Preparation method and application of a ratio polypeptide anti-fouling electrochemical sensor for quantitative detection of furin activity

Abstract

The application discloses a preparation method and application of a ratio polypeptide antifouling electrochemical sensor for quantitative detection of Furin activity. The application also discloses a method for quantitative detection of Furin activity by using the prepared ratio polypeptide antifouling electrochemical sensor, in particular, application of the ratio polypeptide antifouling electrochemical sensor in detection of Furin activity in two kinds of cancer cells, i.e., human breast cancer cell MDA-MB-468 and human glioma cell U251, which overexpress Furin. Experimental results show that the ratio polypeptide antifouling electrochemical sensor disclosed by the application can effectively determine Furin in a biological sample, and the method has high accuracy, sensitivity and practicability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical technology, and relates to a novel ratio polypeptide antifouling electrochemical sensor capable of specifically recognizing and detecting Furin. In particular, the present application relates to a preparation and application of a ratio polypeptide antifouling electrochemical sensor based on a macrocyclic compound and capable of being used for quantitative detection of Furin activity. BACKGROUND

[0002] Furin is a secreted protease mainly existing in the network outside the Golgi apparatus, and is an important endogenous protease in eukaryotic cells. It is responsible for the processing of many precursor proteins, and has a wide range of substrates, including most peptide hormones, growth factor receptors, blood coagulation factors and metal matrix proteases, etc., and has important biological functions and research value. Abnormal expression of Furin is closely related to the occurrence of many diseases. In particular, in the brain, the activation and cleavage of Furin on its substrates affect the progress of human neurophysiological activities, and play an important role in the occurrence of some mental diseases. Studies have shown that Furin can activate alpha-secretase and inhibit beta-secretase, and both of these two enzymes can cleave amyloid precursor protein which causes Alzheimer's disease, thereby affecting the development process of the disease. Therefore, developing a method capable of accurately and effectively detecting the activity of Furin is of great significance for further exploring the physiological functions of Furin and its role in related diseases.

[0003] Furin can recognize specific amino acid sequences, which provides the possibility for electrochemical detection. Polypeptide electrochemical sensors have good application prospects in the detection of biological macromolecules due to the good selectivity and flexible structure of polypeptides. However, in the detection environment of complex biological media, sensor contamination caused by non-specific adsorption of biological macromolecules is one of the problems that must be solved. Therefore, many studies have reported polypeptide antifouling sequences, which are composed of alternating positively and negatively charged amino acids and can resist protein adsorption due to strong hydration. However, the essence of polypeptide is a polymer formed by covalent bonding of amino acids through peptide bonds, and proteases present in real samples can destroy the peptide bonds, catalyze the hydrolysis of polypeptides, and reduce the stability and service life of the sensor. Therefore, the currently developed polypeptide antifouling electrochemical sensors still have certain limitations. SUMMARY

[0004] In order to overcome the above-mentioned defects of the prior art, the present application discloses a preparation method of a ratio polypeptide antifouling electrochemical sensor based on a macrocyclic compound and capable of being used for quantitative detection of Furin activity, which is characterized by the following steps:

[0005] Step 1, a glassy carbon electrode (GCE) with a diameter of 3 mm was polished with alumina powder until the surface was smooth, then ultrasonically cleaned in ethanol and deionized water in turn and dried at room temperature to obtain a pretreated glassy carbon electrode;

[0006] Step 2, the glassy carbon electrode obtained in step 1 was drop-coated with a carbon nanotube dispersion (CNT) dispersed in a solvent of DMF:H2O=1:1 with a concentration of 0.5-5 mg / mL and dried under an infrared lamp to obtain a CNT / GCE; gold nanoparticles (AuNPs) were modified on the surface of the CNT / GCE in a chloroauric acid solution by electrodeposition to obtain an AuNPs / CNT / GCE; the AuNPs / CNT / GCE was immersed in a methylene blue solution (MB) with a concentration of 1 μM for 30 min-5 h to modify the reference molecules and obtain an MB / AuNPs / CNT / GCE; finally, the electrode was immersed in a solution of a specific recognition peptide pretreated with tris(2-carboxyethyl)phosphine (TCEP) with a concentration of 5-30 mM for 8-16 h to obtain a peptide / MB / AuNPs / CNT / GCE modified electrode; the concentration of the chloroauric acid was 5 mM, the electrodeposition potential was-0.2 V, and the electrodeposition time was 10-100 s;

[0007] Step 3, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used as catalysts, the concentration ratio of the EDC and NHS catalysts was 2-6:1, the free carboxyl groups in the polypeptide sequence were activated, the signal molecule aminoferrocene (Fc-NH2) was coordinated to the electrode surface through an amide bond reaction, and then the electrode was placed in a water-soluble tetraphenylsulfate solution (MC) and incubated at 4℃ for 2-48 h to obtain a ratio polypeptide antifouling electrochemical sensor; the concentration of the signal molecule Fc-NH2 was 1 mg / mL, the solvent used was ethanol, and the treatment time was 1-5 h.

[0008] The application also discloses a method for quantitatively detecting Furin activity by using the ratio polypeptide antifouling electrochemical sensor prepared by the above method, which is characterized by the following steps:

[0009] Step 1, a Furin standard sample or a corresponding cell lysate was diluted into different concentrations with a phosphate buffer solution (PBS) with a concentration of 10 mM and a pH of 7.4;

[0010] Step 2, the prepared ratio polypeptide antifouling electrochemical sensor was immersed in the solution prepared in step 1 and incubated for 10-50 min at a temperature of 4℃, so that the Furin cut the recognition site on the polypeptide sequence and release the signal molecule;

[0011] Step 3, after the electrode incubated in step 2 is rinsed with PBS buffer solution, it is used as a working electrode, Ag / AgCl is used as a reference electrode and Pt electrode is used as a counter electrode to form a three-electrode system, and electrochemical measurement is carried out; the electrochemical measurement is to obtain a differential pulse voltammogram (DPV), and the parameter settings are as follows: a scanning range is -0.6 V - 0.6 V; an amplitude is 0.05 V; a pulse period is 0.5 s; and a potential increment is 4.0 mV.

[0012] The application further discloses application of the ratio polypeptide antifouling electrochemical sensor prepared by the method in detection of Furin activity in two Furin overexpressed cancer cells, namely human breast cancer cells MDA-MB-468 and human glioma cells U251, and experimental results show that the results obtained by using the sensor prepared by the method to perform electrochemical detection are very consistent with test results obtained by a traditional enzyme-linked immunosorbent assay, and reliability and practicability of the method are proved.

[0013] The application is described in more detail as follows:

[0014] The application discloses a preparation method of a ratio polypeptide antifouling electrochemical sensor based on a macrocyclic compound and capable of being used for quantitative detection of Furin activity.

[0015] Step 1, an alumina powder is used to polish a glassy carbon electrode until the surface is smooth, the glassy carbon electrode is sequentially ultrasonically cleaned in ethanol and deionized water and then is blown dry at room temperature, and a pretreated glassy carbon electrode is obtained.

[0016] Step 2, a carbon nanotube dispersion solution is dropped on the surface of the glassy carbon electrode obtained in step 1, and the glassy carbon electrode is dried under an infrared lamp to obtain a CNT / GCE; AuNPs are modified on the surface of the CNT / GCE by using an electrodeposition method to obtain an AuNPs / CNT / GCE; the AuNPs / CNT / GCE is soaked in an MB solution to modify a reference molecule, and an MB / AuNPs / CNT / GCE is obtained; finally, the electrode is immersed in a solution of a specific recognition polypeptide (previously treated by using TCEP) for a period of time to obtain a peptide / MB / AuNPs / CNT / GCE modified electrode.

[0017] Step 3, EDC and NHS are used as catalysts to activate free carboxyl groups in the polypeptide sequence, so that a signal molecule Fc-NH2 is coordinated to the surface of the electrode through an amide bond reaction, and then the electrode is placed in a water-soluble tetraphenylsulfate solution and is incubated at 4 DEG C to obtain a ratio polypeptide antifouling electrochemical sensor.

[0018] In the step 1, the diameter of the glassy carbon electrode is 3 mm.

[0019] The carbon nanotube dispersion liquid in step 2 is dispersed by a solvent of DMF:H2O=1:1, and the concentration is 0.5-5 mg / mL, preferably 2 mg / mL.

[0020] The concentration of chloroauric acid in step 2 is 5 mM, the electrodeposition potential is-0.2 V, and the electrodeposition time is 10-100 s, preferably 50 s.

[0021] The concentration of the MB solution in step 2 is 1 μM, and the modification time is 30 min-5 h, preferably 3 h.

[0022] The specific sequence of the polypeptide specifically recognized in step 2 is shown in the literature A ratiometricelectrochemical strategy for sensitive determination of Furinactivity based on dual signal amplification and antifouling nanosurfaces, and the processing time is 8-16 h, preferably 12 h.

[0023] The concentration of TCEP in step 2 is 5-30 mM, preferably 10 mM.

[0024] The concentration ratio of EDC and NHS catalyst in step 3 is 2-6:1, preferably 4:1.

[0025] The concentration of the signal molecule Fc-NH2 in step 3 is 1 mg / mL, the solvent used is ethanol, and the processing time is 1-5 h, preferably 2 h.

[0026] The structure of the water-soluble tetraphenyl sulfate salt in step 3 is shown in formula (I) (see the literature Supramolecular Detoxification of Macromolecular Biotoxin through the Complexation by a Large-Sized Macrocycle):

[0027]

[0028] Formula (I)

[0029] The incubation time in step 3 is 2-48 h, preferably 24 h.

[0030] The application also provides a method for quantitatively detecting Furin activity by using the ratio polypeptide anti-fouling electrochemical sensor, comprising the following specific steps:

[0031] Step 1, diluting the Furin standard sample or the corresponding cell lysate into different concentrations with a PBS buffer solution.

[0032] Step 2, incubating the prepared ratio polypeptide anti-fouling electrochemical sensor in the solution prepared in step 1 to make Furin cut the recognition site on the polypeptide sequence and release the signal molecule.

[0033] Step 3, using the electrode incubated in step 2 as a working electrode, using Ag / AgCl as a reference electrode and using a Pt electrode as a counter electrode to form a three-electrode system, and then performing electrochemical measurement.

[0034] In step 1, the PBS buffer solution has a concentration of 10 mM and a pH of 7.4.

[0035] In step 2, the incubation time is 10-50 min, preferably 30 min, and the incubation temperature is 4℃.

[0036] In step 3, the electrochemical measurement is differential pulse voltammetry (DPV), and the parameter settings are as follows: scan range, -0.6 V - 0.6 V; amplitude, 0.05 V; pulse period, 0.5 s; and potential increment, 4.0 mV.

[0037] The application mainly solves the problem of accurately measuring Furin activity by using an electrochemical method, focuses on the anti-fouling performance and stability of the prepared sensor, and the main difficulty lies in using macrocyclic compound modification to improve the anti-protease hydrolysis performance of the polypeptide sensor.

[0038] The application has the beneficial effects that a new ratio polypeptide anti-fouling electrochemical sensor based on macrocyclic compounds is disclosed, and the preparation and application thereof are disclosed. The sensor realizes quantitative detection of Furin activity by specific recognition and cutting of the recognition site on the polypeptide sequence, and therefore has high specificity and high selectivity. The combination of macrocyclic compounds and polypeptides can protect the polypeptides, and through the spatial structure and charge effect thereof, the sensor has good anti-protein adsorption and anti-enzyme hydrolysis performance, and can further improve the stability and detection sensitivity of the sensor. The method can realize electrochemical detection of Furin, has low cost and simple operation, and also provides a new idea for anti-fouling of polypeptide electrochemical sensors, and has important significance for further exploring the physiological and pathological effects of Furin in the human body. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1DPV curves of sensor modification process in Example 2 of the present application; wherein a is bare GCE, b is CNT / GCE, c is AuNPs / CNT / GCE, d is MB / AuNPs / CNT / GCE, e is peptide-MC / MB / AuNPs / CNT / GCE, f is Furin / peptide-MC / MB / AuNPs / CNT / GCE

[0040] b is CNT / GCE, c is AuNPs / CNT / GCE, d is MB / AuNPs / CNT / GCE, e is peptide-MC / MB / AuNPs / CNT / GCE, f is Furin / peptide-MC / MB / AuNPs / CNT / GCE

[0041] Figure 2 DPV curves and linear relationship plots of electrochemical determination of Furin activity in vitro in Example 3 of the present application; a is DPV curves of Furin detection with different activities, b is linear relationship plots made according to the DPV curves

[0042] Figure 3 DPV curves and bar charts of anti-pollution performance test of the sensor in complex human serum in Example 4 of the present application; a is DPV curves of the polypeptide electrochemical sensor without macrocycle modification tested in different concentrations of human serum, b is DPV curves of the polypeptide electrochemical sensor with macrocycle modification tested in different concentrations of human serum, c is bar chart of anti-pollution performance comparison of the two sensors

[0043] Figure 4 Bar chart of selectivity test of the sensor for target Furin in Example 5 of the present application

[0044] Figure 5 Bar chart of anti-hydrolysis performance test of the sensor under the action of protease in Example 6 of the present application

[0045] Figure 6 Bar chart of Furin activity determination of the sensor in cells in Example 7 of the present application DETAILED DESCRIPTION

[0046] The present application is described in detail below with specific embodiments. Unless otherwise specified, the technical means used in the present application are methods well known to those skilled in the art. In addition, the embodiments should be understood as illustrative, rather than limiting the scope of the present application, the essence and scope of the present application are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and amount in these embodiments without departing from the essence and scope of the present application also belong to the protection scope of the present application. The raw materials and reagents used in the present application are commercially available.

[0047] Example 1

[0048] Preparation of ratio polypeptide anti-pollution electrochemical sensor

[0049] The glassy carbon electrode was polished to a smooth surface using alumina powder, then ultrasonically cleaned sequentially in ethanol and deionized water, and dried at room temperature. A 5 μL (2 mg / mL) carbon nanotube dispersion was drop-coated onto the surface, dried under an infrared lamp, and then immersed in a 5 mM chloroauric acid solution for 50 s electrodeposition to modify AuNPs. After rinsing with deionized water, the electrode was immersed in a 1 μM MB solution for 3 h to modify the reference molecule. Then, the electrode was immersed in a solution of a specific recognition peptide (pre-treated with 10 mM TCCEP) for 12 h to obtain a peptide / MB / AuNPs / CNT / GCE modified electrode. The modified electrode surface was placed in a 1 mg / mL Fc-NH2 ethanol solution (containing 0.4 M EDC and 0.1 M NHS as catalysts to activate the carboxyl groups) for 2 h to allow the Fc signal molecule to coordinate to the peptide sequence. Finally, it was immersed in a water-soluble tetraphenylsulfate solution and incubated at 4 °C for 24 h to obtain a ratiometric peptide antifouling electrochemical sensor. Without using water-soluble tetraphenyl sulfate treatment (all other steps are the same), a peptide electrochemical sensor for comparative testing was obtained.

[0050] Example 2

[0051] Characterization of electrode modification

[0052] The sensor fabrication process described in Example 1 was characterized using a CHI832D electrochemical workstation and differential pulse voltammetry in 10 mM PBS solution. Figure 1 As shown, the bare glassy carbon electrode and the modified carbon nanotubes and gold nanoparticles (curves a, b, and c) only show the background current; no oxidation peak appears due to the lack of a redox-active signal molecule. After modifying the reference molecule, an MB signal peak can be detected at -0.32 V (curve d). After immobilizing the specific recognition peptide, signal molecule, and macrocyclic compound on the electrode surface, a new oxidation peak appears at +0.37 V (curve e). This is attributed to the Fc group in the signal molecule, indicating that the amide bond reaction successfully coordinated Fc-NH2 to the peptide sequence and that the macrocyclic compound modification did not affect the peak elution, thus proving the successful construction of the sensor. After incubating the sensor in a Furin solution for a period of time, it was observed that the oxidation peak intensity of the Fc molecule decreased significantly, while the MB signal peak was almost unaffected (curve f). This indicates that Furin successfully recognized and cleaved the recognition site in the peptide sequence, causing the Fc molecule fixed at one end of the peptide to be released and move away from the electrode surface, resulting in a decrease in the Fc oxidation peak signal. This process did not have a significant impact on the reference molecule, proving that the prepared sensor meets the expected design and can perform ratio sensing of Furin activity.

[0053] Example 3

[0054] Electrochemical detection of furin activity

[0055] The ratio polypeptide anti-fouling electrochemical sensor prepared in Example 1 was immersed in different activity of Furin solution for 30 min, and the corresponding differential pulse voltammetry curves were recorded, as shown in Figure 2 , Figure 2 a, I to XII respectively represent the activity of Furin is 1 U L -1 , 5 U L -1 , 10 U L -1 , 20 U L -1 , 30 U L -1 , 40 U L -1 , 50 U L -1 , 60 U L -1 , 70 U L -1 , 80 U L -1 , 90 U L -1 , 100 U L -1 . From Figure 2 a, it can be seen that with the increase of Furin activity, the reference peak current remains basically unchanged, while the DPV response of Fc oxidation peak gradually decreases, because the increase of Furin activity leads to more polypeptide recognition sites on the sensor interface being cut off, and more Fc signal molecules being released. From Figure 2 b, it can be seen that within the activity range of 1-90 U L -1 , the value of ΔI Fc / I MB and Furin activity presents a good linear relationship, and the detection limit is 0.83 U L -1 . This shows that the ratio polypeptide anti-fouling electrochemical sensor constructed by this method can effectively quantitatively detect Furin activity, and has high accuracy and sensitivity.

[0056] Example 4

[0057] Anti-fouling test

[0058] The ratio polypeptide anti-fouling electrochemical sensor prepared in Example 1 was incubated in different concentrations of complex human serum for 30 min, and the anti-fouling performance of the sensor was evaluated by recording the DPV signal change before and after incubation. As shown in Figure 3 , Figure 3 a is the DPV test curve of the contrast electrode without adding water-soluble tetraphenyl sulfate macrocyclic modification, Figure 3 b is the DPV test curve of the electrode prepared according to the steps described in Example 1, Figure 3c is the signal inhibition histogram of different electrodes in different concentrations of human serum. From the results shown in the figure, after the water-soluble tetraphenyl sulfate macrocycle is modified, the sensor shows better anti-pollution performance in a complex detection environment. Even under the test conditions of 100% human serum, the signal inhibition is only 3.3%. The above results show that the addition of water-soluble tetraphenyl sulfate macrocycle can effectively resist the non-specific adsorption of biological macromolecules, and the ratio polypeptide anti-pollution electrochemical sensor prepared by the method indeed has good anti-pollution performance.

[0059] Example 5

[0060] Selectivity test

[0061] In order to evaluate the selectivity of the sensor for Furin detection, the present application selects a series of metal ions, amino acids and biological macromolecules as potential interferents for testing, and the results are shown in Figure 4 Except for cysteine, the signal interference caused by the rest of the metal ions and amino acids is less than 3%, and the signal interference caused by biological macromolecules BSA, lgG, AFP and CEA is less than 5%. The above results show that the introduced interfering substances have no obvious effect on the detection of the target substance Furin, and the specificity of Furin for recognizing and cutting specific peptide sequence sites makes the ratio polypeptide anti-pollution electrochemical sensor prepared in Example 1 have good selectivity.

[0062] Example 6

[0063] Anti-protease hydrolysis test

[0064] In the detection process of actual samples, in addition to the problem of sensor pollution caused by non-specific adsorption of biological macromolecules, the destruction of peptide bonds in polypeptide sequences by proteolytic enzymes is also one of the problems that must be considered. In order to evaluate the anti-hydrolysis performance of the sensor, the present application uses two common proteolytic enzymes, trypsin and carboxypeptidase Y, for corresponding tests. The sensor prepared in Example 1 is treated in 0.02 mg / mL trypsin solution and carboxypeptidase Y solution for 5 h, and then immersed in 20% human serum sample for 30 min before testing. The results are shown in Figure 5 The signal inhibition of the sensor without macrocycle compound modification in 20% human serum sample after trypsin and carboxypeptidase Y hydrolysis treatment increases significantly to 11.2% and 12.3%, respectively, while the sensor prepared according to the steps described in Example 1 does not show obvious change in signal inhibition after the same treatment, which shows that the introduction of water-soluble tetraphenyl sulfate macrocycle has a good protective effect on the polypeptide sequence, can resist protease hydrolysis, and further enhances the detection stability of the sensor.

[0065] Example 7

[0066] Detection of furin activity in cells

[0067] The ratio polypeptide anti-fouling electrochemical sensor prepared in Example 1 was used to detect the furin activity of two cancer cells, human breast cancer cell MDA-MB-468 and human glioma cell U251, which overexpress furin (the number of cells was 10 4 ), and the test results were compared with those of the traditional enzyme-linked immunosorbent assay, as shown in Figure 6 The experimental results show that the test results obtained by the two methods are very consistent, which indicates that the ratio polypeptide anti-fouling electrochemical sensor disclosed in the present application can effectively detect furin in a biological sample, and the method has certain practicability.

[0068] The protection scope of the present application is not limited to the above examples. Changes and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. A method for the preparation of a ratio polypeptide antifouling electrochemical sensor based on macrocycles for the quantitative detection of Furin activity, characterized by, The following steps are taken: Step 1, a glassy carbon electrode with a diameter of 3 mm is polished with alumina powder until the surface is smooth, then ultrasonic cleaned in ethanol and deionized water in turn, and dried at room temperature to obtain a pretreated glassy carbon electrode; Step 2, drop the carbon nanotube dispersion solution on the surface of the glassy carbon electrode obtained in step 1, the carbon nanotube dispersion solution is dispersed in DMF:H2O=1:1 solvent, the concentration is 0.5-5 mg / mL, and dried under infrared lamp to obtain CNT / GCE; AuNPs are modified on the surface of CNT / GCE in chloroauric acid solution by electrodeposition to obtain AuNPs / CNT / GCE; the AuNPs / CNT / GCE is soaked in a 1 μM MB solution to modify the reference molecule, the modification time is 30 min-5 h, and MB / AuNPs / CNT / GCE is obtained; finally, the electrode is immersed in a solution of specific recognition polypeptide pretreated with a concentration of 5-30 mM TCEP for 8-16 h to obtain a peptide / MB / AuNPs / CNT / GCE modified electrode; the concentration of chloroauric acid is 5 mM, the electrodeposition potential is-0.2 V, and the electrodeposition time is 10-100 s; Step 3, using EDC and NHS as catalysts, the concentration ratio of EDC and NHS catalysts is 2-6:1, the free carboxyl group in the polypeptide sequence is activated, and the signal molecule Fc-NH2 is coordinated to the electrode surface through amide bond reaction, and then it is placed in a water-soluble tetraphenylsulfate solution and incubated at 4℃ for 2-48 h to obtain a ratio polypeptide antifouling electrochemical sensor; the concentration of the signal molecule Fc-NH2 is 1 mg / mL, the solvent used is ethanol, and the treatment time is 1-5 h.

2. A method for the quantitative detection of Furin activity using a ratio polypeptide anti-fouling electrochemical sensor prepared according to the method of claim 1, characterized in that, The following steps are taken: Step 1, dilute the Furin standard sample or the corresponding cell lysate with a phosphate buffer solution (PBS) with a concentration of 10 mM and a pH of 7.4 to different concentrations; Step 2, immerse the prepared ratio polypeptide antifouling electrochemical sensor in the solution prepared in step 1 and incubate for 10-50 min at 4℃ to allow Furin to cleave the recognition site on the polypeptide sequence and release the signal molecule; Step 3, use the PBS buffer solution to rinse the electrode incubated in step 2 as the working electrode, Ag / AgCl as the reference electrode and Pt electrode as the counter electrode to form a three-electrode system for electrochemical measurement; the electrochemical measurement is to obtain a differential pulse voltammogram (DPV) with the following parameters: scan range, -0.6 V - 0.6 V; amplitude, 0.05 V; pulse period, 0.5 s; and potential increment, 4.0 mV.

3. Application of the ratio polypeptide antifouling electrochemical sensor prepared by the method of claim 1 in the detection of Furin activity in specific human breast cancer cell MDA-MB-468 and human glioma cell U251, both of which overexpress Furin. ​

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