mixed with acetylcholinesterase whose activity is inhibited by 90%, and acetylthiocholine chloride is added, and then standing for 30 minutes at 37℃, and then adding nanostar solution and sodium iodide solution, and then standing for 10 minutes at room temperature, and then determining the ultraviolet-visible spectrum and the maximum absorption peak of the solution after reaction, and comparing with the values of the blank control group, to evaluate the recovery effect of the reactivator candidate on cholinesterase activity.

4. The method for preparing the nano-Venus material as described in claim 3, characterized in that, In the morphology regulation of the nanostar material, nanostar etching coloration is a redox reaction between iodine ions and gold atoms, which makes the nanostar change into nanogold ball, and the solution color gradually changes from dark blue to pink.

5. The nanogold star-based cholinesterase application method according to claim 4, wherein, The nano gold star etching inhibition is used for cholinesterase activity detection, and the nano gold star etching inhibition is a thiol molecule which forms a stable gold-sulfur bond through a mercapto group and a gold atom, forms a self-assembled monolayer on the surface of the nano gold star material, and thus blocks the oxidation-reduction reaction between iodine ions and gold atoms.

6. The nanogold star-based cholinesterase application method according to claim 5, wherein, The nano gold star etching inhibition is used for cholinesterase activity detection, and the nano gold star etching inhibition is a thiol molecule which forms a stable gold-sulfur bond through a mercapto group and a gold atom, forms a self-assembled monolayer on the surface of the nano gold star material, and thus blocks the oxidation-reduction reaction between iodine ions and gold atoms.

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