CN113324933A - Method for detecting angiotensin converting enzyme and application thereof - Google Patents
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Abstract
The invention discloses a method for detecting angiotensin converting enzyme and application thereof, wherein ACE is diluted in deionized water to a final concentration of 0.5mU/L to 60mU/L, each 96-pore plate contains 25 mU L of ACE and 40mU L of 5mU M Cys-ang (I) -Cys peptide, incubation is carried out at 37 ℃, then 50 mU L AuNPs is added into the 96-pore plate, an ACE activity signal is amplified by detecting the residual amount of the Cys-ang (I) -Cys peptide, and finally the ultraviolet visible absorption spectrum of the AuNPs solution in the range of 400-800nm is recorded within 5 min. The invention designs a Cys-Ang (I) -Cys peptide based on angiotensin I, wherein each end of the Cys-Ang (I) -Cys peptide is connected with a cysteine (Cys), the Cys-Ang (I) -Cys peptide can be gathered on the surfaces of AuNPs at two ends through Au-S bonds and stimulates the gathering of the AuNPs to be purple or blue, the Cys-Ang (I) -Cys peptide is hydrolyzed after ACE is introduced, and sulfydryl is dissociated from one end of the peptide to cause the AuNPs to be dispersed and red, and the ACE detection is carried out in a rapid and direct mode by realizing the hydrolysis of ACE catalytic peptide and the assembly/decomposition of the AuNPs mediated by the peptide, and the method is operated to screen ACE inhibitors or inhibitory peptides.
Description
Technical Field
The invention relates to the technical field of angiotensin converting enzyme activity detection, in particular to a method for detecting angiotensin converting enzyme and application thereof.
Background
Angiotensin Converting Enzyme (ACE) plays a dominant role in the renin-angiotensin system in regulating blood pressure, the product of which directly affects the pathogenesis of cardiovascular disease (CVD). ACE is essentially a class of zinc-dependent peptidases that hydrolyze angiotensin (Ang) I to angiotensin II, which is further cleaved by ACE2 to form shorter Ang- (1-7) peptides.
In addition to hydrolyzing angiotensin I, ACE hydrolyzes many other polypeptides, such as substance P, enkephalin and bradykinin, affecting many physiological processes such as reproduction, renal metabolism, hematopoiesis, and immune response. Elevated ACE activity leads to the production of large amounts of angiotensin II, a potent vasoconstrictor that makes hypertension a possible and therefore ACE is a potential target for antihypertensive drugs. ACE is composed of two independent catalytic domains with different biological functions, and an inhibitor aiming at a specific domain of the ACE is developed into an effective antihypertensive drug which is proved to have a strong antihypertensive effect. ACE inhibitors, such as food-derived peptides, are being developed, are safer and more beneficial to human health. Therefore, efficient and rapid detection of ACE levels in human serum is crucial for the prevention of hypertension and the discovery of novel inhibitors.
Traditional methods of measuring ACE activity have relied primarily on fluorimetric assays, where ACE catalyzes the hydrolysis of a particular substrate to produce a fluorescent substrate, and spectrophotometric assays to determine the amount of hydrolysate produced. Other methods based on High Performance Liquid Chromatography (HPLC) and mass spectrometry have also been developed. However, most methods of detecting ACE have limited reliability and sensitivity. More importantly, the large reagent requirements, complex operations and advanced instrumentation limit their use in some resource-poor environments. Therefore, there is an urgent need to develop a simple and effective Angiotensin Converting Enzyme (ACE) detection technique, which is of great value in biomedical diagnosis. Rapidly evolving nanotechnology offers a promising platform for the development of biosensing technologies to address these challenges. The unique phenomenon and extraordinary performance of nanostructures on the nanometer scale is an important aid to the performance of biosensors. One notable material is plasmonic gold nanoparticles (AuNPs), which have been widely used in biomedical analysis due to their unique surface chemistry, electronic and tunable optical properties. The plasma sensor based on AuNPs can be effectively, simply and conveniently read by naked eyes without the help of advanced instruments, which brings huge prospect to various biological analysis methods in biomedical diagnosis.
Disclosure of Invention
In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a method for detecting angiotensin converting enzyme and applications thereof, and the technical problems to be solved by the present invention are: the activity of the angiotensin converting enzyme can be detected more simply, conveniently and effectively.
In order to achieve the purpose, the invention provides the following technical scheme: a method for detecting angiotensin converting enzyme, when detecting ACE activity, dilute ACE in deionized water to final concentration from 0.5mU/L to 60mU/L, each 96-well plate contains ACE 25 mU L, Cys-ang (I) -Cys peptide 40mU L, incubate at 37 deg.C, then add 50 mU L AuNPs in 96-well plate, amplify ACE activity signal by detecting the residue of Cys-ang (I) -Cys peptide, finally record the UV-visible absorption spectrum of AuNPs solution in 400-800nm within 5 min;
before the ACE activity is detected, AuNPs need to be synthesized, and the synthesis method of the AuNPs comprises the following steps:
100 ml of 1mM chloroauric acid was weighed into a round-bottom flask, heated to 120 ℃ by continuous stirring, then 10 ml of 38.8mM sodium citrate solution was added rapidly and continuously to ensure complete reduction of chloroauric acid, which when the solution turned from colorless to deep red, indicated the formation of AuNPs, the heat was removed and the AuNPs were cooled at room temperature and ready for use.
In a preferred embodiment, before the ACE activity is detected, aggregation of AuNPs is induced by Cys-Ang (I) -Cys peptide, different concentrations of Cys-Ang (I) -Cys peptide are added into 150 μ L deionized water, 50 μ L AuNPs are added, after 5 minutes of equilibration, the AuNPs solution is aggregated to different degrees due to Au-S interaction, the color of the AuNPs solution changes from red to blue or purple with the increase of the concentration of Cys-Ang (I) -Cys peptide, and the solution is further analyzed and characterized by ultraviolet-visible spectroscopy, dynamic light scattering and transmission electron microscopy, so that the concentration of Cys-Ang (I) -Cys peptide added into each 96-well plate is determined to be 5 μ M.
In a preferred embodiment, the incubation time at 37 ℃ for 30 minutes to ensure adequate hydrolysis of the Cys-Ang (I) -Cys peptide when detecting ACE activity.
In a preferred embodiment, the heat source is removed during the ACE synthesis process, and after cooling, a polyethersulfone membrane is used for removing larger impurities in AuNPs to obtain AuNPs required by experiments.
In a preferred embodiment, the amount of Cys-Ang (I) -Cys peptide added when inducing aggregation of AuNPs using Cys-Ang (I) -Cys peptide is 50. mu.L, ensuring a final volume of 250. mu.L in a 96-well plate with different concentrations of Cys-Ang (I) -Cys peptide in the range of 0-1000 nM.
The invention also provides an application of the method for detecting the angiotensin converting enzyme,
(I): detecting ACE inhibitor, mixing 40mu L of ACE with 40mu L of enalapril and ramipril with different concentrations, incubating at 37 ℃, adding 40mu L of Cys-Ang (I) -Cys peptide and 30mu L of deionized water, testing the residual ACE activity according to the method for detecting ACE activity, then adding 50 mu L of AuNPs as a signal amplification tool, and detecting ACE inhibition activity by adopting 400-800nm AuNPs ultraviolet visible absorption spectrum;
(II): screening ACE inhibitory peptides, using 9 different active peptides and 30mU/mL ACE in deionized water, incubating for 15 minutes, adding 5 μ M Cys-Ang (I) -Cys to react with the inactivated ACE at 37 ℃ for 15 minutes, adding 50 μ L AuNPs into 150 μ L mixture, and screening polypeptides with ACE inhibitory activity by color change.
In a preferred embodiment, the incubation time for detection of ACE inhibitors is 20 minutes at 37 ℃ to ensure adequate inhibition of ACE activity.
In a preferred embodiment, the screening of ACE inhibiting peptides comprises selecting peptides having ACE inhibiting function which induce aggregation of AuNPs to have a purple color, and selecting peptides having no ACE inhibiting function which do not induce aggregation of AuNPs to have a red color; the 9 different active peptides were KAFAF, CAAAA, VPP, IPP, RPP, IPA, LEP, LQP and GSH, respectively, and the concentrations of the 9 active peptides were all 15 μ M.
Compared with the prior art, the invention has the technical effects and advantages that:
the invention designs a Cys-Ang (I) -Cys peptide based on angiotensin I, wherein each end of the Cys-Ang (I) -Cys peptide is connected with a cysteine (Cys), the Cys-Ang (I) -Cys peptide can be gathered on the surfaces of AuNPs at two ends through Au-S bonds and stimulates the gathering of the AuNPs to be purple or blue, the Cys-Ang (I) -Cys peptide is hydrolyzed after ACE is introduced, and sulfydryl is dissociated from one end of the peptide to cause the AuNPs to be dispersed and red, and the ACE detection is carried out in a rapid and direct mode by realizing the hydrolysis of ACE catalytic peptide and the assembly/decomposition of the AuNPs mediated by the peptide, and the method is operated to screen ACE inhibitors or inhibitory peptides.
Drawings
FIG. 1 is a schematic representation of the detection of angiotensin converting enzyme activity and inhibitors or inhibitory peptides according to the present invention;
FIG. 2 is a diagram illustrating the linear range of ACE detection according to the present invention;
FIG. 3 is a graph showing the results of the Ramipril inhibition assay of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in figure 1, a method for detecting angiotensin converting enzyme, when detecting ACE activity, diluting ACE in deionized water to final concentration from 0.5mU/L to 60mU/L, each 96-well plate contains ACE 25 mU L, Cys-ang (I) -Cys peptide 40mU L, incubating at 37 ℃, then adding 50 mU L AuNPs in 96-well plate, amplifying ACE activity signal by detecting residue of Cys-ang (I) -Cys peptide, and finally recording UV-visible absorption spectrum of AuNPs solution in 400-800nm range within 5 min;
before the ACE activity is detected, AuNPs need to be synthesized, and the synthesis method of the AuNPs comprises the following steps:
100 ml of 1mM chloroauric acid was weighed into a round-bottom flask, heated to 120 ℃ by continuous stirring, then 10 ml of 38.8mM sodium citrate solution was added rapidly and continuously to ensure complete reduction of chloroauric acid, which when the solution turned from colorless to deep red, indicated the formation of AuNPs, the heat was removed and the AuNPs were cooled at room temperature and ready for use.
In a preferred embodiment, before the ACE activity is detected, aggregation of AuNPs is induced by Cys-Ang (I) -Cys peptide, different concentrations of Cys-Ang (I) -Cys peptide are added into 150 μ L deionized water, 50 μ L AuNPs are added, after 5 minutes of equilibration, the AuNPs solution is aggregated to different degrees due to Au-S interaction, the color of the AuNPs solution changes from red to blue or purple with the increase of the concentration of Cys-Ang (I) -Cys peptide, and the solution is further analyzed and characterized by ultraviolet-visible spectroscopy, dynamic light scattering and transmission electron microscopy, so that the concentration of Cys-Ang (I) -Cys peptide added into each 96-well plate is determined to be 5 μ M.
In a preferred embodiment, the incubation time at 37 ℃ for 30 minutes to ensure adequate hydrolysis of the Cys-Ang (I) -Cys peptide when detecting ACE activity.
In a preferred embodiment, the heat source is removed during the ACE synthesis process, and after cooling, a polyethersulfone membrane is used for removing larger impurities in AuNPs to obtain AuNPs required by experiments.
In a preferred embodiment, the amount of Cys-Ang (I) -Cys peptide added when inducing aggregation of AuNPs using Cys-Ang (I) -Cys peptide is 50. mu.L, ensuring a final volume of 250. mu.L in a 96-well plate with different concentrations of Cys-Ang (I) -Cys peptide in the range of 0-1000 nM.
The invention also provides an application of the method for detecting the angiotensin converting enzyme,
(I): detecting ACE inhibitor, mixing 40mu L of ACE with 40mu L of enalapril and ramipril with different concentrations, incubating at 37 ℃, adding 40mu L of Cys-Ang (I) -Cys peptide and 30mu L of deionized water, testing the residual ACE activity according to the method for detecting ACE activity, then adding 50 mu L of AuNPs as a signal amplification tool, and detecting ACE inhibition activity by adopting 400-800nm AuNPs ultraviolet visible absorption spectrum;
(II): screening ACE inhibitory peptides, using 9 different active peptides and 30mU/mL ACE in deionized water, incubating for 15 minutes, adding 5 μ M Cys-Ang (I) -Cys to react with the inactivated ACE at 37 ℃ for 15 minutes, adding 50 μ L AuNPs into 150 μ L mixture, and screening polypeptides with ACE inhibitory activity by color change.
In a preferred embodiment, the incubation time for detection of ACE inhibitors is 20 minutes at 37 ℃ to ensure adequate inhibition of ACE activity.
In a preferred embodiment, the screening of ACE inhibiting peptides comprises selecting peptides having ACE inhibiting function which induce aggregation of AuNPs to have a purple color, and selecting peptides having no ACE inhibiting function which do not induce aggregation of AuNPs to have a red color; the 9 different active peptides were KAFAF, CAAAA, VPP, IPP, RPP, IPA, LEP, LQP and GSH, respectively, and the concentrations of the 9 active peptides were all 15 μ M.
The ACE activity is detected by an assembly sensor containing polypeptide-mediated AuNPs, and the Cys-ang (I) -Cys peptide based on angiotensin I is designed, each end of the peptide is connected with a cysteine, the cysteine contains a sulfydryl and can be connected to the AuNPs through an Au-S bond, so that the Cys-ang (I) -Cys peptide can induce the assembly of the AuNPs, and due to the Surface Plasmon Resonance (SPR) characteristic of the AuNPs, the dispersed AuNPs and the aggregated AuNPs show different Surface Plasmon Resonance (SPR) absorption in solution and are accompanied by color change which can be visually observed by naked eyes. Cys-Ang (I) -Cys peptide can gather on the surface of AuNPs at two ends through Au-S bonds and stimulate the gathering of the AuNPs to be purple or blue, and after ACE is introduced, the Cys-Ang (I) -Cys peptide is hydrolyzed, and sulfhydryl is dissociated from one end of the peptide, so that the AuNPs are dispersed and red. By realizing hydrolysis of ACE catalytic peptide and assembly/decomposition of AuNPs mediated by peptide, ACE detection is carried out in a rapid and direct mode, and the plasma biosensor can also be used for screening high-selectivity ACE inhibitors and developing antihypertensive drugs in consideration of the inhibitory effect of the ACE inhibitors on the ACE catalytic activity.
As shown in fig. 2: the UV-Vis spectrum of the invention shows that as the ACE concentration is increased from 5mU/mL to 60mU/mL, the SPR absorption peak is red-shifted, the solution gradually turns red, A580/A520Values were used to evaluate the aggregation status of AuNPs for ACE detection according to A580/A520Values for ACE detection were linear ranging from 5-40mU/mL, R2 ═ 0.9743, and detection limit of ACE was 0.40mU/mL (S/N ═ 3).
The plasma nanosensor was further used to detect the ACE inhibitor ramipril because they can inhibit the enzyme activity of ACE, resulting in the peptide inducing aggregation of AuNPs, which changed color from red to blue after the ACE inhibitor was added, indicating that ACE-catalyzed hydrolysis was inhibited, the remaining Cys-ang (i) -Cys causing aggregation of AuNPs, a580/A520The values increased with increasing ramipril concentration and ACE activity was completely inhibited when the ramipril concentration reached 31.62 μ M, the median Inhibitory Concentration (IC) of ramipril50Value) was 6.65. mu.M, as shown in FIG. 3.
The points to be finally explained are: first, in the description of the present application, it should be noted that, unless otherwise specified and limited, the terms "mounted," "connected," and "connected" should be understood broadly, and may be a mechanical connection or an electrical connection, or a communication between two elements, and may be a direct connection, and "upper," "lower," "left," and "right" are only used to indicate a relative positional relationship, and when the absolute position of the object to be described is changed, the relative positional relationship may be changed;
secondly, the method comprises the following steps: in the drawings of the disclosed embodiments of the invention, only the structures related to the disclosed embodiments are referred to, other structures can refer to common designs, and the same embodiment and different embodiments of the invention can be combined with each other without conflict;
and finally: the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that are within the spirit and principle of the present invention are intended to be included in the scope of the present invention.
Claims (8)
1. A method for detecting angiotensin converting enzyme, comprising:
when the ACE activity is detected, diluting the ACE in deionized water to a final concentration of 0.5mU/L to 60mU/L, wherein each 96-well plate contains 25 mU L, Cys-ang (I) -40mU L of Cys peptide, incubating at 37 ℃, adding 50 mU L of AuNPs in the 96-well plate, amplifying an ACE activity signal by detecting the residual amount of the Cys-ang (I) -Cys peptide, and finally recording the ultraviolet visible absorption spectrum of the AuNPs solution in the range of 400-800nm within 5 min;
before the ACE activity is detected, AuNPs need to be synthesized, and the synthesis method of the AuNPs comprises the following steps:
100 ml of 1mM chloroauric acid was weighed into a round-bottom flask, heated to 120 ℃ by continuous stirring, then 10 ml of 38.8mM sodium citrate solution was added rapidly and continuously to ensure complete reduction of chloroauric acid, which when the solution turned from colorless to deep red, indicated the formation of AuNPs, the heat was removed and the AuNPs were cooled at room temperature and ready for use.
2. The method for detecting angiotensin converting enzyme according to claim 1 wherein: before the ACE activity is detected, Cys-Ang (I) -Cys peptide is used for inducing aggregation of AuNPs, Cys-Ang (I) -Cys peptide with different concentrations is added into 150 mu L of deionized water, then 50 mu L of AuNPs is added, after 5 minutes of equilibrium, AuNPs solution is aggregated to different degrees due to Au-S interaction, the color of the AuNPs solution changes from red to blue or purple along with the increase of the concentration of the Cys-Ang (I) -Cys peptide, then the solution is further analyzed and characterized by utilizing ultraviolet-visible spectrum, dynamic light scattering and a transmission electron microscope, and the concentration of the Cys-Ang (I) -Cys peptide added into each 96-well plate is determined to be 5mu M.
3. The method for detecting angiotensin converting enzyme according to claim 1 wherein: incubation at 37 ℃ for 30 minutes to ensure adequate hydrolysis of the Cys-Ang (I) -Cys peptide for ACE activity.
4. The method for detecting angiotensin converting enzyme according to claim 1 wherein: and removing a heat source in the ACE synthesis process, cooling, and removing larger impurities in AuNPs by adopting a polyether sulfone membrane to obtain the AuNPs required by the experiment.
5. The method for detecting angiotensin converting enzyme according to claim 2 wherein: the addition amount of Cys-Ang (I) -Cys peptide was 50. mu.L when inducing aggregation of AuNPs using Cys-Ang (I) -Cys peptide, ensuring a final volume of 250. mu.L in 96-well plate with concentration of Cys-Ang (I) -Cys peptide in the range of 0-1000nM at different concentrations in 96-well plate.
6. Use of a method for the detection of angiotensin converting enzyme according to any of claims 1 to 5, characterized in that:
(I): detecting ACE inhibitor, mixing 40mu L of ACE with 40mu L of enalapril and ramipril with different concentrations, incubating at 37 ℃, adding 40mu L of Cys-Ang (I) -Cys peptide and 30mu L of deionized water, testing the residual ACE activity according to the method for detecting ACE activity, then adding 50 mu L of AuNPs as a signal amplification tool, and detecting ACE inhibition activity by adopting 400-800nm AuNPs ultraviolet visible absorption spectrum;
(II): screening ACE inhibitory peptides, using 9 different active peptides and 30mU/mL ACE in deionized water, incubating for 15 minutes, adding 5 μ M Cys-Ang (I) -Cys to react with the inactivated ACE at 37 ℃ for 15 minutes, adding 50 μ L AuNPs into 150 μ L mixture, and screening polypeptides with ACE inhibitory activity by color change.
7. The use of a method according to claim 6 for the detection of angiotensin converting enzyme wherein: the incubation time for detection of ACE inhibitors was 20 minutes at 37 ℃ to ensure sufficient inhibition of ACE activity.
8. The use of a method according to claim 6 for the detection of angiotensin converting enzyme wherein: when the ACE inhibitory peptides are screened, the peptides with the ACE inhibitory function can induce AuNPs to aggregate and are purple, while the peptides without the ACE inhibitory function can not induce the AuNPs to aggregate and are red; the 9 different active peptides were KAFAF, CAAAA, VPP, IPP, RPP, IPA, LEP, LQP and GSH, respectively, and the concentrations of the 9 active peptides were all 15 μ M.
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