CN109187508B - Transpeptidase detection method based on electrochemiluminescence characteristic sensing - Google Patents

Abstract

The invention discloses a transpeptidase detection method based on electrochemiluminescence characteristic sensing, which specifically includes the following steps: preparation of hydrated iridium complexes, ligation reaction, signal probe labeling and electrochemiluminescence detection, using metal cyclized iridium The electrochemiluminescence characteristic of the complex is sensed, and the concentration and activity changes of the transpeptidase are judged by the change of the electrochemiluminescence intensity; the invention can quickly detect the concentration and activity of the transpeptidase, and the detection process is simple and easy to operate. In addition, the reagents and equipment used in the detection are inexpensive, and the detection process is environmentally friendly and pollution-free.

Description

Detection method of transpeptidase based on electrochemiluminescence property sensing

technical field

The invention belongs to the technical field of analytical chemistry and biological sensing, in particular to a method for detecting transpeptidase based on electrochemiluminescence characteristic sensing.

Background technique

Sortase A is a group of transpeptidase proteases that exist in Gram-positive bacteria and can mediate the covalent binding of surface proteins to the cell wall. It is mainly responsible for the modification of cell surface proteins and the construction of bacterial flagella. Sortase A of Staphylococcus aureus has been widely studied because it can catalyze the ligation reaction of substrate polypeptides containing LPxTG (x represents any amino acid residue) sequence at the C-terminus with substrates containing oligoglycine sequences at the N-terminus; On the one hand, the inter-polypeptide ligation reaction based on the action of Sortase A has the characteristics of high specificity, convenience and efficiency, and can be used for the connection of peptides and peptides, proteins, nucleic acid analogs, polysaccharides and other active substances and the immobilization of proteins. Good application prospects; on the other hand, Sortase A is an enzyme that plays a key role in the process of bacterial surface protein secretion and localization to the cell wall, and is closely related to the pathogenicity of bacteria. Connectivity is important.

Electrochemiluminescence detection method is a method for detecting target compounds by utilizing the phenomenon of chemiluminescence caused by electrochemical reaction. It has the characteristics of simple operation, low background signal, easy control and high specificity; metal cyclized iridium complexes have good electrochemical performance. Luminescence and fluorescence properties, it can be used as an electrochemiluminescence tracer, and triethylamine (TPA) is used as a co-reactant to construct a related electrochemiluminescence sensing system; histidine and polypeptides containing histidine residues can be passed through The imidazolyl group is coordinated with the hydrated iridium complex (Ir(OH ₂ ) ₂ ) to form a histidine-metallic cyclized iridium complex. Using this coordination property, the hydrated iridium complex can be used to realize the synthesis of histidine-containing residues. Based on the electrochemical probe labeling of the polypeptide, combined with the electrochemiluminescence properties of the metallocyclized iridium complex, it can be used to detect Sortase A.

At present, the quantitative detection of transpeptidase mainly adopts immunoblotting method, radioisotope labeling method, etc. The immunoblotting method requires expensive biological reagents, takes a long time, and the detection equipment is relatively high and expensive, while the radioisotope labeling method requires radioisotope Labeling, expensive reagents and radioactive contamination, therefore, it is of great significance to design a simple, rapid, low-cost, environmentally friendly and pollution-free detection method for transpeptidase.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a transpeptidase detection method based on electrochemiluminescence characteristic sensing, so as to realize the simple and rapid detection of transpeptidase, the reagents and equipment used in the detection are inexpensive, the detection time is short, and the detection is environmentally friendly and pollution-free. .

The technical solution adopted in the present invention is that the detection method of transpeptidase based on electrochemiluminescence characteristic sensing specifically includes the following steps:

Step 1: Preparation of Hydrated Iridium Complex

a. Dissolve [Ir(bpy) ₂ Cl] ₂ in dichloromethane;

b. Take AgOTf and dissolve it in methanol solution;

c. The solution prepared by b is slowly added dropwise to the solution prepared by a in stirring;

d, react at room temperature for 1 hour, filter under reduced pressure, and wash with dichloromethane;

e, take the filtrate, dichloromethane and methanol are removed by distillation under reduced pressure, and obtaining yellow powder is reaction product Ir(OH ₂ ) ₂ ;

Step 2: Ligation and Signal Probe Labeling

a. Peptide ligation reaction

The reaction substrates P1, P2 and Sortase A were added to the reaction buffer, mixed evenly, and the peptide was connected for 2h to generate P3;

Wherein P1: Ac-GALPHTGAT-CH ₃ , P2: GGGHGA-CH ₃ , P3: Ac-GALPHTGGGHGA-CH ₃ ;

b. Add 500 μM of Ir(OH ₂ ) ₂ prepared in step 1 to the reaction system of a, mix well, generate metal cyclized iridium complexes, incubate for 30 min in a constant temperature incubator at 37°C, and conduct electroporation on the metal cyclized iridium complexes. chemical probe labeling;

Step 3: Electrochemiluminescence detection

Take 5 μL of the metal cyclized iridium complex labeled with the electrochemical probe, and inject it into the ITO working electrode detection cell containing 400 μL of luminescence detection solution. The platinum wire is used as the counter electrode, and the Ag/AgCl electrode is used as the reference electrode. A voltage of 0.2V to 1.25V is applied on it, the capillary high voltage is 750V, and the amplification stage is 3 times to detect the electrochemiluminescence signal on the working electrode.

Further, the concentration of [Ir(bpy) ₂ Cl] ₂ in the solution prepared in step 1a was 10.72 mg mL ^-1 .

Further, the concentration of AgOTf in the solution prepared in step 1b was 5.24 mg mL ^-1 .

Further, in step 2a, the volume ratio of P1, P2, Sortase A and the reaction buffer is 2:2:1:15, and the concentration ratio of P1 and P2 is 1:4.

Further, in step 2a, the reaction buffer consists of 130 mM NaCl, 10 mM CaCl ₂ , pH=7.5, and 50 mM tris.

Further, the volume of Ir(OH ₂ ) ₂ added in step 2b is a quarter of the volume of the reaction system in step 2a.

Further, the luminescence detection solution in step 3 was prepared by adding 2 μL, 80 mM TPA to 398 μL, 0.2 M, pH=8.0 PBS buffer solution to prepare a PBS buffer solution with a final concentration of 400 μM TPA.

The beneficial effects of the present invention are as follows: Sortase A contains histidine residues, which can be combined with hydrated iridium to construct an electrochemiluminescence system, and then combined with the electrochemiluminescence properties of the metal cyclized iridium complex to perform electrochemiluminescence detection; the present invention has the following advantages: The experimental operation is simple, the detection process is easy to control, the time-consuming is short, the cost is low, and the accuracy is high; it can directly detect and analyze the effect of the transpeptidase, quantitatively and qualitatively determine the concentration and activity of the transpeptidase, and judge accordingly. Effects of transpeptidase influencing factors.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Figure 1 (A) is the cyclic voltammetry curve of the metal iridium complex; (B) is the corresponding electrochemiluminescence diagram;

Fig. 2 is the electrochemiluminescence contrast diagram with or without Sortase A;

Figure 3 is a real-time fluorescence curve of the binding of different polypeptides to hydrated iridium complexes (Ir(OH ₂ ) ₂ );

Fig. 4 is the change curve of ECL intensity with Sortase A reaction time;

Figure 5 is a graph showing the relationship between the concentration ratio of the substrate polypeptide and the ECL intensity of the reaction product;

Fig. 6 (A) is the electrochemiluminescence intensity curve of metal cyclized iridium complexes under different Sortase A concentrations; (B) is the linear fitting curve after corresponding signal processing;

Fig. 7 is the relation curve of berberine hydrochloride concentration and signal-to-background ratio;

Figure 8 is a graph of the ECL intensity of Sortase A at different concentrations in cell lysates.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The detection method of transpeptidase based on electrochemiluminescence characteristic sensing specifically includes the following steps:

Step 1: Preparation of Hydrated Iridium Complex

a. Dissolve [Ir(bpy) ₂ Cl] ₂ in dichloromethane to prepare a solution with a concentration of [Ir(bpy) ₂ Cl] ₂ of 10.72 mg mL ^-1 ;

b. Dissolve AgOTf in methanol solution to prepare a solution with AgOTf concentration of 5.24 mg mL ^-1 ;

c. The solution prepared by b is slowly added dropwise to the solution prepared by a in stirring;

d, react at room temperature for 1 hour, filter under reduced pressure, and wash with dichloromethane;

e, take the filtrate, dichloromethane and methanol are removed by distillation under reduced pressure, and obtaining yellow powder is reaction product Ir(OH ₂ ) ₂ ;

Step 2: Ligation and Signal Probe Labeling

a. Peptide ligation reaction

Add the reaction substrate 2μL, 10μM P1, 2μL, 40μM P2 and 1μL, 200nM Sortase A into 15μL reaction buffer to form a total of 20μL system and mix evenly, the peptide is connected for 2h to generate P3;

The reaction buffer is composed of 130mM NaCl, 10mM CaCl ₂ , pH=7.5, and 50mM tris;

Wherein P1: Ac-GALPHTGAT-CH ₃ , P2: GGGHGA-CH ₃ , P3: Ac-GALPHTGGGHGA-CH ₃ ;

b. Add 5 μL, 500 μM of Ir(OH ₂ ) ₂ prepared in step 1 to the reaction system of a, mix well to generate metallcyclized iridium complexes, incubate in a constant temperature incubator at 37°C for 30min, and treat the metallcyclized iridium complexes for 30 minutes. Carry out electrochemical probe labeling, and its reaction formula is as formula (1);

Step 3: Electrochemiluminescence detection

Take 5 μL of the metal cyclized iridium complex labeled with the electrochemical probe, and inject it into the ITO working electrode detection cell containing 400 μL of luminescence detection solution. The platinum wire is used as the counter electrode, and the Ag/AgCl electrode is used as the reference electrode. Apply a voltage of 0.2V to 1.25V, the capillary high voltage is 750V, the amplification stage is 3 times, and the electrochemiluminescence signal on the working electrode is detected;

The luminescence detection solution was prepared by adding 2 μL, 80 mM TPA to 398 μL, 0.2 M, pH=8.0 PBS buffer solution to prepare a PBS buffer solution with a final concentration of 400 μM TPA.

Example 1

To check the electrochemiluminescence properties of metal cyclized iridium complexes, the specific operation steps are as follows:

(1) Mix 20 μL, 10 μM P3 with 5 μL, 500 μM Ir(OH ₂ ) ₂ , incubate in a 37°C incubator for 30 min, and carry out electrochemical probe labeling;

(2) Take 5 μL of the reaction solution labeled with the electrochemical probe and inject it into the ITO working electrode detection cell for detection. The experimental results are shown in Figure 1;

After incubation of P3 with Ir(OH ₂ ) ₂ , the reaction product can see obvious green fluorescence under UV lamp irradiation, indicating the formation of metal cyclized iridium complexes, as shown in Figure 1(A), metal cyclized iridium complexes The compound has an obvious oxidation peak at 1.05V; Figure 1(B) shows that the metal cyclized iridium complex has a strong electrochemiluminescence intensity, the initial luminescence potential is at 0.9V, and the maximum luminescence intensity appears at 1.05V. The potential is consistent with the oxidation peak potential in Fig. 1(A), indicating that the metal cyclized iridium complex has good electrochemiluminescence properties.

Example 2

The feasibility of electrochemiluminescence sensing is discussed, and the specific experimental process is as follows:

Add 2 μL of reaction substrate, 1.0 μM P1, 2 μL, and 4.0 μM P2 to 16 μL of reaction buffer to form a total of 20 μL of system and mix well, and let stand for 2 h; add 5 μL, 500 μM of Ir(OH ₂ ) ₂ to the reaction system and mix Evenly, incubate in a constant temperature incubator at 37°C for 30 min to generate metal cyclized iridium complexes, and carry out electrochemical probe labeling on the metal cyclized iridium complexes; take 5 μL of the labeled metal cyclized iridium complexes and inject them into 400 μL of metal cyclized iridium complexes. The detection is carried out in the ITO working electrode detection cell of the luminescent detection solution;

The detection results are shown in Figure 2. In the absence of Sortase A, a certain luminescence signal can also be detected. This is because both P1 and P2 contain histidine residues, which can also coordinate with Ir(OH ₂ ) ₂ to form a metal Cyclization of the iridium complex, resulting in a certain background electrochemiluminescence signal.

Example 3

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 4.0μM P2 and 1μL, 20nM Sortase A into 15μL reaction buffer to form a total of 20μL system and mix well, the peptide ligation reaction 2h; add 5μL, 500μM to the reaction system Ir(OH ₂ ) ₂ was mixed evenly, incubated in a constant temperature incubator at 37°C for 30 min to generate metallcyclized iridium complexes, and electrochemical probe labeling was carried out on the metallcyclized iridium complexes; 5 μL of labeled metallcyclized iridium complexes were taken. The complex was injected into the ITO working electrode detection cell containing 400 μL of luminescence detection solution for detection;

The detection results are shown in Figure 2. After the addition of Sortase A and the ligation reaction, the electrochemiluminescence intensity has been significantly enhanced, and the signal-to-background ratio has increased. This is because the metal cyclized iridium formed by the coordination of P3 and Ir(OH ₂ ) ₂ The complex has good electrochemiluminescence efficiency;

Combining Example 2 and Example 3, it can be seen that the polypeptide can coordinate with Ir(OH ₂ ) ₂ to form a metal cyclized iridium complex, but the P3 generated after adding Sortase A, and the complex formed by Ir(OH ₂ ) ₂ coordination. The electrochemiluminescence intensity of metal cyclized iridium complexes is stronger than that of the metal cyclized iridium complexes formed by the coordination of P1, P2 and Ir(OH ₂ ) ₂ when Sortase A is not added in the control group, and the signal-to-background ratio also increases, Therefore, the effect of Sortase A can be detected by the change of electrochemiluminescence intensity.

Example 4

Add 5 μL of reaction substrate and 1.0 μM P1 to 15 μL of buffer to form a total of 20 μL of system, mix well, and let stand for 2 h; then add 5 μL, 500 μM Ir(OH ₂ ) ₂ and 75 μL of water and mix well to form 100 μL of fluorescence The liquid to be tested, and finally the liquid to be tested is added to the fluorescence cuvette for real-time fluorescence intensity detection, and the excitation wavelength is 375 nm;

The detection results are shown in Figure ₃ , indicating that Ir(OH ₂ ) ₂ can combine with P1 to form metal cyclized iridium complexes, so there is a certain background signal _; increase, the fluorescence intensity increases, and the fluorescence intensity stabilizes after 30 min, so 30 min is selected as the incubation time for the experiment.

Example 5

Add 5 μL of reaction substrate and 4.0 μM P2 to 15 μL of the above buffer to form a total of 20 μL of system, mix well, and let stand for 2 h; then add 5 μL, 500 μM Ir(OH ₂ )2 and 75 μL water and mix well to form 100 μL Fluorescence liquid to be tested, finally adding the liquid to be tested into a fluorescence cuvette for real-time fluorescence intensity detection, and the excitation wavelength is 375 nm;

The detection results are shown in Figure ₃ , indicating that Ir(OH ₂ ) ₂ can combine with P2 to form metal cyclized iridium complexes, so there is a certain background signal _; increase, the fluorescence intensity increases, and the fluorescence intensity stabilizes after 30 min, so 30 min is selected as the incubation time for the experiment.

Example 6

5 μL of reaction substrate and 1.0 μM P3 were added to 15 μL of the above buffer to form a total of 20 μL of system and mixed evenly, and allowed to stand for 2 h; 5 μL, 500 μM Ir(OH ₂ ) ₂ and 75 μL of water were added and mixed to form 100 μL of fluorescence. The liquid to be tested, and finally the liquid to be tested is added to the fluorescence cuvette for real-time fluorescence intensity detection, and the excitation wavelength is 375 nm;

The detection results are shown in Figure 3, indicating that Ir(OH ₂ ) ₂ can combine with P3 to form a metal cyclized iridium complex, and a strong electrochemiluminescence signal can be detected; at the same time, with the coordination of P3 and Ir(OH ₂ ) ₂ With the increase of the binding time, the fluorescence intensity increased, and the fluorescence intensity reached a stable level after 30 min, so 30 min was selected as the incubation time for the experiment.

Example 7

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 4.0μM P2 and 1μL, 20nM Sortase A to 15μL of the above buffer to form a total of 20μL system and mix well, the peptide ligation reaction 2h; then add 5μL, 500μM Ir to it (OH ₂ ) ₂ and 75 μL of water were mixed evenly to form 100 μL of the fluorescent solution to be tested. Finally, the above-mentioned solution to be tested was added to the fluorescence cuvette for real-time fluorescence intensity detection, and the excitation wavelength was 375 nm;

_The detection results are shown in Figure ₃ , indicating that Ir(OH ₂ ) ₂ can combine with P1, P2, and P3 to form metal cyclized iridium complexes. With the increase of time, the fluorescence intensity increased, and the fluorescence intensity stabilized after 30 min, so 30 min was chosen as the incubation time for the experiment.

Example 8

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 4.0μM P2 and 1μL, 10nM Sortase A to 15μL buffer to form a total of 20μL system and mix well, and the peptides are ligated for a period of time; then add 5μL, 500μMIr ( OH ₂ ) ₂ was mixed evenly, incubated in a constant temperature incubator at 37°C for 30 min, and the metal cyclized iridium complex was electrochemically labeled with probes. Finally, 5 μL of the labeled reaction solution was injected into the ITO working electrode detection cell for detection;

The electrochemiluminescence intensity of the reaction solution of the peptide ligation reaction at different times was detected. The detection results are shown in Figure 4. With the increase of the reaction time of Sortase A, the signal-to-background ratio increased, indicating that the amount of P3 continued to increase with the reaction time, and the reaction was 2 hours. After the reaction, the signal-to-background ratio reached the maximum, even if the reaction time was extended to 32h, the signal-to-background ratio remained basically unchanged, so the time for the Sortase A ligation reaction was selected to be 2h.

Example 9

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 1.0μM P2 and 1μL, 20nM Sortase A to 15μL buffer to form a total of 20μL system and mix well, the peptide ligation reaction 2h; then add 5μL, 500μM Ir( OH ₂ ) ₂ was mixed evenly, incubated in a 37°C constant temperature incubator for 30 min, and the metal cyclized iridium complex was electrochemically labeled with probes. Finally, 5 μL of the labeled reaction solution was injected into the ITO working electrode detection cell for detection.

Example 10

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 2.0μM P2 and 1μL, 20nM Sortase A to 15μL buffer to form a total of 20μL system and mix well, the peptide ligation reaction 2h; then add 5μL, 500μM Ir( OH ₂ ) ₂ was mixed evenly, incubated in a 37°C constant temperature incubator for 30 min, and the metal cyclized iridium complex was electrochemically labeled with probes. Finally, 5 μL of the labeled reaction solution was injected into the ITO working electrode detection cell for detection.

Example 11

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 4.0μM P2 and 1μL, 20nM Sortase A to 15μL buffer to form a total of 20μL system and mix well, the peptide ligation reaction 2h; then add 5μL, 500μM Ir( OH ₂ ) ₂ was mixed evenly, incubated in a constant temperature incubator at 37°C for 30 min, and the metal cyclized iridium complex was electrochemically labeled with probes. Finally, 5 μL of the labeled reaction solution was injected into the ITO working electrode detection cell for detection;

The experimental results of Examples 9-11 are shown in Figure 5. With the increase of P2 concentration, the signal-to-background ratio increases, indicating that the enzymatic efficiency of SortaseA is improved. When the P2 concentration is 4 times the P1 concentration, the signal-to-background ratio is the largest, and does not follow The reaction time was prolonged; therefore, the concentration ratio of P2:P1 was chosen to be 4:1 in the experiment.

Example 12

Add 2 μL of reaction substrates, 1.0 μM P1, 2 μL, 4.0 μM P2 and 1 μL Sortase A to 15 μL of buffer to form a total of 20 μL of system and mix them evenly, and the peptide is ligated for 2 h; then add 5 μL, 500 μM Ir(OH ₂ ) ₂ Mix evenly, incubate for 30 min in a 37°C constant temperature incubator, and perform electrochemical probe labeling on the metal cyclized iridium complex; finally, take 5 μL of the labeled reaction solution and inject it into the ITO working electrode detection cell for detection;

The concentrations of Sortase A were 0.125nM, 0.25nM, 1.25nM, 2.5nM, 6.25nM, and 12.5nM, respectively. The detection results are shown in Figure 6. It can be seen from Figure 6(A) that the higher the Sortase A concentration, the higher the electrical conductivity. The stronger the chemiluminescence intensity, the higher the signal-to-background ratio. It can be seen from Figure 6(B) that in the range of Sortase A concentration from 1.25nM to 12.5nM, the SortaseA concentration has a linear relationship with the ECL signal, and its regression equation is y=0.553x +2.884, the fitting degree is 0.998, and the detection limit is 0.75nM, which provides a new method for the detection of Sortase A.

Example 13

Add the reaction substrate 2μL, 1.0μM P1, 2μL, 4.0μM P2 and 1μL, 12.5nM Sortase A into 15μL buffer to form a total of 20μL system and mix evenly, the peptide ligation reaction 2h; then add 5μL, 500 μM Ir(OH ₂ ) ₂ was mixed evenly, incubated in a constant temperature incubator at 37°C for 30 min, and the metal cyclized iridium complex was labeled with electrochemical probes; finally, 5 μL of the labeled reaction solution was injected into the ITO working electrode detection cell for detection. ;

Berberine hydrochloride is an inhibitor of Sortase A and has a strong inhibitory effect on Sortase A. Different concentrations of berberine hydrochloride were added during the peptide ligation reaction, and other experimental conditions were unchanged. The activity of berberine hydrochloride on Sortase A was detected. Influence, as shown in Figure 7, as the concentration of berberine hydrochloride increases, the signal-to-background ratio, that is, the electrochemiluminescence intensity, decreases, and the inhibitory effect of berberine hydrochloride on Sortase A can be qualitatively judged. The inhibitory concentration value _IC50 is approximately 9 μM at the micromolar level.

Example 14

Add 2 μL of reaction substrates, 1.0 μM P1, 2 μL, 4.0 μM P2 and 1 μL Sortase A to 15 μL of buffer to form a total of 20 μL of system and mix them evenly, and the peptide is ligated for 2 h; then add 5 μL, 500 μM Ir(OH ₂ ) ₂ Mix evenly, incubate for 30 min in a 37°C constant temperature incubator, carry out electrochemical probe labeling on the metal cyclized iridium complex, and finally inject 5 μL of the labeled reaction solution into the ITO working electrode detection cell for detection;

The experiments were carried out at a constant cell lysate concentration of 15,000 cells and different Sortase A concentrations: 0nM, 2.5nM, 6.25nM, 12.5nM, and 18.5nM. The electrochemiluminescence intensity, the experimental results are shown in Figure 8, as the concentration of Sortase A increases from 0 to 18.5nM, the electrochemiluminescence intensity increases, which is due to the increase of the ligation product P3 of Sortase A; The electrochemiluminescence intensity of the reaction solution in the cell lysate can qualitatively judge the effect of the cell lysate on Sortase A, and detect the activity of Sortase A in the cell lysate.

Each embodiment in this specification is described in a related manner, and the same and similar parts between the various embodiments may be referred to each other, and each embodiment focuses on the differences from other embodiments. In particular, as for the system embodiments, since they are basically similar to the method embodiments, the description is relatively simple, and for related parts, please refer to the partial descriptions of the method embodiments.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in 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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