CN109187508B - Transpeptidase detection method based on electrochemiluminescence characteristic sensing - Google Patents
Transpeptidase detection method based on electrochemiluminescence characteristic sensing Download PDFInfo
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Abstract
The invention discloses a method for detecting transpeptidase based on electrochemiluminescence characteristic sensing, which specifically comprises the following steps: preparing a hydrated iridium complex, performing a connection reaction, labeling a signal probe and detecting electrochemiluminescence, sensing by using the electrochemiluminescence characteristic of the metal cyclized iridium complex, and judging the concentration and activity change of the transpeptidase according to the change of the electrochemiluminescence intensity; the invention can rapidly detect the concentration and activity of the transpeptidase, has simple detection process, easy operation, low price of reagents and equipment used for detection, and environmental protection and no pollution in the detection process.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry and biosensing, and particularly relates to a transpeptidase detection method based on electrochemiluminescence characteristic sensing.
Background
The Sortase A is a group of transpeptidase existing in gram-positive bacteria and capable of mediating covalent binding of surface protein and cell wall, and is mainly responsible for modification of cell surface protein and construction of bacterial flagellum, at present, the Sortase A derived from staphylococcus aureus can catalyze substrate polypeptide containing LPxTG (x represents any amino acid residue) sequence at C terminal to perform a connection reaction with substrate containing oligoglycine sequence at N terminal, so that extensive research is obtained; on one hand, the peptide-peptide connection reaction based on the Sortase A effect has the characteristics of high specificity, convenience and high efficiency, can be used for the connection of peptides and active substances such as peptides, proteins, nucleic acid analogues, polysaccharides and the like and the immobilization of proteins, and shows good application prospect; on the other hand, Sortase A is an enzyme playing a key role in the process of secreting and positioning bacterial surface protein to a cell wall, and is closely related to pathogenicity of bacteria, so that detection of Sortase A has important significance for understanding bacterial infection mechanism and related connection action.
The electrochemical luminescence detection method is a method for detecting a target compound by utilizing the phenomenon of chemiluminescence caused by an electrochemical reaction, and has the advantages of simple and convenient operation, low background signal and easy operationControl, specificity and the like; the metal cyclized iridium complex has good electrochemical luminescence and fluorescence properties, can be used as an electrochemical luminescence tracer, and takes Triethylamine (TPA) as a co-reactant to construct a related electrochemical luminescence sensing system; histidine and polypeptides containing histidine residues can be reacted with iridium complex hydrate (Ir (OH)2)2) And (2) performing coordination binding to form a histidine-metal cyclized iridium complex, using the coordination property, using the iridium complex hydrate to realize electrochemical probe labeling on the polypeptide containing histidine residues, and combining the electrochemiluminescence property of the iridium complex metallated to detect Sortase A.
At present, the quantitative detection of the transpeptidase mainly adopts an immunoblotting method, a radioactive isotope labeling method and the like, the immunoblotting method needs expensive biological reagents and takes a long time, a detection instrument has high requirements and high price, the radioactive isotope labeling method needs radioactive isotope labeling, the reagents are expensive and have radioactive pollution, and therefore, the design of the simple, rapid, environment-friendly and pollution-free transpeptidase detection method with low cost has important significance.
Disclosure of Invention
The invention aims to provide a method for detecting transpeptidase based on electrochemiluminescence characteristic sensing, which is used for simply and quickly detecting the transpeptidase, and has the advantages of low price of reagents and equipment used for detection, short detection time consumption, environmental protection and no pollution.
The invention adopts the technical scheme that the method for detecting the transpeptidase based on the electrochemiluminescence characteristic sensing specifically comprises the following steps:
step 1: preparation of hydrated iridium complexes
a. Taking [ Ir (bpy)2Cl]2Dissolving in dichloromethane;
b. dissolving AgOTf in a methanol solution;
c. slowly dripping the solution prepared in the step b into the solution prepared in the step a while stirring;
d. reacting for 1 hour at room temperature, filtering under reduced pressure, and washing with dichloromethane;
e. taking the filtrate, distilling under reduced pressureRemoving dichloromethane and methanol to obtain yellow powder which is the reaction product Ir (OH)2)2；
Step 2: ligation and Signal Probe labeling
a. Polypeptide ligation reactions
Adding reaction substrates P1, P2 and Sortase A into a reaction buffer solution, uniformly mixing, and carrying out polypeptide ligation reaction for 2h to generate P3;
wherein P1: Ac-GALPHTGAT-CH3，P2：GGGHGA-CH3，P3：Ac-GALPHTGGGHGA-CH3；
b. To the reaction system of a, 500. mu.M of Ir (OH) prepared in step 1 was added2)2Uniformly mixing to generate a metal cyclized iridium complex, incubating for 30min in a constant-temperature incubator at 37 ℃, and carrying out electrochemical probe labeling on the metal cyclized iridium complex;
and step 3: electrochemiluminescence detection
Injecting 5 mu L of metal cyclized iridium complex marked by an electrochemical probe into an ITO working electrode detection pool containing 400 mu L of luminescence detection liquid, applying a voltage of 0.2-1.25V to the working electrode by taking a platinum wire as a counter electrode and an Ag/AgCl electrode as a reference electrode, applying a capillary high voltage of 750V, amplifying the voltage by 3 times, and detecting an electrochemical luminescence signal on the working electrode.
Further, in the solution prepared in step 1a [ Ir (bpy) ]2Cl]2Is 10.72mg mL-1。
Further, the concentration of AgOTf in the solution prepared in step 1b was 5.24mg mL-1。
Further, in step 2a, the volume ratio of P1, P2, Sortase A and reaction buffer is 2:2:1:15, and the concentration ratio of P1 to P2 is 1: 4.
Further, the reaction buffer in step 2a is prepared from 130mM NaCl, 10mM CaCl2pH 7.5, 50mM Tris solution.
Further, Ir (OH) added in step 2b2)2The volume is one fourth of the volume of the reaction system in step 2 a.
Further, the luminescence detection solution in step 3 was prepared by adding 2. mu.L of 80mM TPA to 398. mu.L of 0.2M, pH-8.0 PBS buffer solution to a final concentration of 400. mu.M TPA in PBS buffer solution.
The invention has the beneficial effects that: the Sortase A containing histidine residues can be matched with hydrated iridium to construct an electrochemiluminescence system, and then electrochemiluminescence detection is carried out by combining the electrochemiluminescence property of the metal cyclized iridium complex; the method has the advantages of simple experiment operation, easily controlled detection process, short time consumption, low cost and high accuracy; the method can directly detect and analyze the action effect of the transpeptidase, quantitatively and qualitatively judge the concentration and the activity of the transpeptidase, and accordingly judge the effect of the transpeptidase influence factors.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1(A) is a cyclic voltammogram of a metal iridium complex; (B) is the corresponding electrochemiluminescence diagram;
FIG. 2 is a comparison of electrochemiluminescence with and without Sortase A;
FIG. 3 shows different polypeptides with iridium hydrate complex (Ir (OH)2)2) A combined real-time fluorescence profile;
FIG. 4 is a graph of ECL intensity as a function of Sortase A reaction time;
FIG. 5 is a graph of substrate polypeptide concentration ratio versus reaction product ECL intensity;
FIG. 6(A) is an electrochemiluminescence intensity curve of the metal cyclized iridium complex at different Sortase A concentrations; (B) is a linear fitting curve after corresponding signal processing;
FIG. 7 is a graph of berberine hydrochloride concentration versus signal-to-back ratio;
FIG. 8 is a graph showing ECL intensity of different concentrations of Sortase A in cell lysates.
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.
The method for detecting the transpeptidase based on the electrochemiluminescence characteristic sensing specifically comprises the following steps:
step 1: preparation of hydrated iridium complexes
a. Taking [ Ir (bpy)2Cl]2Dissolving in dichloromethane to obtain [ Ir (bpy)2Cl]2The concentration was 10.72mg mL-1The solution of (1);
b. dissolving AgOTf in methanol solution to obtain AgOTf with concentration of 5.24mg mL-1The solution of (1);
c. slowly dripping the solution prepared in the step b into the solution prepared in the step a while stirring;
d. reacting for 1 hour at room temperature, filtering under reduced pressure, and washing with dichloromethane;
e. taking the filtrate, distilling under reduced pressure to remove dichloromethane and methanol to obtain yellow powder, namely a reaction product Ir (OH)2)2；
Step 2: ligation and Signal Probe labeling
a. Polypeptide ligation reactions
Adding reaction substrates 2 mu L, 10 mu M P1, 2 mu L, 40 mu M P2 and 1 mu L and 200nM Sortase A into 15 mu L reaction buffer solution to form a total 20 mu L system, uniformly mixing, and performing polypeptide ligation reaction for 2h to generate P3;
wherein the reaction buffer solution is prepared from 130mM NaCl and 10mM CaCl2pH 7.5, 50mM Tris solution;
wherein P1: Ac-GALPHTGAT-CH3，P2：GGGHGA-CH3，P3：Ac-GALPHTGGGHGA-CH3；
b. Adding into the reaction system of aAdd 5. mu.L of 500. mu.M Ir (OH) prepared in step 12)2Uniformly mixing to generate a metal cyclized iridium complex, incubating for 30min in a constant-temperature incubator at 37 ℃, and carrying out electrochemical probe labeling on the metal cyclized iridium complex, wherein the reaction formula is shown as formula (1);
and step 3: electrochemiluminescence detection
Injecting 5 mu L of metal cyclized iridium complex marked by an electrochemical probe into an ITO working electrode detection pool containing 400 mu L of luminescence detection liquid, applying a voltage of 0.2-1.25V, a capillary high voltage of 750V and an amplification stage of 3 times on a working electrode by taking a platinum wire as a counter electrode and an Ag/AgCl electrode as a reference electrode, and detecting an electrochemical luminescence signal on the working electrode;
the luminescence detection solution was prepared by adding 2. mu.L of 80mM TPA to 398. mu.L of 0.2M, pH ═ 8.0PBS buffer solution, and then prepared into PBS buffer solution with a final concentration of 400. mu.M TPA.
Example 1
The electrochemiluminescence characteristic of the metal cyclized iridium complex is tested, and the specific operation steps are as follows:
first, 20 μ L, 10 μ M P3, 5 μ L, and 500 μ M Ir (OH) were taken2)2Mixing, incubating in a constant temperature oven at 37 deg.C for 30min, and marking with electrochemical probe;
injecting 5 mu L of reaction solution marked by the electrochemical probe into an ITO working electrode detection pool for detection, wherein the experimental result is shown in figure 1;
p3 with Ir (OH)2)2After incubation, the reaction product can see obvious green fluorescence under the irradiation of an ultraviolet lamp, which indicates that the metal cyclized iridium complex is formed, and as shown in fig. 1(a), the metal cyclized iridium complex has an obvious oxidation peak at 1.05V; FIG. 1(B) shows that the metallocycle iridium complex has a strong electrochemiluminescence intensity, the initial luminescence potential is 0.9V, and the maximum luminescence intensity appears at 1.05V, which is consistent with the oxidation peak potential of FIG. 1(A), indicating that the metallocycle iridium complex has a good electrochemiluminescence property.
Example 2
The electrochemical luminescence sensing feasibility study has the following specific experimental process:
adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L and 4.0 mu M P2 into 16 mu L of reaction buffer solution to form a system of 20 mu L in total, uniformly mixing, and standing for 2 hours; 5 μ L of 500 μ M Ir (OH) was added to the reaction system2)2Uniformly mixing, incubating for 30min in a constant-temperature incubator at 37 ℃ to generate a metal cyclized iridium complex, and carrying out electrochemical probe labeling on the metal cyclized iridium complex; injecting 5 mu L of marked metal cyclized iridium complex into an ITO working electrode detection pool containing 400 mu L of luminescence detection liquid for detection;
as shown in FIG. 2, a certain luminescence signal was detected without the presence of Sortase A, since both P1 and P2 contain histidine residues and are also capable of reacting with Ir (OH)2)2The coordination forms a metal cyclized iridium complex, thereby causing a certain background electrochemiluminescence signal.
Example 3
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2, 1 mu L and 20nM Sortase A into 15 mu L reaction buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; 5 μ L of 500 μ M Ir (OH) was added to the reaction system2)2Uniformly mixing, incubating for 30min in a constant-temperature incubator at 37 ℃ to generate a metal cyclized iridium complex, and carrying out electrochemical probe labeling on the metal cyclized iridium complex; injecting 5 mu L of marked metal cyclized iridium complex into an ITO working electrode detection pool containing 400 mu L of luminescence detection liquid for detection;
as shown in FIG. 2, the electrochemical luminescence intensity is obviously enhanced and the signal-to-back ratio is increased after the ligation reaction is carried out by adding Sortase A, because P3 and Ir (OH)2)2The metal cyclized iridium complex formed by coordination has good electrochemical luminescence efficiency;
the combination of example 2 and example 3 shows that both polypeptides are capable of binding Ir (OH)2)2The metal formed by coordination cyclizes the iridium complex, but P3 generated after adding Sortase A is reacted with Ir(OH2)2The electrochemical luminescence intensity of the metal cyclized iridium complex formed by coordination is stronger than that of P1, P2 and Ir (OH) in a control group without adding Sortase A2)2The signal-to-back ratio of the coordinated and formed metal cyclized iridium complex is increased, so that the effect of the Sortase A can be detected through the change of the electrochemiluminescence intensity.
Example 4
Adding reaction substrates of 5 mu L and 1.0 mu M P1 into 15 mu L buffer solution to form a total of 20 mu L system, uniformly mixing, and standing for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Mixing with 75 μ L water to obtain 100 μ L fluorescence solution to be detected, and adding the solution to be detected into a fluorescence cuvette for real-time fluorescence intensity detection with excitation wavelength of 375 nm;
the results of the detection are shown in FIG. 3, which illustrates Ir (OH)2)2The compound can be combined with P1 to form a metal cyclized iridium complex, so that a certain background signal is generated; simultaneously with P1 and Ir (OH)2)2And the fluorescence intensity is increased due to the increase of the coordination binding time, and the fluorescence intensity is stable after 30min, so that 30min is selected as the incubation time for experiments.
Example 5
Adding reaction substrates of 5 mu L and 4.0 mu M P2 into 15 mu L of the buffer solution to form a system of 20 mu L in total, uniformly mixing, and standing for 2 hours; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2 and 75 mu L of water are uniformly mixed to form 100 mu L of fluorescence solution to be detected, and finally the solution to be detected is added into a fluorescence cuvette for real-time fluorescence intensity detection, wherein the excitation wavelength is 375 nm;
the results of the detection are shown in FIG. 3, which illustrates Ir (OH)2)2The compound can be combined with P2 to form a metal cyclized iridium complex, so that a certain background signal is generated; simultaneously with P2 and Ir (OH)2)2And the fluorescence intensity is increased due to the increase of the coordination binding time, and the fluorescence intensity is stable after 30min, so that 30min is selected as the incubation time for experiments.
Example 6
Adding reaction substrate 5 μ L, 1.0 μ M m P3 into the above buffer solution 15 μ L to form a total of 20 μ L system and mixingUniformly standing for 2 hours; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Mixing with 75 μ L water to obtain 100 μ L fluorescence solution to be detected, and adding the solution to be detected into a fluorescence cuvette for real-time fluorescence intensity detection with excitation wavelength of 375 nm;
the results of the detection are shown in FIG. 3, which illustrates Ir (OH)2)2Can be combined with P3 to form a metal cyclized iridium complex, and can detect a strong electrochemiluminescence signal; simultaneously with P3 and Ir (OH)2)2And the fluorescence intensity is increased due to the increase of the coordination binding time, and the fluorescence intensity is stable after 30min, so that 30min is selected as the incubation time for experiments.
Example 7
Adding reaction substrates 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2, 1 mu L and 20nM Sortase A into 15 mu L of the buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Mixing with 75 μ L water to obtain 100 μ L fluorescence solution to be detected, and adding the solution to be detected into a fluorescence cuvette for real-time fluorescence intensity detection with excitation wavelength of 375 nm;
the results of the detection are shown in FIG. 3, which illustrates Ir (OH)2)2Can be combined with P1, P2 and P3 to form a metal cyclized iridium complex, and simultaneously, the metal cyclized iridium complex is combined with P1, P2 and P3 and Ir (OH)2)2And the fluorescence intensity is increased due to the increase of the coordination binding time, and the fluorescence intensity is stable after 30min, so that 30min is selected as the incubation time for experiments.
Example 8
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2, 1 mu L and 10nM Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for a period of time; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Uniformly mixing, incubating in a constant-temperature incubator at 37 ℃ for 30min, carrying out electrochemical probe labeling on the metal cyclized iridium complex, and finally injecting 5 mu L of labeled reaction solution into an ITO working electrode detection pool for detection;
and (3) detecting the electrochemiluminescence intensity of the reaction solution of the polypeptide ligation reaction at different times, wherein the detection result is shown in figure 4, the signal-to-back ratio is increased along with the increase of the reaction time of the Sortase A, which shows that the amount of P3 is continuously increased along with the reaction time, the signal-to-back ratio is maximum after the reaction is carried out for 2 hours, and the signal-to-back ratio is basically unchanged even if the reaction time is prolonged to 32 hours, so that the time of the Sortase A ligation reaction is selected to be 2 hours.
Example 9
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 1.0 mu M P2, 1 mu L and 20nM Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Mixing uniformly, incubating for 30min in a constant-temperature incubator at 37 ℃, carrying out electrochemical probe labeling on the metal cyclized iridium complex, and finally injecting 5 mu L of labeled reaction solution into an ITO working electrode detection pool for detection.
Example 10
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 2.0 mu M P2, 1 mu L and 20nM Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Mixing uniformly, incubating for 30min in a constant-temperature incubator at 37 ℃, carrying out electrochemical probe labeling on the metal cyclized iridium complex, and finally injecting 5 mu L of labeled reaction solution into an ITO working electrode detection pool for detection.
Example 11
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2, 1 mu L and 20nM Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Uniformly mixing, incubating in a constant-temperature incubator at 37 ℃ for 30min, carrying out electrochemical probe labeling on the metal cyclized iridium complex, and finally injecting 5 mu L of labeled reaction solution into an ITO working electrode detection pool for detection;
the experimental results of examples 9-11 are shown in FIG. 5, and the signal-to-back ratio increases with the increase of the concentration of P2, indicating that the enzymatic efficiency of Sortase A is improved, and the signal-to-back ratio is maximum when the concentration of P2 is 4 times that of P1 and does not change with the increase of the reaction time; thus, the concentration ratio of P2 to P1 was chosen to be 4:1 in the experiment.
Example 12
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2 and 1 mu L Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Uniformly mixing, incubating for 30min in a constant-temperature incubator at 37 ℃, and carrying out electrochemical probe labeling on the metal cyclized iridium complex; finally, injecting 5 mu L of marked reaction solution into an ITO working electrode detection pool for detection;
wherein the concentration of Sortase a is 0.125nM, 0.25nM, 1.25nM, 2.5nM, 6.25nM and 12.5nM, respectively, and the detection result is shown in fig. 6, as shown in fig. 6(a), it can be seen that the higher the concentration of Sortase a, the stronger the electrochemiluminescence intensity, the higher the signal-to-back ratio, as shown in fig. 6(B), the concentration of Sortase a and the ECL signal are in a linear relationship in the range of 1.25nM to 12.5nM, the regression equation is 0.553x +2.884, the fitting degree is 0.998, the detection limit is 0.75nM, and a new method is provided for detecting Sortase a.
Example 13
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2, 1 mu L and 12.5nM Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total and uniformly mixing, and performing polypeptide ligation reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added to the reaction system2)2Uniformly mixing, incubating for 30min in a constant-temperature incubator at 37 ℃, and carrying out electrochemical probe labeling on the metal cyclized iridium complex; finally, injecting 5 mu L of marked reaction solution into an ITO working electrode detection pool for detection;
berberine hydrochloride is an inhibitor of Sortase A, has strong inhibiting effect on Sortase A, berberine hydrochloride with different concentrations is added during polypeptide connection reaction, other experimental conditions are unchanged, and the influence of berberine hydrochloride on the activity of Sortase A is detected, as shown in figure 7, along with the increase of berberine hydrochloride concentration, the signal-to-back ratio, namely electrochemical luminescence intensity is reduced, the inhibiting effect of berberine hydrochloride on Sortase A and the maximum half-inhibiting concentration value IC of berberine hydrochloride on Sortase A can be qualitatively judged50About 9 μ M, inMicromolar concentration levels.
Example 14
Adding reaction substrates of 2 mu L, 1.0 mu M P1, 2 mu L, 4.0 mu M P2 and 1 mu L Sortase A into 15 mu L buffer solution to form a system of 20 mu L in total, uniformly mixing, and performing polypeptide connection reaction for 2 h; then, 5. mu.L of 500. mu.M Ir (OH) was added thereto2)2Uniformly mixing, incubating in a constant-temperature incubator at 37 ℃ for 30min, carrying out electrochemical probe labeling on the metal cyclized iridium complex, and finally injecting 5 mu L of labeled reaction solution into an ITO working electrode detection pool for detection;
the experiment was performed at a constant cell lysate concentration of 1.5 ten thousand cells and at different Sortase a concentrations: the electrochemiluminescence intensity of Sortase A in the cell lysate is tested at 0nM, 2.5nM, 6.25nM, 12.5nM and 18.5nM, and the experimental results are shown in FIG. 8, wherein the electrochemiluminescence intensity increases as the concentration of Sortase A increases from 0 to 18.5nM, which is due to the increase of the ligation product P3 of Sortase A; by comparing the electrochemiluminescence intensity of the reaction solution when the cell lysate is added with the reaction solution when the cell lysate is not added, the effect of the cell lysate on Sortase A can be qualitatively judged, and the activity of the Sortase A in the cell lysate can be detected.
All the embodiments in the present specification are described in a related manner, and the same and similar parts among the embodiments may be referred to each other, and each embodiment focuses on the differences from the other embodiments. In particular, for the system embodiment, since it is substantially similar to the method embodiment, the description is simple, and for the relevant points, reference may be made to the partial description of the method embodiment.
The above description is only for the preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (6)
1. The method for detecting the transpeptidase based on the electrochemiluminescence characteristic sensing is characterized by comprising the following steps:
step 1: preparation of hydrated iridium complexes
1
Getting [ Ir (bpy)2Cl]2Dissolving in dichloromethane;
1b, dissolving AgOTf in a methanol solution;
1c, slowly dripping the solution prepared in the step 1b into the solution prepared in the step 1a while stirring;
1d, reacting for 1 hour at room temperature, filtering under reduced pressure, and washing with dichloromethane;
1e, taking the filtrate, and distilling under reduced pressure to remove dichloromethane and methanol to obtain yellow powder, namely a reaction product Ir (OH)2)2，
Step 2: ligation and Signal Probe labeling
2a, polypeptide ligation reaction
Adding reaction substrates P1, P2 and Sortase A into a reaction buffer solution, uniformly mixing, and carrying out polypeptide ligation reaction for 2h to generate P3;
wherein P1: Ac-GALPHTGAT-CH3，P2：GGGHGA-CH3，P3：Ac-GALPHTGGGHGA-CH3；
2b, adding 500. mu.M of Ir (OH) prepared in the step 1 into the reaction system in the step 2a2)2Uniformly mixing to generate a metal cyclized iridium complex Ir-P3, incubating for 30min in a constant-temperature incubator at 37 ℃, and carrying out electrochemical probe labeling on the metal cyclized iridium complex;
and step 3: electrochemiluminescence detection
Injecting 5 mu L of the metal cyclized iridium complex marked by the electrochemical probe into an ITO working electrode detection cell containing 400 mu L of luminescence detection liquid, adding 2 mu L of the luminescence detection liquid and 80mM TPA into 398 mu L of PBS buffer solution with the concentration of 0.2M, pH =8.0 to prepare PBS buffer solution with the final concentration of 400 mu M TPA, applying 0.2V-1.25V of voltage to the working electrode by taking a platinum wire as a counter electrode and an Ag/AgCl electrode as a reference electrode, applying 750V of capillary high voltage and amplifying the level by 3 times, and detecting an electrochemical luminescence signal on the working electrode.
2. The method for detecting a transpeptidase based on electrochemiluminescence characteristics sensor of claim 1, wherein the solution prepared in step 1a is [ Ir (bpy)2Cl]2Is 10.72mg mL-1。
3. The method for detecting the transpeptidase based on the electrochemiluminescence characteristic sensing of claim 1, wherein the concentration of AgOTf in the solution prepared in the step 1b is 5.24mg mL-1。
4. The method for detecting the electrochemiluminescence property-based sensing of the transpeptidase, according to claim 1, wherein the volume ratio of the P1, the P2, the Sortase A and the reaction buffer in the step 2a is 2:2:1:15, and the mass concentration ratio of the P1 to the P2 is 1: 4.
5. The method for detecting a transpeptidase based on electrochemiluminescence characteristics sensing of claim 1, wherein the reaction buffer solution in step 2a is prepared from 130mM NaCl, 10mM CaCl2pH =7.5, 50mM tris solution.
6. The method for detecting a transpeptidase based on electrochemiluminescence property sensor according to claim 1, wherein Ir (OH) is added in the step 2b2)2The volume is one fourth of the volume of the reaction system in step 2 a.
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| WO2015042526A1 (en) * | 2013-09-23 | 2015-03-26 | Quest Diagnostics Investments Incorporated | Detection of methicillin-resistant staphylococcus aureus in biological samples |
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| WO2015042526A1 (en) * | 2013-09-23 | 2015-03-26 | Quest Diagnostics Investments Incorporated | Detection of methicillin-resistant staphylococcus aureus in biological samples |
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